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Sample records for methylation status correlated

  1. Axin gene methylation status correlates with radiosensitivity of lung cancer cells

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    Yang, Lian-He; Stoecker, Maggie; Wang, Endi; Xu, Ke; Wang, En-Hua; Han, Yang; Li, Guang; Xu, Hong-Tao; Jiang, Gui-Yang; Miao, Yuan; Zhang, Xiu-Peng; Zhao, Huan-Yu; Xu, Zheng-Fan

    2013-01-01

    We previously reported that Axin1 (Axin) is down-regulated in many cases of lung cancer, and X-ray irradiation increased Axin expression and inhibited lung cancer cells. The mechanisms, however, were not clear. Four lung cancer cell lines were used to detect the methylation status of Axin with or without X-ray treatment. Real-time PCR was used to quantify the expression of Axin, and western blot analysis was applied to measure protein levels of Axin, β-catenin, Cyclin D1, MMP-7, DNMTS, MeCP2 and acetylated histones. Flow cytometric analysis, colony formation assay, transwell assay and xenograft growth experiment were used to study the biological behavior of the cells with hypermethylated or unmethylated Axin gene after X-ray treatment. Hypermethylated Axin gene was detected in 2 of 4 cell lines, and it correlated inversely with Axin expression. X-ray treatment significantly up-regulated Axin expression in H446 and H157 cells, which possess intrinsic hypermethylation of the Axin gene (P<0.01), but did not show up-regulation in LTE and H460 cells, which have unmethylated Axin gene. 2Gy X-ray significantly reduced colony formation (from 71% to 10.5%) in H157 cells, while the reduction was lower in LTE cells (from 71% to 20%). After X-ray irradiation, xenograft growth was significantly decreased in H157 cells (from 1.15 g to 0.28 g) in comparison with LTE cells (from 1.06 g to 0.65 g). Significantly decreased cell invasiveness and increased apoptosis were also observed in H157 cells treated with X-ray irradiation (P<0.01). Down-regulation of DNMTs and MeCP2 and up-regulation of acetylated histones could be detected in lung cancer cells. X-ray-induced inhibition of lung cancer cells may be mediated by enhanced expression of Axin via genomic DNA demethylation and histone acetylation. Lung cancer cells with a different methylation status of the Axin gene showed different radiosensitivity, suggesting that the methylation status of the Axin gene may be one important factor

  2. MRI texture features as biomarkers to predict MGMT methylation status in glioblastomas

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    Korfiatis, Panagiotis; Kline, Timothy L.; Erickson, Bradley J., E-mail: bje@mayo.edu [Department of Radiology, Mayo Clinic, 200 1st Street SW, Rochester, Minnesota 55905 (United States); Coufalova, Lucie [Department of Radiology, Mayo Clinic, 200 1st Street SW, Rochester, Minnesota 55905 (United States); Department of Neurosurgery of First Faculty of Medicine, Charles University in Prague, Military University Hospital, Prague 128 21 (Czech Republic); International Clinical Research Center, St. Anne’s University Hospital Brno, Brno 656 91 (Czech Republic); Lachance, Daniel H. [Department of Neurology, Mayo Clinic, 200 1st Street SW, Rochester, Minnesota 55905 (United States); Parney, Ian F. [Department of Neurologic Surgery, Mayo Clinic, 200 1st Street SW, Rochester, Minnesota 55905 (United States); Carter, Rickey E. [Department of Health Sciences Research, Mayo Clinic, 200 1st Street SW, Rochester, Minnesota 55905 (United States); Buckner, Jan C. [Department of Medical Oncology, Mayo Clinic, 200 1st Street SW, Rochester, Minnesota 55905 (United States)

    2016-06-15

    Purpose: Imaging biomarker research focuses on discovering relationships between radiological features and histological findings. In glioblastoma patients, methylation of the O{sup 6}-methylguanine methyltransferase (MGMT) gene promoter is positively correlated with an increased effectiveness of current standard of care. In this paper, the authors investigate texture features as potential imaging biomarkers for capturing the MGMT methylation status of glioblastoma multiforme (GBM) tumors when combined with supervised classification schemes. Methods: A retrospective study of 155 GBM patients with known MGMT methylation status was conducted. Co-occurrence and run length texture features were calculated, and both support vector machines (SVMs) and random forest classifiers were used to predict MGMT methylation status. Results: The best classification system (an SVM-based classifier) had a maximum area under the receiver-operating characteristic (ROC) curve of 0.85 (95% CI: 0.78–0.91) using four texture features (correlation, energy, entropy, and local intensity) originating from the T2-weighted images, yielding at the optimal threshold of the ROC curve, a sensitivity of 0.803 and a specificity of 0.813. Conclusions: Results show that supervised machine learning of MRI texture features can predict MGMT methylation status in preoperative GBM tumors, thus providing a new noninvasive imaging biomarker.

  3. MRI texture features as biomarkers to predict MGMT methylation status in glioblastomas

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    Korfiatis, Panagiotis; Kline, Timothy L.; Erickson, Bradley J.; Coufalova, Lucie; Lachance, Daniel H.; Parney, Ian F.; Carter, Rickey E.; Buckner, Jan C.

    2016-01-01

    Purpose: Imaging biomarker research focuses on discovering relationships between radiological features and histological findings. In glioblastoma patients, methylation of the O 6 -methylguanine methyltransferase (MGMT) gene promoter is positively correlated with an increased effectiveness of current standard of care. In this paper, the authors investigate texture features as potential imaging biomarkers for capturing the MGMT methylation status of glioblastoma multiforme (GBM) tumors when combined with supervised classification schemes. Methods: A retrospective study of 155 GBM patients with known MGMT methylation status was conducted. Co-occurrence and run length texture features were calculated, and both support vector machines (SVMs) and random forest classifiers were used to predict MGMT methylation status. Results: The best classification system (an SVM-based classifier) had a maximum area under the receiver-operating characteristic (ROC) curve of 0.85 (95% CI: 0.78–0.91) using four texture features (correlation, energy, entropy, and local intensity) originating from the T2-weighted images, yielding at the optimal threshold of the ROC curve, a sensitivity of 0.803 and a specificity of 0.813. Conclusions: Results show that supervised machine learning of MRI texture features can predict MGMT methylation status in preoperative GBM tumors, thus providing a new noninvasive imaging biomarker.

  4. Subsets of microsatellite-unstable colorectal cancers exhibit discordance between the CpG island methylator phenotype and MLH1 methylation status.

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    Kim, Jung H; Rhee, Ye-Y; Bae, Jeong-M; Kwon, Hyeong-J; Cho, Nam-Y; Kim, Mi J; Kang, Gyeong H

    2013-07-01

    Although the presence of MLH1 methylation in microsatellite-unstable colorectal cancer generally indicates involvement of the CpG island methylator phenotype (CIMP) in the development of the tumor, these two conditions do not always correlate. A minority of microsatellite-unstable colorectal cancers exhibit discordance between CIMP and MLH1 methylation statuses. However, the clinicopathological features of such microsatellite-unstable colorectal cancers with discrepant MLH1 methylation and CIMP statuses remain poorly studied. Microsatellite-unstable colorectal cancers (n=220) were analyzed for CIMP and MLH1 methylation statuses using the MethyLight assay. Based on the combinatorial CIMP and MLH1 methylation statuses, the microsatellite-unstable colorectal cancers were grouped into four subtypes (CIMP-high (CIMP-H) MLH1 methylation-positive (MLH1m+), CIMP-H MLH1 methylation-negative, CIMP-low/0 (CIMP-L/0) MLH1m+, and CIMP-L/0 MLH1 methylation-negative), which were compared in terms of their associations with clinicopathological and molecular features. The CIMP-L/0 MLH1 methylation-negative and CIMP-H MLH1m+ subtypes were predominant, comprising 63.6 and 24.1% of total microsatellite-unstable colorectal cancers, respectively. The discordant subtypes, CIMP-H MLH1 methylation-negative and CIMP-L/0 MLH1m+, were found in 5 and 7% of microsatellite-unstable colorectal cancers, respectively. The CIMP-H MLH1 methylation-negative subtype exhibited elevated incidence rates in male patients and was associated with larger tumor size, more frequent loss of MSH2 expression, increased frequency of KRAS mutation, and advanced cancer stage. The CIMP-L/0 MLH1m+ subtype was associated with onset at an earlier age, a predominance of MLH1 loss, and earlier cancer stage. None of the CIMP-L/0 MLH1m+ subtype patients succumbed to death during the follow-up. Our findings suggest that the discordant subtypes of colorectal cancers exhibit distinct clinicopathological and molecular features

  5. A common mutation in the 5,10-methylenetetrahydrofolate reductase gene affects genomic DNA methylation through an interaction with folate status

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    Friso, Simonetta; Choi, Sang-Woon; Girelli, Domenico; Mason, Joel B.; Dolnikowski, Gregory G.; Bagley, Pamela J.; Olivieri, Oliviero; Jacques, Paul F.; Rosenberg, Irwin H.; Corrocher, Roberto; Selhub, Jacob

    2002-01-01

    DNA methylation, an essential epigenetic feature of DNA that modulates gene expression and genomic integrity, is catalyzed by methyltransferases that use the universal methyl donor S-adenosyl-l-methionine. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the synthesis of 5-methyltetrahydrofolate (5-methylTHF), the methyl donor for synthesis of methionine from homocysteine and precursor of S-adenosyl-l-methionine. In the present study we sought to determine the effect of folate status on genomic DNA methylation with an emphasis on the interaction with the common C677T mutation in the MTHFR gene. A liquid chromatography/MS method for the analysis of nucleotide bases was used to assess genomic DNA methylation in peripheral blood mononuclear cell DNA from 105 subjects homozygous for this mutation (T/T) and 187 homozygous for the wild-type (C/C) MTHFR genotype. The results show that genomic DNA methylation directly correlates with folate status and inversely with plasma homocysteine (tHcy) levels (P < 0.01). T/T genotypes had a diminished level of DNA methylation compared with those with the C/C wild-type (32.23 vs.62.24 ng 5-methylcytosine/μg DNA, P < 0.0001). When analyzed according to folate status, however, only the T/T subjects with low levels of folate accounted for the diminished DNA methylation (P < 0.0001). Moreover, in T/T subjects DNA methylation status correlated with the methylated proportion of red blood cell folate and was inversely related to the formylated proportion of red blood cell folates (P < 0.03) that is known to be solely represented in those individuals. These results indicate that the MTHFR C677T polymorphism influences DNA methylation status through an interaction with folate status. PMID:11929966

  6. Relationship between methylation status of vitamin D-related genes, vitamin D levels, and methyl-donor biochemistry

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    Emma Louise Beckett

    2016-12-01

    Full Text Available Vitamin D is known for its role in the regulation of gene expression via the vitamin D receptor, a nuclear transcription factor. More recently, a role for vitamin D in regulating DNA methylation has been identified as an additional mechanism of modulation of gene expression. How methylation status influences vitamin D metabolism and response pathways is not yet clear. Therefore, we aimed to assess the relationship between plasma 25-hydroxycholecalciferol (25(OHD and the methylation status of vitamin D metabolism enzyme genes (CYP2R1, CYP27B1 and CYP24A1 and the vitamin D receptor gene (VDR. This analysis was conducted in the context of dietary vitamin D, and background methyl donor related biochemistry, with adjustment for several dietary and lifestyle variables. Percentage methylation at CpG sites was assessed in peripheral blood cells using methylation sensitive and dependent enzymes and qPCR. Standard analytical techniques were used to determine plasma 25(OHD and homocysteine, and serum folate and B12, with the relationship to methylation status assessed using multi-variable regression analysis. CYP2R1 and VDR methylation were found to be independent predictors of plasma 25(OHD, when adjusted for vitamin D intake and other lifestyle variables. CYP24A1 was related to plasma 25(OHD directly, but not in the context of vitamin D intake. Methyl-group donor biochemistry was associated with the methylation status of some genes, but did not alter the relationship between methylation and plasma 25(OHD. Modulation of methylation status of CYP2R1, CYP24A1 and VDR in response to plasma 25(OHD may be part of feedback loops involved in maintaining vitamin D homeostasis, and may explain a portion of the variance in plasma 25(OHD levels in response to intake and sun exposure. Methyl-group donor biochemistry, while a potential independent modulator, did not alter this effect.

  7. Differential DNA methylation patterns define status epilepticus and epileptic tolerance.

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    Miller-Delaney, Suzanne F C; Das, Sudipto; Sano, Takanori; Jimenez-Mateos, Eva M; Bryan, Kenneth; Buckley, Patrick G; Stallings, Raymond L; Henshall, David C

    2012-02-01

    Prolonged seizures (status epilepticus) produce pathophysiological changes in the hippocampus that are associated with large-scale, wide-ranging changes in gene expression. Epileptic tolerance is an endogenous program of cell protection that can be activated in the brain by previous exposure to a non-harmful seizure episode before status epilepticus. A major transcriptional feature of tolerance is gene downregulation. Here, through methylation analysis of 34,143 discrete loci representing all annotated CpG islands and promoter regions in the mouse genome, we report the genome-wide DNA methylation changes in the hippocampus after status epilepticus and epileptic tolerance in adult mice. A total of 321 genes showed altered DNA methylation after status epilepticus alone or status epilepticus that followed seizure preconditioning, with >90% of the promoters of these genes undergoing hypomethylation. These profiles included genes not previously associated with epilepsy, such as the polycomb gene Phc2. Differential methylation events generally occurred throughout the genome without bias for a particular chromosomal region, with the exception of a small region of chromosome 4, which was significantly overrepresented with genes hypomethylated after status epilepticus. Surprisingly, only few genes displayed differential hypermethylation in epileptic tolerance. Nevertheless, gene ontology analysis emphasized the majority of differential methylation events between the groups occurred in genes associated with nuclear functions, such as DNA binding and transcriptional regulation. The present study reports select, genome-wide DNA methylation changes after status epilepticus and in epileptic tolerance, which may contribute to regulating the gene expression environment of the seizure-damaged hippocampus.

  8. Prognostic role of APC and RASSF1A promoter methylation status in cell free circulating DNA of operable gastric cancer patients.

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    Balgkouranidou, I; Matthaios, D; Karayiannakis, A; Bolanaki, H; Michailidis, P; Xenidis, N; Amarantidis, K; Chelis, L; Trypsianis, G; Chatzaki, E; Lianidou, E S; Kakolyris, S

    2015-08-01

    Gastric carcinogenesis is a multistep process including not only genetic mutations but also epigenetic alterations. The best known and more frequent epigenetic alteration is DNA methylation affecting tumor suppressor genes that may be involved in various carcinogenetic pathways. The aim of the present study was to investigate the methylation status of APC promoter 1A and RASSF1A promoter in cell free DNA of operable gastric cancer patients. Using methylation specific PCR, we examined the methylation status of APC promoter 1A and RASSF1A promoter in 73 blood samples obtained from patients with gastric cancer. APC and RASSF1A promoters were found to be methylated in 61 (83.6%) and 50 (68.5%) of the 73 gastric cancer samples examined, but in none of the healthy control samples (p APC promoter and elevated CEA (p = 0.033) as well as CA-19.9 (p = 0.032) levels, was noticed. The Kaplan-Meier estimates of survival, significantly favored patients with a non-methylated APC promoter status (p = 0.008). No other significant correlations between APC and RASSF1A methylation status and different tumor variables examined was observed. Serum RASSF1A and APC promoter hypermethylation is a frequent epigenetic event in patients with early operable gastric cancer. The observed correlations between APC promoter methylation status and survival as well as between a hypermethylated RASSF1A promoter and nodal positivity may be indicative of a prognostic role for those genes in early operable gastric cancer. Additional studies, in a larger cohort of patients are required to further explore whether these findings could serve as potential molecular biomarkers of survival and/or response to specific treatments. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. EG-15THE METHYLATION STATUS OF MGMT IN MEDULLOBLASTOMA

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    Shimizu, Yuzaburo; Kurimoto, Tomoko; Kondo, Akihide; Arai, Hajime

    2014-01-01

    BACKGROUND: Medulloblastoma is a highly malignant brain tumor in childhood. Some studies reported that alkylating chemotherapeutic drugs are effective agents in the treatment of patients with medulloblastoma. O6-methylguanine-DNA methyltransferase (MGMT) is one of the DNA repair enzymes and plays a significant role in tumor resistance to alkylating agents. Low MGMT expression or MGMT promoter methylation have been found to be associated with favorable outcomes in malignant glioma patients treated with alkylating agents such as temozolomide. However, impact of MGMT status on clinical outcomes in medulloblastoma patients is not fully evaluated. OBJECTIVE: The objective of this study is to investigate the association between MGMT status and the response for chemotherapy in pediatric patients with medulloblastoma. METHODS: Patients with medulloblastoma treated at our institution between 1995 and 2012 were reviewed retrospectively. Relevant clinical information including current disease status, tumor response to chemotherapy was obtained from the hospital charts. To evaluate the MGMT status, we performed bisulfite sequencing analysis to determine the methylation status of the MGMT promoter. RESULTS: Tumor material and detailed clinical information were available in 22 patients. Of them, 13 patients were alive (11 in CR), seven died of disease and two were lost to follow up. Five patients were with dissemination at diagnosis. We succeeded to evaluate both the MGMT status of tumors and the number of methylation sites in MGMT promoter. CONCLUSIONS: We studied the prognostic value of MGMT promoter methylation in medulloblastoma children.

  10. Methylation screening of the TGFBI promoter in human lung and prostate cancer by methylation-specific PCR

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    Shah, Jinesh N; Shao, Genze; Hei, Tom K; Zhao, Yongliang

    2008-01-01

    Hypermethylation of the TGFBI promoter has been shown to correlate with decreased expression of this gene in human tumor cell lines. In this study, we optimized a methylation-specific polymerase chain reaction (MSP) method and investigated the methylation status of the TGFBI promoter in human lung and prostate cancer specimens. Methylation-specific primers were designed based on the methylation profiles of the TGFBI promoter in human tumor cell lines, and MSP conditions were optimized for accurate and efficient amplification. Genomic DNA was isolated from lung tumors and prostatectomy tissues of prostate cancer patients, bisulfite-converted, and analyzed by MSP. Among 50 lung cancer samples, 44.0% (22/50) harbored methylated CpG sites in the TGFBI promoter. An analysis correlating gene methylation status with clinicopathological cancer features revealed that dense methylation of the TGFBI promoter was associated with a metastatic phenotype, with 42.9% (6/14) of metastatic lung cancer samples demonstrating dense methylation vs. only 5.6% (2/36) of primary lung cancer samples (p < 0.05). Similar to these lung cancer results, 82.0% (41/50) of prostate cancer samples harbored methylated CpG sites in the TGFBI promoter, and dense methylation of the promoter was present in 38.9% (7/18) of prostate cancer samples with the feature of locoregional invasiveness vs. only 19.4% (6/31) of prostate cancer samples without locoregional invasiveness (p < 0.05). Furthermore, promoter hypermethylation correlated with highly reduced expression of the TGFBI gene in human lung and prostate tumor cell lines. We successfully optimized a MSP method for the precise and efficient screening of TGFBI promoter methylation status. Dense methylation of the TGFBI promoter correlated with the extent of TGFBI gene silencing in tumor cell lines and was related to invasiveness of prostate tumors and metastatic status of lung cancer tumors. Thus, TGFBI promoter methylation can be used as a potential

  11. Methylation status of individual CpG sites within Alu elements in the human genome and Alu hypomethylation in gastric carcinomas

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    Xiang, Shengyan; Liu, Zhaojun; Zhang, Baozhen; Zhou, Jing; Zhu, Bu-Dong; Ji, Jiafu; Deng, Dajun

    2010-01-01

    Alu methylation is correlated with the overall level of DNA methylation and recombination activity of the genome. However, the maintenance and methylation status of each CpG site within Alu elements (Alu) and its methylation status have not well characterized. This information is useful for understanding natural status of Alu in the genome and helpful for developing an optimal assay to quantify Alu hypomethylation. Bisulfite clone sequencing was carried out in 14 human gastric samples initially. A Cac8I COBRA-DHPLC assay was developed to detect methylated-Alu proportion in cell lines and 48 paired gastric carcinomas and 55 gastritis samples. DHPLC data were statistically interpreted using SPSS version 16.0. From the results of 427 Alu bisulfite clone sequences, we found that only 27.2% of CpG sites within Alu elements were preserved (4.6 of 17 analyzed CpGs, A ~ Q) and that 86.6% of remaining-CpGs were methylated. Deamination was the main reason for low preservation of methylation targets. A high correlation coefficient of methylation was observed between Alu clones and CpG site J (0.963), A (0.950), H (0.946), D (0.945). Comethylation of the sites H and J were used as an indicator of the proportion of methylated-Alu in a Cac8I COBRA-DHPLC assay. Validation studies showed that hypermethylation or hypomethylation of Alu elements in human cell lines could be detected sensitively by the assay after treatment with 5-aza-dC and M.SssI, respectively. The proportion of methylated-Alu copies in gastric carcinomas (3.01%) was significantly lower than that in the corresponding normal samples (3.19%) and gastritis biopsies (3.23%). Most Alu CpG sites are deaminated in the genome. 27% of Alu CpG sites represented in our amplification products. 87% of the remaining CpG sites are methylated. Alu hypomethylation in primary gastric carcinomas could be detected with the Cac8I COBRA-DHPLC assay quantitatively

  12. Residual Deep Convolutional Neural Network Predicts MGMT Methylation Status.

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    Korfiatis, Panagiotis; Kline, Timothy L; Lachance, Daniel H; Parney, Ian F; Buckner, Jan C; Erickson, Bradley J

    2017-10-01

    Predicting methylation of the O6-methylguanine methyltransferase (MGMT) gene status utilizing MRI imaging is of high importance since it is a predictor of response and prognosis in brain tumors. In this study, we compare three different residual deep neural network (ResNet) architectures to evaluate their ability in predicting MGMT methylation status without the need for a distinct tumor segmentation step. We found that the ResNet50 (50 layers) architecture was the best performing model, achieving an accuracy of 94.90% (+/- 3.92%) for the test set (classification of a slice as no tumor, methylated MGMT, or non-methylated). ResNet34 (34 layers) achieved 80.72% (+/- 13.61%) while ResNet18 (18 layers) accuracy was 76.75% (+/- 20.67%). ResNet50 performance was statistically significantly better than both ResNet18 and ResNet34 architectures (p deep neural architectures can be used to predict molecular biomarkers from routine medical images.

  13. Correlation between the methylation of APC gene promoter and colon cancer.

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    Li, Bing-Qiang; Liu, Peng-Peng; Zhang, Cai-Hua

    2017-08-01

    The present study was planned to explore the correlation between the methylation of APC (adenomatous polyposis coli) and colon carcinogenesis. Colon cancer tissues and tumor-adjacent normal tissues of 60 colon cancer patients (who received surgical operation in our hospital from January 2012 to December 2014) were collected. SW1116 cells in human colon cancer tissues were selected for culturing. 5-aza-2c-deoxycytidine (5-aza-dC) was utilized as an inhibitor of the methylation for APC gene. Methylation specific PCR (MSP) was utilized for detection of APC methylation in SW1116 cells. The MTT and Transwell assays were performed to detect the effect of the methylation of APC gene on the proliferation and invasive abilities of SW1116 cells. The correlation between the methylation of APC gene and pathological parameters of colon cancer patients was analyzed. MSP results revealed that 41 cases (68.33%) showed methylation of APC gene in colon cancer tissues. No methylation of APC gene was found in tumor-adjacent normal tissues. 5-aza-dC was able to inhibit the methylation of APC gene in SW1116 cells. APC gene methylation was correlated with tumor size, differentiation degree, lymph node metastasis and Dukes staging. In conclusion, the levels of the methylation of APC in colon cancer tissues and SW1116 cells are relatively high. The methylation of APC promoted the proliferation and invasion abilities of SW1116 cells. Furthermore, methylation is correlated with a variety of clinicopathological features of colon cancer patients.

  14. Clinical and prognosis value of the CIMP status combined with MLH1 or p16 INK4a methylation in colorectal cancer.

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    Saadallah-Kallel, Amana; Abdelmaksoud-Dammak, Rania; Triki, Mouna; Charfi, Slim; Khabir, Abdelmajid; Sallemi-Boudawara, Tahia; Mokdad-Gargouri, Raja

    2017-08-01

    Aberrant DNA methylation of CpG islands occurred frequently in CRC and associated with transcriptional silencing of key genes. In this study, the CIMP combined with MLH1 or p16 INK4a methylation status was determined in CRC patients and correlated with clinicopathological parameters and overall survival. Our data showed that CIMP+ CRCs were identified in 32.9% of cases and that CACNAG1 is the most frequently methylated promoter. When we combined the CIMP with the MLH1 or the p16 INK4a methylation status, we found that CIMP-/MLH1-U (37.8%) and CIMP-/p16 INK4a -U (35.4%) tumors were the most frequent among the four subtypes. Statistical analysis showed that tumor location, lymphovascular invasion, TNM stage, and MSI differed among the group of patients. Kaplan-Meier analyses revealed differences in overall survival according to the CIMP combined with MLH1 or p16 INK4a methylation status. In a multivariate analysis, CIMP/MLH1 and CIMP/p16 INK4a methylation statuses were predictive of prognosis, and the OS was longer for patients with tumors CIMP-/MLH1-M, as well as CIMP-/p16 INK4a -M. Furthermore, DNMT1 is significantly overexpressed in tumors than in normal tissues as well as in CIMP+ than CIMP- tumors. Our results suggest that tumor classification based on the CIMP status combined with MLH1 or p16 INK4a methylation is useful to predict prognosis in CRC patients.

  15. Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1).

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    Nwaobi, Sinifunanya E; Olsen, Michelle L

    2015-09-26

    DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

  16. Endometrial tumour BRAF mutations and MLH1 promoter methylation as predictors of germline mismatch repair gene mutation status: a literature review.

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    Metcalf, Alexander M; Spurdle, Amanda B

    2014-03-01

    Colorectal cancer (CRC) that displays high microsatellite instability (MSI-H) can be caused by either germline mutations in mismatch repair (MMR) genes, or non-inherited transcriptional silencing of the MLH1 promoter. A correlation between MLH1 promoter methylation, specifically the 'C' region, and BRAF V600E status has been reported in CRC studies. Germline MMR mutations also greatly increase risk of endometrial cancer (EC), but no systematic review has been undertaken to determine if these tumour markers may be useful predictors of MMR mutation status in EC patients. Endometrial cancer cohorts meeting review inclusion criteria encompassed 2675 tumours from 20 studies for BRAF V600E, and 447 tumours from 11 studies for MLH1 methylation testing. BRAF V600E mutations were reported in 4/2675 (0.1%) endometrial tumours of unknown MMR mutation status, and there were 7/823 (0.9%) total sequence variants in exon 11 and 27/1012 (2.7%) in exon 15. Promoter MLH1 methylation was not observed in tumours from 32 MLH1 mutation carriers, or for 13 MSH2 or MSH6 mutation carriers. MMR mutation-negative individuals with tumour MLH1 and PMS2 IHC loss displayed MLH1 methylation in 48/51 (94%) of tumours. We have also detailed specific examples that show the importance of MLH1 promoter region, assay design, and quantification of methylation. This review shows that BRAF mutations occurs so infrequently in endometrial tumours they can be discounted as a useful marker for predicting MMR-negative mutation status, and further studies of endometrial cohorts with known MMR mutation status are necessary to quantify the utility of tumour MLH1 promoter methylation as a marker of negative germline MMR mutation status in EC patients.

  17. Widespread promoter methylation of synaptic plasticity genes in long-term potentiation in the adult brain in vivo.

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    Maag, Jesper L V; Kaczorowski, Dominik C; Panja, Debabrata; Peters, Timothy J; Bramham, Clive R; Wibrand, Karin; Dinger, Marcel E

    2017-03-23

    DNA methylation is a key modulator of gene expression in mammalian development and cellular differentiation, including neurons. To date, the role of DNA modifications in long-term potentiation (LTP) has not been explored. To investigate the occurrence of DNA methylation changes in LTP, we undertook the first detailed study to describe the methylation status of all known LTP-associated genes during LTP induction in the dentate gyrus of live rats. Using a methylated DNA immunoprecipitation (MeDIP)-array, together with previously published matched RNA-seq and public histone modification data, we discover widespread changes in methylation status of LTP-genes. We further show that the expression of many LTP-genes is correlated with their methylation status. We show that these correlated genes are enriched for RNA-processing, active histone marks, and specific transcription factors. These data reveal that the synaptic activity-evoked methylation changes correlates with pre-existing activation of the chromatin landscape. Finally, we show that methylation of Brain-derived neurotrophic factor (Bdnf) CpG-islands correlates with isoform switching from transcripts containing exon IV to exon I. Together, these data provide the first evidence of widespread regulation of methylation status in LTP-associated genes.

  18. The global DNA methylation surrogate LINE-1 methylation is correlated with MGMT promoter methylation and is a better prognostic factor for glioma.

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    Fumiharu Ohka

    Full Text Available Gliomas are the most frequently occurring primary brain tumor in the central nervous system of adults. Glioblastoma multiformes (GBMs, WHO grade 4 have a dismal prognosis despite the use of the alkylating agent, temozolomide (TMZ, and even low grade gliomas (LGGs, WHO grade 2 eventually transform to malignant secondary GBMs. Although GBM patients benefit from promoter hypermethylation of the O(6-methylguanine-DNA methyltransferase (MGMT that is the main determinant of resistance to TMZ, recent studies suggested that MGMT promoter methylation is of prognostic as well as predictive significance for the efficacy of TMZ. Glioma-CpG island methylator phenotype (G-CIMP in the global genome was shown to be a significant predictor of improved survival in patients with GBM. Collectively, we hypothesized that MGMT promoter methylation might reflect global DNA methylation. Additionally in LGGs, the significance of MGMT promoter methylation is still undetermined. In the current study, we aimed to determine the correlation between clinical, genetic, and epigenetic profiles including LINE-1 and different cancer-related genes and the clinical outcome in newly diagnosed 57 LGG and 54 GBM patients. Here, we demonstrated that (1 IDH1/2 mutation is closely correlated with MGMT promoter methylation and 1p/19q codeletion in LGGs, (2 LINE-1 methylation levels in primary and secondary GBMs are lower than those in LGGs and normal brain tissues, (3 LINE-1 methylation is proportional to MGMT promoter methylation in gliomas, and (4 higher LINE-1 methylation is a favorable prognostic factor in primary GBMs, even compared to MGMT promoter methylation. As a global DNA methylation marker, LINE-1 may be a promising marker in gliomas.

  19. Skin score correlates with global DNA methylation and GSTO1 A140D polymorphism in arsenic-affected population of Eastern India.

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    Majumder, Moumita; Dasgupta, Uma B; Guha Mazumder, D N; Das, Nilansu

    2017-07-01

    Arsenic is a potent environmental toxicant causing serious public health concerns in India, Bangladesh and other parts of the world. Gene- and promoter-specific hypermethylation has been reported in different arsenic-exposed cell lines, whereas whole genome DNA methylation study suggested genomic hypo- and hypermethylation after arsenic exposure in in vitro and in vivo studies. Along with other characteristic biomarkers, arsenic toxicity leads to typical skin lesions. The present study demonstrates significant correlation between severities of skin manifestations with their whole genome DNA methylation status as well as with a particular polymorphism (Ala 140 Asp) status in arsenic metabolizing enzyme Glutathione S-transferase Omega-1 (GSTO1) in arsenic-exposed population of the district of Nadia, West Bengal, India.

  20. Correlation of beta-catenin localization with cyclooxygenase-2 expression and CpG island methylator phenotype (CIMP) in colorectal cancer.

    Science.gov (United States)

    Kawasaki, Takako; Nosho, Katsuhiko; Ohnishi, Mutsuko; Suemoto, Yuko; Kirkner, Gregory J; Dehari, Reiko; Meyerhardt, Jeffrey A; Fuchs, Charles S; Ogino, Shuji

    2007-07-01

    The WNT/beta-catenin (CTNNB1) pathway is commonly activated in the carcinogenic process. Cross-talks between the WNT and cyclooxygenase-2 (COX-2 or PTGS2)/prostaglandin pathways have been suggested. The relationship between beta-catenin activation and microsatellite instability (MSI) in colorectal cancer has been controversial. The CpG island methylator phenotype (CIMP or CIMP-high) with widespread promoter methylation is a distinct epigenetic phenotype in colorectal cancer, which is associated with MSI-high. However, no study has examined the relationship between beta-catenin activation and CIMP status. Using 832 population-based colorectal cancer specimens, we assessed beta-catenin localization by immunohistochemistry. We quantified DNA methylation in eight CIMP-specific promoters [CACNA1G, CDKN2A(p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1] by real-time polymerase chain reaction (MethyLight). MSI-high, CIMP-high, and BRAF mutation were associated inversely with cytoplasmic and nuclear beta-catenin expressions (i.e., beta-catenin activation) and associated positively with membrane expression. The inverse relation between beta-catenin activation and CIMP was independent of MSI. COX-2 overexpression correlated with cytoplasmic beta-catenin expression (even after tumors were stratified by CIMP status), but did not correlate significantly with nuclear or membrane expression. In conclusion, beta-catenin activation is inversely associated with CIMP-high independent of MSI status. Cytoplasmic beta-catenin is associated with COX-2 overexpression, supporting the role of cytoplasmic beta-catenin in stabilizing PTGS2 (COX-2) mRNA.

  1. Correlation of β-Catenin Localization with Cyclooxygenase-2 Expression and CpG Island Methylator Phenotype (CIMP in Colorectal Cancer

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    Takako Kawasaki

    2007-07-01

    Full Text Available The WNT/β-catenin (CTNNB1 pathway is commonly activated in the carcinogenic process. Cross-talks between the WNT and cyclooxygenase-2 (COX-2 or PTGS2/prostaglandin pathways have been suggested. The relationship between (3-catenin activation and microsatellite instability (MSI in colorectal cancer has been controversial. The CpG island methylator phenotype (CIMP or CIMP-high with widespread promoter methylation is a distinct epigenetic phenotype in colorectal cancer, which is associated with MSI-high. However, no study has examined the relationship between (β-catenin activation and CIMP status. Using 832 population-based colorectal cancer specimens, we assessed (3-catenin localization by immunohistochemistry. We quantified DNA methylation in eight CIMP-specific promoters [CACNA1G, CDKN2A(p16, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1] by real-time polymerase chain reaction (MethyLight. MSI-high, CIMP-high, and BRAF mutation were associated inversely with cytoplasmic and nuclear (β-catenin expressions (i.e., β-catenin activation and associated positively with membrane expression. The inverse relation between (β-catenin activation and CIMP was independent of MSI. COX-2 overexpression correlated with cytoplasmic (β-catenin expression (even after tumors were stratified by CIMP status, but did not correlate significantly with nuclear or membrane expression. In conclusion, β-catenin activation is inversely associated with CIMP-high independent of MSI status. Cytoplasmic β-catenin is associated with COX-2 overexpression, supporting the role of cytoplasmic β-catenin in stabilizing PTGS2(COX-2 mRNA.

  2. Clinical Significance of MLH1 Methylation and CpG Island Methylator Phenotype as Prognostic Markers in Patients with Gastric Cancer

    Science.gov (United States)

    Shigeyasu, Kunitoshi; Nagasaka, Takeshi; Mori, Yoshiko; Yokomichi, Naosuke; Kawai, Takashi; Fuji, Tomokazu; Kimura, Keisuke; Umeda, Yuzo; Kagawa, Shunsuke; Goel, Ajay; Fujiwara, Toshiyoshi

    2015-01-01

    Background To improve the outcome of patients suffering from gastric cancer, a better understanding of underlying genetic and epigenetic events in this malignancy is required. Although CpG island methylator phenotype (CIMP) and microsatellite instability (MSI) have been shown to play pivotal roles in gastric cancer pathogenesis, the clinical significance of these events on survival outcomes in patients with gastric cancer remains unknown. Methods This study included a patient cohort with pathologically confirmed gastric cancer who had surgical resections. A cohort of 68 gastric cancers was analyzed. CIMP and MSI statuses were determined by analyzing promoter CpG island methylation status of 28 genes/loci, and genomic instability at 10 microsatellite markers, respectively. A Cox’s proportional hazards model was performed for multivariate analysis including age, stage, tumor differentiation, KRAS mutation status, and combined CIMP/MLH1 methylation status in relation to overall survival (OS). Results By multivariate analysis, longer OS was significantly correlated with lower pathologic stage (P = 0.0088), better tumor differentiation (P = 0.0267) and CIMP-high and MLH1 3' methylated status (P = 0.0312). Stratification of CIMP status with regards to MLH1 methylation status further enabled prediction of gastric cancer prognosis. Conclusions CIMP and/or MLH1 methylation status may have a potential to be prognostic biomarkers for patients with gastric cancer. PMID:26121593

  3. Clinical Significance of MLH1 Methylation and CpG Island Methylator Phenotype as Prognostic Markers in Patients with Gastric Cancer.

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    Kunitoshi Shigeyasu

    Full Text Available To improve the outcome of patients suffering from gastric cancer, a better understanding of underlying genetic and epigenetic events in this malignancy is required. Although CpG island methylator phenotype (CIMP and microsatellite instability (MSI have been shown to play pivotal roles in gastric cancer pathogenesis, the clinical significance of these events on survival outcomes in patients with gastric cancer remains unknown.This study included a patient cohort with pathologically confirmed gastric cancer who had surgical resections. A cohort of 68 gastric cancers was analyzed. CIMP and MSI statuses were determined by analyzing promoter CpG island methylation status of 28 genes/loci, and genomic instability at 10 microsatellite markers, respectively. A Cox's proportional hazards model was performed for multivariate analysis including age, stage, tumor differentiation, KRAS mutation status, and combined CIMP/MLH1 methylation status in relation to overall survival (OS.By multivariate analysis, longer OS was significantly correlated with lower pathologic stage (P = 0.0088, better tumor differentiation (P = 0.0267 and CIMP-high and MLH1 3' methylated status (P = 0.0312. Stratification of CIMP status with regards to MLH1 methylation status further enabled prediction of gastric cancer prognosis.CIMP and/or MLH1 methylation status may have a potential to be prognostic biomarkers for patients with gastric cancer.

  4. DGKI methylation status modulates the prognostic value of MGMT in glioblastoma patients treated with combined radio-chemotherapy with temozolomide.

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    Amandine Etcheverry

    Full Text Available Consistently reported prognostic factors for glioblastoma (GBM are age, extent of surgery, performance status, IDH1 mutational status, and MGMT promoter methylation status. We aimed to integrate biological and clinical prognostic factors into a nomogram intended to predict the survival time of an individual GBM patient treated with a standard regimen. In a previous study we showed that the methylation status of the DGKI promoter identified patients with MGMT-methylated tumors that responded poorly to the standard regimen. We further evaluated the potential prognostic value of DGKI methylation status.399 patients with newly diagnosed GBM and treated with a standard regimen were retrospectively included in this study. Survival modelling was performed on two patient populations: intention-to-treat population of all included patients (population 1 and MGMT-methylated patients (population 2. Cox proportional hazard models were fitted to identify the main prognostic factors. A nomogram was developed for population 1. The prognostic value of DGKI promoter methylation status was evaluated on population 1 and population 2.The nomogram-based stratification of the cohort identified two risk groups (high/low with significantly different median survival. We validated the prognostic value of DGKI methylation status for MGMT-methylated patients. We also demonstrated that the DGKI methylation status identified 22% of poorly responding patients in the low-risk group defined by the nomogram.Our results improve the conventional MGMT stratification of GBM patients receiving standard treatment. These results could help the interpretation of published or ongoing clinical trial outcomes and refine patient recruitment in the future.

  5. DGKI methylation status modulates the prognostic value of MGMT in glioblastoma patients treated with combined radio-chemotherapy with temozolomide.

    Science.gov (United States)

    Etcheverry, Amandine; Aubry, Marc; Idbaih, Ahmed; Vauleon, Elodie; Marie, Yannick; Menei, Philippe; Boniface, Rachel; Figarella-Branger, Dominique; Karayan-Tapon, Lucie; Quillien, Veronique; Sanson, Marc; de Tayrac, Marie; Delattre, Jean-Yves; Mosser, Jean

    2014-01-01

    Consistently reported prognostic factors for glioblastoma (GBM) are age, extent of surgery, performance status, IDH1 mutational status, and MGMT promoter methylation status. We aimed to integrate biological and clinical prognostic factors into a nomogram intended to predict the survival time of an individual GBM patient treated with a standard regimen. In a previous study we showed that the methylation status of the DGKI promoter identified patients with MGMT-methylated tumors that responded poorly to the standard regimen. We further evaluated the potential prognostic value of DGKI methylation status. 399 patients with newly diagnosed GBM and treated with a standard regimen were retrospectively included in this study. Survival modelling was performed on two patient populations: intention-to-treat population of all included patients (population 1) and MGMT-methylated patients (population 2). Cox proportional hazard models were fitted to identify the main prognostic factors. A nomogram was developed for population 1. The prognostic value of DGKI promoter methylation status was evaluated on population 1 and population 2. The nomogram-based stratification of the cohort identified two risk groups (high/low) with significantly different median survival. We validated the prognostic value of DGKI methylation status for MGMT-methylated patients. We also demonstrated that the DGKI methylation status identified 22% of poorly responding patients in the low-risk group defined by the nomogram. Our results improve the conventional MGMT stratification of GBM patients receiving standard treatment. These results could help the interpretation of published or ongoing clinical trial outcomes and refine patient recruitment in the future.

  6. Differential DNA methylation profiles in gynecological cancers and correlation with clinico-pathological data

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    Tsang Percy CK

    2006-08-01

    Full Text Available Abstract Background Epigenetic gene silencing is one of the major causes of carcinogenesis. Its widespread occurrence in cancer genome could inactivate many cellular pathways including DNA repair, cell cycle control, apoptosis, cell adherence, and detoxification. The abnormal promoter methylation might be a potential molecular marker for cancer management. Methods For rapid identification of potential targets for aberrant methylation in gynecological cancers, methylation status of the CpG islands of 34 genes was determined using pooled DNA approach and methylation-specific PCR. Pooled DNA mixture from each cancer type (50 cervical cancers, 50 endometrial cancers and 50 ovarian cancers was made to form three test samples. The corresponding normal DNA from the patients of each cancer type was also pooled to form the other three control samples. Methylated alleles detected in tumors, but not in normal controls, were indicative of aberrant methylation in tumors. Having identified potential markers, frequencies of methylation were further analyzed in individual samples. Markers identified are used to correlate with clinico-pathological data of tumors using χ2 or Fisher's exact test. Results APC and p16 were hypermethylated across the three cancers. MINT31 and PTEN were hypermethylated in cervical and ovarian cancers. Specific methylation was found in cervical cancer (including CDH1, DAPK, MGMT and MINT2, endometrial cancer (CASP8, CDH13, hMLH1 and p73, and ovarian cancer (BRCA1, p14, p15, RIZ1 and TMS1. The frequencies of occurrence of hypermethylation in 4 candidate genes in individual samples of each cancer type (DAPK, MGMT, p16 and PTEN in 127 cervical cancers; APC, CDH13, hMLH1 and p16 in 60 endometrial cancers; and BRCA1, p14, p16 and PTEN in 49 ovarian cancers were examined for further confirmation. Incidence varied among different genes and in different cancer types ranging from the lowest 8.2% (PTEN in ovarian cancer to the highest 56

  7. Differential DNA methylation profiles in gynecological cancers and correlation with clinico-pathological data

    International Nuclear Information System (INIS)

    Yang, Hui-Juan; Liu, Vincent WS; Wang, Yue; Tsang, Percy CK; Ngan, Hextan YS

    2006-01-01

    Epigenetic gene silencing is one of the major causes of carcinogenesis. Its widespread occurrence in cancer genome could inactivate many cellular pathways including DNA repair, cell cycle control, apoptosis, cell adherence, and detoxification. The abnormal promoter methylation might be a potential molecular marker for cancer management. For rapid identification of potential targets for aberrant methylation in gynecological cancers, methylation status of the CpG islands of 34 genes was determined using pooled DNA approach and methylation-specific PCR. Pooled DNA mixture from each cancer type (50 cervical cancers, 50 endometrial cancers and 50 ovarian cancers) was made to form three test samples. The corresponding normal DNA from the patients of each cancer type was also pooled to form the other three control samples. Methylated alleles detected in tumors, but not in normal controls, were indicative of aberrant methylation in tumors. Having identified potential markers, frequencies of methylation were further analyzed in individual samples. Markers identified are used to correlate with clinico-pathological data of tumors using χ 2 or Fisher's exact test. APC and p16 were hypermethylated across the three cancers. MINT31 and PTEN were hypermethylated in cervical and ovarian cancers. Specific methylation was found in cervical cancer (including CDH1, DAPK, MGMT and MINT2), endometrial cancer (CASP8, CDH13, hMLH1 and p73), and ovarian cancer (BRCA1, p14, p15, RIZ1 and TMS1). The frequencies of occurrence of hypermethylation in 4 candidate genes in individual samples of each cancer type (DAPK, MGMT, p16 and PTEN in 127 cervical cancers; APC, CDH13, hMLH1 and p16 in 60 endometrial cancers; and BRCA1, p14, p16 and PTEN in 49 ovarian cancers) were examined for further confirmation. Incidence varied among different genes and in different cancer types ranging from the lowest 8.2% (PTEN in ovarian cancer) to the highest 56.7% (DAPK in cervical cancer). Aberrant methylation

  8. Maternal global methylation status and risk of congenital heart diseases

    NARCIS (Netherlands)

    van Driel, Lydi M. J. W.; de Jonge, Robert; Helbing, Willem A.; van Zelst, Bertrand D.; Ottenkamp, Jaap; Steegers, Eric A. P.; Steegers-Theunissen, Regine P. M.

    2008-01-01

    OBJECTIVE: To investigate whether the association between the maternal methylation status as reflected by low S-adenosylmethionine and high S-adenosylhomocysteine, is detrimental for cardiogenesis and congenital heart disease (CHD) in the offspring. METHODS: As part of a case-control study in the

  9. Quantitative DNA methylation analysis improves epigenotype-phenotype correlations in Beckwith-Wiedemann syndrome

    Science.gov (United States)

    Calvello, Mariarosaria; Tabano, Silvia; Colapietro, Patrizia; Maitz, Silvia; Pansa, Alessandra; Augello, Claudia; Lalatta, Faustina; Gentilin, Barbara; Spreafico, Filippo; Calzari, Luciano; Perotti, Daniela; Larizza, Lidia; Russo, Silvia; Selicorni, Angelo; Sirchia, Silvia M; Miozzo, Monica

    2013-01-01

    Beckwith-Wiedemann syndrome (BWS) is a rare disorder characterized by overgrowth and predisposition to embryonal tumors. BWS is caused by various epigenetic and/or genetic alterations that dysregulate the imprinted genes on chromosome region 11p15.5. Molecular analysis is required to reinforce the clinical diagnosis of BWS and to identify BWS patients with cancer susceptibility. This is particularly crucial prenatally because most signs of BWS cannot be recognized in utero. We established a reliable molecular assay by pyrosequencing to quantitatively evaluate the methylation profiles of ICR1 and ICR2. We explored epigenotype-phenotype correlations in 19 patients that fulfilled the clinical diagnostic criteria for BWS, 22 patients with suspected BWS, and three fetuses with omphalocele. Abnormal methylation was observed in one prenatal case and 19 postnatal cases, including seven suspected BWS. Seven cases showed ICR1 hypermethylation, five cases showed ICR2 hypomethylation, and eight cases showed abnormal methylation of ICR1 and ICR2 indicating paternal uniparental disomy (UPD). More cases of ICR1 alterations and UPD were found than expected. This is likely due to the sensitivity of this approach, which can detect slight deviations in methylation from normal levels. There was a significant correlation (p < 0.001) between the percentage of ICR1 methylation and BWS features: severe hypermethylation (range: 75–86%) was associated with macroglossia, macrosomia, and visceromegaly, whereas mild hypermethylation (range: 55–59%) was associated with umbilical hernia and diastasis recti. Evaluation of ICR1 and ICR2 methylation by pyrosequencing in BWS can improve epigenotype-phenotype correlations, detection of methylation alterations in suspected cases, and identification of UPD. PMID:23917791

  10. Methylation status and protein expression of RASSF1A in breast cancer patients.

    Science.gov (United States)

    Hagrass, Hoda A; Pasha, Heba F; Shaheen, Mohamed A; Abdel Bary, Eman H; Kassem, Rasha

    2014-01-01

    Recently genetics and epigenetics alterations have been found to be characteristic of malignancy and hence can be used as targets for detection of neoplasia. RAS association domain family protein 1A (RASSF1A) gene hypermethylation has been a subject of interest in recent researches on cancer breast patients. The aim of the present study was to evaluate whether RASSF1A methylation status and RASSF1A protein expression are associated with the major clinico-pathological parameters. One hundred and twenty breast cancer Egyptian patients and 100-control subjects diagnosed with benign lesions of the breast were enrolled in this study. We evaluated RASSF1A methylation status in tissue and serum samples using Methyl specific PCR together with RASSF1A protein expression in tissues by immunohistochemistry. Results were studied in relation to known prognostic clinicopathological features in breast cancer. Frequency of RASSF1A methylation in tissues and serum were 70 and 63.3 % respectively and RASSF1A protein expression showed frequency of 46.7 %. There was an association between RASSF1A methylation in tissues, serum and loss of protein expression in tissues with invasive carcinoma, advanced stage breast cancer, L.N. metastasis, ER/PR and HER2 negativity. RASSF1A methylation in serum showed high degree of concordance with methylation in tissues (Kappa = 0.851, P < 0.001). RASSF1A hypermethylation in tissues and serum and its protein expression may be a valid, reliable and sensitive tool for detection and follow up of breast cancer patients.

  11. Recurrence in oral and pharyngeal cancer is associated with quantitative MGMT promoter methylation

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    Taioli, Emanuela; Ragin, Camille; Wang, Xiao-hong; Chen, Jiangying; Langevin, Scott M; Brown, Ashley R; Gollin, Susanne M; Garte, Seymour; Sobol, Robert W

    2009-01-01

    Biomarkers that predict clinical response, tumor recurrence or patient survival are severely lacking for most cancers, particularly for oral and pharyngeal cancer. This study examines whether gene-promoter methylation of tumor DNA correlates with survival and recurrence rates in a population of patients with oral or pharyngeal cancer. The promoter methylation status of the DNA repair gene MGMT and the tumor suppressor genes CDKN2A and RASSF1 were evaluated by methylation-specific PCR in 88 primary oral and pharyngeal tumors and correlated with survival and tumor recurrence. Quantitative MGMT methylation was also assessed. 29.6% of the tumors presented with MGMT methylation, 11.5% with CDKN2A methylation and 12.1% with RASSF1 methylation. MGMT promoter methylation was significantly associated with poorer overall and disease-free survival. No differences in methylation status of MGMT and RASSF1 with HPV infection, smoking or drinking habits were observed. A significant inverse trend with the amount of MGMT methylation and overall and disease-free survival was observed (p trend = 0.002 and 0.001 respectively). These results implicate MGMT promoter methylation as a possible biomarker for oral and pharyngeal cancer prognosis. The critical role of MGMT in DNA repair suggests that defective DNA repair may be correlative in the observed association between MGMT promoter methylation and tumor recurrence. Follow-up studies should include further quantitative MSP-PCR measurement, global methylation profiling and detailed analysis of downstream DNA repair genes regulated by promoter methylation

  12. Homogeneous MGMT immunoreactivity correlates with an unmethylated MGMT promoter status in brain metastases of various solid tumors.

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    Barbara Ingold

    Full Text Available The O(6-methylguanine-methyltransferase (MGMT promoter methylation status is a predictive parameter for the response of malignant gliomas to alkylating agents such as temozolomide. First clinical reports on treating brain metastases with temozolomide describe varying effects. This may be due to the fact that MGMT promoter methylation of brain metastases has not yet been explored in depth. Therefore, we assessed MGMT promoter methylation of various brain metastases including those derived from lung (n = 91, breast (n = 72 kidney (n = 49 and from malignant melanomas (n = 113 by methylation-specific polymerase chain reaction (MS-PCR and MGMT immunoreactivity. Fifty-nine of 199 brain metastases (29.6% revealed a methylated MGMT promoter. The methylation rate was the highest in brain metastases derived from lung carcinomas (46.5% followed by those from breast carcinoma (28.8%, malignant melanoma (24.7% and from renal carcinoma (20%. A significant correlation of homogeneous MGMT-immunoreactivity (>95% MGMT positive tumor cells and an unmethylated MGMT promoter was found. Promoter methylation was detected in 26 of 61 (43% tumors lacking MGMT immunoreactivity, in 17 of 63 (27% metastases with heterogeneous MGMT expression, but only in 5 of 54 brain metastases (9% showing a homogeneous MGMT immunoreactivity. Our results demonstrate that a significant number of brain metastases reveal a methylated MGMT-promoter. Based on an obvious correlation between homogeneous MGMT immunoreactivity and unmethylated MGMT promoter, we hypothesize that immunohistochemistry for MGMT may be a helpful diagnostic tool to identify those tumors that probably will not benefit from the use of alkylating agents. The discrepancy between promoter methylation and a lack of MGMT immunoreactivity argues for assessing MGMT promoter methylation both by immunohistochemical as well as by molecular approaches for diagnostic purposes.

  13. Molecular correlates with MGMT promoter methylation and silencing support CpG island methylator phenotype-low (CIMP-low) in colorectal cancer.

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    Ogino, Shuji; Kawasaki, Takako; Kirkner, Gregory J; Suemoto, Yuko; Meyerhardt, Jeffrey A; Fuchs, Charles S

    2007-11-01

    The CpG island methylator phenotype (CIMP or CIMP-high) with widespread promoter methylation is a distinct epigenetic phenotype in colorectal cancer. In contrast, a phenotype with less widespread promoter methylation (CIMP-low) has not been well characterised. O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation and silencing have been associated with G>A mutations and microsatellite instability-low (MSI-low). To examine molecular correlates with MGMT methylation/silencing in colorectal cancer. Utilising MethyLight technology, we quantified DNA methylation in MGMT and eight other markers (a CIMP-diagnostic panel; CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1) in 920 population-based colorectal cancers. Tumours with both MGMT methylation and loss were correlated positively with MSI-low (p = 0.02), CIMP-high (>or=6/8 methylated CIMP markers, p = 0.005), CIMP-low (1/8-5/8 methylated CIMP markers, p = 0.002, compared to CIMP-0 with 0/8 methylated markers), KRAS G>A mutation (p = 0.02), and inversely with 18q loss of heterozygosity (p = 0.0002). Tumours were classified into nine MSI/CIMP subtypes. Among the CIMP-low group, tumours with both MGMT methylation and loss were far more frequent in MSI-low tumours (67%, 12/18) than MSI-high tumours (5.6%, 1/18; p = 0.0003) and microsatellite stable (MSS) tumours (33%, 52/160; p = 0.008). However, no such relationship was observed among the CIMP-high or CIMP-0 groups. The relationship between MGMT methylation/silencing and MSI-low is limited to only CIMP-low tumours, supporting the suggestion that CIMP-low in colorectal cancer may be a different molecular phenotype from CIMP-high and CIMP-0. Our data support a molecular difference between MSI-low and MSS in colorectal cancer, and a possible link between CIMP-low, MSI-low, MGMT methylation/loss and KRAS mutation.

  14. Quantitative correlation between promoter methylation and messenger RNA levels of the reduced folate carrier

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    Kheradpour Albert

    2008-05-01

    Full Text Available Abstract Background Methotrexate (MTX uptake is mediated by the reduced folate carrier (RFC. Defective drug uptake in association with decreased RFC expression is a common mechanism of MTX resistance in many tumor types. Heavy promoter methylation was previously identified as a basis for the complete silencing of RFC in MDA-MB-231 breast cancer cells, its role and prevalence in RFC transcription regulation are, however, not widely studied. Methods In the current study, RFC promoter methylation was assessed using methylation specific PCR in a panel of malignant cell lines (n = 8, including MDA-MB-231, and M805, a MTX resistant cell line directly established from the specimen of a patient with malignant fibrohistocytoma, whom received multiple doses of MTX. A quantitative approach of real-time PCR for measuring the extent of RFC promoter methylation was developed, and was validated by direct bisulfite genomic sequencing. RFC mRNA levels were determined by quantitative real-time RT-PCR and were related to the extent of promoter methylation in these cell lines. Results A partial promoter methylation and RFC mRNA down-regulation were observed in M805. Using the quantitative approach, a reverse correlation (correlation coefficient = -0.59, p Conclusion This study further suggests that promoter methylation is a potential basis for MTX resistance. The quantitative correlation identified in this study implies that promoter methylation is possibly a mechanism involved in the fine regulation of RFC transcription.

  15. pETM: a penalized Exponential Tilt Model for analysis of correlated high-dimensional DNA methylation data.

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    Sun, Hokeun; Wang, Ya; Chen, Yong; Li, Yun; Wang, Shuang

    2017-06-15

    DNA methylation plays an important role in many biological processes and cancer progression. Recent studies have found that there are also differences in methylation variations in different groups other than differences in methylation means. Several methods have been developed that consider both mean and variance signals in order to improve statistical power of detecting differentially methylated loci. Moreover, as methylation levels of neighboring CpG sites are known to be strongly correlated, methods that incorporate correlations have also been developed. We previously developed a network-based penalized logistic regression for correlated methylation data, but only focusing on mean signals. We have also developed a generalized exponential tilt model that captures both mean and variance signals but only examining one CpG site at a time. In this article, we proposed a penalized Exponential Tilt Model (pETM) using network-based regularization that captures both mean and variance signals in DNA methylation data and takes into account the correlations among nearby CpG sites. By combining the strength of the two models we previously developed, we demonstrated the superior power and better performance of the pETM method through simulations and the applications to the 450K DNA methylation array data of the four breast invasive carcinoma cancer subtypes from The Cancer Genome Atlas (TCGA) project. The developed pETM method identifies many cancer-related methylation loci that were missed by our previously developed method that considers correlations among nearby methylation loci but not variance signals. The R package 'pETM' is publicly available through CRAN: http://cran.r-project.org . sw2206@columbia.edu. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

  16. Multiple correlation analyses revealed complex relationship between DNA methylation and mRNA expression in human peripheral blood mononuclear cells.

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    Xie, Fang-Fei; Deng, Fei-Yan; Wu, Long-Fei; Mo, Xing-Bo; Zhu, Hong; Wu, Jian; Guo, Yu-Fan; Zeng, Ke-Qin; Wang, Ming-Jun; Zhu, Xiao-Wei; Xia, Wei; Wang, Lan; He, Pei; Bing, Peng-Fei; Lu, Xin; Zhang, Yong-Hong; Lei, Shu-Feng

    2018-01-01

    DNA methylation is an important regulator on the mRNA expression. However, a genome-wide correlation pattern between DNA methylation and mRNA expression in human peripheral blood mononuclear cells (PBMCs) is largely unknown. The comprehensive relationship between mRNA and DNA methylation was explored by using four types of correlation analyses and a genome-wide methylation-mRNA expression quantitative trait locus (eQTL) analysis in PBMCs in 46 unrelated female subjects. An enrichment analysis was performed to detect biological function for the detected genes. Single pair correlation coefficient (r T1 ) between methylation level and mRNA is moderate (-0.63-0.62) in intensity, and the negative and positive correlations are nearly equal in quantity. Correlation analysis on each gene (T4) found 60.1% genes showed correlations between mRNA and gene-based methylation at P correlation (R T4  > 0.8). Methylation sites have regulation effects on mRNA expression in eQTL analysis, with more often observations in region of transcription start site (TSS). The genes under significant methylation regulation both in correlation analysis and eQTL analysis tend to cluster to the categories (e.g., transcription, translation, regulation of transcription) that are essential for maintaining the basic life activities of cells. Our findings indicated that DNA methylation has predictive regulation effect on mRNA with a very complex pattern in PBMCs. The results increased our understanding on correlation of methylation and mRNA and also provided useful clues for future epigenetic studies in exploring biological and disease-related regulatory mechanisms in PBMC.

  17. CpG island methylator phenotype and its association with malignancy in sporadic duodenal adenomas.

    Science.gov (United States)

    Sun, Lifeng; Guzzetta, Angela A; Fu, Tao; Chen, Jinming; Jeschke, Jana; Kwak, Ruby; Vatapalli, Rajita; Baylin, Stephen B; Iacobuzio-Donahue, Christine A; Wolfgang, Christopher L; Ahuja, Nita

    2014-05-01

    CpG island methylator phenotype (CIMP) has been found in multiple precancerous and cancerous lesions, including colorectal adenomas, colorectal cancers, and duodenal adenocarcinomas. There are no reports in the literature of a relationship between CIMP status and clinicopathologic features of sporadic duodenal adenomas. This study sought to elucidate the role of methylation in duodenal adenomas and correlate it with KRAS and BRAF mutations. CIMP+ (with more than 2 markers methylated) was seen in 33.3% of duodenal adenomas; 61% of these CIMP+ adenomas were CIMP-high (with more than 3 markers methylated). Furthermore, CIMP+ status significantly correlated with older age of patients, larger size and villous type of tumor, coexistent dysplasia and periampullary location. MLH1 methylation was seen in 11.1% of duodenal adenomas and was significantly associated with CIMP+ tumors, while p16 methylation was an infrequent event. KRAS mutations were frequent and seen in 26.3% of adenomas; however, no BRAF mutations were detected. Furthermore, CIMP-high status was associated with larger size and villous type of tumor and race (non-white). These results suggest that CIMP+ duodenal adenomas may have a higher risk for developing malignancy and may require more aggressive management and surveillance.

  18. Urine mercury levels correlate with DNA methylation of imprinting gene H19 in the sperm of reproductive-aged men.

    Directory of Open Access Journals (Sweden)

    Zhaoxu Lu

    Full Text Available Mercury (Hg is a well-recognized environmental pollutant known by its toxicity of development and neurotoxicity, which results in adverse health outcomes. However, the mechanisms underlying the teratogenic effects of Hg are not well understood. Imprinting genes are emerging regulators for fetal development subjecting to environmental pollutants impacts. In this study, we examined the association between preconceptional Hg exposure and the alteration of DNA methylation of imprinting genes H19, Meg3, and Peg3 in human sperm DNA.A total of 616 men, aged from 22 to 59, were recruited from Reproductive Medicine Clinic of Maternal and Child Care Service Center and the Urologic Surgery Clinic of Shanxi Academy of Medical Sciences during April 2015 and March 2016. Demographic information was collected through questionnaires. Urine was collected and urinary Hg concentrations were measured using a fully-automatic double-channel hydride generation atomic fluorescence spectrometer. Methylation of imprinting genes H19, Meg3 and Peg3 of sperm DNA from 242 participants were examined by bisulfite pyrosequencing. Spearman's rank and multivariate regression analysis were used for correlation analysis between sperm DNA methylation status of imprinting genes and urinary Hg levels.The median concentration of Hg for 616 participants was 9.14μg/l (IQR: 5.56-12.52 μg/l; ranging 0.16-71.35μg/l. A total of 42.7% of the participants are beyond normal level for non-occupational exposure according to the criterion of Hg poisoning (≥10 μg/L. Spearman's rank analysis indicated a negative correlation between urinary Hg concentrations and average DNA methylation levels of imprinted genes H19 (rs = -0.346, p <0.05, but there was no such a correlation for Peg3 and Meg3. Further, we analyzed the correlation between methylation level at individual CpG site of H19 and urinary Hg level. The results showed a negative correlation between urinary Hg concentrations and three out of

  19. Correlation of SHOX2 Gene Amplification and DNA Methylation in Lung Cancer Tumors

    International Nuclear Information System (INIS)

    Schneider, Katja U; Liebenberg, Volker; Kneip, Christoph; Seegebarth, Anke; Erdogan, Fikret; Rappold, Gudrun; Schmidt, Bernd; Dietrich, Dimo; Fleischhacker, Michael; Leschber, Gunda; Merk, Johannes; Schäper, Frank; Stapert, Henk R; Vossenaar, Erik R; Weickmann, Sabine

    2011-01-01

    DNA methylation in the SHOX2 locus was previously used to reliably detect lung cancer in a group of critical controls, including 'cytologically negative' samples with no visible tumor cell content, at a high specificity based on the analysis of bronchial lavage samples. This study aimed to investigate, if the methylation correlates with SHOX2 gene expression and/or copy number alterations. An amplification of the SHOX2 gene locus together with the observed tumor-specific hypermethylation might explain the good performance of this marker in bronchial lavage samples. SHOX2 expression, gene copy number and DNA methylation were determined in lung tumor tissues and matched morphologically normal adjacent tissues (NAT) from 55 lung cancer patients. Quantitative HeavyMethyl (HM) real-time PCR was used to detect SHOX2 DNA methylation levels. SHOX2 expression was assayed with quantitative real-time PCR, and copy numbers alterations were measured with conventional real-time PCR and array CGH. A hypermethylation of the SHOX2 locus in tumor tissue as compared to the matched NAT from the same patient was detected in 96% of tumors from a group of 55 lung cancer patients. This correlated highly significantly with the frequent occurrence of copy number amplification (p < 0.0001), while the expression of the SHOX2 gene showed no difference. Frequent gene amplification correlated with hypermethylation of the SHOX2 gene locus. This concerted effect qualifies SHOX2 DNA methylation as a biomarker for lung cancer diagnosis, especially when sensitive detection is needed, i.e. in bronchial lavage or blood samples

  20. Methylation Status of miR-182 Promoter in Lung Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Yongwen LI

    2015-05-01

    Full Text Available Background and objective It has been proven that the abnormal expression of miR-182 was related to the occurrence and development of tumors. The aim of this study is to explore the relationship between the methylation of miR-182 promoter and its expression in lung cancer cell lines. Methods Real-time quantitative PCR and methylation-specific PCR were used to detect the expression level of miR-182 and its promoter methylation status in five lung cancer cell lines (A549, L9981, NL9980, 95C and 95D. DNA sequencing was used to confirm the methylation results. Results The level of miR-182 expression significantly differs among these lung cancer cell lines. The highly metastatic human lung cancer cell lines, namely, A549 and L9981, demonstrate a relatively lower expression level of miR-182 compared with the lowly metastatic human lung cancer cell line 95C. Methylation-specific PCR and DNA sequencing assay results indicate that these lung cancer cell lines present different levels of miR-182 promoter methylation, and the highest methylation level is observed in A549 cells. Furthermore, the expression of miR-182 in these cell lines significantly increases when treated with 10 μM 5’-Aza-dC. Conclusion DNA methylation occurs in the miR-182 promoter region in lung cancer cell lines. This methylation can regulate the expression level of miR-182. Further study must be conducted to explore the function of miR-182 promoter methylation in lung cancer occurrence and development.

  1. Methylation status of the APC and RASSF1A promoter in cell-free circulating DNA and its prognostic role in patients with colorectal cancer.

    Science.gov (United States)

    Matthaios, Dimitrios; Balgkouranidou, Ioanna; Karayiannakis, Anastasios; Bolanaki, Helen; Xenidis, Nikolaos; Amarantidis, Kyriakos; Chelis, Leonidas; Romanidis, Konstantinos; Chatzaki, Aikaterini; Lianidou, Evi; Trypsianis, Grigorios; Kakolyris, Stylianos

    2016-07-01

    DNA methylation is the most frequent epigenetic alteration. Using methylation-specific polymerase chain reaction (MSP), the methylation status of the adenomatous polyposis coli ( APC ) and Ras association domain family 1 isoform A ( RASSF1A ) genes was examined in cell-free circulating DNA from 155 plasma samples obtained from patients with early and advanced colorectal cancer (CRC). APC and RASSF1A hypermethylation was frequently observed in both early and advanced disease, and was significantly associated with a poorer disease outcome. The methylation status of the APC and RASSF1A promoters was investigated in cell-free DNA of patients with CRC. Using MSP, the promoter methylation status of APC and RASSF1A was examined in 155 blood samples obtained from patients with CRC, 88 of whom had operable CRC (oCRC) and 67 had metastatic CRC (mCRC). The frequency of APC methylation in patients with oCRC was 33%. Methylated APC promoter was significantly associated with older age (P=0.012), higher stage (P=0.014) and methylated RASSF1A status (P=0.050). The frequency of APC methylation in patients with mCRC was 53.7%. In these patients, APC methylation was significantly associated with methylated RASSF1A status (P=0.016). The frequency of RASSF1A methylation in patients with oCRC was 25%. Methylated RASSF1A in oCRC was significantly associated with higher stage (P=0.021). The frequency of RASSF1A methylation in mCRC was 44.8%. Methylated RASSF1A in mCRC was associated with moderate differentiation (P=0.012), high levels of carcinoembryonic antigen (P=0.023) and methylated APC status (P=0.016). Patients with an unmethylated APC gene had better survival in both early (81±5 vs. 27±4 months, PAPC . Patients with an unmethylated RASSF1A gene had better survival in both early (71±6 vs. 46±8 months, PAPC and RASSF1A promoter methylation status and survival may be indicative of a prognostic role for these genes in CRC, which requires additional testing in larger studies.

  2. The Prognostic Roles of Gender and O6-Methylguanine-DNA Methyltransferase Methylation Status in Glioblastoma Patients: The Female Power.

    Science.gov (United States)

    Franceschi, Enrico; Tosoni, Alicia; Minichillo, Santino; Depenni, Roberta; Paccapelo, Alexandro; Bartolini, Stefania; Michiara, Maria; Pavesi, Giacomo; Urbini, Benedetta; Crisi, Girolamo; Cavallo, Michele A; Tosatto, Luigino; Dazzi, Claudio; Biasini, Claudia; Pasini, Giuseppe; Balestrini, Damiano; Zanelli, Francesca; Ramponi, Vania; Fioravanti, Antonio; Giombelli, Ermanno; De Biase, Dario; Baruzzi, Agostino; Brandes, Alba A

    2018-04-01

    Clinical and molecular factors are essential to define the prognosis in patients with glioblastoma (GBM). O6-methylguanine-DNA methyltransferase (MGMT) methylation status, age, Karnofsky Performance Status (KPS), and extent of surgical resection are the most relevant prognostic factors. Our investigation of the role of gender in predicting prognosis shows a slight survival advantage for female patients. We performed a prospective evaluation of the Project of Emilia Romagna on Neuro-Oncology (PERNO) registry to identify prognostic factors in patients with GBM who received standard treatment. A total of 169 patients (99 males [58.6%] and 70 females [41.4%]) were evaluated prospectively. MGMT methylation was evaluable in 140 patients. Among the male patients, 36 were MGMT methylated (25.7%) and 47 were unmethylated (33.6%); among the female patients, 32 were methylated (22.9%) and 25 were unmethylated (17.9%). Survival was longer in the methylated females compared with the methylated males (P = 0.028) but was not significantly different between the unmethylated females and the unmethylated males (P = 0.395). In multivariate analysis, gender and MGMT methylation status considered together (methylated females vs. methylated males; hazard ratio [HR], 0.459; 95% confidence interval [CI], 0.242-0.827; P = 0.017), age (HR, 1.025; 95% CI, 1.002-1.049; P = 0.032), and KPS (HR, 0.965; 95% CI, 0.948-0.982; P factor. Copyright © 2018 Elsevier Inc. All rights reserved.

  3. Fluorescence-labeled methylation-sensitive amplified fragment length polymorphism (FL-MS-AFLP) analysis for quantitative determination of DNA methylation and demethylation status.

    Science.gov (United States)

    Kageyama, Shinji; Shinmura, Kazuya; Yamamoto, Hiroko; Goto, Masanori; Suzuki, Koichi; Tanioka, Fumihiko; Tsuneyoshi, Toshihiro; Sugimura, Haruhiko

    2008-04-01

    The PCR-based DNA fingerprinting method called the methylation-sensitive amplified fragment length polymorphism (MS-AFLP) analysis is used for genome-wide scanning of methylation status. In this study, we developed a method of fluorescence-labeled MS-AFLP (FL-MS-AFLP) analysis by applying a fluorescence-labeled primer and fluorescence-detecting electrophoresis apparatus to the existing method of MS-AFLP analysis. The FL-MS-AFLP analysis enables quantitative evaluation of more than 350 random CpG loci per run. It was shown to allow evaluation of the differences in methylation level of blood DNA of gastric cancer patients and evaluation of hypermethylation and hypomethylation in DNA from gastric cancer tissue in comparison with adjacent non-cancerous tissue.

  4. Human Papillomavirus 16, 18, 31 and 45 viral load, integration and methylation status stratified by cervical disease stage

    International Nuclear Information System (INIS)

    Marongiu, Luigi; Godi, Anna; Parry, John V; Beddows, Simon

    2014-01-01

    Persistent infection with oncogenic Human Papillomavirus (HPV) is associated with the development of cervical cancer with each genotype differing in their relative contribution to the prevalence of cervical disease. HPV DNA testing offers improved sensitivity over cytology testing alone but is accompanied by a generally low specificity. Potential molecular markers of cervical disease include type-specific viral load (VL), integration of HPV DNA into the host genome and methylation of the HPV genome. The aim of this study was to evaluate the relationship between HPV type-specific viral load, integration and methylation status and cervical disease stage in samples harboring HPV16, HPV18, HPV31 or HPV45. Samples singly infected with HPV16 (n = 226), HPV18 (n = 32), HPV31 (n = 75) or HPV45 (n = 29) were selected from a cohort of 4,719 women attending cervical screening in England. Viral load and integration status were determined by real-time PCR while 3’L1-URR methylation status was determined by pyrosequencing or sequencing of multiple clones derived from each sample. Viral load could differentiate between normal and abnormal cytology with a sensitivity of 75% and a specificity of 80% (odds ratio [OR] 12.4, 95% CI 6.2–26.1; p < 0.001) with some variation between genotypes. Viral integration was poorly associated with cervical disease. Few samples had fully integrated genomes and these could be found throughout the course of disease. Overall, integration status could distinguish between normal and abnormal cytology with a sensitivity of 72% and a specificity of 50% (OR 2.6, 95% CI 1.0–6.8; p = 0.054). Methylation levels were able to differentiate normal and low grade cytology from high grade cytology with a sensitivity of 64% and a specificity of 82% (OR 8.2, 95% CI 3.8–18.0; p < 0.001). However, methylation varied widely between genotypes with HPV18 and HPV45 exhibiting a broader degree and higher magnitude of methylated CpG sites than HPV16 and HPV31. This

  5. Highly efficient PCR assay to discriminate allelic DNA methylation status using whole genome amplification

    Directory of Open Access Journals (Sweden)

    Ito Takashi

    2011-06-01

    Full Text Available Abstract Background We previously developed a simple method termed HpaII-McrBC PCR (HM-PCR to discriminate allelic methylation status of the genomic sites of interest, and successfully applied it to a comprehensive analysis of CpG islands (CGIs on human chromosome 21q. However, HM-PCR requires 200 ng of genomic DNA to examine one target site, thereby precluding its application to such samples that are limited in quantity. Findings We developed HpaII-McrBC whole-genome-amplification PCR (HM-WGA-PCR that uses whole-genome-amplified DNA as the template. HM-WGA-PCR uses only 1/100th the genomic template material required for HM-PCR. Indeed, we successfully analyzed 147 CGIs by HM-WGA-PCR using only ~300 ng of DNA, whereas previous HM-PCR study had required ~30 μg. Furthermore, we confirmed that allelic methylation status revealed by HM-WGA-PCR is identical to that by HM-PCR in every case of the 147 CGIs tested, proving high consistency between the two methods. Conclusions HM-WGA-PCR would serve as a reliable alternative to HM-PCR in the analysis of allelic methylation status when the quantity of DNA available is limited.

  6. Quantitative analysis of DNA methylation in chronic lymphocytic leukemia patients.

    Science.gov (United States)

    Lyko, Frank; Stach, Dirk; Brenner, Axel; Stilgenbauer, Stephan; Döhner, Hartmut; Wirtz, Michaela; Wiessler, Manfred; Schmitz, Oliver J

    2004-06-01

    Changes in the genomic DNA methylation level have been found to be closely associated with tumorigenesis. In order to analyze the relation of aberrant DNA methylation to clinical and biological risk factors, we have determined the cytosine methylation level of 81 patients diagnosed with chronic lymphocytic leukemia (CLL). The analysis was based on DNA hydrolysis followed by derivatization of the 2'-desoxyribonucleoside-3'-monophosphates with BODIPY FL EDA. Derivatives were separated by micellar electrokinetic chromatography, and laser-induced fluorescence was used for detection. We analyzed potential correlations between DNA methylation levels and numerous patient parameters, including clinical observations and biological data. As a result, we observed a significant correlation with the immunoglobulin variable heavy chain gene (VH) mutation status. This factor has been repeatedly proposed as a reliable prognostic marker for CLL, which suggests that the methylation level might be a valuable factor in determining the prognostic outcome of CLL. We are now in the process of refining our method to broaden its application potential. In this context, we show here that the oxidation of the fluorescence marker in the samples and the evaporation of methanol in the electrolytes can be prevented by a film of paraffin oil. In summary, our results thus establish capillary electrophoresis as a valuable tool for analyzing the DNA methylation status of clinical samples.

  7. Propofol effectively inhibits lithium-pilocarpine- induced status epilepticus in rats via downregulation of N-methyl-D-aspartate receptor 2B subunit expression

    Science.gov (United States)

    Wang, Henglin; Wang, Zhuoqiang; Mi, Weidong; Zhao, Cong; Liu, Yanqin; Wang, Yongan; Sun, Haipeng

    2012-01-01

    Status epilepticus was induced via intraperitoneal injection of lithium-pilocarpine. The inhibitory effects of propofol on status epilepticus in rats were judged based on observation of behavior, electroencephalography and 24-hour survival rate. Propofol (12.5–100 mg/kg) improved status epilepticus in a dose-dependent manner, and significantly reduced the number of deaths within 24 hours of lithium-pilocarpine injection. Western blot results showed that, 24 hours after induction of status epilepticus, the levels of N-methyl-D-aspartate receptor 2A and 2B subunits were significantly increased in rat cerebral cortex and hippocampus. Propofol at 50 mg/kg significantly suppressed the increase in N-methyl-D-aspartate receptor 2B subunit levels, but not the increase in N-methyl-D-aspartate receptor 2A subunit levels. The results suggest that propofol can effectively inhibit status epilepticus induced by lithium-pilocarpine. This effect may be associated with downregulation of N-methyl-D-aspartate receptor 2B subunit expression after seizures. PMID:25737709

  8. Assignment of methyl NMR resonances of a 52 kDa protein with residue-specific 4D correlation maps

    International Nuclear Information System (INIS)

    Mishra, Subrata H.; Frueh, Dominique P.

    2015-01-01

    Methyl groups have become key probes for structural and functional studies by nuclear magnetic resonance. However, their NMR signals cluster in a small spectral region and assigning their resonances can be a tedious process. Here, we present a method that facilitates assignment of methyl resonances from assigned amide groups. Calculating the covariance between sensitive methyl and amide 3D spectra, each providing correlations to C α and C β separately, produces 4D correlation maps directly correlating methyl groups to amide groups. Optimal correlation maps are obtained by extracting residue-specific regions, applying derivative to the dimensions subject to covariance, and multiplying 4D maps stemming from different 3D spectra. The latter procedure rescues weak signals that may be missed in traditional assignment procedures. Using these covariance correlation maps, nearly all assigned isoleucine, leucine, and valine amide resonances of a 52 kDa nonribosomal peptide synthetase cyclization domain were paired with their corresponding methyl groups

  9. DNA methylation signatures of educational attainment

    Science.gov (United States)

    van Dongen, Jenny; Bonder, Marc Jan; Dekkers, Koen F.; Nivard, Michel G.; van Iterson, Maarten; Willemsen, Gonneke; Beekman, Marian; van der Spek, Ashley; van Meurs, Joyce B. J.; Franke, Lude; Heijmans, Bastiaan T.; van Duijn, Cornelia M.; Slagboom, P. Eline; Boomsma, Dorret I.; BIOS consortium

    2018-03-01

    Educational attainment is a key behavioural measure in studies of cognitive and physical health, and socioeconomic status. We measured DNA methylation at 410,746 CpGs (N = 4152) and identified 58 CpGs associated with educational attainment at loci characterized by pleiotropic functions shared with neuronal, immune and developmental processes. Associations overlapped with those for smoking behaviour, but remained after accounting for smoking at many CpGs: Effect sizes were on average 28% smaller and genome-wide significant at 11 CpGs after adjusting for smoking and were 62% smaller in never smokers. We examined sources and biological implications of education-related methylation differences, demonstrating correlations with maternal prenatal folate, smoking and air pollution signatures, and associations with gene expression in cis, dynamic methylation in foetal brain, and correlations between blood and brain. Our findings show that the methylome of lower-educated people resembles that of smokers beyond effects of their own smoking behaviour and shows traces of various other exposures.

  10. The development and validation of EpiComet-Chip, a modified high-throughput comet assay for the assessment of DNA methylation status.

    Science.gov (United States)

    Townsend, Todd A; Parrish, Marcus C; Engelward, Bevin P; Manjanatha, Mugimane G

    2017-08-01

    DNA damage and alterations in global DNA methylation status are associated with multiple human diseases and are frequently correlated with clinically relevant information. Therefore, assessing DNA damage and epigenetic modifications, including DNA methylation, is critical for predicting human exposure risk of pharmacological and biological agents. We previously developed a higher-throughput platform for the single cell gel electrophoresis (comet) assay, CometChip, to assess DNA damage and genotoxic potential. Here, we utilized the methylation-dependent endonuclease, McrBC, to develop a modified alkaline comet assay, "EpiComet," which allows single platform evaluation of genotoxicity and global DNA methylation [5-methylcytosine (5-mC)] status of single-cell populations under user-defined conditions. Further, we leveraged the CometChip platform to create an EpiComet-Chip system capable of performing quantification across simultaneous exposure protocols to enable unprecedented speed and simplicity. This system detected global methylation alterations in response to exposures which included chemotherapeutic and environmental agents. Using EpiComet-Chip on 63 matched samples, we correctly identified single-sample hypermethylation (≥1.5-fold) at 87% (20/23), hypomethylation (≥1.25-fold) at 100% (9/9), with a 4% (2/54) false-negative rate (FNR), and 10% (4/40) false-positive rate (FPR). Using a more stringent threshold to define hypermethylation (≥1.75-fold) allowed us to correctly identify 94% of hypermethylation (17/18), but increased our FPR to 16% (7/45). The successful application of this novel technology will aid hazard identification and risk characterization of FDA-regulated products, while providing utility for investigating epigenetic modes of action of agents in target organs, as the assay is amenable to cultured cells or nucleated cells from any tissue. Environ. Mol. Mutagen. 58:508-521, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  11. Histone modification alteration coordinated with acquisition of promoter DNA methylation during Epstein-Barr virus infection.

    Science.gov (United States)

    Funata, Sayaka; Matsusaka, Keisuke; Yamanaka, Ryota; Yamamoto, Shogo; Okabe, Atsushi; Fukuyo, Masaki; Aburatani, Hiroyuki; Fukayama, Masashi; Kaneda, Atsushi

    2017-08-15

    Aberrant DNA hypermethylation is a major epigenetic mechanism to inactivate tumor suppressor genes in cancer. Epstein-Barr virus positive gastric cancer is the most frequently hypermethylated tumor among human malignancies. Herein, we performed comprehensive analysis of epigenomic alteration during EBV infection, by Infinium HumanMethylation 450K BeadChip for DNA methylation and ChIP-sequencing for histone modification alteration during EBV infection into gastric cancer cell line MKN7. Among 7,775 genes with increased DNA methylation in promoter regions, roughly half were "DNA methylation-sensitive" genes, which acquired DNA methylation in the whole promoter regions and thus were repressed. These included anti-oncogenic genes, e.g. CDKN2A . The other half were "DNA methylation-resistant" genes, where DNA methylation is acquired in the surrounding of promoter regions, but unmethylated status is protected in the vicinity of transcription start site. These genes thereby retained gene expression, and included DNA repair genes. Histone modification was altered dynamically and coordinately with DNA methylation alteration. DNA methylation-sensitive genes significantly correlated with loss of H3K27me3 pre-marks or decrease of active histone marks, H3K4me3 and H3K27ac. Apoptosis-related genes were significantly enriched in these epigenetically repressed genes. Gain of active histone marks significantly correlated with DNA methylation-resistant genes. Genes related to mitotic cell cycle and DNA repair were significantly enriched in these epigenetically activated genes. Our data show that orchestrated epigenetic alterations are important in gene regulation during EBV infection, and histone modification status in promoter regions significantly associated with acquisition of de novo DNA methylation or protection of unmethylated status at transcription start site.

  12. Arabidopsis phyllotaxis is controlled by the methyl-esterification status of cell-wall pectins.

    Science.gov (United States)

    Peaucelle, Alexis; Louvet, Romain; Johansen, Jorunn N; Höfte, Herman; Laufs, Patrick; Pelloux, Jérome; Mouille, Grégory

    2008-12-23

    Plant organs are produced from meristems in a characteristic pattern. This pattern, referred to as phyllotaxis, is thought to be generated by local gradients of an information molecule, auxin. Some studies propose a key role for the mechanical properties of the cell walls in the control of organ outgrowth. A major cell-wall component is the linear alpha-1-4-linked D-GalAp pectic polysaccharide homogalacturonan (HG), which plays a key role in cell-to-cell cohesion. HG is deposited in the cell wall in a highly (70%-80%) methyl-esterified form and is subsequently de-methyl-esterified by pectin methyl-esterases (PME, EC 3.1.1.11). PME activity is itself regulated by endogenous PME inhibitor (PMEI) proteins. PME action modulates cell-wall-matrix properties and plays a role in the control of cell growth. Here, we show that the formation of flower primordia in the Arabidopsis shoot apical meristem is accompanied by the de-methyl-esterification of pectic polysaccharides in the cell walls. In addition, experimental perturbation of the methyl-esterification status of pectins within the meristem dramatically alters the phyllotactic pattern. These results demonstrate that regulated de-methyl-esterification of pectins is a key event in the outgrowth of primordia and possibly also in phyllotactic patterning.

  13. High resolution melt curve analysis based on methylation status for human semen identification.

    Science.gov (United States)

    Fachet, Caitlyn; Quarino, Lawrence; Karnas, K Joy

    2017-03-01

    A high resolution melt curve assay to differentiate semen from blood, saliva, urine, and vaginal fluid based on methylation status at the Dapper Isoform 1 (DACT1) gene was developed. Stains made from blood, saliva, urine, semen, and vaginal fluid were obtained from volunteers and DNA was isolated using either organic extraction (saliva, urine, and vaginal fluid) or Chelex ® 100 extraction (blood and semen). Extracts were then subjected to bisulfite modification in order to convert unmethylated cytosines to uracil, consequently creating sequences whose amplicons have melt curves that vary depending on their initial methylation status. When primers designed to amplify the promoter region of the DACT1 gene were used, DNA from semen samples was distinguishable from other fluids by a having a statistically significant lower melting temperature. The assay was found to be sperm-significant since semen from a vasectomized man produced a melting temperature similar to the non-semen body fluids. Blood and semen stains stored up to 5 months and tested at various intervals showed little variation in melt temperature indicating the methylation status was stable during the course of the study. The assay is a more viable method for forensic science practice than most molecular-based methods for body fluid stain identification since it is time efficient and utilizes instrumentation common to forensic biology laboratories. In addition, the assay is advantageous over traditional presumptive chemical methods for body fluid identification since results are confirmatory and the assay offers the possibility of multiplexing which may test for multiple body fluids simultaneously.

  14. Transgenerational epigenetics: Inheritance of global cytosine methylation and methylation-related epigenetic markers in the shrub Lavandula latifolia.

    Science.gov (United States)

    Herrera, Carlos M; Alonso, Conchita; Medrano, Mónica; Pérez, Ricardo; Bazaga, Pilar

    2018-04-01

    The ecological and evolutionary significance of natural epigenetic variation (i.e., not based on DNA sequence variants) variation will depend critically on whether epigenetic states are transmitted from parents to offspring, but little is known on epigenetic inheritance in nonmodel plants. We present a quantitative analysis of transgenerational transmission of global DNA cytosine methylation (= proportion of all genomic cytosines that are methylated) and individual epigenetic markers (= methylation status of anonymous MSAP markers) in the shrub Lavandula latifolia. Methods based on parent-offspring correlations and parental variance component estimation were applied to epigenetic features of field-growing plants ('maternal parents') and greenhouse-grown progenies. Transmission of genetic markers (AFLP) was also assessed for reference. Maternal parents differed significantly in global DNA cytosine methylation (range = 21.7-36.7%). Greenhouse-grown maternal families differed significantly in global methylation, and their differences were significantly related to maternal origin. Methylation-sensitive amplified polymorphism (MSAP) markers exhibited significant transgenerational transmission, as denoted by significant maternal variance component of marker scores in greenhouse families and significant mother-offspring correlations of marker scores. Although transmission-related measurements for global methylation and MSAP markers were quantitatively lower than those for AFLP markers taken as reference, this study has revealed extensive transgenerational transmission of genome-wide global cytosine methylation and anonymous epigenetic markers in L. latifolia. Similarity of results for global cytosine methylation and epigenetic markers lends robustness to this conclusion, and stresses the value of considering both types of information in epigenetic studies of nonmodel plants. © 2018 Botanical Society of America.

  15. Relative expression of rRNA transcripts and 45S rDNA promoter methylation status are dysregulated in tumors in comparison with matched-normal tissues in breast cancer.

    Science.gov (United States)

    Karahan, Gurbet; Sayar, Nilufer; Gozum, Gokcen; Bozkurt, Betul; Konu, Ozlen; Yulug, Isik G

    2015-06-01

    Ribosomal RNA (rRNA) expression, one of the most important factors regulating ribosome production, is primarily controlled by a CG-rich 45 S rDNA promoter. However, the DNA methylation state of the 45 S rDNA promoter, as well as its effect on rRNA gene expression in types of human cancers is controversial. In the present study we analyzed the methylation status of the rDNA promoter (-380 to +53 bp) as well as associated rRNA expression levels in breast cancer cell lines and breast tumor-normal tissue pairs. We found that the aforementioned regulatory region was extensively methylated (74-96%) in all cell lines and in 68% (13/19 tumor-normal pairs) of the tumors. Expression levels of rRNA transcripts 18 S, 28 S, 5.8 S and 45 S external transcribed spacer (45 S ETS) greatly varied in the breast cancer cell lines regardless of their methylation status. Analyses of rRNA transcript expression levels in the breast tumor and normal matched tissues showed no significant difference when normalized with TBP. On the other hand, using the geometric mean of the rRNA expression values (GM-rRNA) as reference enabled us to identify significant changes in the relative expression of rRNAs in the tissue samples. We propose GM-rRNA normalization as a novel strategy to analyze expression differences between rRNA transcripts. Accordingly, the 18S rRNA/GM-rRNA ratio was significantly higher whereas the 5.8S rRNA/GM-rRNA ratio was significantly lower in breast tumor samples than this ratio in the matched normal samples. Moreover, the 18S rRNA/GM-rRNA ratio was negatively correlated with the 45 S rDNA promoter methylation level in the normal breast tissue samples, yet not in the breast tumors. Significant correlations observed between the expression levels of rRNA transcripts in the normal samples were lost in the tumor samples. We showed that the expression of rRNA transcripts may not be based solely on promoter methylation. Carcinogenesis may cause dysregulation of the correlation

  16. Analysis of deuterium relaxation-derived methyl axis order parameters and correlation with local structure

    International Nuclear Information System (INIS)

    Mittermaier, Anthony; Kay, Lewis E.; Forman-Kay, Julie D.

    1999-01-01

    Methyl axis (S2axis) and backbone NH (S2NH) order parameters derived from eight proteins have been analyzed. Similar distribution profiles for Ala S2axis and S2NH order parameters were observed. A good correlation between the two S2axis values of Val and Leu methyl groups is noted, although differences between order parameters can arise. The relation of S2axis or S2NH to solvent accessibility and packing density has also been investigated. Correlations are weak, likely reflecting the importance of collective, non-local motions in proteins. The lack of correlation between these simple structural parameters and dynamics emphasizes the importance of motional studies to fully characterize proteins

  17. Methylation status regulates lipoprotein lipase expression in chronic lymphocytic leukemia.

    Science.gov (United States)

    Abreu, Cecilia; Moreno, Pilar; Palacios, Florencia; Borge, Mercedes; Morande, Pablo; Landoni, Ana Inés; Gabus, Raul; Dighiero, Guillermo; Giordano, Mirta; Gamberale, Romina; Oppezzo, Pablo

    2013-08-01

    Among different prognostic factors in chronic lymphocytic leukemia (CLL), we previously demonstrated that lipoprotein lipase (LPL) is associated with an unmutated immunoglobulin profile and clinical poor outcome. Despite the usefulness of LPL for CLL prognosis, its functional role and the molecular mechanism regulating its expression are still open questions. Interaction of CLL B-cells with the tissue microenvironment favors disease progression by promoting malignant B-cell growth. Since tissue methylation can be altered by environmental factors, we investigated the methylation status of the LPL gene and the possibility that overexpression could be associated with microenvironment signals. Our results show that a demethylated state of the LPL gene is responsible for its anomalous expression in unmutated CLL cases and that this expression is dependent on microenvironment signals. Overall, this work proposes that an epigenetic mechanism, triggered by the microenvironment, regulates LPL expression in CLL disease.

  18. Global DNA methylation analysis using methyl-sensitive amplification polymorphism (MSAP).

    Science.gov (United States)

    Yaish, Mahmoud W; Peng, Mingsheng; Rothstein, Steven J

    2014-01-01

    DNA methylation is a crucial epigenetic process which helps control gene transcription activity in eukaryotes. Information regarding the methylation status of a regulatory sequence of a particular gene provides important knowledge of this transcriptional control. DNA methylation can be detected using several methods, including sodium bisulfite sequencing and restriction digestion using methylation-sensitive endonucleases. Methyl-Sensitive Amplification Polymorphism (MSAP) is a technique used to study the global DNA methylation status of an organism and hence to distinguish between two individuals based on the DNA methylation status determined by the differential digestion pattern. Therefore, this technique is a useful method for DNA methylation mapping and positional cloning of differentially methylated genes. In this technique, genomic DNA is first digested with a methylation-sensitive restriction enzyme such as HpaII, and then the DNA fragments are ligated to adaptors in order to facilitate their amplification. Digestion using a methylation-insensitive isoschizomer of HpaII, MspI is used in a parallel digestion reaction as a loading control in the experiment. Subsequently, these fragments are selectively amplified by fluorescently labeled primers. PCR products from different individuals are compared, and once an interesting polymorphic locus is recognized, the desired DNA fragment can be isolated from a denaturing polyacrylamide gel, sequenced and identified based on DNA sequence similarity to other sequences available in the database. We will use analysis of met1, ddm1, and atmbd9 mutants and wild-type plants treated with a cytidine analogue, 5-azaC, or zebularine to demonstrate how to assess the genetic modulation of DNA methylation in Arabidopsis. It should be noted that despite the fact that MSAP is a reliable technique used to fish for polymorphic methylated loci, its power is limited to the restriction recognition sites of the enzymes used in the genomic

  19. Correlation of MLH1 and MGMT expression and promoter methylation with genomic instability in patients with thyroid carcinoma

    International Nuclear Information System (INIS)

    Santos, Juliana Carvalho; Bastos, André Uchimura; Cerutti, Janete Maria; Ribeiro, Marcelo Lima

    2013-01-01

    Gene silencing of the repair genes MLH1 and MGMT was shown to be a mechanism underlying the development of microsatellite instability (MSI), a phenotype frequently associated with various human malignancies. Recently, aberrant methylation of MLH1, MGMT and MSI were shown to be associated with mutations in genes such as BRAF, RAS and IDH1 in colon and brain tumours. Little is known about the methylation status of MLH1 and MGMT in thyroid tumours and its association with MSI and mutational status. In a series of 96 thyroid tumours whose mutational profiles of BRAF, IDH1 and NRAS mutations and RET/PTC were previously determined, we investigated MLH1 and MGMT expression and methylation status by qPCR and methylation-specific PCR after bisulphite treatment, respectively. MSI was determined by PCR using seven standard microsatellite markers. Samples with point mutations (BRAF, IDH1 and NRAS) show a decrease in MLH1 expression when compared to negative samples. Additionally, malignant lesions show a higher MSI pattern than benign lesions. The MSI phenotype was also associated with down-regulation of MLH1. The results of this study allow us to conclude that low expression of MLH1 is associated with BRAF V600E mutations, RET/PTC rearrangements and transitions (IDH1 and NRAS) in patients with thyroid carcinoma. In addition, a significant relationship between MSI status and histological subtypes was found

  20. Clinical Neuropathology practice news 1-2014: Pyrosequencing meets clinical and analytical performance criteria for routine testing of MGMT promoter methylation status in glioblastoma

    Science.gov (United States)

    Preusser, Matthias; Berghoff, Anna S.; Manzl, Claudia; Filipits, Martin; Weinhäusel, Andreas; Pulverer, Walter; Dieckmann, Karin; Widhalm, Georg; Wöhrer, Adelheid; Knosp, Engelbert; Marosi, Christine; Hainfellner, Johannes A.

    2014-01-01

    Testing of the MGMT promoter methylation status in glioblastoma is relevant for clinical decision making and research applications. Two recent and independent phase III therapy trials confirmed a prognostic and predictive value of the MGMT promoter methylation status in elderly glioblastoma patients. Several methods for MGMT promoter methylation testing have been proposed, but seem to be of limited test reliability. Therefore, and also due to feasibility reasons, translation of MGMT methylation testing into routine use has been protracted so far. Pyrosequencing after prior DNA bisulfite modification has emerged as a reliable, accurate, fast and easy-to-use method for MGMT promoter methylation testing in tumor tissues (including formalin-fixed and paraffin-embedded samples). We performed an intra- and inter-laboratory ring trial which demonstrates a high analytical performance of this technique. Thus, pyrosequencing-based assessment of MGMT promoter methylation status in glioblastoma meets the criteria of high analytical test performance and can be recommended for clinical application, provided that strict quality control is performed. Our article summarizes clinical indications, practical instructions and open issues for MGMT promoter methylation testing in glioblastoma using pyrosequencing. PMID:24359605

  1. Tumor suppressor genes are frequently methylated in lymph node metastases of breast cancers

    Directory of Open Access Journals (Sweden)

    Xu Jia

    2010-07-01

    Full Text Available Abstract Introduction Metastasis represents a major adverse step in the progression of breast carcinoma. Lymph node invasion is the most relevant prognostic factor; however little is known on the molecular events associated with lymph node metastasis process. This study is to investigate the status and role of methylation in lymph node metastatic tumors. Materials and methods Bisulfite pyrosequencing is used to screen 6 putative tumor suppressor genes (HIN-1, RASSF1A, RIL, CDH13, RARβ2 and E-cadherin in 38 pairs of primary breast tumors and lymph node metastases. Results We found that HIN-1, CDH13, RIL, RASSF1A and RARβ2 were frequently methylated both in primary and metastatic tissues (range: 55.3%~89.5%. E-cadherin was not frequently methylated in either setting (range: 18.4%~23.7%. The methylation status of HIN-1, CDH13, RIL, and RARβ2 in lymph nodes metastasis were correlated with that in primary tumors. The Pearson correlation values ranged from 0.624 to 0.472 (p values HIN-1 methylation and hormone status in metastatic lymph nodes. Hypermethylation of HIN-1 in metastasis lymph nodes was significantly associated with expression of ER (odds ratio, 1.070; P = 0.024 and with PR (odds ratio, 1.046; P = 0.026. Conclusions This study suggests that hypermethylation of tumor suppressor genes is extended from primary to metastatic tumors during tumor progression.

  2. Amyloid protein-mediated differential DNA methylation status regulates gene expression in Alzheimer’s disease model cell line

    International Nuclear Information System (INIS)

    Sung, Hye Youn; Choi, Eun Nam; Ahn Jo, Sangmee; Oh, Seikwan; Ahn, Jung-Hyuck

    2011-01-01

    Highlights: ► Genome-wide DNA methylation pattern in Alzheimer’s disease model cell line. ► Integrated analysis of CpG methylation and mRNA expression profiles. ► Identify three Swedish mutant target genes; CTIF, NXT2 and DDR2 gene. ► The effect of Swedish mutation on alteration of DNA methylation and gene expression. -- Abstract: The Swedish mutation of amyloid precursor protein (APP-sw) has been reported to dramatically increase beta amyloid production through aberrant cleavage at the beta secretase site, causing early-onset Alzheimer’s disease (AD). DNA methylation has been reported to be associated with AD pathogenesis, but the underlying molecular mechanism of APP-sw-mediated epigenetic alterations in AD pathogenesis remains largely unknown. We analyzed genome-wide interplay between promoter CpG DNA methylation and gene expression in an APP-sw-expressing AD model cell line. To identify genes whose expression was regulated by DNA methylation status, we performed integrated analysis of CpG methylation and mRNA expression profiles, and identified three target genes of the APP-sw mutant; hypomethylated CTIF (CBP80/CBP20-dependent translation initiation factor) and NXT2 (nuclear exporting factor 2), and hypermethylated DDR2 (discoidin domain receptor 2). Treatment with the demethylating agent 5-aza-2′-deoxycytidine restored mRNA expression of these three genes, implying methylation-dependent transcriptional regulation. The profound alteration in the methylation status was detected at the −435, −295, and −271 CpG sites of CTIF, and at the −505 to −341 region in the promoter of DDR2. In the promoter region of NXT2, only one CpG site located at −432 was differentially unmethylated in APP-sw cells. Thus, we demonstrated the effect of the APP-sw mutation on alteration of DNA methylation and subsequent gene expression. This epigenetic regulatory mechanism may contribute to the pathogenesis of AD.

  3. Association between methylation of the glucocorticoid receptor gene, childhood maltreatment, and clinical severity in borderline personality disorder.

    Science.gov (United States)

    Martín-Blanco, Ana; Ferrer, Marc; Soler, Joaquim; Salazar, Juliana; Vega, Daniel; Andión, Oscar; Sanchez-Mora, Cristina; Arranz, Maria Jesús; Ribases, Marta; Feliu-Soler, Albert; Pérez, Víctor; Pascual, Juan Carlos

    2014-10-01

    The hypothalamus-pituitary-adrenal axis (HPA) is essential in the regulation of stress responses. Increased methylation of the promoter region of the glucocorticoid receptor gene (NR3C1) has been described both in subjects with history of childhood trauma and in patients with Borderline Personality Disorder (BPD). However, no data on the possible association between a higher methylation of this gene and clinical severity is available. The aim of this study was to evaluate the association between NR3C1 methylation status, the history of childhood trauma, and current clinical severity in subjects with BPD. A sample of 281 subjects with BPD (diagnosed by SCID-II and DIB-R semi-structured diagnostic interviews) was recruited. Clinical variables included previous hospitalizations, self-injurious behavior, and self-reported history of childhood trauma. DNA was extracted from peripheral blood. The results indicated a significant positive correlation between NR3C1 methylation status and childhood maltreatment (specifically physical abuse). In addition, a positive correlation between methylation status and clinical severity (DIB-R total score and hospitalizations) was observed. These findings suggest that NR3C1 methylation in subjects with BPD may be associated not only with childhood trauma but also with clinical severity, adding new evidence to the involvement of gene-environment interactions in this disorder. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Generation of Five Human Lactoferrin Transgenic Cloned Goats Using Fibroblast Cells and Their Methylation Status of Putative Differential Methylation Regions of IGF2R and H19 Imprinted Genes

    Science.gov (United States)

    Sun, Yanyan; Zhang, Yanli; Wang, Ziyu; Song, Yang; Wang, Feng

    2013-01-01

    Background Somatic cell nuclear transfer (SCNT) is a promising technique to produce transgenic cloned mammalian, including transgenic goats which may produce Human Lactoferrin (hLF). However, success percentage of SCNT is low, because of gestational and neonatal failure of transgenic embryos. According to the studies on cattle and mice, DNA methylation of some imprinted genes, which plays a vital role in the reprogramming of embryo in NT maybe an underlying mechanism. Methodology/Principal Findings Fibroblast cells were derived from the ear of a two-month-old goat. The vector expressing hLF was constructed and transfected into fibroblasts. G418 selection, EGFP expression, PCR, and cell cycle distribution were applied sequentially to select transgenic cells clones. After NT and embryo transfer, five transgenic cloned goats were obtained from 240 cloned transgenic embryos. These transgenic goats were identified by 8 microsatellites genotyping and southern blot. Of the five transgenic goats, 3 were lived after birth, while 2 were dead during gestation. We compared differential methylation regions (DMR) pattern of two paternally imprinted genes (H19 and IGF2R) of the ear tissues from the lived transgenic goats, dead transgenic goats, and control goats from natural reproduction. Hyper-methylation pattern appeared in cloned aborted goats, while methylation status was relatively normal in cloned lived goats compared with normal goats. Conclusions/Significance In this study, we generated five hLF transgenic cloned goats by SCNT. This is the first time the DNA methylation of lived and dead transgenic cloned goats was compared. The results demonstrated that the methylation status of DMRs of H19 and IGF2R were different in lived and dead transgenic goats and therefore this may be potentially used to assess the reprogramming status of transgenic cloned goats. Understanding the pattern of gene imprinting may be useful to improve cloning techniques in future. PMID:24204972

  5. Altered mucosal DNA methylation in parallel with highly active Helicobacter pylori-related gastritis.

    Science.gov (United States)

    Yoshida, Takeichi; Kato, Jun; Maekita, Takao; Yamashita, Satoshi; Enomoto, Shotaro; Ando, Takayuki; Niwa, Tohru; Deguchi, Hisanobu; Ueda, Kazuki; Inoue, Izumi; Iguchi, Mikitaka; Tamai, Hideyuki; Ushijima, Toshikazu; Ichinose, Masao

    2013-10-01

    Chronic inflammation triggered by Helicobacter pylori causes altered DNA methylation in stomach mucosae, which is deeply involved in gastric carcinogenesis. This study aimed to elucidate the correlation between altered mucosal DNA methylation levels and activity of H. pylori-related gastritis, because inflammatory activity shows particular correlations with the development of diffuse-type cancer. Methylation levels in stomach mucosae of 78 healthy volunteers were determined by real-time methylation-specific PCR or bisulfite pyrosequencing. Examined loci were the promoter CpG islands of six genes (FLNc, HAND1, THBD, p41ARC, HRASLS, and LOX) and the CpG sites of non-coding repetitive elements (Alu and Satα) that are reportedly altered by H. pylori infection. Activity of H. pylori-related gastritis was evaluated using two serum markers: H. pylori antibody titer and pepsinogen II. Methylation levels of the six CpG islands were consistently increased, and those of the two repetitive elements were consistently decreased in a stepwise manner with the activity of gastric inflammation as represented by serum marker levels. Each serum marker level was well correlated with the overall DNA methylation status of stomach mucosa, and these two serologic markers were additive in the detection of the mucosa with severely altered DNA methylation. Alteration in mucosal DNA methylation level was closely correlated with activity of H. pylori-related gastritis as evaluated by serum markers. The observed correlation between altered DNA methylation levels and activity of H. pylori-related gastritis appears to be one of the relevant molecular mechanisms underlying the development of diffuse-type cancer.

  6. Associations between methylation of paternally expressed gene 3 (PEG3, cervical intraepithelial neoplasia and invasive cervical cancer.

    Directory of Open Access Journals (Sweden)

    Monica D Nye

    Full Text Available Cytology-based screening for invasive cervical cancer (ICC lacks sensitivity and specificity to discriminate between cervical intraepithelial neoplasia (CIN likely to persist or progress from cases likely to resolve. Genome-wide approaches have been used to identify DNA methylation marks associated with CIN persistence or progression. However, associations between DNA methylation marks and CIN or ICC remain weak and inconsistent. Between 2008-2009, we conducted a hospital-based, case-control study among 213 Tanzania women with CIN 1/2/3 or ICC. We collected questionnaire data, biopsies, peripheral blood, cervical scrapes, Human papillomavirus (HPV and HIV-1 infection status. We assessed PEG3 methylation status by bisulfite pyrosequencing. Multinomial logistic regression was used to estimate odds ratios (OR and confidence intervals (CI 95% for associations between PEG3 methylation status and CIN or ICC. After adjusting for age, gravidity, hormonal contraceptive use and HPV infection, a 5% increase in PEG3 DNA methylation was associated with increased risk for ICC (OR = 1.6; 95% CI 1.2-2.1. HPV infection was associated with a higher risk of CIN1-3 (OR = 15.7; 95% CI 5.7-48.6 and ICC (OR = 29.5, 95% CI 6.3-38.4. Infection with high risk HPV was correlated with mean PEG3 differentially methylated regions (DMRs methylation (r = 0.34 p<0.0001, while the correlation with low risk HPV infection was weaker (r = 0.16 p = 0.047. Although small sample size limits inference, these data support that PEG3 methylation status has potential as a molecular target for inclusion in CIN screening to improve prediction of progression. Impact statement: We present the first evidence that aberrant methylation of the PEG3 DMR is an important co-factor in the development of Invasive cervical carcinoma (ICC, especially among women infected with high risk HPV. Our results show that a five percent increase in DNA methylation of PEG3 is associated with

  7. DNA methylation profiling reveals the presence of population-specific signatures correlating with phenotypic characteristics.

    Science.gov (United States)

    Giri, Anil K; Bharadwaj, Soham; Banerjee, Priyanka; Chakraborty, Shraddha; Parekatt, Vaisak; Rajashekar, Donaka; Tomar, Abhishek; Ravindran, Aarthi; Basu, Analabha; Tandon, Nikhil; Bharadwaj, Dwaipayan

    2017-06-01

    Phenotypic characteristics are known to vary substantially among different ethnicities around the globe. These variations are mediated by number of stochastic events and cannot be attributed to genetic architecture alone. DNA methylation is a well-established mechanism that sculpts our epigenome influencing phenotypic variation including disease manifestation. Since DNA methylation is an important determinant for health issues of a population, it demands a thorough investigation of the natural differences in genome wide DNA methylation patterns across different ethnic groups. This study is based on comparative analyses of methylome from five different ethnicities with major focus on Indian subjects. The current study uses hierarchical clustering approaches, principal component analysis and locus specific differential methylation analysis on Illumina 450K methylation data to compare methylome of different ethnic subjects. Our data indicates that the variations in DNA methylation patterns of Indians are less among themselves compared to other global population. It empirically correlated with dietary, cultural and demographical divergences across different ethnic groups. Our work further suggests that Indians included in this study, despite their genetic similarity with the Caucasian population, are in close proximity with Japanese in terms of their methylation signatures.

  8. Correlation of MLH1 and MGMT methylation levels between peripheral blood leukocytes and colorectal tissue DNA samples in colorectal cancer patients.

    Science.gov (United States)

    Li, Xia; Wang, Yibaina; Zhang, Zuoming; Yao, Xiaoping; Ge, Jie; Zhao, Yashuang

    2013-11-01

    CpG island methylation in the promoter regions of the DNA mismatch repair gene mutator L homologue 1 ( MLH1 ) and DNA repair gene O 6 -methylguanine-DNA methyltransferase ( MGMT ) genes has been shown to occur in the leukocytes of peripheral blood and colorectal tissue. However, it is unclear whether the methylation levels in the blood leukocytes and colorectal tissue are correlated. The present study analyzed and compared the levels of MGMT and MLH1 gene methylation in the leukocytes of peripheral blood and colorectal tissues obtained from patients with colorectal cancer (CRC). The methylation levels of MGMT and MLH1 were examined using methylation-sensitive high-resolution melting (MS-HRM) analysis. A total of 44 patients with CRC were selected based on the MLH1 and MGMT gene methylation levels in the leukocytes of the peripheral blood. Corresponding colorectal tumor and normal tissues were obtained from each patient and the DNA methylation levels were determined. The correlation coefficients were evaluated using Spearman's rank test. Agreement was determined by generalized κ-statistics. Spearman's rank correlation coefficients (r) for the methylation levels of the MGMT and MLH1 genes in the leukocytes of the peripheral blood and normal colorectal tissue were 0.475 and 0.362, respectively (P=0.001 and 0.016, respectively). The agreement of the MGMT and MLH1 gene methylation levels in the leukocytes of the peripheral blood and normal colorectal tissue were graded as fair and poor (κ=0.299 and 0.126, respectively). The methylation levels of MGMT and MLH1 were moderately and weakly correlated between the patient-matched leukocytes and the normal colorectal tissue, respectively. Blood-derived DNA methylation measurements may not always represent the levels of normal colorectal tissue methylation.

  9. FKBP5 methylation as a possible marker for cortisol state and transient cortisol exposure in healthy human subjects.

    Science.gov (United States)

    Winkler, Britta K; Lehnert, Hendrik; Oster, Henrik; Kirchner, Henriette; Harbeck, Birgit

    2017-10-01

    Current glucocorticoid replacement regimens, in adrenal insufficiency, fail to mimic the physiological cortisol secretion, thereby fostering serious side effects. To experimentally evaluate the impact of CpG methylation within the FKBP5 gene as a possible short- and long-term marker for cortisol exposure in humans. An ACTH-stimulation test was carried out and methylation status of the FKBP5 gene in leukocytes was determined. A negative correlation between basal levels of methylation and serum cortisol was observed. Individual changes in FKBP5 methylation after 24 h correlated with cortisol responses. Considering previous studies conducted with murine leucocytes, FKBP5 methylation may be suitable as a long-term biomarker, rather than acute glucocorticoid exposure, also in humans.

  10. Catalytic hydrodeoxygenation of methyl-substituted phenols: correlations of kinetic parameters with molecular properties.

    Science.gov (United States)

    Massoth, F E; Politzer, P; Concha, M C; Murray, J S; Jakowski, J; Simons, Jack

    2006-07-27

    The hydrodeoxygenation of methyl-substituted phenols was carried out in a flow microreactor at 300 degrees C and 2.85 MPa hydrogen pressure over a sulfided CoMo/Al(2)O(3) catalyst. The primary reaction products were methyl-substituted benzene, cyclohexene, cyclohexane, and H(2)O. Analysis of the results suggests that two independent reaction paths are operative, one leading to aromatics and the other to partially or completely hydrogenated cyclohexanes. The reaction data were analyzed using Langmuir-Hinshelwood kinetics to extract the values of the reactant-to-catalyst adsorption constant and of the rate constants characterizing the two reaction paths. The adsorption constant was found to be the same for both reactions, suggesting that a single catalytic site center is operative in both reactions. Ab initio electronic structure calculations were used to evaluate the electrostatic potentials and valence orbital ionization potentials for all of the substituted phenol reactants. Correlations were observed between (a) the adsorption constant and the two reaction rate constants measured for various methyl-substitutions and (b) certain moments of the electrostatic potentials and certain orbitals' ionization potentials of the isolated phenol molecules. On the basis of these correlations to intrinsic reactant-molecule properties, a reaction mechanism is proposed for each pathway, and it is suggested that the dependencies of adsorption and reaction rates upon methyl-group substitution are a result of the substituents' effects on the electrostatic potential and orbitals rather than geometric (steric) effects.

  11. Double-labelling immunohistochemistry for MGMT and a "cocktail" of non-tumourous elements is a reliable, quick and easy technique for inferring methylation status in glioblastomas and other primary brain tumours.

    Science.gov (United States)

    Burke, Elinor; Grobler, Mariana; Elderfield, Kay; Bond, Frances; Crocker, Matthew; Taylor, Rohan; Bridges, Leslie R

    2013-06-10

    Our aim was to develop a new protocol for MGMT immunohistochemistry with good agreement between observers and good correlation with molecular genetic tests of tumour methylation. We examined 40 primary brain tumours (30 glioblastomas and 10 oligodendroglial tumours) with our new technique, namely double-labelling immunohistochemistry for MGMT and a "cocktail" of non-tumour antigens (CD34, CD45 and CD68). We compared the results with single-labelling immunohistochemistry for MGMT and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA, a recognised molecular genetic technique which we applied as the gold-standard for the methylation status). Double-labelling immunohistochemistry for MGMT produced a visual separation of tumourous and non-tumourous elements on the same histological slide, making it quick and easy to determine whether tumour cell nuclei were MGMT-positive or MGMT-negative (and thereby infer the methylation status of the tumour). We found good agreement between observers (kappa 0.76) and within observer (kappa 0.84). Furthermore, double-labelling showed good specificity (80%), sensitivity (73.33%), positive predictive value (PPV, 83.33%) and negative predictive value (NPV, 68.75%) compared to MS-MLPA. Double-labelling was quicker and easier to assess than single-labelling and it outperformed quantitative computerised image analysis of MGMT single-labelling in terms of sensitivity, specificity, PPV and NPV. Double-labelling immunohistochemistry for MGMT and a cocktail of non-tumourous elements provides a "one look" method for determining whether tumour cell nuclei are MGMT-positive or MGMT-negative. This can be used to infer the methylation status of the tumour. There is good observer agreement and good specificity, sensitivity, PPV and NPV compared to a molecular gold-standard.

  12. Sleep quality and methylation status of selected tumor suppressor genes among nurses and midwives.

    Science.gov (United States)

    Bukowska-Damska, Agnieszka; Reszka, Edyta; Kaluzny, Pawel; Wieczorek, Edyta; Przybek, Monika; Zienolddiny, Shanbeh; Peplonska, Beata

    2018-01-01

    Chronic sleep restriction may affect metabolism, hormone secretion patterns and inflammatory responses. Limited reports suggest also epigenetic effects, such as changes in DNA methylation profiles. The study aims to assess the potential association between poor sleep quality or sleep duration and the levels of 5-methylcytosine in the promoter regions of selected tumor suppressor genes. A cross-sectional study was conducted on 710 nurses and midwives aged 40-60 years. Data from interviews regarding sleep habits and potential confounders were used. The methylation status of tumor suppressor genes was determined via qMSP reactions using DNA samples derived from leucocytes. No significant findings were observed in the total study population or in the two subgroups of women stratified by the current system of work. A borderline significance association was observed between a shorter duration of sleep and an increased methylation level in CDKN2A among day working nurses and midwives. Further studies are warranted to explore this under-investigated topic.

  13. Whole-genome DNA methylation status associated with clinical PTSD measures of OIF/OEF veterans

    Science.gov (United States)

    Hammamieh, R; Chakraborty, N; Gautam, A; Muhie, S; Yang, R; Donohue, D; Kumar, R; Daigle, B J; Zhang, Y; Amara, D A; Miller, S-A; Srinivasan, S; Flory, J; Yehuda, R; Petzold, L; Wolkowitz, O M; Mellon, S H; Hood, L; Doyle, F J; Marmar, C; Jett, M

    2017-01-01

    Emerging knowledge suggests that post-traumatic stress disorder (PTSD) pathophysiology is linked to the patients’ epigenetic changes, but comprehensive studies examining genome-wide methylation have not been performed. In this study, we examined genome-wide DNA methylation in peripheral whole blood in combat veterans with and without PTSD to ascertain differentially methylated probes. Discovery was initially made in a training sample comprising 48 male Operation Enduring Freedom (OEF)/Operation Iraqi Freedom (OIF) veterans with PTSD and 51 age/ethnicity/gender-matched combat-exposed PTSD-negative controls. Agilent whole-genome array detected ~5600 differentially methylated CpG islands (CpGI) annotated to ~2800 differently methylated genes (DMGs). The majority (84.5%) of these CpGIs were hypermethylated in the PTSD cases. Functional analysis was performed using the DMGs encoding the promoter-bound CpGIs to identify networks related to PTSD. The identified networks were further validated by an independent test set comprising 31 PTSD+/29 PTSD− veterans. Targeted bisulfite sequencing was also used to confirm the methylation status of 20 DMGs shown to be highly perturbed in the training set. To improve the statistical power and mitigate the assay bias and batch effects, a union set combining both training and test set was assayed using a different platform from Illumina. The pathways curated from this analysis confirmed 65% of the pool of pathways mined from training and test sets. The results highlight the importance of assay methodology and use of independent samples for discovery and validation of differentially methylated genes mined from whole blood. Nonetheless, the current study demonstrates that several important epigenetically altered networks may distinguish combat-exposed veterans with and without PTSD. PMID:28696412

  14. Correlation of EEG with neuropsychological status in children with epilepsy.

    Science.gov (United States)

    Hsu, David A; Rayer, Katherine; Jackson, Daren C; Stafstrom, Carl E; Hsu, Murielle; Ferrazzano, Peter A; Dabbs, Kevin; Worrell, Gregory A; Jones, Jana E; Hermann, Bruce P

    2016-02-01

    To determine correlations of the EEG frequency spectrum with neuropsychological status in children with idiopathic epilepsy. Forty-six children ages 8-18 years old with idiopathic epilepsy were retrospectively identified and analyzed for correlations between EEG spectra and neuropsychological status using multivariate linear regression. In addition, the theta/beta ratio, which has been suggested as a clinically useful EEG marker of attention-deficit hyperactivity disorder (ADHD), and an EEG spike count were calculated for each subject. Neuropsychological status was highly correlated with posterior alpha (8-15 Hz) EEG activity in a complex way, with both positive and negative correlations at lower and higher alpha frequency sub-bands for each cognitive task in a pattern that depends on the specific cognitive task. In addition, the theta/beta ratio was a specific but insensitive indicator of ADHD status in children with epilepsy; most children both with and without epilepsy have normal theta/beta ratios. The spike count showed no correlations with neuropsychological status. (1) The alpha rhythm may have at least two sub-bands which serve different purposes. (2) The theta/beta ratio is not a sensitive indicator of ADHD status in children with epilepsy. (3) The EEG frequency spectrum correlates more robustly with neuropsychological status than spike count analysis in children with idiopathic epilepsy. (1) The role of posterior alpha rhythms in cognition is complex and can be overlooked if EEG spectral resolution is too coarse or if neuropsychological status is assessed too narrowly. (2) ADHD in children with idiopathic epilepsy may involve different mechanisms from those in children without epilepsy. (3) Reliable correlations with neuropsychological status require longer EEG samples when using spike count analysis than when using frequency spectra. Copyright © 2015 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights

  15. Huntington's disease accelerates epigenetic aging of human brain and disrupts DNA methylation levels.

    Science.gov (United States)

    Horvath, Steve; Langfelder, Peter; Kwak, Seung; Aaronson, Jeff; Rosinski, Jim; Vogt, Thomas F; Eszes, Marika; Faull, Richard L M; Curtis, Maurice A; Waldvogel, Henry J; Choi, Oi-Wa; Tung, Spencer; Vinters, Harry V; Coppola, Giovanni; Yang, X William

    2016-07-01

    Age of Huntington's disease (HD) motoric onset is strongly related to the number of CAG trinucleotide repeats in the huntingtin gene, suggesting that biological tissue age plays an important role in disease etiology. Recently, a DNA methylation based biomarker of tissue age has been advanced as an epigenetic aging clock. We sought to inquire if HD is associated with an accelerated epigenetic age. DNA methylation data was generated for 475 brain samples from various brain regions of 26 HD cases and 39 controls. Overall, brain regions from HD cases exhibit a significant epigenetic age acceleration effect (p=0.0012). A multivariate model analysis suggests that HD status increases biological age by 3.2 years. Accelerated epigenetic age can be observed in specific brain regions (frontal lobe, parietal lobe, and cingulate gyrus). After excluding controls, we observe a negative correlation (r=-0.41, p=5.5×10-8) between HD gene CAG repeat length and the epigenetic age of HD brain samples. Using correlation network analysis, we identify 11 co-methylation modules with a significant association with HD status across 3 broad cortical regions. In conclusion, HD is associated with an accelerated epigenetic age of specific brain regions and more broadly with substantial changes in brain methylation levels.

  16. Expression profiling of O6 methylguanine-DNA-methyl transferase in prolactinomas: a correlative study of promoter methylation and pathological features in 136 cases

    International Nuclear Information System (INIS)

    Jiang, Xiao-Bing; Hu, Bin; He, Dong-Sheng; Mao, Zhi-Gang; Wang, Xin; Song, Bing-Bing; Zhu, Yong-Hong; Wang, Hai-Jun

    2015-01-01

    Low-level expression of O 6 methylguanine-DNA-methyl transferase (MGMT) prolactinomas has been noted previously in case reports, although what modulates MGMT expression remains unclear. This study therefore aimed to delineate the factors regulating MGMT expression in prolactinomas. We retrospectively reviewed 136 prolactinoma patients who were treated in our center between January 2000 and September 2013. Expression of MGMT, Ki-67, and p53 protein were examined by immunohistochemical staining, and MGMT promoter methylation evaluated with methylation-specific PCR. MGMT immunopositivity was <25 % in 106/136 tumor specimens (77.94 %). MGMT immunoexpression was positively correlated with age (r = 0.251, p = 0.003), but inversely correlated with p53 staining (r = −0.153, p = 0.021). Moreover, reduced MGMT expression was more frequent in atypical prolactinomas (p = 0.044). Methylated MGMT promoter was confirmed in 10/46 specimens (21.7 %), all of which had low level or absent MGMT staining. Both p53 protein (r = −0.33, p = 0.025) and promoter methylation (r = −0.331, p = 0.025) were negatively associated with MGMT expression. Multivariate logistic analysis indicated that age (odds ratio [OR] = 1.127. 95 % confidence interval [CI] 1.027–1.236, p = 0.012) and p53 (OR = 0.116. 95 % CI 0.018–0.761, p = 0.025) staining were independent determents of MGMT expression. The majority of prolactinomas, especially atypical prolactinomas, showed low-level or no MGMT immunoexpression, providing a rationale for the utility of temozolomide as an alternative to managing prolactinomas. In summary, epigenetic and transcriptional regulation are involved in silencing MGMT expression

  17. Promoter Methylation and mRNA Expression of Response Gene to Complement 32 in Breast Carcinoma

    International Nuclear Information System (INIS)

    Nasab, E. E.; Nasab, E. E.; Hashemi, M.; Rafighdoost, F.

    2016-01-01

    Response gene to complement 32 (RGC32), induced by activation of complements, has been characterized as a cell cycle regulator; however, its role in carcinogenesis is still controversial. In the present study we compared RGC32 promoter methylation patterns and mRNA expression in breast cancerous tissues and adjacent normal tissues. Materials and Methods. Sixty-three breast cancer tissues and 63 adjacent non neoplastic tissues were included in our study. Design. Nested methylation-specific polymerase chain reaction (Nested-MSP) and quantitative PCR (qPCR) were used to determine RGC32 promoter methylation status and its mRNA expression levels, respectively. Results. RGC32 methylation pattern was not different between breast cancerous tissue and adjacent non neoplastic tissue (OR=2.30, 95% CI=0.95-5.54). However, qPCR analysis displayed higher levels of RGC32 mRNA in breast cancerous tissues than in noncancerous tissues (1.073 versus 0.959; P=0.001), irrespective of the promoter methylation status. The expression levels and promoter methylation of RGC32 were not correlated with any of patients’ clinical characteristics (P>0.05).

  18. Evaluation of promoter methylation status of MLH1 gene in Iranian patients with colorectal tumors and adenoma polyps.

    Science.gov (United States)

    Zarandi, Ashkan; Irani, Shiva; Savabkar, Sanaz; Chaleshi, Vahid; Ghavideldarestani, Maryam; Mirfakhraie, Reza; Khodadoostan, Mahsa; Nazemalhosseini-Mojarad, Ehsan; Asadzadeh Aghdaei, Hamid

    2017-01-01

    The aim of this study was to evaluate the methylation status of the promoter region of MLH1 gene in colorectal cancer (CRC) and its precursor lesions as well as elucidate its association with various clinicopathological characteristics among Iranian population. Epigenetic silencing of mismatch repair genes, such as MLH1 , by methylation of CpG islands of their promoter region has been proved to be an important mechanism in colorectal carcinogenesis. Fifty colorectal cancer and polyp tissue samples including 13 Primary colorectal tumor and 37 Adenoma polyp samples were enrolled in this study. Methylation-specific polymerase chain reaction (MSP) was performed to find the frequency of MLH1 Promoter Methylation. Promoter methylation of MLH1 gene was detected in 5 out of 13 tumor tissues and 4 out of 37 adenoma polyp. The frequency of MLH1 methylation in tumor samples was significantly higher compared to that in polyp tissues (P= 0.026). No significant association was observed between MLH1 promoter methylation and clinicopathological characteristics of the patients. The frequency of  MLH1  promoter methylation in CRC and colon polyp was 18%. Our findings indicated that methylation of MLH1 promoter region alone cannot be considered as a biomarker for early detection of CRC.

  19. Methylation of SOCS3 is inversely associated with metabolic syndrome in an epigenome-wide association study of obesity.

    Science.gov (United States)

    Ali, Omar; Cerjak, Diana; Kent, Jack W; James, Roland; Blangero, John; Carless, Melanie A; Zhang, Yi

    2016-09-01

    Epigenetic mechanisms, including DNA methylation, mediate the interaction between gene and environment and may play an important role in the obesity epidemic. We assessed the relationship between DNA methylation and obesity in peripheral blood mononuclear cells (PBMCs) at 485,000 CpG sites across the genome in family members (8-90 y of age) using a discovery cohort (192 individuals) and a validation cohort (1,052 individuals) of Northern European ancestry. After Bonferroni-correction (P α=0.05 = 1.31 × 10 -7 ) for genome-wide significance, we identified 3 loci, cg18181703 (SOCS3), cg04502490 (ZNF771), and cg02988947 (LIMD2), where methylation status was associated with body mass index percentile (BMI%), a clinical index for obesity in children, adolescents, and adults. These sites were also associated with multiple metabolic syndrome (MetS) traits, including central obesity, fat depots, insulin responsiveness, and plasma lipids. The SOCS3 methylation locus was also associated with the clinical definition of MetS. In the validation cohort, SOCS3 methylation status was found to be inversely associated with BMI% (P = 1.75 × 10 -6 ), waist to height ratio (P = 4.18 × 10 -7 ), triglycerides (P = 4.01 × 10 -4 ), and MetS (P = 4.01 × 10 -7 ), and positively correlated with HDL-c (P = 4.57 × 10 -8 ). Functional analysis in a sub cohort (333 individuals) demonstrated SOCS3 methylation and gene expression in PBMCs were inversely correlated (P = 2.93 × 10 -4 ) and expression of SOCS3 was positively correlated with status of MetS (P = 0.012). We conclude that epigenetic modulation of SOCS3, a gene involved in leptin and insulin signaling, may play an important role in obesity and MetS.

  20. ESR1 gene promoter region methylation in free circulating DNA and its correlation with estrogen receptor protein expression in tumor tissue in breast cancer patients

    International Nuclear Information System (INIS)

    Martínez-Galán, Joaquina; Ríos, Sandra; Delgado, Juan Ramón; Torres-Torres, Blanca; Núñez, María Isabel; López-Peñalver, Jesús; Del Moral, Rosario; Ruiz De Almodóvar, José Mariano; Menjón, Salomón; Concha, Ángel; Chamorro, Clara

    2014-01-01

    Tumor expression of estrogen receptor (ER) is an important marker of prognosis, and is predictive of response to endocrine therapy in breast cancer. Several studies have observed that epigenetic events, such methylation of cytosines and deacetylation of histones, are involved in the complex mechanisms that regulate promoter transcription. However, the exact interplay of these factors in transcription activity is not well understood. In this study, we explored the relationship between ER expression status in tumor tissue samples and the methylation of the 5′ CpG promoter region of the estrogen receptor gene (ESR1) isolated from free circulating DNA (fcDNA) in plasma samples from breast cancer patients. Patients (n = 110) with non-metastatic breast cancer had analyses performed of ER expression (luminal phenotype in tumor tissue, by immunohistochemistry method), and the ESR1-DNA methylation status (fcDNA in plasma, by quantitative methylation specific PCR technique). Our results showed a significant association between presence of methylated ESR1 in patients with breast cancer and ER negative status in the tumor tissue (p = 0.0179). There was a trend towards a higher probability of ESR1-methylation in those phenotypes with poor prognosis i.e. 80% of triple negative patients, 60% of HER2 patients, compared to 28% and 5.9% of patients with better prognosis such as luminal A and luminal B, respectively. Silencing, by methylation, of the promoter region of the ESR1 affects the expression of the estrogen receptor protein in tumors of breast cancer patients; high methylation of ESR1-DNA is associated with estrogen receptor negative status which, in turn, may be implicated in the patient’s resistance to hormonal treatment in breast cancer. As such, epigenetic markers in plasma may be of interest as new targets for anticancer therapy, especially with respect to endocrine treatment

  1. Methylation of MGMT Is Associated with Poor Prognosis in Patients with Stage III Duodenal Adenocarcinoma.

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    Tao Fu

    Full Text Available O6-methylguanine-DNA methyltransferase (MGMT methylation status has not been extensively investigated in duodenal adenocarcinoma (DA. The aim of this study was to evaluate the MGMT methylation status and examine its possible prognostic value in patients with stage III DA.Demographics, tumor characteristics and survival were available for 64 patients with stage III DA. MGMT methylation was detected by using MethyLight. A Cox proportional hazard model was built to predict survival, adjusted for clinicopathological characteristics and tumor molecular features, including the CpG island methylator phenotype (CIMP, microsatellite instability (MSI, and KRAS mutations.MGMT methylation was detected in 17 of 64 (26.6% patients, and was not correlated with sex, age, tumor differentiation, CIMP, MSI, or KRAS mutations. MGMT methylation was the only one factor associated with both overall survival (OS and disease-free survival (DFS on both univariate and multivariate analyses. In patients treated with surgery alone, MGMT-methylated group had worse OS and DFS when compared with MGMT-unmethylated group. However, in patients treated with chemotherapy/radiotherapy, outcomes became comparable between the two groups.Our results demonstrate MGMT methylation is a reliable and independent prognostic factor in DAs. Methylation of MGMT is associated with poor prognosis in patients with stage III DAs.

  2. Characterization of the Methylation Status of Pax7 and Myogenic Regulator Factors in Cell Myogenic Differentiation.

    Science.gov (United States)

    Chao, Zhe; Zheng, Xin-Li; Sun, Rui-Ping; Liu, Hai-Long; Huang, Li-Li; Cao, Zong-Xi; Deng, Chang-Yan; Wang, Feng

    2016-07-01

    Epigenetic processes in the development of skeletal muscle have been appreciated for over a decade. DNA methylation is a major epigenetic modification important for regulating gene expression and suppressing spurious transcription. Up to now, the importance of epigenetic marks in the regulation of Pax7 and myogenic regulatory factors (MRFs) expression is far less explored. In the present study, semi-quantitative the real-time polymerase chain reaction (RT-PCR) analyses showed MyoD and Myf5 were expressed in activated and quiescent C2C12 cells. MyoG was expressed in a later stage of myogenesis. Pax7 was weakly expressed in differentiated C2C12 cells. To further understand the regulation of expression of these genes, the DNA methylation status of Pax7, MyoD, and Myf5 was determined by bisulfite sequencing PCR. During the C2C12 myoblasts fusion process, the changes of promoter and exon 1 methylation of Pax7, MyoD, and Myf5 genes were observed. In addition, an inverse relationship of low methylation and high expression was found. These results suggest that DNA methylation may be an important mechanism regulating Pax7 and MRFs transcription in cell myogenic differentiation.

  3. MGMT methylation analysis of glioblastoma on the Infinium methylation BeadChip identifies two distinct CpG regions associated with gene silencing and outcome, yielding a prediction model for comparisons across datasets, tumor grades, and CIMP-status

    NARCIS (Netherlands)

    P. Bady (Pierre); D. Sciuscio (Davide); A.C. Diserens; J. Bloch (Jocelyne); M.J. van den Bent (Martin); C. Marosi (Christine); P-Y. Dietrich (Pierre Yves); M. Weller (Michael); L. Mariani (Luigi); F.L. Heppner (Frank ); D.R. Mcdonald (David ); D. Lacombe (Denis); R. Stupp (Roger); M. Delorenzi (Mauro); M.E. Hegi (Monika)

    2012-01-01

    textabstractThe methylation status of the O6-methylguanine- DNA methyltransferase (MGMT) gene is an important predictive biomarker for benefit from alkylating agent therapy in glioblastoma. Recent studies in anaplastic glioma suggest a prognostic value for MGMT methylation. Investigation of

  4. Double-labelling immunohistochemistry for MGMT and a “cocktail” of non-tumourous elements is a reliable, quick and easy technique for inferring methylation status in glioblastomas and other primary brain tumours

    Science.gov (United States)

    2013-01-01

    Background Our aim was to develop a new protocol for MGMT immunohistochemistry with good agreement between observers and good correlation with molecular genetic tests of tumour methylation. We examined 40 primary brain tumours (30 glioblastomas and 10 oligodendroglial tumours) with our new technique, namely double-labelling immunohistochemistry for MGMT and a "cocktail" of non-tumour antigens (CD34, CD45 and CD68). We compared the results with single-labelling immunohistochemistry for MGMT and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA, a recognised molecular genetic technique which we applied as the gold-standard for the methylation status). Results Double-labelling immunohistochemistry for MGMT produced a visual separation of tumourous and non-tumourous elements on the same histological slide, making it quick and easy to determine whether tumour cell nuclei were MGMT-positive or MGMT-negative (and thereby infer the methylation status of the tumour). We found good agreement between observers (kappa 0.76) and within observer (kappa 0.84). Furthermore, double-labelling showed good specificity (80%), sensitivity (73.33%), positive predictive value (PPV, 83.33%) and negative predictive value (NPV, 68.75%) compared to MS-MLPA. Double-labelling was quicker and easier to assess than single-labelling and it outperformed quantitative computerised image analysis of MGMT single-labelling in terms of sensitivity, specificity, PPV and NPV. Conclusions Double-labelling immunohistochemistry for MGMT and a cocktail of non-tumourous elements provides a "one look" method for determining whether tumour cell nuclei are MGMT-positive or MGMT-negative. This can be used to infer the methylation status of the tumour. There is good observer agreement and good specificity, sensitivity, PPV and NPV compared to a molecular gold-standard. PMID:24252243

  5. Using the apparent diffusion coefficient to identifying MGMT promoter methylation status early in glioblastoma: importance of analytical method

    Energy Technology Data Exchange (ETDEWEB)

    Rundle-Thiele, Dayle [Centre for Clinical Research, University of Queensland, Brisbane, Queensland (Australia); Day, Bryan; Stringer, Brett [Brain Cancer Research Unit, Queensland Institute of Medical Research, Brisbane, Queensland (Australia); Fay, Michael [Department of Radiation Oncology, Royal Brisbane and Women' s Hospital, Brisbane, Queensland (Australia); Martin, Jennifer [Discipline of Clinical Pharmacology, School of Medicine and Public Health, University of Newcastle, Newcastle, New South Wales (Australia); Jeffree, Rosalind L [Department of Neurosurgery, Royal Brisbane and Women' s Hospital, Brisbane, Queensland (Australia); Thomas, Paul [Queensland PET Service, Royal Brisbane and Women' s Hospital, Brisbane, Queensland (Australia); Bell, Christopher [Centre for Clinical Research, University of Queensland, Brisbane, Queensland (Australia); Salvado, Olivier [CSIRO Digital Productivity Flagship, CSIRO, Herston, Queensland (Australia); Gal, Yaniv [Centre for Medical Diagnostic Technologies in Queensland, University of Queensland, Brisbane, Queensland (Australia); Coulthard, Alan [Discipline of Medical Imaging, University of Queensland, St Lucia, Queensland (Australia); Department of Medical Imaging, Royal Brisbane and Women' s Hospital, Brisbane, Queensland (Australia); Crozier, Stuart [Centre for Medical Diagnostic Technologies in Queensland, University of Queensland, Brisbane, Queensland (Australia); Rose, Stephen, E-mail: stephen.rose@csiro.au [CSIRO Digital Productivity Flagship, CSIRO, Herston, Queensland (Australia); Centre for Clinical Research, University of Queensland, Brisbane, Queensland (Australia)

    2015-06-15

    Accurate knowledge of O{sup 6}-methylguanine methyltransferase (MGMT) gene promoter subtype in patients with glioblastoma (GBM) is important for treatment. However, this test is not always available. Pre-operative diffusion MRI (dMRI) can be used to probe tumour biology using the apparent diffusion coefficient (ADC); however, its ability to act as a surrogate to predict MGMT status has shown mixed results. We investigated whether this was due to variations in the method used to analyse ADC. We undertook a retrospective study of 32 patients with GBM who had MGMT status measured. Matching pre-operative MRI data were used to calculate the ADC within contrast enhancing regions of tumour. The relationship between ADC and MGMT was examined using two published ADC methods. A strong trend between a measure of ‘minimum ADC’ and methylation status was seen. An elevated minimum ADC was more likely in the methylated compared to the unmethylated MGMT group (U = 56, P = 0.0561). In contrast, utilising a two-mixture model histogram approach, a significant reduction in mean measure of the ‘low ADC’ component within the histogram was associated with an MGMT promoter methylation subtype (P < 0.0246). This study shows that within the same patient cohort, the method selected to analyse ADC measures has a significant bearing on the use of that metric as a surrogate marker of MGMT status. Thus for dMRI data to be clinically useful, consistent methods of data analysis need to be established prior to establishing any relationship with genetic or epigenetic profiling.

  6. Using the apparent diffusion coefficient to identifying MGMT promoter methylation status early in glioblastoma: importance of analytical method

    International Nuclear Information System (INIS)

    Rundle-Thiele, Dayle; Day, Bryan; Stringer, Brett; Fay, Michael; Martin, Jennifer; Jeffree, Rosalind L; Thomas, Paul; Bell, Christopher; Salvado, Olivier; Gal, Yaniv; Coulthard, Alan; Crozier, Stuart; Rose, Stephen

    2015-01-01

    Accurate knowledge of O 6 -methylguanine methyltransferase (MGMT) gene promoter subtype in patients with glioblastoma (GBM) is important for treatment. However, this test is not always available. Pre-operative diffusion MRI (dMRI) can be used to probe tumour biology using the apparent diffusion coefficient (ADC); however, its ability to act as a surrogate to predict MGMT status has shown mixed results. We investigated whether this was due to variations in the method used to analyse ADC. We undertook a retrospective study of 32 patients with GBM who had MGMT status measured. Matching pre-operative MRI data were used to calculate the ADC within contrast enhancing regions of tumour. The relationship between ADC and MGMT was examined using two published ADC methods. A strong trend between a measure of ‘minimum ADC’ and methylation status was seen. An elevated minimum ADC was more likely in the methylated compared to the unmethylated MGMT group (U = 56, P = 0.0561). In contrast, utilising a two-mixture model histogram approach, a significant reduction in mean measure of the ‘low ADC’ component within the histogram was associated with an MGMT promoter methylation subtype (P < 0.0246). This study shows that within the same patient cohort, the method selected to analyse ADC measures has a significant bearing on the use of that metric as a surrogate marker of MGMT status. Thus for dMRI data to be clinically useful, consistent methods of data analysis need to be established prior to establishing any relationship with genetic or epigenetic profiling

  7. Epigenetic regulation during fetal femur development: DNA methylation matters.

    Directory of Open Access Journals (Sweden)

    María C de Andrés

    Full Text Available Epigenetic modifications are heritable changes in gene expression without changes in DNA sequence. DNA methylation has been implicated in the control of several cellular processes including differentiation, gene regulation, development, genomic imprinting and X-chromosome inactivation. Methylated cytosine residues at CpG dinucleotides are commonly associated with gene repression; conversely, strategic loss of methylation during development could lead to activation of lineage-specific genes. Evidence is emerging that bone development and growth are programmed; although, interestingly, bone is constantly remodelled throughout life. Using human embryonic stem cells, human fetal bone cells (HFBCs, adult chondrocytes and STRO-1(+ marrow stromal cells from human bone marrow, we have examined a spectrum of developmental stages of femur development and the role of DNA methylation therein. Using pyrosequencing methodology we analysed the status of methylation of genes implicated in bone biology; furthermore, we correlated these methylation levels with gene expression levels using qRT-PCR and protein distribution during fetal development evaluated using immunohistochemistry. We found that during fetal femur development DNA methylation inversely correlates with expression of genes including iNOS (NOS2 and COL9A1, but not catabolic genes including MMP13 and IL1B. Furthermore, significant demethylation was evident in the osteocalcin promoter between the fetal and adult developmental stages. Increased TET1 expression and decreased expression of DNA (cytosine-5--methyltransferase 1 (DNMT1 in adult chondrocytes compared to HFBCs could contribute to the loss of methylation observed during fetal development. HFBC multipotency confirms these cells to be an ideal developmental system for investigation of DNA methylation regulation. In conclusion, these findings demonstrate the role of epigenetic regulation, specifically DNA methylation, in bone development

  8. Quantitative Evaluation of MMP-9 and TIMP-1 Promoter Methylation in Chronic Periodontitis.

    Science.gov (United States)

    Li, Xiting; Lu, Jiaxuan; Teng, Wei; Zhao, Chuanjiang; Ye, Xiaolei

    2018-03-01

    In this study, we investigated the promoter DNA methylation (DNAm) status of the MMP-9 and TIMP-1 genes in patients with chronic periodontitis to evaluate disease progression. Using pyrosequencing technology, DNAm levels of MMP-9 and TIMP-1 CpG islands were measured in 88 chronic periodontitis patients and 15 healthy controls. We found a positive correlation between methylation levels of MMP-9 CpG islands and the severity of chronic periodontitis. Methylated CpG islands were also closely associated with the duration of chronic periodontitis. Moreover, female patients exhibited lower methylation levels of MMP-9 but higher methylation levels of TIMP-1 compared with male patients, and the methylation levels of TIMP-1 gradually decreased with age. The findings of gender disparity in the DNAm of MMP-9 and TIMP-1 genes provide novel insights into chronic periodontitis.

  9. Epigenetics in type 1 diabetes: TNFa gene promoter methylation status in Chilean patients with type 1 diabetes mellitus.

    Science.gov (United States)

    Arroyo-Jousse, Viviana; Garcia-Diaz, Diego F; Codner, Ethel; Pérez-Bravo, Francisco

    2016-12-01

    TNF-α is a pro-inflammatory cytokine that is involved in type 1 diabetes (T1D) pathogenesis. The TNFa gene is subject of epigenetic regulation in which folate and homocysteine are important molecules because they participate in the methionine cycle where the most important methyl group donor (S-adenosylmethionine) is formed. We investigated whether TNFa gene promoter methylation status in T1D patients was related to blood folate, homocysteine and TNF-α in a transversal case-control study. We studied T1D patients (n 25, mean=13·7 years) and healthy control subjects (n 25, mean=31·1 years), without T1D and/or other autoimmune diseases or direct family history of these diseases. A blood sample was obtained for determination of serum folate, plasma homocysteine and TNF-α concentrations. Whole blood was used for the extraction of DNA to determine the percentage of methylation by real-time PCR and melting-curve analysis. Results are expressed as means and standard deviations for parametric variables and as median (interquartile range) for non-parametric variables. T1D patients showed a higher TNFa gene promoter methylation (39·2 (sd 19·5) %) when compared with control subjects (25·4 (sd 13·7) %) (P=0·008). TNFa gene promoter methylation was positively associated only with homocysteine levels in T1D patients (r 0·55, P=0·007), but not in control subjects (r -0·122, P=0·872). To our knowledge, this is the first work that reports the methylation status of the TNFa gene promoter and its relationship with homocysteine metabolism in Chilean T1D patients without disease complications.

  10. Suppression of prolactin gene expression in GH cells correlates with site-specific DNA methylation.

    Science.gov (United States)

    Zhang, Z X; Kumar, V; Rivera, R T; Pasion, S G; Chisholm, J; Biswas, D K

    1989-10-01

    Prolactin- (PRL) producing and nonproducing subclones of the GH line of (rat) pituitary tumor cells have been compared to elucidate the regulatory mechanisms of PRL gene expression. Particular emphasis was placed on delineating the molecular basis of the suppressed state of the PRL gene in the prolactin-nonproducing (PRL-) GH subclone (GH(1)2C1). We examined six methylatable cytosine residues (5, -CCGG- and 1, -GCGC-) within the 30-kb region of the PRL gene in these subclones. This analysis revealed that -CCGG-sequences of the transcribed region, and specifically, one in the fourth exon of the PRL gene, were heavily methylated in the PRL-, GH(1)2C1 cells. Furthermore, the inhibition of PRL gene expression in GH(1)2C1 was reversed by short-term treatment of the cells with a sublethal concentration of azacytidine (AzaC), an inhibitor of DNA methylation. The reversion of PRL gene expression by AzaC was correlated with the concurrent demethylation of the same -CCGG- sequences in the transcribed region of PRL gene. An inverse correlation between PRL gene expression and the level of methylation of the internal -C- residues in the specific -CCGG-sequence of the transcribed region of the PRL gene was demonstrated. The DNase I sensitivity of these regions of the PRL gene in PRL+, PRL-, and AzaC-treated cells was also consistent with an inverse relationship between methylation state, a higher order of structural modification, and gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Histone Lysine Methylation and Neurodevelopmental Disorders

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    Jeong-Hoon Kim

    2017-06-01

    Full Text Available Methylation of several lysine residues of histones is a crucial mechanism for relatively long-term regulation of genomic activity. Recent molecular biological studies have demonstrated that the function of histone methylation is more diverse and complex than previously thought. Moreover, studies using newly available genomics techniques, such as exome sequencing, have identified an increasing number of histone lysine methylation-related genes as intellectual disability-associated genes, which highlights the importance of accurate control of histone methylation during neurogenesis. However, given the functional diversity and complexity of histone methylation within the cell, the study of the molecular basis of histone methylation-related neurodevelopmental disorders is currently still in its infancy. Here, we review the latest studies that revealed the pathological implications of alterations in histone methylation status in the context of various neurodevelopmental disorders and propose possible therapeutic application of epigenetic compounds regulating histone methylation status for the treatment of these diseases.

  12. 18q loss of heterozygosity in microsatellite stable colorectal cancer is correlated with CpG island methylator phenotype-negative (CIMP-0) and inversely with CIMP-low and CIMP-high.

    Science.gov (United States)

    Ogino, Shuji; Kawasaki, Takako; Kirkner, Gregory J; Ohnishi, Mutsuko; Fuchs, Charles S

    2007-05-02

    The CpG island methylator phenotype (CIMP) with widespread promoter methylation is a distinct epigenetic phenotype in colorectal cancer, associated with microsatellite instability-high (MSI-high) and BRAF mutations. 18q loss of heterozygosity (LOH) commonly present in colorectal cancer with chromosomal instability (CIN) is associated with global hypomethylation in tumor cell. A recent study has shown an inverse correlation between CIN and CIMP (determined by MINTs, p16, p14 and MLH1 methylation) in colorectal cancer. However, no study has examined 18q LOH in relation to CIMP-high, CIMP-low (less extensive promoter methylation) and CIMP-0 (CIMP-negative), determined by quantitative DNA methylation analysis. Utilizing MethyLight technology (real-time PCR), we quantified DNA methylation in 8 CIMP-specific promoters {CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1} in 758 non-MSI-high colorectal cancers obtained from two large prospective cohorts. Using four 18q microsatellite markers (D18S55, D18S56, D18S67 and D18S487) and stringent criteria for 18q LOH, we selected 374 tumors (236 LOH-positive tumors with > or = 2 markers showing LOH; and 138 LOH-negative tumors with > or = 3 informative markers and no LOH). CIMP-0 (0/8 methylated promoters) was significantly more common in 18q LOH-positive tumors (59% = 139/236, p = 0.002) than 18q LOH-negative tumors (44% = 61/138), while CIMP-low/high (1/8-8/8 methylated promoters) was significantly more common (56%) in 18q LOH-negative tumors than 18q LOH-positive tumors (41%). These relations persisted after stratification by sex, location, or the status of MSI, p53 expression (by immunohistochemistry), or KRAS/BRAF mutation. 18q LOH is correlated positively with CIMP-0 and inversely with CIMP-low and CIMP-high. Our findings provide supporting evidence for relationship between CIMP-0 and 18q LOH as well as a molecular difference between CIMP-0 and CIMP-low in colorectal cancer.

  13. Comprehensive analysis of preeclampsia-associated DNA methylation in the placenta.

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    Tianjiao Chu

    Full Text Available A small number of recent reports have suggested that altered placental DNA methylation may be associated with early onset preeclampsia. It is important that further studies be undertaken to confirm and develop these findings. We therefore undertook a systematic analysis of DNA methylation patterns in placental tissue from 24 women with preeclampsia and 24 with uncomplicated pregnancy outcome.We analyzed the DNA methylation status of approximately 27,000 CpG sites in placental tissues in a massively parallel fashion using an oligonucleotide microarray. Follow up analysis of DNA methylation at specific CpG loci was performed using the Epityper MassArray approach and high-throughput bisulfite sequencing.Preeclampsia-specific DNA methylation changes were identified in placental tissue samples irrespective of gestational age of delivery. In addition, we identified a group of CpG sites within specific gene sequences that were only altered in early onset-preeclampsia (EOPET although these DNA methylation changes did not correlate with altered mRNA transcription. We found evidence that fetal gender influences DNA methylation at autosomal loci but could find no clear association between DNA methylation and gestational age.Preeclampsia is associated with altered placental DNA methylation. Fetal gender should be carefully considered during the design of future studies in which placental DNA is analyzed at the level of DNA methylation. Further large-scale analyses of preeclampsia-associated DNA methylation are necessary.

  14. O6-Methylguanine-DNA methyltransferase protein expression by immunohistochemistry in brain and non-brain systemic tumours: systematic review and meta-analysis of correlation with methylation-specific polymerase chain reaction

    International Nuclear Information System (INIS)

    Brell, Marta; Ibáñez, Javier; Tortosa, Avelina

    2011-01-01

    The DNA repair protein O 6 -Methylguanine-DNA methyltransferase (MGMT) confers resistance to alkylating agents. Several methods have been applied to its analysis, with methylation-specific polymerase chain reaction (MSP) the most commonly used for promoter methylation study, while immunohistochemistry (IHC) has become the most frequently used for the detection of MGMT protein expression. Agreement on the best and most reliable technique for evaluating MGMT status remains unsettled. The aim of this study was to perform a systematic review and meta-analysis of the correlation between IHC and MSP. A computer-aided search of MEDLINE (1950-October 2009), EBSCO (1966-October 2009) and EMBASE (1974-October 2009) was performed for relevant publications. Studies meeting inclusion criteria were those comparing MGMT protein expression by IHC with MGMT promoter methylation by MSP in the same cohort of patients. Methodological quality was assessed by using the QUADAS and STARD instruments. Previously published guidelines were followed for meta-analysis performance. Of 254 studies identified as eligible for full-text review, 52 (20.5%) met the inclusion criteria. The review showed that results of MGMT protein expression by IHC are not in close agreement with those obtained with MSP. Moreover, type of tumour (primary brain tumour vs others) was an independent covariate of accuracy estimates in the meta-regression analysis beyond the cut-off value. Protein expression assessed by IHC alone fails to reflect the promoter methylation status of MGMT. Thus, in attempts at clinical diagnosis the two methods seem to select different groups of patients and should not be used interchangeably

  15. Correlation between Food Intake and Health Status with the Nutritional Status of School Children Age 9-11 in Semarang City

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    Aiman Farag Mohammed Ali

    2016-12-01

    Full Text Available Malnutrition, a major risk factor for a number of infectious diseases, including acute upper respiratory tract infections (AURTI, is common in developing countries. Nutritional status is an important index of the quality of life. Objectives:To analyze the correlation between food intake and health status to nutritional status of 9-11 years old children in Semarang. The study was a correlation study carried among school children in Semarang aged 9-11 years old. Data are presented in the descriptive analyses and Spearman correlation. Overall, food intake (energy and protein of 9-11 years old children in Semarang is normal with ≥ 90% RDA, health status of them was satisfactory (very low AURTI incidence,and their nutritional status were mostly normal. There was a correlation between energy intake with nutritional status with indicators BMI, and z-score of W/A and H/A, but there was no correlation between protein intake and AURTI with nutritional status. Energy and food intake of the children correlate with all nutritional status being studied. It should be suggested to parents to implement balanced diet, to avoid the development of obesity among elementary school children through nutrition education to prevent malnutrition as well as obesity.How to CiteAli, A. F. M., Muis, S. F., & Suhartono, S. (2016. Correlation between Food Intake and Health Status with The Nutritional Status of School Children Age 9-11 in Semarang City. Biosaintifika: Journal of Biology & Biology Education, 8(3, 249-256. 

  16. Correlation of nutritional status with academic achievement in adolescents

    Science.gov (United States)

    Sinurat, R. S.; Sembiring, T.; Azlin, E.; Faranita, T.; Pratita, W.

    2018-03-01

    Malnutrition is considered a problem that limits learning ability (cognitive function), which is related to poor academic achievement results. This study aimed to determine the relationship of nutritional status with academic achievement in adolescents. A cross-sectional study was conducted on 126 junior high school students ranging from 12 to 15 years in Batubara, North Sumatra in January 2015. Nutritional status is determined by weight for height. Academic achievement was recorded from the final results of their school exams. The value of intelligence quotient (IQ) was assessed by using the Aptitude Test. Data were then analyzed by using Spearman correlation and Chi-Square test. In conclusion, there was no significant difference between nutritional status with IQ score (p=0,540) but showed a significant relationship (p=0.003) between normal nutritional status with the total value of the report card with positive weak correlation strength (r=0.342). There was also a significant difference (p=0.020) and moderate positive correlation (r=0.541) between overweight with academic achievement based on mathematics.

  17. Identification of methylated genes associated with aggressive clinicopathological features in mantle cell lymphoma.

    Directory of Open Access Journals (Sweden)

    Anna Enjuanes

    Full Text Available BACKGROUND: Mantle cell lymphoma (MCL is genetically characterized by the t(11;14(q13;q32 translocation and a high number of secondary chromosomal alterations. The contribution of DNA methylation to MCL lymphomagenesis is not well known. We sought to identify epigenetically silenced genes in these tumours that might have clinical relevance. METHODOLOGY/PRINCIPAL FINDINGS: To identify potential methylated genes in MCL we initially investigated seven MCL cell lines treated with epigenetic drugs and gene expression microarray profiling. The methylation status of selected candidate genes was validated by a quantitative assay and subsequently analyzed in a series of primary MCL (n = 38. After pharmacological reversion we identified 252 potentially methylated genes. The methylation analysis of a subset of these genes (n = 25 in the MCL cell lines and normal B lymphocytes confirmed that 80% of them were methylated in the cell lines but not in normal lymphocytes. The subsequent analysis in primary MCL identified five genes (SOX9, HOXA9, AHR, NR2F2, and ROBO1 frequently methylated in these tumours. The gene methylation events tended to occur in the same primary neoplasms and correlated with higher proliferation, increased number of chromosomal abnormalities, and shorter survival of the patients. CONCLUSIONS: We have identified a set of genes whose methylation degree and gene expression levels correlate with aggressive clinicopathological features of MCL. Our findings also suggest that a subset of MCL might show a CpG island methylator phenotype (CIMP that may influence the behaviour of the tumours.

  18. Genetic and non-genetic influences during pregnancy on infant global and site specific DNA methylation: role for folate gene variants and vitamin B12.

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    Jill A McKay

    Full Text Available Inter-individual variation in patterns of DNA methylation at birth can be explained by the influence of environmental, genetic and stochastic factors. This study investigates the genetic and non-genetic determinants of variation in DNA methylation in human infants. Given its central role in provision of methyl groups for DNA methylation, this study focuses on aspects of folate metabolism. Global (LUMA and gene specific (IGF2, ZNT5, IGFBP3 DNA methylation were quantified in 430 infants by Pyrosequencing®. Seven polymorphisms in 6 genes (MTHFR, MTRR, FOLH1, CβS, RFC1, SHMT involved in folate absorption and metabolism were analysed in DNA from both infants and mothers. Red blood cell folate and serum vitamin B(12 concentrations were measured as indices of vitamin status. Relationships between DNA methylation patterns and several covariates viz. sex, gestation length, maternal and infant red cell folate, maternal and infant serum vitamin B(12, maternal age, smoking and genotype were tested. Length of gestation correlated positively with IGF2 methylation (rho = 0.11, p = 0.032 and inversely with ZNT5 methylation (rho = -0.13, p = 0.017. Methylation of the IGFBP3 locus correlated inversely with infant vitamin B(12 concentration (rho = -0.16, p = 0.007, whilst global DNA methylation correlated inversely with maternal vitamin B(12 concentrations (rho = 0.18, p = 0.044. Analysis of common genetic variants in folate pathway genes highlighted several associations including infant MTRR 66G>A genotype with DNA methylation (χ(2 = 8.82, p = 0.003 and maternal MTHFR 677C>T genotype with IGF2 methylation (χ(2 = 2.77, p = 0.006. These data support the hypothesis that both environmental and genetic factors involved in one-carbon metabolism influence DNA methylation in infants. Specifically, the findings highlight the importance of vitamin B(12 status, infant MTRR genotype and maternal MTHFR genotype, all of which may influence the supply of methyl groups for

  19. Differential DNA methylation profile of key genes in malignant prostate epithelial cells transformed by inorganic arsenic or cadmium.

    Science.gov (United States)

    Pelch, Katherine E; Tokar, Erik J; Merrick, B Alex; Waalkes, Michael P

    2015-08-01

    Previous work shows altered methylation patterns in inorganic arsenic (iAs)- or cadmium (Cd)-transformed epithelial cells. Here, the methylation status near the transcriptional start site was assessed in the normal human prostate epithelial cell line (RWPE-1) that was malignantly transformed by 10μM Cd for 11weeks (CTPE) or 5μM iAs for 29weeks (CAsE-PE), at which time cells showed multiple markers of acquired cancer phenotype. Next generation sequencing of the transcriptome of CAsE-PE cells identified multiple dysregulated genes. Of the most highly dysregulated genes, five genes that can be relevant to the carcinogenic process (S100P, HYAL1, NTM, NES, ALDH1A1) were chosen for an in-depth analysis of the DNA methylation profile. DNA was isolated, bisulfite converted, and combined bisulfite restriction analysis was used to identify differentially methylated CpG sites, which was confirmed with bisulfite sequencing. Four of the five genes showed differential methylation in transformants relative to control cells that was inversely related to altered gene expression. Increased expression of HYAL1 (>25-fold) and S100P (>40-fold) in transformants was correlated with hypomethylation near the transcriptional start site. Decreased expression of NES (>15-fold) and NTM (>1000-fold) in transformants was correlated with hypermethylation near the transcriptional start site. ALDH1A1 expression was differentially expressed in transformed cells but was not differentially methylated relative to control. In conclusion, altered gene expression observed in Cd and iAs transformed cells may result from altered DNA methylation status. Published by Elsevier Inc.

  20. Acceleration of age-associated methylation patterns in HIV-1-infected adults.

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    Tammy M Rickabaugh

    Full Text Available Patients with treated HIV-1-infection experience earlier occurrence of aging-associated diseases, raising speculation that HIV-1-infection, or antiretroviral treatment, may accelerate aging. We recently described an age-related co-methylation module comprised of hundreds of CpGs; however, it is unknown whether aging and HIV-1-infection exert negative health effects through similar, or disparate, mechanisms. We investigated whether HIV-1-infection would induce age-associated methylation changes. We evaluated DNA methylation levels at >450,000 CpG sites in peripheral blood mononuclear cells (PBMC of young (20-35 and older (36-56 adults in two separate groups of participants. Each age group for each data set consisted of 12 HIV-1-infected and 12 age-matched HIV-1-uninfected samples for a total of 96 samples. The effects of age and HIV-1 infection on methylation at each CpG revealed a strong correlation of 0.49, p<1 x 10(-200 and 0.47, p<1 x 10(-200. Weighted gene correlation network analysis (WGCNA identified 17 co-methylation modules; module 3 (ME3 was significantly correlated with age (cor=0.70 and HIV-1 status (cor=0.31. Older HIV-1+ individuals had a greater number of hypermethylated CpGs across ME3 (p=0.015. In a multivariate model, ME3 was significantly associated with age and HIV status (Data set 1: βage=0.007088, p=2.08 x 10(-9; βHIV=0.099574, p=0.0011; Data set 2: βage=0.008762, p=1.27 x 10(-5; βHIV=0.128649, p=0.0001. Using this model, we estimate that HIV-1 infection accelerates age-related methylation by approximately 13.7 years in data set 1 and 14.7 years in data set 2. The genes related to CpGs in ME3 are enriched for polycomb group target genes known to be involved in cell renewal and aging. The overlap between ME3 and an aging methylation module found in solid tissues is also highly significant (Fisher-exact p=5.6 x 10(-6, odds ratio=1.91. These data demonstrate that HIV-1 infection is associated with methylation patterns that

  1. Correlation of sense of coherence with oral health behaviors, socioeconomic status, and periodontal status.

    Science.gov (United States)

    Reddy, Kommuri Sahithi; Doshi, Dolar; Kulkarni, Suhas; Reddy, Bandari Srikanth; Reddy, Madupu Padma

    2016-01-01

    The sense of coherence (SOC) has been suggested to be highly applicable concept in the public health area because a strong SOC is stated to decrease the likelihood of perceiving the social environment as stressful. This reduces the susceptibility to the health-damaging effect of chronic stress by lowering the likelihood of repeated negative emotions to stress perception. The demographic data and general information of subjects' oral health behaviors such as frequency of cleaning teeth, aids used to clean teeth, and dental attendance were recorded in the self-administered questionnaire. The SOC-related data were obtained using the short version of Antonovsky's SOC scale. The periodontal status was recorded based on the modified World Health Organization 1997 pro forma. The total of 780 respondents comprising 269 (34.5%) males and 511 (65.5%) females participated in the study. A significant difference was noted among the subjects for socioeconomic status based on gender ( P = 0.000). The healthy periodontal status (community periodontal index [CPI] code 0) was observed for 67 (24.9%) males and 118 (23.1%) females. The overall SOC showed statistically negative correlation with socioeconomic status scale ( r = -0.287). The CPI and loss of attachment (periodontal status) were significantly and negatively correlated with SOC. The present study concluded that a high level of SOC was associated with good oral health behaviors, periodontal status, and socioeconomic status.

  2. O6-Methylguanine-DNA methyltransferase protein expression by immunohistochemistry in brain and non-brain systemic tumours: systematic review and meta-analysis of correlation with methylation-specific polymerase chain reaction

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    Ibáñez Javier

    2011-01-01

    Full Text Available Abstract Background The DNA repair protein O6-Methylguanine-DNA methyltransferase (MGMT confers resistance to alkylating agents. Several methods have been applied to its analysis, with methylation-specific polymerase chain reaction (MSP the most commonly used for promoter methylation study, while immunohistochemistry (IHC has become the most frequently used for the detection of MGMT protein expression. Agreement on the best and most reliable technique for evaluating MGMT status remains unsettled. The aim of this study was to perform a systematic review and meta-analysis of the correlation between IHC and MSP. Methods A computer-aided search of MEDLINE (1950-October 2009, EBSCO (1966-October 2009 and EMBASE (1974-October 2009 was performed for relevant publications. Studies meeting inclusion criteria were those comparing MGMT protein expression by IHC with MGMT promoter methylation by MSP in the same cohort of patients. Methodological quality was assessed by using the QUADAS and STARD instruments. Previously published guidelines were followed for meta-analysis performance. Results Of 254 studies identified as eligible for full-text review, 52 (20.5% met the inclusion criteria. The review showed that results of MGMT protein expression by IHC are not in close agreement with those obtained with MSP. Moreover, type of tumour (primary brain tumour vs others was an independent covariate of accuracy estimates in the meta-regression analysis beyond the cut-off value. Conclusions Protein expression assessed by IHC alone fails to reflect the promoter methylation status of MGMT. Thus, in attempts at clinical diagnosis the two methods seem to select different groups of patients and should not be used interchangeably.

  3. CpG Island Methylator Phenotype in Primary Gastric Carcinoma

    OpenAIRE

    TOJO Masayuki:筆頭著者; KONISHI Kazuo; YANO Yuichiro; KATAGIRI Atsushi; NOZAWA Hisako; KUBOTA Yutaro; MURAMOTO Takashi; KONDA Kenichi; SHINMURA Kensuke; TAKIMOTO Masafumi; IMAWARI Michio; YOSHIDA Hitoshi

    2013-01-01

    Gastric cancers (GC) with methylation of multiple CpG islands have a CpG island methylator phenotype (CIMP) and they can have different biological features. The aim of this study was to investigate the DNA methylation status of GCs and its association with their clinicopathological features. We evaluated the methylation status of four genes (MINT1, MINT2, MINT25 and MINT31) in 105 primary GCs using bisulfite-pyrosequencing analysis. We classified tumors as CIMP-high (CIMP-H), CIMP-low (CIMP-L...

  4. Ancestral TCDD exposure promotes epigenetic transgenerational inheritance of imprinted gene Igf2: Methylation status and DNMTs

    International Nuclear Information System (INIS)

    Ma, Jing; Chen, Xi; Liu, Yanan; Xie, Qunhui; Sun, Yawen; Chen, Jingshan; Leng, Ling; Yan, Huan; Zhao, Bin; Tang, Naijun

    2015-01-01

    Ancestral TCDD exposure could induce epigenetic transgenerational phenotypes, which may be mediated in part by imprinted gene inheritance. The aim of our study was to evaluate the transgenerational effects of ancestral TCDD exposure on the imprinted gene insulin-like growth factor-2 (Igf2) in rat somatic tissue. TCDD was administered daily by oral gavage to groups of F0 pregnant SD rats at dose levels of 0 (control), 200 or 800 ng/kg bw during gestation day 8–14. Animal transgenerational model of ancestral exposure to TCDD was carefully built, avoiding sibling inbreeding. Hepatic Igf2 expression of the TCDD male progeny was decreased concomitantly with hepatic damage and increased activities of serum hepatic enzymes both in the F1 and F3 generation. Imprinted Control Region (ICR) of Igf2 manifested a hypermethylated pattern, whereas methylation status in the Differentially Methylated Region 2 (DMR2) showed a hypomethylated manner in the F1 generation. These epigenetic alterations in these two regions maintained similar trends in the F3 generation. Meanwhile, the expressions of DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) changed in a non-monotonic manner both in the F1 and F3 generation. This study provides evidence that ancestral TCDD exposure may promote epigenetic transgenerational alterations of imprinted gene Igf2 in adult somatic tissue. - Highlights: • Ancestral TCDD exposure induces epigenetic transgenerational inheritance. • Ancestral TCDD exposure affects methylation status in ICR and DMR2 region of Igf2. • DNMTs play a role in TCDD induced epigenetic transgenerational changes of Igf2.

  5. Ancestral TCDD exposure promotes epigenetic transgenerational inheritance of imprinted gene Igf2: Methylation status and DNMTs

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Jing; Chen, Xi; Liu, Yanan [Department of Occupational and Environmental Health, School of Public Health, Tianjin Medical University, Tianjin 300070 (China); Xie, Qunhui [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085 (China); Sun, Yawen; Chen, Jingshan; Leng, Ling; Yan, Huan [Department of Occupational and Environmental Health, School of Public Health, Tianjin Medical University, Tianjin 300070 (China); Zhao, Bin, E-mail: binzhao@rcees.ac.cn [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085 (China); Tang, Naijun, E-mail: tangnaijun@tijmu.edu.cn [Department of Occupational and Environmental Health, School of Public Health, Tianjin Medical University, Tianjin 300070 (China)

    2015-12-01

    Ancestral TCDD exposure could induce epigenetic transgenerational phenotypes, which may be mediated in part by imprinted gene inheritance. The aim of our study was to evaluate the transgenerational effects of ancestral TCDD exposure on the imprinted gene insulin-like growth factor-2 (Igf2) in rat somatic tissue. TCDD was administered daily by oral gavage to groups of F0 pregnant SD rats at dose levels of 0 (control), 200 or 800 ng/kg bw during gestation day 8–14. Animal transgenerational model of ancestral exposure to TCDD was carefully built, avoiding sibling inbreeding. Hepatic Igf2 expression of the TCDD male progeny was decreased concomitantly with hepatic damage and increased activities of serum hepatic enzymes both in the F1 and F3 generation. Imprinted Control Region (ICR) of Igf2 manifested a hypermethylated pattern, whereas methylation status in the Differentially Methylated Region 2 (DMR2) showed a hypomethylated manner in the F1 generation. These epigenetic alterations in these two regions maintained similar trends in the F3 generation. Meanwhile, the expressions of DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) changed in a non-monotonic manner both in the F1 and F3 generation. This study provides evidence that ancestral TCDD exposure may promote epigenetic transgenerational alterations of imprinted gene Igf2 in adult somatic tissue. - Highlights: • Ancestral TCDD exposure induces epigenetic transgenerational inheritance. • Ancestral TCDD exposure affects methylation status in ICR and DMR2 region of Igf2. • DNMTs play a role in TCDD induced epigenetic transgenerational changes of Igf2.

  6. 18q loss of heterozygosity in microsatellite stable colorectal cancer is correlated with CpG island methylator phenotype-negative (CIMP-0 and inversely with CIMP-low and CIMP-high

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    Kirkner Gregory J

    2007-05-01

    Full Text Available Abstract Background: The CpG island methylator phenotype (CIMP with widespread promoter methylation is a distinct epigenetic phenotype in colorectal cancer, associated with microsatellite instability-high (MSI-high and BRAF mutations. 18q loss of heterozygosity (LOH commonly present in colorectal cancer with chromosomal instability (CIN is associated with global hypomethylation in tumor cell. A recent study has shown an inverse correlation between CIN and CIMP (determined by MINTs, p16, p14 and MLH1 methylation in colorectal cancer. However, no study has examined 18q LOH in relation to CIMP-high, CIMP-low (less extensive promoter methylation and CIMP-0 (CIMP-negative, determined by quantitative DNA methylation analysis. Methods: Utilizing MethyLight technology (real-time PCR, we quantified DNA methylation in 8 CIMP-specific promoters {CACNA1G, CDKN2A (p16, CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1} in 758 non-MSI-high colorectal cancers obtained from two large prospective cohorts. Using four 18q microsatellite markers (D18S55, D18S56, D18S67 and D18S487 and stringent criteria for 18q LOH, we selected 374 tumors (236 LOH-positive tumors with ≥ 2 markers showing LOH; and 138 LOH-negative tumors with ≥ 3 informative markers and no LOH. Results: CIMP-0 (0/8 methylated promoters was significantly more common in 18q LOH-positive tumors (59% = 139/236, p = 0.002 than 18q LOH-negative tumors (44% = 61/138, while CIMP-low/high (1/8–8/8 methylated promoters was significantly more common (56% in 18q LOH-negative tumors than 18q LOH-positive tumors (41%. These relations persisted after stratification by sex, location, or the status of MSI, p53 expression (by immunohistochemistry, or KRAS/BRAF mutation. Conclusion: 18q LOH is correlated positively with CIMP-0 and inversely with CIMP-low and CIMP-high. Our findings provide supporting evidence for relationship between CIMP-0 and 18q LOH as well as a molecular difference between CIMP-0 and CIMP-low in

  7. Homogalacturonan methyl-esterification and plant development.

    Science.gov (United States)

    Wolf, Sebastian; Mouille, Grégory; Pelloux, Jérome

    2009-09-01

    The ability of a plant cell to expand is largely defined by the physical constraints imposed by its cell wall. Accordingly, cell wall properties have to be regulated during development. The pectic polysaccharide homogalacturonan is a major component of the plant primary walls. Biosynthesis and in muro modification of homogalacturonan have recently emerged as key determinants of plant development, controlling cell adhesion, organ development, and phyllotactic patterning. This review will focus on recent findings regarding impact of homogalacturonan content and methyl-esterification status of this polymer on plant life. De-methyl-esterification of homogalacturonan occurs through the action of the ubiquitous enzyme 'pectin methyl-esterase'. We here describe various strategies developed by the plant to finely tune the methyl-esterification status of homogalacturonan along key events of the plant lifecycle.

  8. Nucleosomes correlate with in vivo progression pattern of de novo methylation of p16 CpG islands in human gastric carcinogenesis.

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    Zhe-Ming Lu

    Full Text Available BACKGROUND: The exact relationship between nucleosome positioning and methylation of CpG islands in human pathogenesis is unknown. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we characterized the nucleosome position within the p16 CpG island and established a seeding methylation-specific PCR (sMSP assay based on bisulfite modification to enrich the p16 alleles containing methylated-CpG at the methylation "seeding" sites within its intron-1 in gastric carcinogenesis. The sMSP-positive rate in primary gastric carcinoma (GC samples (36/40 was significantly higher than that observed in gastritis (19/45 or normal samples (7/13 (P<0.01. Extensive clone sequencing of these sMSP products showed that the density of methylated-CpGs in p16 CpG islands increased gradually along with the severity of pathological changes in gastric tissues. In gastritis lesions the methylation was frequently observed in the region corresponding to the exon-1 coding-nucleosome and the 5'UTR-nucleosome; the methylation was further extended to the region corresponding to the promoter-nucleosome in GC samples. Only few methylated-CpG sites were randomly detected within p16 CpG islands in normal tissues. The significantly inversed relationship between the p16 exon-1 methylation and its transcription was observed in GC samples. An exact p16 promoter-specific 83 bp-MSP assay confirms the result of sMSP (33/55 vs. 1/6, P<0.01. In addition, p16 methylation in chronic gastritis lesions significantly correlated with H. pylori infection; however, such correlation was not observed in GC specimens. CONCLUSIONS/SIGNIFICANCE: It was determined that de novo methylation was initiated in the coding region of p16 exon-1 in gastritis, then progressed to its 5'UTR, and ultimately to the proximal promoter in GCs. Nucleosomes may function as the basic extension/progression unit of de novo methylation of p16 CpG islands in vivo.

  9. Wp specific methylation of highly proliferated LCLs

    International Nuclear Information System (INIS)

    Park, Jung-Hoon; Jeon, Jae-Pil; Shim, Sung-Mi; Nam, Hye-Young; Kim, Joon-Woo; Han, Bok-Ghee; Lee, Suman

    2007-01-01

    The epigenetic regulation of viral genes may be important for the life cycle of EBV. We determined the methylation status of three viral promoters (Wp, Cp, Qp) from EBV B-lymphoblastoid cell lines (LCLs) by pyrosequencing. Our pyrosequencing data showed that the CpG region of Wp was methylated, but the others were not. Interestingly, Wp methylation was increased with proliferation of LCLs. Wp methylation was as high as 74.9% in late-passage LCLs, but 25.6% in early-passage LCLs. From two Burkitt's lymphoma cell lines, Wp specific hypermethylation was also found (>80%). Interestingly, the expression of EBNA2 gene which located directly next to Wp was associated with its methylation. Our data suggested that Wp specific methylation may be important for the indicator of the proliferation status of LCLs, and the epigenetic viral gene regulation of EBNA2 gene by Wp should be further defined possibly with other biological processes

  10. Integrated analysis of gene expression, CpG island methylation, and gene copy number in breast cancer cells by deep sequencing.

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    Zhifu Sun

    Full Text Available We used deep sequencing technology to profile the transcriptome, gene copy number, and CpG island methylation status simultaneously in eight commonly used breast cell lines to develop a model for how these genomic features are integrated in estrogen receptor positive (ER+ and negative breast cancer. Total mRNA sequence, gene copy number, and genomic CpG island methylation were carried out using the Illumina Genome Analyzer. Sequences were mapped to the human genome to obtain digitized gene expression data, DNA copy number in reference to the non-tumor cell line (MCF10A, and methylation status of 21,570 CpG islands to identify differentially expressed genes that were correlated with methylation or copy number changes. These were evaluated in a dataset from 129 primary breast tumors. Gene expression in cell lines was dominated by ER-associated genes. ER+ and ER- cell lines formed two distinct, stable clusters, and 1,873 genes were differentially expressed in the two groups. Part of chromosome 8 was deleted in all ER- cells and part of chromosome 17 amplified in all ER+ cells. These loci encoded 30 genes that were overexpressed in ER+ cells; 9 of these genes were overexpressed in ER+ tumors. We identified 149 differentially expressed genes that exhibited differential methylation of one or more CpG islands within 5 kb of the 5' end of the gene and for which mRNA abundance was inversely correlated with CpG island methylation status. In primary tumors we identified 84 genes that appear to be robust components of the methylation signature that we identified in ER+ cell lines. Our analyses reveal a global pattern of differential CpG island methylation that contributes to the transcriptome landscape of ER+ and ER- breast cancer cells and tumors. The role of gene amplification/deletion appears to more modest, although several potentially significant genes appear to be regulated by copy number aberrations.

  11. A review on environmental factors regulating arsenic methylation in humans

    International Nuclear Information System (INIS)

    Tseng, C.-H.

    2009-01-01

    Subjects exposed to arsenic show significant inter-individual variation in urinary patterns of arsenic metabolites but insignificant day-to-day intra-individual variation. The inter-individual variation in arsenic methylation can be partly responsible for the variation in susceptibility to arsenic toxicity. Wide inter-ethnic variation and family correlation in urinary arsenic profile suggest a genetic effect on arsenic metabolism. In this paper the environmental factors affecting arsenic metabolism are reviewed. Methylation capacity might reduce with increasing dosage of arsenic exposure. Furthermore, women, especially at pregnancy, have better methylation capacity than their men counterparts, probably due to the effect of estrogen. Children might have better methylation capacity than adults and age shows inconsistent relevance in adults. Smoking and alcohol consumption might be associated with a poorer methylation capacity. Nutritional status is important in the methylation capacity and folate may facilitate the methylation and excretion of arsenic. Besides, general health conditions and medications might influence the arsenic methylation capacity; and technical problems can cause biased estimates. The consumption of seafood, seaweed, rice and other food with high arsenic contents and the extent of cooking and arsenic-containing water used in food preparation may also interfere with the presentation of the urinary arsenic profile. Future studies are necessary to clarify the effects of the various arsenic metabolites including the trivalent methylated forms on the development of arsenic-induced human diseases with the consideration of the effects of confounding factors and the interactions with other effect modifiers

  12. DNA methylation-histone modification relationships across the desmin locus in human primary cells

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    Clelland Gayle K

    2009-05-01

    Full Text Available Abstract Background We present here an extensive epigenetic analysis of a 500 kb region, which encompasses the human desmin gene (DES and its 5' locus control region (LCR, the only muscle-specific transcriptional regulatory element of this type described to date. These data complement and extend Encyclopaedia of DNA Elements (ENCODE studies on region ENr133. We analysed histone modifications and underlying DNA methylation patterns in physiologically relevant DES expressing (myoblast/myotube and non-expressing (peripheral blood mononuclear primary human cells. Results We found that in expressing myoblast/myotube but not peripheral blood mononuclear cell (PBMC cultures, histone H4 acetylation displays a broadly distributed enrichment across a gene rich 200 kb region whereas H3 acetylation localizes at the transcriptional start site (TSS of genes. We show that the DES LCR and TSS of DES are enriched with hyperacetylated domains of acetylated histone H3, with H3 lysine 4 di- and tri-methylation (H3K4me2 and me3 exhibiting a different distribution pattern across this locus. The CpG island that extends into the first intron of DES is methylation-free regardless of the gene's expression status and in non-expressing PBMCs is marked with histone H3 lysine 27 tri-methylation (H3K27me3. Conclusion Overall, our results constitute the first study correlating patterns of histone modifications and underlying DNA methylation of a muscle-specific LCR and its associated downstream gene region whilst additionally placing this within a much broader genomic context. Our results clearly show that there are distinct patterns of histone H3 and H4 acetylation and H3 methylation at the DES LCR, promoter and intragenic region. In addition, the presence of H3K27me3 at the DES methylation-free CpG only in non-expressing PBMCs may serve to silence this gene in non-muscle tissues. Generally, our work demonstrates the importance of using multiple, physiologically relevant

  13. Identification of Differentially Methylated Sites with Weak Methylation Effects

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    Hong Tran

    2018-02-01

    Full Text Available Deoxyribonucleic acid (DNA methylation is an epigenetic alteration crucial for regulating stress responses. Identifying large-scale DNA methylation at single nucleotide resolution is made possible by whole genome bisulfite sequencing. An essential task following the generation of bisulfite sequencing data is to detect differentially methylated cytosines (DMCs among treatments. Most statistical methods for DMC detection do not consider the dependency of methylation patterns across the genome, thus possibly inflating type I error. Furthermore, small sample sizes and weak methylation effects among different phenotype categories make it difficult for these statistical methods to accurately detect DMCs. To address these issues, the wavelet-based functional mixed model (WFMM was introduced to detect DMCs. To further examine the performance of WFMM in detecting weak differential methylation events, we used both simulated and empirical data and compare WFMM performance to a popular DMC detection tool methylKit. Analyses of simulated data that replicated the effects of the herbicide glyphosate on DNA methylation in Arabidopsis thaliana show that WFMM results in higher sensitivity and specificity in detecting DMCs compared to methylKit, especially when the methylation differences among phenotype groups are small. Moreover, the performance of WFMM is robust with respect to small sample sizes, making it particularly attractive considering the current high costs of bisulfite sequencing. Analysis of empirical Arabidopsis thaliana data under varying glyphosate dosages, and the analysis of monozygotic (MZ twins who have different pain sensitivities—both datasets have weak methylation effects of <1%—show that WFMM can identify more relevant DMCs related to the phenotype of interest than methylKit. Differentially methylated regions (DMRs are genomic regions with different DNA methylation status across biological samples. DMRs and DMCs are essentially the same

  14. Dietary Chromium Restriction of Pregnant Mice Changes the Methylation Status of Hepatic Genes Involved with Insulin Signaling in Adult Male Offspring.

    Science.gov (United States)

    Zhang, Qian; Sun, Xiaofang; Xiao, Xinhua; Zheng, Jia; Li, Ming; Yu, Miao; Ping, Fan; Wang, Zhixin; Qi, Cuijuan; Wang, Tong; Wang, Xiaojing

    2017-01-01

    Maternal undernutrition is linked with an elevated risk of diabetes mellitus in offspring regardless of the postnatal dietary status. This is also found in maternal micro-nutrition deficiency, especial chromium which is a key glucose regulator. We investigated whether maternal chromium restriction contributes to the development of diabetes in offspring by affecting DNA methylation status in liver tissue. After being mated with control males, female weanling 8-week-old C57BL mice were fed a control diet (CON, 1.19 mg chromium/kg diet) or a low chromium diet (LC, 0.14 mg chromium/kg diet) during pregnancy and lactation. After weaning, some offspring were shifted to the other diet (CON-LC, or LC-CON), while others remained on the same diet (CON-CON, or LC-LC) for 29 weeks. Fasting blood glucose, serum insulin, and oral glucose tolerance test was performed to evaluate the glucose metabolism condition. Methylation differences in liver from the LC-CON group and CON-CON groups were studied by using a DNA methylation array. Bisulfite sequencing was carried out to validate the results of the methylation array. Maternal chromium limitation diet increased the body weight, blood glucose, and serum insulin levels. Even when switched to the control diet after weaning, the offspring also showed impaired glucose tolerance and insulin resistance. DNA methylation profiling of the offspring livers revealed 935 differentially methylated genes in livers of the maternal chromium restriction diet group. Pathway analysis identified the insulin signaling pathway was the main process affected by hypermethylated genes. Bisulfite sequencing confirmed that some genes in insulin signaling pathway were hypermethylated in livers of the LC-CON and LC-LC group. Accordingly, the expression of genes in insulin signaling pathway was downregulated. There findings suggest that maternal chromium restriction diet results in glucose intolerance in male offspring through alterations in DNA methylation which

  15. Dietary Chromium Restriction of Pregnant Mice Changes the Methylation Status of Hepatic Genes Involved with Insulin Signaling in Adult Male Offspring.

    Directory of Open Access Journals (Sweden)

    Qian Zhang

    Full Text Available Maternal undernutrition is linked with an elevated risk of diabetes mellitus in offspring regardless of the postnatal dietary status. This is also found in maternal micro-nutrition deficiency, especial chromium which is a key glucose regulator. We investigated whether maternal chromium restriction contributes to the development of diabetes in offspring by affecting DNA methylation status in liver tissue. After being mated with control males, female weanling 8-week-old C57BL mice were fed a control diet (CON, 1.19 mg chromium/kg diet or a low chromium diet (LC, 0.14 mg chromium/kg diet during pregnancy and lactation. After weaning, some offspring were shifted to the other diet (CON-LC, or LC-CON, while others remained on the same diet (CON-CON, or LC-LC for 29 weeks. Fasting blood glucose, serum insulin, and oral glucose tolerance test was performed to evaluate the glucose metabolism condition. Methylation differences in liver from the LC-CON group and CON-CON groups were studied by using a DNA methylation array. Bisulfite sequencing was carried out to validate the results of the methylation array. Maternal chromium limitation diet increased the body weight, blood glucose, and serum insulin levels. Even when switched to the control diet after weaning, the offspring also showed impaired glucose tolerance and insulin resistance. DNA methylation profiling of the offspring livers revealed 935 differentially methylated genes in livers of the maternal chromium restriction diet group. Pathway analysis identified the insulin signaling pathway was the main process affected by hypermethylated genes. Bisulfite sequencing confirmed that some genes in insulin signaling pathway were hypermethylated in livers of the LC-CON and LC-LC group. Accordingly, the expression of genes in insulin signaling pathway was downregulated. There findings suggest that maternal chromium restriction diet results in glucose intolerance in male offspring through alterations in DNA

  16. Quantitative DNA methylation analyses reveal stage dependent DNA methylation and association to clinico-pathological factors in breast tumors

    International Nuclear Information System (INIS)

    Klajic, Jovana; Tost, Jörg; Kristensen, Vessela N; Fleischer, Thomas; Dejeux, Emelyne; Edvardsen, Hege; Warnberg, Fredrik; Bukholm, Ida; Lønning, Per Eystein; Solvang, Hiroko; Børresen-Dale, Anne-Lise

    2013-01-01

    Aberrant DNA methylation of regulatory genes has frequently been found in human breast cancers and correlated to clinical outcome. In the present study we investigate stage specific changes in the DNA methylation patterns in order to identify valuable markers to understand how these changes affect breast cancer progression. Quantitative DNA methylation analyses of 12 candidate genes ABCB1, BRCCA1, CDKN2A, ESR1, GSTP1, IGF2, MGMT, HMLH1, PPP2R2B, PTEN, RASSF1A and FOXC1 was performed by pyrosequencing a series of 238 breast cancer tissue samples from DCIS to invasive tumors stage I to IV. Significant differences in methylation levels between the DCIS and invasive stage II tumors were observed for six genes RASSF1A, CDKN2A, MGMT, ABCB1, GSTP1 and FOXC1. RASSF1A, ABCB1 and GSTP1 showed significantly higher methylation levels in late stage compared to the early stage breast carcinoma. Z-score analysis revealed significantly lower methylation levels in DCIS and stage I tumors compared with stage II, III and IV tumors. Methylation levels of PTEN, PPP2R2B, FOXC1, ABCB1 and BRCA1 were lower in tumors harboring TP53 mutations then in tumors with wild type TP53. Z-score analysis showed that TP53 mutated tumors had significantly lower overall methylation levels compared to tumors with wild type TP53. Methylation levels of RASSF1A, PPP2R2B, GSTP1 and FOXC1 were higher in ER positive vs. ER negative tumors and methylation levels of PTEN and CDKN2A were higher in HER2 positive vs. HER2 negative tumors. Z-score analysis also showed that HER2 positive tumors had significantly higher z-scores of methylation compared to the HER2 negative tumors. Univariate survival analysis identifies methylation status of PPP2R2B as significant predictor of overall survival and breast cancer specific survival. In the present study we report that the level of aberrant DNA methylation is higher in late stage compared with early stage of invasive breast cancers and DCIS for genes mentioned above

  17. Transcription of hepatitis B virus covalently closed circular DNA is regulated by CpG methylation during chronic infection.

    Directory of Open Access Journals (Sweden)

    Yongmei Zhang

    Full Text Available The persistence of hepatitis B virus (HBV infection is maintained by the nuclear viral covalently closed circular DNA (cccDNA, which serves as transcription template for viral mRNAs. Previous studies suggested that cccDNA contains methylation-prone CpG islands, and that the minichromosome structure of cccDNA is epigenetically regulated by DNA methylation. However, the regulatory effect of each CpG island methylation on cccDNA activity remains elusive. In the present study, we analyzed the distribution of CpG methylation within cccDNA in patient samples and investigated the impact of CpG island methylation on cccDNA-driven virus replication. Our study revealed the following observations: 1 Bisulfite sequencing of cccDNA from chronic hepatitis B patients indicated that CpG island I was seldom methylated, 2 CpG island II methylation was correlated to the low level of serum HBV DNA in patients, and in vitro methylation studies confirmed that CpG island II methylation markedly reduced cccDNA transcription and subsequent viral core DNA replication, 3 CpG island III methylation was associated with low serum HBsAg titers, and 4 Furthermore, we found that HBV genotype, HBeAg positivity, and patient age and liver fibrosis stage were also relevant to cccDNA CpG methylation status. Therefore, we clearly demonstrated that the status of cccDNA methylation is connected to the biological behavior of HBV. Taken together, our study provides a complete profile of CpG island methylation within HBV cccDNA and new insights for the function of CpG methylation in regulating HBV cccDNA transcription.

  18. Distinct features between MLH1-methylated and unmethylated colorectal carcinomas with the CpG island methylator phenotype: implications in the serrated neoplasia pathway.

    Science.gov (United States)

    Kim, Jung Ho; Bae, Jeong Mo; Cho, Nam-Yun; Kang, Gyeong Hoon

    2016-03-22

    The presence or absence of MLH1 methylation may critically affect the heterogeneity of colorectal carcinoma (CRC) with the CpG island methylator phenotype (CIMP). Here, we investigated the differential characteristics of CIMP-high (CIMP-H) CRCs according to MLH1 methylation status. To further confirm the MLH1-dependent features in CIMP-H CRC, an independent analysis was performed using data from The Cancer Genome Atlas (TCGA). In our CIMP-H CRC samples, MLH1-methylated tumors were characterized by older patient age, proximal colonic location, mucinous histology, intense lymphoid reactions, RUNX3/SOCS1 promoter methylation, BRAF mutations, and microsatellite instability-high (MSI-H) status. By contrast, MLH1-unmethylated tumors were associated with earlier age of onset, increased distal colorectal localization, adverse pathologic features, and KRAS mutations. In the TCGA dataset, the MLH1-silenced CIMP-H CRC demonstrated proximal location, MSI-H status, hypermutated phenotype, and frequent BRAF mutations, but the MLH1-non-silenced CIMP-H CRC was significantly associated with high frequencies of KRAS and APC mutations. In conclusion, the differential nature of CIMP-H CRCs depends primarily on the MLH1 methylation status. Based on the current knowledge, the sessile serrated adenoma/polyp may be the major precursor of MLH1-methylated CIMP-H CRCs, whereas MLH1-unmethylated CIMP-H CRCs may develop predominantly from KRAS-mutated traditional serrated adenomas and less commonly from BRAF-mutated traditional serrated adenomas and/or sessile serrated adenomas/polyps.

  19. Myopodin methylation is a prognostic biomarker and predicts antiangiogenic response in advanced kidney cancer.

    Science.gov (United States)

    Pompas-Veganzones, N; Sandonis, V; Perez-Lanzac, Alberto; Beltran, M; Beardo, P; Juárez, A; Vazquez, F; Cozar, J M; Alvarez-Ossorio, J L; Sanchez-Carbayo, Marta

    2016-10-01

    Myopodin is a cytoskeleton protein that shuttles to the nucleus depending on the cellular differentiation and stress. It has shown tumor suppressor functions. Myopodin methylation status was useful for staging bladder and colon tumors and predicting clinical outcome. To our knowledge, myopodin has not been tested in kidney cancer to date. The purpose of this study was to evaluate whether myopodin methylation status could be clinically useful in renal cancer (1) as a prognostic biomarker and 2) as a predictive factor of response to antiangiogenic therapy in patients with metastatic disease. Methylation-specific polymerase chain reactions (MS-PCR) were used to evaluate myopodin methylation in 88 kidney tumors. These belonged to patients with localized disease and no evidence of disease during follow-up (n = 25) (group 1), and 63 patients under antiangiogenic therapy (sunitinib, sorafenib, pazopanib, and temsirolimus), from which group 2 had non-metastatic disease at diagnosis (n = 32), and group 3 showed metastatic disease at diagnosis (n = 31). Univariate and multivariate Cox analyses were utilized to assess outcome and response to antiangiogenic agents taking progression, disease-specific survival, and overall survival as clinical endpoints. Myopodin was methylated in 50 out of the 88 kidney tumors (56.8 %). Among the 88 cases analyzed, 10 of them recurred (11.4 %), 51 progressed (57.9 %), and 40 died of disease (45.4 %). Myopodin methylation status correlated to MSKCC Risk score (p = 0.050) and the presence of distant metastasis (p = 0.039). Taking all patients, an unmethylated myopodin identified patients with shorter progression-free survival, disease-specific survival, and overall survival. Using also in univariate and multivariate models, an unmethylated myopodin predicted response to antiangiogenic therapy (groups 2 and 3) using progression-free survival, disease-specific, and overall survival as clinical endpoints. Myopodin was revealed

  20. Association between human papillomavirus and Epstein - Barr virus DNA and gene promoter methylation of RB1 and CDH1 in the cervical lesions: a transversal study.

    Science.gov (United States)

    McCormick, Thaís M; Canedo, Nathalie H S; Furtado, Yara L; Silveira, Filomena A; de Lima, Roberto J; Rosman, Andréa D F; Almeida Filho, Gutemberg L; Carvalho, Maria da Glória da C

    2015-06-02

    Human papillomavirus (HPV) inactivates the retinoblastoma 1 (RB1) gene by promoter methylation and reduces cellular E-cadherin expression by overexpression of DNA methyltransferase 1 (DNMT1). The Epstein-Barr virus (EBV) is an oncogenic virus that may be related to cervical carcinogenesis. In gastric cancer, it has been demonstrated that E-cadherin gene (CDH1) hypermethylation is associated with DNMT1 overexpression by EBV infection. Our aim was to analyze the gene promoter methylation frequency of RB1 and CDH1 and verify the association between that methylation frequency and HPV and EBV infection in cervical lesions. Sixty-five samples were obtained from cervical specimens: 15 normal cervices, 17 low-grade squamous intraepithelial lesions (LSIL), 15 high-grade squamous intraepithelial lesions (HSIL), and 18 cervical cancers. HPV and EBV DNA testing was performed by PCR, and the methylation status was verified by MSP. HPV frequency was associated with cervical cancer cases (p = 0.005) but not EBV frequency (p = 0.732). Viral co-infection showed a statistically significant correlation with cancer (p = 0.027). No viral infection was detected in 33.3% (5/15) of controls. RB1 methylated status was associated with cancer (p = 0.009) and HPV infection (p = 0.042). CDH1 methylation was not associated with cancer (p = 0.181). Controls and LSIL samples did not show simultaneous methylation, while both genes were methylated in 27.8% (5/18) of cancer samples. In the presence of EBV, CDH1 methylation was present in 27.8% (5/18) of cancer samples. Only cancer cases presented RB1 promoter methylation in the presence of HPV and EBV (33.3%). The methylation status of both genes increased with disease progression. With EBV, RB1 methylation was a tumor-associated event because only the cancer group presented methylated RB1 with HPV infection. HPV infection was shown to be significantly correlated with cancer conditions. The global methylation frequency was

  1. Acceleration of Age-Associated Methylation Patterns in HIV-1-Infected Adults

    Science.gov (United States)

    Sehl, Mary; Sinsheimer, Janet S.; Hultin, Patricia M.; Hultin, Lance E.; Quach, Austin; Martínez-Maza, Otoniel; Horvath, Steve; Vilain, Eric; Jamieson, Beth D.

    2015-01-01

    Patients with treated HIV-1-infection experience earlier occurrence of aging-associated diseases, raising speculation that HIV-1-infection, or antiretroviral treatment, may accelerate aging. We recently described an age-related co-methylation module comprised of hundreds of CpGs; however, it is unknown whether aging and HIV-1-infection exert negative health effects through similar, or disparate, mechanisms. We investigated whether HIV-1-infection would induce age-associated methylation changes. We evaluated DNA methylation levels at >450,000 CpG sites in peripheral blood mononuclear cells (PBMC) of young (20-35) and older (36-56) adults in two separate groups of participants. Each age group for each data set consisted of 12 HIV-1-infected and 12 age-matched HIV-1-uninfected samples for a total of 96 samples. The effects of age and HIV-1 infection on methylation at each CpG revealed a strong correlation of 0.49, pmodules; module 3 (ME3) was significantly correlated with age (cor=0.70) and HIV-1 status (cor=0.31). Older HIV-1+ individuals had a greater number of hypermethylated CpGs across ME3 (p=0.015). In a multivariate model, ME3 was significantly associated with age and HIV status (Data set 1: βage= 0.007088, p=2.08 x 10-9; βHIV= 0.099574, p=0.0011; Data set 2: βage= 0.008762, p=1.27x 10-5; βHIV= 0.128649, p= 0.0001). Using this model, we estimate that HIV-1 infection accelerates age-related methylation by approximately 13.7 years in data set 1 and 14.7 years in data set 2. The genes related to CpGs in ME3 are enriched for polycomb group target genes known to be involved in cell renewal and aging. The overlap between ME3 and an aging methylation module found in solid tissues is also highly significant (Fisher-exact p=5.6 x 10-6, odds ratio=1.91). These data demonstrate that HIV-1 infection is associated with methylation patterns that are similar to age-associated patterns and suggest that general aging and HIV-1 related aging work through some common cellular

  2. Heterogeneity of DNA methylation in multifocal prostate cancer.

    Science.gov (United States)

    Serenaite, Inga; Daniunaite, Kristina; Jankevicius, Feliksas; Laurinavicius, Arvydas; Petroska, Donatas; Lazutka, Juozas R; Jarmalaite, Sonata

    2015-01-01

    Most prostate cancer (PCa) cases are multifocal, and separate foci display histological and molecular heterogeneity. DNA hypermethylation is a frequent alteration in PCa, but interfocal heterogeneity of these changes has not been extensively investigated. Ten pairs of foci from multifocal PCa and 15 benign prostatic hyperplasia (BPH) samples were obtained from prostatectomy specimens, resulting altogether in 35 samples. Methylation-specific PCR (MSP) was used to evaluate methylation status of nine tumor suppressor genes (TSGs), and a set of selected TSGs was quantitatively analyzed for methylation intensity by pyrosequencing. Promoter sequences of the RASSF1 and ESR1 genes were methylated in all paired PCa foci, and frequent (≥75 %) DNA methylation was detected in RARB, GSTP1, and ABCB1 genes. MSP revealed different methylation status of at least one gene in separate foci in 8 out of 10 multifocal tumors. The mean methylation level of ESR1, GSTP1, RASSF1, and RARB differed between the paired foci of all PCa cases. The intensity of DNA methylation in these TSGs was significantly higher in PCa cases than in BPH (p epigenetic profile of recurrent tumors can be inferred from our data.

  3. Methylation-Specific PCR Unraveled

    Directory of Open Access Journals (Sweden)

    Sarah Derks

    2004-01-01

    Full Text Available Methylation‐specific PCR (MSP is a simple, quick and cost‐effective method to analyze the DNA methylation status of virtually any group of CpG sites within a CpG island. The technique comprises two parts: (1 sodium bisulfite conversion of unmethylated cytosine's to uracil under conditions whereby methylated cytosines remains unchanged and (2 detection of the bisulfite induced sequence differences by PCR using specific primer sets for both unmethylated and methylated DNA. This review discusses the critical parameters of MSP and presents an overview of the available MSP variants and the (clinical applications.

  4. Differential DNA methylation profile of key genes in malignant prostate epithelial cells transformed by inorganic arsenic or cadmium

    International Nuclear Information System (INIS)

    Pelch, Katherine E.; Tokar, Erik J.; Merrick, B. Alex; Waalkes, Michael P.

    2015-01-01

    Previous work shows altered methylation patterns in inorganic arsenic (iAs)- or cadmium (Cd)-transformed epithelial cells. Here, the methylation status near the transcriptional start site was assessed in the normal human prostate epithelial cell line (RWPE-1) that was malignantly transformed by 10 μM Cd for 11 weeks (CTPE) or 5 μM iAs for 29 weeks (CAsE-PE), at which time cells showed multiple markers of acquired cancer phenotype. Next generation sequencing of the transcriptome of CAsE-PE cells identified multiple dysregulated genes. Of the most highly dysregulated genes, five genes that can be relevant to the carcinogenic process (S100P, HYAL1, NTM, NES, ALDH1A1) were chosen for an in-depth analysis of the DNA methylation profile. DNA was isolated, bisulfite converted, and combined bisulfite restriction analysis was used to identify differentially methylated CpG sites, which was confirmed with bisulfite sequencing. Four of the five genes showed differential methylation in transformants relative to control cells that was inversely related to altered gene expression. Increased expression of HYAL1 (> 25-fold) and S100P (> 40-fold) in transformants was correlated with hypomethylation near the transcriptional start site. Decreased expression of NES (> 15-fold) and NTM (> 1000-fold) in transformants was correlated with hypermethylation near the transcriptional start site. ALDH1A1 expression was differentially expressed in transformed cells but was not differentially methylated relative to control. In conclusion, altered gene expression observed in Cd and iAs transformed cells may result from altered DNA methylation status. - Highlights: • Cd and iAs are known human carcinogens, yet neither appears directly mutagenic. • Prior data suggest epigenetic modification plays a role in Cd or iAs induced cancer. • Altered methylation of four misregulated genes was found in Cd or iAs transformants. • The resulting altered gene expression may be relevant to cellular

  5. Differential DNA methylation profile of key genes in malignant prostate epithelial cells transformed by inorganic arsenic or cadmium

    Energy Technology Data Exchange (ETDEWEB)

    Pelch, Katherine E.; Tokar, Erik J. [National Toxicology Program Laboratory, Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 (United States); Merrick, B. Alex [Molecular Toxicology and Informatics Group, Biomolecular Screening Branch, Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Morrisville, NC 27560 (United States); Waalkes, Michael P., E-mail: waalkes@niehs.nih.gov [National Toxicology Program Laboratory, Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 (United States)

    2015-08-01

    Previous work shows altered methylation patterns in inorganic arsenic (iAs)- or cadmium (Cd)-transformed epithelial cells. Here, the methylation status near the transcriptional start site was assessed in the normal human prostate epithelial cell line (RWPE-1) that was malignantly transformed by 10 μM Cd for 11 weeks (CTPE) or 5 μM iAs for 29 weeks (CAsE-PE), at which time cells showed multiple markers of acquired cancer phenotype. Next generation sequencing of the transcriptome of CAsE-PE cells identified multiple dysregulated genes. Of the most highly dysregulated genes, five genes that can be relevant to the carcinogenic process (S100P, HYAL1, NTM, NES, ALDH1A1) were chosen for an in-depth analysis of the DNA methylation profile. DNA was isolated, bisulfite converted, and combined bisulfite restriction analysis was used to identify differentially methylated CpG sites, which was confirmed with bisulfite sequencing. Four of the five genes showed differential methylation in transformants relative to control cells that was inversely related to altered gene expression. Increased expression of HYAL1 (> 25-fold) and S100P (> 40-fold) in transformants was correlated with hypomethylation near the transcriptional start site. Decreased expression of NES (> 15-fold) and NTM (> 1000-fold) in transformants was correlated with hypermethylation near the transcriptional start site. ALDH1A1 expression was differentially expressed in transformed cells but was not differentially methylated relative to control. In conclusion, altered gene expression observed in Cd and iAs transformed cells may result from altered DNA methylation status. - Highlights: • Cd and iAs are known human carcinogens, yet neither appears directly mutagenic. • Prior data suggest epigenetic modification plays a role in Cd or iAs induced cancer. • Altered methylation of four misregulated genes was found in Cd or iAs transformants. • The resulting altered gene expression may be relevant to cellular

  6. Prostate tumor DNA methylation is associated with cigarette smoking and adverse prostate cancer outcomes.

    Science.gov (United States)

    Shui, Irene M; Wong, Chao-Jen; Zhao, Shanshan; Kolb, Suzanne; Ebot, Ericka M; Geybels, Milan S; Rubicz, Rohina; Wright, Jonathan L; Lin, Daniel W; Klotzle, Brandy; Bibikova, Marina; Fan, Jian-Bing; Ostrander, Elaine A; Feng, Ziding; Stanford, Janet L

    2016-07-15

    DNA methylation has been hypothesized as a mechanism for explaining the association between smoking and adverse prostate cancer (PCa) outcomes. This study was aimed at assessing whether smoking is associated with prostate tumor DNA methylation and whether these alterations may explain in part the association of smoking with PCa recurrence and mortality. A total of 523 men had radical prostatectomy as their primary treatment, detailed smoking history data, long-term follow-up for PCa outcomes, and tumor tissue profiled for DNA methylation. Ninety percent of the men also had matched tumor gene expression data. A methylome-wide analysis was conducted to identify differentially methylated regions (DMRs) by smoking status. To select potential functionally relevant DMRs, their correlation with the messenger RNA (mRNA) expression of corresponding genes was evaluated. Finally, a smoking-related methylation score based on the top-ranked DMRs was created to assess its association with PCa outcomes. Forty DMRs were associated with smoking status, and 10 of these were strongly correlated with mRNA expression (aldehyde oxidase 1 [AOX1], claudin 5 [CLDN5], early B-cell factor 1 [EBF1], homeobox A7 [HOXA7], lectin galactoside-binding soluble 3 [LGALS3], microtubule-associated protein τ [MAPT], protocadherin γ A [PCDHGA]/protocadherin γ B [PCDHGB], paraoxonase 3 [PON3], synaptonemal complex protein 2 like [SYCP2L], and zinc finger and SCAN domain containing 12 [ZSCAN12]). Men who were in the highest tertile for the smoking-methylation score derived from these DMRs had a higher risk of recurrence (odds ratio [OR], 2.29; 95% confidence interval [CI], 1.42-3.72) and lethal disease (OR, 4.21; 95% CI, 1.65-11.78) in comparison with men in the lower 2 tertiles. This integrative molecular epidemiology study supports the hypothesis that smoking-associated tumor DNA methylation changes may explain at least part of the association between smoking and adverse PCa outcomes. Future studies

  7. Correlation between Food Intake and Health Status with the Nutritional Status of School Children Age 9-11 in Semarang City

    OpenAIRE

    Aiman Farag Mohammed Ali; Siti Fatimah Muis; Suhartono Suhartono

    2016-01-01

    Malnutrition, a major risk factor for a number of infectious diseases, including acute upper respiratory tract infections (AURTI), is common in developing countries. Nutritional status is an important index of the quality of life. Objectives:To analyze the correlation between food intake and health status to nutritional status of 9-11 years old children in Semarang. The study was a correlation study carried among school children in Semarang aged 9-11 years old. Data are presented in the descr...

  8. CpG Island Methylator Phenotype Positive Tumors in the Absence of MLH1 Methylation Constitute a Distinct Subset of Duodenal Adenocarcinomas and Are Associated with Poor Prognosis

    Science.gov (United States)

    Fu, Tao; Pappou, Emmanouil P.; Guzzetta, Angela A.; Jeschke, Jana; Kwak, Ruby; Dave, Pujan; Hooker, Craig M.; Morgan, Richard; Baylin, Stephen B.; Iacobuzio-Donahue, Christine A.; Wolfgang, Christopher L.; Ahuja, Nita

    2012-01-01

    Purpose Little information is available on genetic and epigenetic changes in duodenal adenocarcinomas. The purpose was to identify possible subsets of duodenal adenocarcinomas based on microsatellite instability (MSI), DNA methylation, mutations in the KRAS and BRAF genes, clinicopathologic features, and prognosis. Experimental Design Demographics, tumor characteristics and survival were available for 99 duodenal adenocarcinoma patients. Testing for KRAS and BRAF mutations, MSI, MLH1 methylation and CpG island methylator phenotype (CIMP) status was performed. A Cox proportional hazard model was built to predict survival. Results CIMP+ was detected in 27 of 99 (27.3%) duodenal adenocarcinomas, and was associated with MSI (P = 0.011) and MLH1 methylation (P CIMP− tumors. No BRAF V600E mutation was detected. Among the CIMP+ tumors, 15 (55.6%) were CIMP+/MLH1-unmethylated (MLH1-U). Kaplan-Meier analysis showed tumors classified by CIMP, CIMP/MLH1 methylation status or CIMP/MSI status could predict overall survival (OS; P = 0.047, 0.002, and 0.002, respectively), while CIMP/MLH1 methylation status could also predict time-to-recurrence (TTR; P = 0.016). In multivariate analysis, CIMP/MLH1 methylation status showed a significant prognostic value regarding both OS (P CIMP+/MLH1-U tumors had the worst OS and TTR. Conclusions Our results demonstrate existence of CIMP in duodenal adenocarcinomas. The combination of CIMP+/MLH1-U appears to be independently associated with poor prognosis in patients with duodenal adenocarcinomas. This study also suggests that BRAF mutations are not involved in duodenal tumorigenesis, MSI or CIMP development. PMID:22825585

  9. Reduced MeCP2 expression is frequent in autism frontal cortex and correlates with aberrant MECP2 promoter methylation.

    Science.gov (United States)

    Nagarajan, Raman P; Hogart, Amber R; Gwye, Ynnez; Martin, Michelle R; LaSalle, Janine M

    2006-01-01

    Mutations in MECP2, encoding methyl CpG binding protein 2 (MeCP2), cause most cases of Rett syndrome (RTT), an X-linked neurodevelopmental disorder. Both RTT and autism are "pervasive developmental disorders" and share a loss of social, cognitive and language skills and a gain in repetitive stereotyped behavior, following apparently normal perinatal development. Although MECP2 coding mutations are a rare cause of autism, MeCP2 expression defects were previously found in autism brain. To further study the role of MeCP2 in autism spectrum disorders (ASDs), we determined the frequency of MeCP2 expression defects in brain samples from autism and other ASDs. We also tested the hypotheses that MECP2 promoter mutations or aberrant promoter methylation correlate with reduced expression in cases of idiopathic autism. MeCP2 immunofluorescence in autism and other neurodevelopmental disorders was quantified by laser scanning cytometry and compared with control postmortem cerebral cortex samples on a large tissue microarray. A significant reduction in MeCP2 expression compared to age-matched controls was found in 11/14 autism (79%), 9/9 RTT (100%), 4/4 Angelman syndrome (100%), 3/4 Prader-Willi syndrome (75%), 3/5 Down syndrome (60%), and 2/2 attention deficit hyperactivity disorder (100%) frontal cortex samples. One autism female was heterozygous for a rare MECP2 promoter variant that correlated with reduced MeCP2 expression. A more frequent occurrence was significantly increased MECP2 promoter methylation in autism male frontal cortex compared to controls. Furthermore, percent promoter methylation of MECP2 significantly correlated with reduced MeCP2 protein expression. These results suggest that both genetic and epigenetic defects lead to reduced MeCP2 expression and may be important in the complex etiology of autism.

  10. The correlation between uptake of methyl green and Feulgen staining intensity of cell nuclei. An image analysis study

    DEFF Research Database (Denmark)

    Lyon, H; Schulte, E; Hoyer, P E

    1989-01-01

    were stored in the computer, making it possible to measure the same cells in the Feulgen-restained sections. Image analysis gave results which invalidate the sequential methods as opposed to the simultaneous method. Mean optical densities were significantly increased for both dyes with the simultaneous...... method after formaldehyde fixation as compared to Carnoy fixation. The quantitative correlation of Methyl Green and DNA in the simultaneous technique was found to parallel exactly that of the Feulgen stain. In conclusion, the simultaneous Methyl Green-Pyronin technique is recommended while the sequential......Paraffin sections of rat tissue fixed in either formaldehyde solution (3.6% w/v) or in Carnoy's fluid were stained using standardized Methyl Green-Pyronin procedures with the dyes used either simultaneously or in sequence. The sections were evaluated for the uptake of the two dyes by cell nuclei...

  11. Epigenomic diversity of colorectal cancer indicated by LINE-1 methylation in a database of 869 tumors

    Directory of Open Access Journals (Sweden)

    Schernhammer Eva S

    2010-05-01

    Full Text Available Abstract Background Genome-wide DNA hypomethylation plays a role in genomic instability and carcinogenesis. LINE-1 (L1 retrotransposon constitutes a substantial portion of the human genome, and LINE-1 methylation correlates with global DNA methylation status. LINE-1 hypomethylation in colon cancer has been strongly associated with poor prognosis. However, whether LINE-1 hypomethylators constitute a distinct cancer subtype remains uncertain. Recent evidence for concordant LINE-1 hypomethylation within synchronous colorectal cancer pairs suggests the presence of a non-stochastic mechanism influencing tumor LINE-1 methylation level. Thus, it is of particular interest to examine whether its wide variation can be attributed to clinical, pathologic or molecular features. Design Utilizing a database of 869 colorectal cancers in two prospective cohort studies, we constructed multivariate linear and logistic regression models for LINE-1 methylation (quantified by Pyrosequencing. Variables included age, sex, body mass index, family history of colorectal cancer, smoking status, tumor location, stage, grade, mucinous component, signet ring cells, tumor infiltrating lymphocytes, CpG island methylator phenotype (CIMP, microsatellite instability, expression of TP53 (p53, CDKN1A (p21, CTNNB1 (β-catenin, PTGS2 (cyclooxygenase-2, and FASN, and mutations in KRAS, BRAF, and PIK3CA. Results Tumoral LINE-1 methylation ranged from 23.1 to 90.3 of 0-100 scale (mean 61.4; median 62.3; standard deviation 9.6, and distributed approximately normally except for extreme hypomethylators [LINE-1 methylation Conclusions LINE-1 extreme hypomethylators appear to constitute a previously-unrecognized, distinct subtype of colorectal cancers, which needs to be confirmed by additional studies. Our tumor LINE-1 methylation data indicate enormous epigenomic diversity of individual colorectal cancers.

  12. Sleep quality and methylation status of core circadian rhythm genes among nurses and midwives.

    Science.gov (United States)

    Bukowska-Damska, Agnieszka; Reszka, Edyta; Kaluzny, Pawel; Wieczorek, Edyta; Przybek, Monika; Zienolddiny, Shanbeh; Peplonska, Beata

    2017-01-01

    ABSTARCT Poor sleep quality or sleep restriction is associated with sleepiness and concentration problems. Moreover, chronic sleep restriction may affect metabolism, hormone secretion patterns and inflammatory responses. Limited recent reports suggest a potential link between sleep deprivation and epigenetic effects such as changes in DNA methylation profiles. The aim of the present study was to assess the potential association between poor sleep quality or sleep duration and the levels of 5-methylcytosine in the promoter regions of PER1, PER2, PER3, BMAL1, CLOCK, CRY1 CRY2 and NPAS2 genes, taking into account rotating night work and chronotype as potential confounders or modifiers. A cross-sectional study was conducted on 710 nurses and midwives (347 working on rotating nights and 363 working only during the day) aged 40-60 years. Data from in-person interviews about sleep quality, chronotype and potential confounders were used. Sleep quality and chronotype were assessed using Pittsburgh Sleep Quality Questionnaire (PSQI) and Morningness-Eveningness Questionnaire (MEQ), respectively. Morning blood samples were collected. The methylation status of the circadian rhythm genes was determined via quantitative methylation-specific real-time PCR assays (qMSP) reactions using DNA samples derived from leucocytes. The proportional odds regression model was fitted to quantify the relationship between methylation index (MI) as the dependent variable and sleep quality or sleep duration as the explanatory variable. Analyses were carried out for the total population as well as for subgroups of women stratified by the current system of work (rotating night shift/day work) and chronotype (morning type/intermediate type/evening type). A potential modifying effect of the system of work or the chronotype was examined using the likelihood ratio test. No significant findings were observed in the total study population. Subgroup analyses revealed two statistically significant

  13. Simultaneous CXCL12 and ESR1 CpG island hypermethylation correlates with poor prognosis in sporadic breast cancer

    International Nuclear Information System (INIS)

    Ramos, Edneia AS; Klassen, Giseli; Camargo, Anamaria A; Braun, Karin; Slowik, Renata; Cavalli, Iglenir J; Ribeiro, Enilze MSF; Pedrosa, Fábio de O; Souza, Emanuel M de; Costa, Fabrício F

    2010-01-01

    CXCL12 is a chemokine that is constitutively expressed in many organs and tissues. CXCL12 promoter hypermethylation has been detected in primary breast tumours and contributes to their metastatic potential. It has been shown that the oestrogen receptor α (ESR1) gene can also be silenced by DNA methylation. In this study, we used methylation-specific PCR (MSP) to analyse the methylation status in two regions of the CXCL12 promoter and ESR1 in tumour cell lines and in primary breast tumour samples, and correlated our results with clinicopathological data. First, we analysed CXCL12 expression in breast tumour cell lines by RT-PCR. We also used 5-aza-2'-deoxycytidine (5-aza-CdR) treatment and DNA bisulphite sequencing to study the promoter methylation for a specific region of CXCL12 in breast tumour cell lines. We evaluated CXCL12 and ESR1 methylation in primary tumour samples by methylation-specific PCR (MSP). Finally, promoter hypermethylation of these genes was analysed using Fisher's exact test and correlated with clinicopathological data using the Chi square test, Kaplan-Meier survival analysis and Cox regression analysis. CXCL12 promoter hypermethylation in the first region (island 2) and second region (island 4) was correlated with lack of expression of the gene in tumour cell lines. In the primary tumours, island 2 was hypermethylated in 14.5% of the samples and island 4 was hypermethylated in 54% of the samples. The ESR1 promoter was hypermethylated in 41% of breast tumour samples. In addition, the levels of ERα protein expression diminished with increased frequency of ESR1 methylation (p < 0.0001). This study also demonstrated that CXCL12 island 4 and ESR1 methylation occur simultaneously at a high frequency (p = 0.0220). This is the first study showing a simultaneous involvement of epigenetic regulation for both CXCL12 and ESR1 genes in Brazilian women. The methylation status of both genes was significantly correlated with histologically advanced

  14. Correlation between Calorie Intake and Nutritional Status of Autism Spectrum Disorder in Children

    Directory of Open Access Journals (Sweden)

    Aryo Windaru

    2016-06-01

    Full Text Available Background: Autism Spectrum Disorder (ASD is a severe pervasive developmental disorder with prevalence as high as one in sixty-eight children. Children diagnosed with ASD may have food intake problem and might affect their nutritional status in the future. The objective of this study was to analyze the correlation between total calorie intake and nutritional status of ASD children. Methods: This cross-sectional study was conducted in Indigrow Child Development and Autism Center involving 16 patients from October to November 2015. Total calorie intake was assessed by 24-hour food recall and nutritional status was measured by Z-score. Correlation was analyzed using Spearman’s Rho. Results: Overweight and obesity were found in 10 out of 16 ASD children assessed. Total calorie intake was not significantly correlated with nutritional status of ASD children (r=0.021, p=0.940. Conclusions: There is no significant relevance between total calorie intake and nutritional status in ASD children at Indigrow Child Development and Autism Center.

  15. SKA2 Methylation is associated with Decreased Prefrontal Cortical Thickness and Greater PTSD Severity among Trauma-Exposed Veterans

    Science.gov (United States)

    Sadeh, Naomi; Spielberg, Jeffrey M.; Logue, Mark W.; Wolf, Erika J.; Smith, Alicia K.; Lusk, Joanna; Hayes, Jasmeet P.; Sperbeck, Emily; Milberg, William P.; McGlinchey, Regina E.; Salat, David H.; Carter, Weleetka C.; Stone, Annjanette; Schichman, Steven A.; Humphries, Donald E.; Miller, Mark W.

    2015-01-01

    Methylation of the SKA2 gene has recently been identified as a promising biomarker of suicide risk. Based on this finding, we examined associations between SKA2 methylation, cortical thickness, and psychiatric phenotypes linked to suicide in trauma-exposed veterans. 200 trauma-exposed white non-Hispanic veterans of the recent conflicts in Iraq and Afghanistan (91% male) underwent clinical assessment and had blood drawn for genotyping and methylation analysis. 145 participants also had neuroimaging data available. Based on previous research, we examined DNA methylation at the CpG locus cg13989295 as well as DNA methylation adjusted for genotype at the methylation-associated SNP (rs7208505) in relationship to whole-brain cortical thickness, posttraumatic stress disorder symptoms (PTSD), and depression symptoms. Whole-brain vertex-wise analyses identified three clusters in prefrontal cortex that were associated with genotype-adjusted SKA2 DNA methylation (methylationadj). Specifically, DNA methylationadj was associated with bilateral reductions of cortical thickness in frontal pole and superior frontal gyrus, and similar effects were found in the right orbitofrontal cortex and right inferior frontal gyrus. PTSD symptom severity was positively correlated with SKA2 DNA methylationadj and negatively correlated with cortical thickness in these regions. Mediation analyses showed a significant indirect effect of PTSD on cortical thickness via SKA2 methylation status. Results suggest that DNA methylationadj of SKA2 in blood indexes stress-related psychiatric phenotypes and neurobiology, pointing to its potential value as a biomarker of stress exposure and susceptibility. PMID:26324104

  16. NGX6 gene mediated by promoter methylation as a potential molecular marker in colorectal cancer

    Directory of Open Access Journals (Sweden)

    Shen Shourong

    2010-04-01

    Full Text Available Abstract Background Nasopharyngeal carcinoma associated gene 6 (NGX6 is down-regulated in most colon cancer cell lines and tumor tissues when compared with their normal tissue samples. As a novel suppress tumor gene, it could inhibit colon cancer cell growth and cell cycle progression. However, little is known about the transcriptional mechanisms controlling NGX6 gene expression. Recent findings suggest that epigenetic inactivation of multiple tumor suppressor genes plays an important role in the tumorigenesis of colorectal carcinoma (CRC. In this study, we explored the role of DNA methylation in regulation of NGX6 transcription. Methods In the present study, we cloned the NGX6 promoter with characteristics of a CpG island by luciferase reporter assay. Then, the CpG methylation status around the NGX6 promoter region in colon cancer cell lines and colorectal tumor tissues was examined by methylation-specific PCR and bisulfite DNA sequencing. Finally, 5-Aza-2'-deoxycytidine (5-Aza-dC treatment was used to confirm the correlation between NGX6 promoter methylation and its gene inactivation. Results The sequence spanning positions -157 to +276 was identified as the NGX6 promoter, in which no canonical TATA boxes were found, while two CAAT boxes and GC boxes were discovered. Methylation status was observed more frequently in 40 colorectal cancer samples than in 40 adjacent normal mucosa samples (18/40 versus 7/40; P Conclusions Down-regulation of NGX6 gene is related to the promoter methylation. DNA methylation of NGX6 promoter might be a potential molecular marker for diagnosis or prognosis, or serve as a therapeutic target.

  17. Inductive effect of methyl group in a series of methylated indoles: A ...

    Indian Academy of Sciences (India)

    Vol. 125, No. 4, July 2013, pp. 905–912. c Indian Academy of Sciences. Inductive effect of methyl group in a series of methylated indoles: A graph theoretical analysis in the light of density functional theory and correlation with experimental charge transfer transition energies. AMIT S TIWARYa,∗ and ASOK K MUKHERJEEb.

  18. DNA methylation differences at growth related genes correlate with birth weight: a molecular signature linked to developmental origins of adult disease?

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    Turan Nahid

    2012-04-01

    Full Text Available Abstract Background Infant birth weight is a complex quantitative trait associated with both neonatal and long-term health outcomes. Numerous studies have been published in which candidate genes (IGF1, IGF2, IGF2R, IGF binding proteins, PHLDA2 and PLAGL1 have been associated with birth weight, but these studies are difficult to reproduce in man and large cohort studies are needed due to the large inter individual variance in transcription levels. Also, very little of the trait variance is explained. We decided to identify additional candidates without regard for what is known about the genes. We hypothesize that DNA methylation differences between individuals can serve as markers of gene "expression potential" at growth related genes throughout development and that these differences may correlate with birth weight better than single time point measures of gene expression. Methods We performed DNA methylation and transcript profiling on cord blood and placenta from newborns. We then used novel computational approaches to identify genes correlated with birth weight. Results We identified 23 genes whose methylation levels explain 70-87% of the variance in birth weight. Six of these (ANGPT4, APOE, CDK2, GRB10, OSBPL5 and REG1B are associated with growth phenotypes in human or mouse models. Gene expression profiling explained a much smaller fraction of variance in birth weight than did DNA methylation. We further show that two genes, the transcriptional repressor MSX1 and the growth factor receptor adaptor protein GRB10, are correlated with transcriptional control of at least seven genes reported to be involved in fetal or placental growth, suggesting that we have identified important networks in growth control. GRB10 methylation is also correlated with genes involved in reactive oxygen species signaling, stress signaling and oxygen sensing and more recent data implicate GRB10 in insulin signaling. Conclusions Single time point measurements of gene

  19. Decreased expression level of BER genes in Alzheimer's disease patients is not derivative of their DNA methylation status.

    Science.gov (United States)

    Sliwinska, Agnieszka; Sitarek, Przemysław; Toma, Monika; Czarny, Piotr; Synowiec, Ewelina; Krupa, Renata; Wigner, Paulina; Bialek, Katarzyna; Kwiatkowski, Dominik; Korycinska, Anna; Majsterek, Ireneusz; Szemraj, Janusz; Galecki, Piotr; Sliwinski, Tomasz

    2017-10-03

    Neurodegeneration in Alzheimer's disease can be caused by accumulation of oxidative DNA damage resulting from altered expression of genes involved in the base excision repair system (BER). Promoter methylation can affect the profile of BER genes expression. Decreased expression of BER genes was observed in the brains of AD patients. The aim of our study was to compare the expression and methylation profiles of six genes coding for proteins involved in BER, namely: hOGG1, APE1, MUTYH, NEIL1, PARP1 and XRCC1, in the peripheral blood cells of AD patients and healthy volunteers. The study consisted of 100 persons diagnosed with Alzheimer's disease according to DSM-IV criteria, and 110 healthy volunteers. DNA and total RNA were isolated from venous blood cells. Promoter methylation profiles were obtained by High Resolution Melting (HRM) analysis of bisulfide converted DNA samples. Real-time PCR with TaqMan probes was employed for gene expression analysis. APE1, hOGG1, MUTYH, PARP1 and NEIL1 were significantly (pgenes. The methylation status of promoters is not associated with downregulation of BER genes. Our results show that downregulation of BER genes detected in peripheral blood samples could reflect the changes occurring in the brain of patients with AD, and may be a useful biomarker of this disease. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Effect of DNA methylation on identification of aggressive prostate cancer.

    Science.gov (United States)

    Alumkal, Joshi J; Zhang, Zhe; Humphreys, Elizabeth B; Bennett, Christina; Mangold, Leslie A; Carducci, Michael A; Partin, Alan W; Garrett-Mayer, Elizabeth; DeMarzo, Angelo M; Herman, James G

    2008-12-01

    Biochemical (prostate-specific antigen) recurrence of prostate cancer after radical prostatectomy remains a major problem. Better biomarkers are needed to identify high-risk patients. DNA methylation of promoter regions leads to gene silencing in many cancers. In this study, we assessed the effect of DNA methylation on the identification of recurrent prostate cancer. We studied the methylation status of 15 pre-specified genes using methylation-specific polymerase chain reaction on tissue samples from 151 patients with localized prostate cancer and at least 5 years of follow-up after prostatectomy. On multivariate logistic regression analysis, a high Gleason score and involvement of the capsule, lymph nodes, seminal vesicles, or surgical margin were associated with an increased risk of biochemical recurrence. Methylation of CDH13 by itself (odds ratio 5.50, 95% confidence interval [CI] 1.34 to 22.67; P = 0.02) or combined with methylation of ASC (odds ratio 5.64, 95% CI 1.47 to 21.7; P = 0.01) was also associated with an increased risk of biochemical recurrence. The presence of methylation of ASC and/or CDH13 yielded a sensitivity of 72.3% (95% CI 57% to 84.4%) and negative predictive value of 79% (95% CI 66.8% to 88.3%), similar to the weighted risk of recurrence (determined from the lymph node status, seminal vesicle status, surgical margin status, and postoperative Gleason score), a powerful clinicopathologic prognostic score. However, 34% (95% CI 21% to 49%) of the patients with recurrence were identified by the methylation profile of ASC and CDH13 rather than the weighted risk of recurrence. The results of our study have shown that methylation of CDH13 alone or combined with methylation of ASC is independently associated with an increased risk of biochemical recurrence after radical prostatectomy even considering the weighted risk of recurrence score. These findings should be validated in an independent, larger cohort of patients with prostate cancer who have

  1. Cord blood buffy coat DNA methylation is comparable to whole cord blood methylation.

    Science.gov (United States)

    Dou, John; Schmidt, Rebecca J; Benke, Kelly S; Newschaffer, Craig; Hertz-Picciotto, Irva; Croen, Lisa A; Iosif, Ana-Maria; LaSalle, Janine M; Fallin, M Daniele; Bakulski, Kelly M

    2018-01-01

    Cord blood DNA methylation is associated with numerous health outcomes and environmental exposures. Whole cord blood DNA reflects all nucleated blood cell types, while centrifuging whole blood separates red blood cells, generating a white blood cell buffy coat. Both sample types are used in DNA methylation studies. Cell types have unique methylation patterns and processing can impact cell distributions, which may influence comparability. We evaluated differences in cell composition and DNA methylation between cord blood buffy coat and whole cord blood samples. Cord blood DNA methylation was measured with the Infinium EPIC BeadChip (Illumina) in eight individuals, each contributing buffy coat and whole blood samples. We analyzed principal components (PC) of methylation, performed hierarchical clustering, and computed correlations of mean-centered methylation between pairs. We conducted moderated t-tests on single sites and estimated cell composition. DNA methylation PCs were associated with individual (P PC1 = 1.4 × 10 -9 ; P PC2 = 2.9 × 10 -5 ; P PC3 = 3.8 × 10 -5 ; P PC4 = 4.2 × 10 -6 ; P PC5 = 9.9 × 10 -13 , P PC6 = 1.3 × 10 -11 ) and not with sample type (P PC1-6 >0.7). Samples hierarchically clustered by individual. Pearson correlations of mean-centered methylation between paired samples ranged from r = 0.66 to r = 0.87. No individual site significantly differed between buffy coat and whole cord blood when adjusting for multiple comparisons (five sites had unadjusted Pcoat and whole cord blood are much lower than inter-individual variation, demonstrating that both sample preparation types can be analytically combined and compared.

  2. Estimates of methyl 13C and 1H CSA values (Δσ) in proteins from cross-correlated spin relaxation

    International Nuclear Information System (INIS)

    Tugarinov, Vitali; Scheurer, Christoph; Brueschweiler, Rafael; Kay, Lewis E.

    2004-01-01

    Simple pulse schemes are presented for the measurement of methyl 13 C and 1 H CSA values from 1 H- 13 C dipole/ 13 C CSA and 1 H- 13 C dipole/ 1 H CSA cross-correlated relaxation. The methodology is applied to protein L and malate synthase G. Average 13 C CSA values are considerably smaller for Ile than Leu/Val (17 vs 25 ppm) and are in good agreement with previous solid state NMR studies of powders of amino acids and dipeptides and in reasonable agreement with quantum-chemical DFT calculations of methyl carbon CSA values in peptide fragments. Small averaged 1 H CSA values on the order of 1 ppm are measured, consistent with a solid state NMR determination of the methyl group 1 H CSA in dimethylmalonic acid

  3. The application of methylation specific electrophoresis (MSE to DNA methylation analysis of the 5' CpG island of mucin in cancer cells

    Directory of Open Access Journals (Sweden)

    Yokoyama Seiya

    2012-02-01

    Full Text Available Abstract Background Methylation of CpG sites in genomic DNA plays an important role in gene regulation and especially in gene silencing. We have reported mechanisms of epigenetic regulation for expression of mucins, which are markers of malignancy potential and early detection of human neoplasms. Epigenetic changes in promoter regions appear to be the first step in expression of mucins. Thus, detection of promoter methylation status is important for early diagnosis of cancer, monitoring of tumor behavior, and evaluating the response of tumors to targeted therapy. However, conventional analytical methods for DNA methylation require a large amount of DNA and have low sensitivity. Methods Here, we report a modified version of the bisulfite-DGGE (denaturing gradient gel electrophoresis using a nested PCR approach. We designated this method as methylation specific electrophoresis (MSE. The MSE method is comprised of the following steps: (a bisulfite treatment of genomic DNA, (b amplification of the target DNA by a nested PCR approach and (c applying to DGGE. To examine whether the MSE method is able to analyze DNA methylation of mucin genes in various samples, we apply it to DNA obtained from state cell lines, ethanol-fixed colonic crypts and human pancreatic juices. Result The MSE method greatly decreases the amount of input DNA. The lower detection limit for distinguishing different methylation status is Conclusions The MSE method can provide a qualitative information of methylated sequence profile. The MSE method allows sensitive and specific analysis of the DNA methylation pattern of almost any block of multiple CpG sites. The MSE method can be applied to analysis of DNA methylation status in many different clinical samples, and this may facilitate identification of new risk markers.

  4. DNA methylation levels associated with race and childhood asthma severity.

    Science.gov (United States)

    Chan, Marcia A; Ciaccio, Christina E; Gigliotti, Nicole M; Rezaiekhaligh, Mo; Siedlik, Jacob A; Kennedy, Kevin; Barnes, Charles S

    2017-10-01

    Asthma is a common chronic childhood disease worldwide. Socioeconomic status, genetic predisposition and environmental factors contribute to its incidence and severity. A disproportionate number of children with asthma are economically disadvantaged and live in substandard housing with potential indoor environmental exposures such as cockroaches, dust mites, rodents and molds. These exposures may manifest through epigenetic mechanisms that can lead to changes in relevant gene expression. We examined the association of global DNA methylation levels with socioeconomic status, asthma severity and race/ethnicity. We measured global DNA methylation in peripheral blood of children with asthma enrolled in the Kansas City Safe and Healthy Homes Program. Inclusion criteria included residing in the same home for a minimum of 4 days per week and total family income of less than 80% of the Kansas City median family income. DNA methylation levels were quantified by an immunoassay that assessed the percentage of 5-methylcytosine. Our results indicate that overall, African American children had higher levels of global DNA methylation than children of other races/ethnicities (p = 0.029). This difference was more pronounced when socioeconomic status and asthma severity were coupled with race/ethnicity (p = 0.042) where low-income, African American children with persistent asthma had significantly elevated methylation levels relative to other races/ethnicities in the same context (p = 0.006, Hedges g = 1.14). Our study demonstrates a significant interaction effect among global DNA methylation levels, asthma severity, race/ethnicity, and socioeconomic status.

  5. Lack of death receptor 4 (DR4) expression through gene promoter methylation in gastric carcinoma.

    Science.gov (United States)

    Lee, Kyung Hwa; Lim, Sang Woo; Kim, Ho Gun; Kim, Dong Yi; Ryu, Seong Yeob; Joo, Jae Kyun; Kim, Jung Chul; Lee, Jae Hyuk

    2009-07-01

    To determine the underlying mechanism for the differential expression, the extent of promoter methylation in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-related genes acting downstream of TRAIL was examined in early and advanced gastric carcinomas. The extent of promoter methylation in the DR4, DR5, DcR1, DcR2, and CASP8 genes was quantified using bisulfite modification and methylation-specific polymerase chain reaction. The promoters for DcR1, DcR2, and CASP8 were largely unmethylated in early gastric carcinoma, advanced gastric carcinoma, and controls, with no significant difference among them. Protein levels of DR4, DcR1, and DcR2 as revealed by immunohistochemistry correlated with the extent of the respective promoter methylation (P < 0.05 in all cases). Hypomethylation, rather than hypermethylation, of the DR4 promoter was noted in invasive gastric malignancies, with statistical significance (P = 0.003). The promoter methylation status of TRAIL receptors in gastric carcinoma may have clinical implications for improving therapeutic strategies in patients with gastric carcinoma.

  6. Correlates of weight status among Norwegian 11-year-olds: The HEIA study

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    Grydeland May

    2012-12-01

    Full Text Available Abstract Background The underlying mechanisms of overweight and obesity in adolescents are still not fully understood. The aim of this study was to investigate modifiable and non-modifiable correlates of weight status among 1103 Norwegian 11-year-old adolescents in the HEalth in Adolescents (HEIA study, including demographic factors such as gender and parental education, and behavioral factors such as intake of sugar-sweetened beverages, snacks and breakfast consumption, watching TV and playing computer games, physical activity and sedentary time. Methods Weight and height were measured objectively, body mass index (BMI was calculated and International Obesity Task Force cut-offs were used to define weight status. Physical activity and sedentary time were measured by accelerometers. Other behavioral correlates and pubertal status were self-reported by questionnaires. Parental education was reported by the parents on the consent form for their child. Associations were investigated using logistic regressions. Results There were gender differences in behavioral correlates of weight status but not for weight status itself. Adolescents with parents in the highest education category had a 46% reduced odds of being overweight compared to adolescents with parents in the lowest education category. Adolescents with parents with medium education had 42% lower odds of being overweight than adolescents with parents with the lowest education category. Level of parental education, breakfast consumption and moderate to vigorous physical activity were positively associated with being normal weight, and time watching TV was positively associated with being overweight for the total sample. Gender differences were detected; boys had a doubled risk of being overweight for every additional hour of watching TV per week, while for girls there was no association. Conclusions The present study showed a social gradient in weight status in 11-year-olds. Both breakfast

  7. Clinical Utility of promoter methylation of the tumor suppressor ...

    African Journals Online (AJOL)

    Aim: Aim is to examine the potential usefulness of blood based methylation specific polymerase chain reaction (MSP) of methylated DKK3 and RASSF1A genes in early detection of breast cancer. Method: Methylation status of DKK3 and RASSF1 was investigated in forty breast cancer patients, twenty fibroadenoma patients ...

  8. MGMT methylation analysis of glioblastoma on the Infinium methylation BeadChip identifies two distinct CpG regions associated with gene silencing and outcome, yielding a prediction model for comparisons across datasets, tumor grades, and CIMP-status.

    Science.gov (United States)

    Bady, Pierre; Sciuscio, Davide; Diserens, Annie-Claire; Bloch, Jocelyne; van den Bent, Martin J; Marosi, Christine; Dietrich, Pierre-Yves; Weller, Michael; Mariani, Luigi; Heppner, Frank L; Mcdonald, David R; Lacombe, Denis; Stupp, Roger; Delorenzi, Mauro; Hegi, Monika E

    2012-10-01

    The methylation status of the O(6)-methylguanine-DNA methyltransferase (MGMT) gene is an important predictive biomarker for benefit from alkylating agent therapy in glioblastoma. Recent studies in anaplastic glioma suggest a prognostic value for MGMT methylation. Investigation of pathogenetic and epigenetic features of this intriguingly distinct behavior requires accurate MGMT classification to assess high throughput molecular databases. Promoter methylation-mediated gene silencing is strongly dependent on the location of the methylated CpGs, complicating classification. Using the HumanMethylation450 (HM-450K) BeadChip interrogating 176 CpGs annotated for the MGMT gene, with 14 located in the promoter, two distinct regions in the CpG island of the promoter were identified with high importance for gene silencing and outcome prediction. A logistic regression model (MGMT-STP27) comprising probes cg12434587 [corrected] and cg12981137 provided good classification properties and prognostic value (kappa = 0.85; log-rank p CIMP) positive tumors was found in glioblastomas from The Cancer Genome Atlas than in low grade and anaplastic glioma cohorts, while in CIMP-negative gliomas MGMT was classified as methylated in approximately 50 % regardless of tumor grade. The proposed MGMT-STP27 prediction model allows mining of datasets derived on the HM-450K or HM-27K BeadChip to explore effects of distinct epigenetic context of MGMT methylation suspected to modulate treatment resistance in different tumor types.

  9. Characterization of human gastric carcinoma-related methylation of 9 miR CpG islands and repression of their expressions in vitro and in vivo

    International Nuclear Information System (INIS)

    Du, Yantao; Liu, Zhaojun; Gu, Liankun; Zhou, Jing; Zhu, Bu-dong; Ji, Jiafu; Deng, Dajun

    2012-01-01

    Many miR genes are located within or around CpG islands. It is unclear whether methylation of these CpG islands represses miR transcription regularly. The aims of this study are to characterize gastric carcinoma (GC)-related methylation of miR CpG islands and its relationship with miRNA expression. Methylation status of 9 representative miR CpG islands in a panel of cell lines and human gastric samples (including 13 normal biopsies, 38 gastritis biopsies, 112 pairs of GCs and their surgical margin samples) was analyzed by bisulfite-DHPLC and sequencing. Mature miRNA levels were determined with quantitative RT-PCR. Relationships between miR methylation, transcription, GC development, and clinicopathological characteristics were statistically analyzed. Methylation frequency of 5 miR CpG islands (miR-9-1, miR-9-3, miR-137, miR-34b, and miR-210) gradually increased while the proportion of methylated miR-200b gradually decreased during gastric carcinogenesis (Ps < 0.01). More miR-9-1 methylation was detected in 62%-64% of the GC samples and 4% of the normal or gastritis samples (18/28 versus 2/48; Odds ratio, 41.4; P < 0.01). miR-210 methylation showed high correlation with H. pylori infection. miR-375, miR-203, and miR-193b methylation might be host adaptation to the development of GCs. Methylation of these miR CpG islands was consistently shown to significantly decrease the corresponding miRNA levels presented in human cell lines. The inverse relationship was also observed for miR-9-1, miR-9-3, miR-137, and miR-200b in gastric samples. Among 112 GC patients, miR-9-1 methylation was an independent favourable predictor of overall survival of GC patients in both univariate and multivariate analysis (P < 0.02). In conclusion, alteration of methylation status of 6 of 9 tested miR CpG islands was characterized in gastric carcinogenesis. miR-210 methylation correlated with H. pylori infection. miR-9-1 methylation may be a GC-specific event. Methylation of miR CpG islands may

  10. Methylated genes as new cancer biomarkers.

    LENUS (Irish Health Repository)

    Duffy, M J

    2012-02-01

    Aberrant hypermethylation of promoter regions in specific genes is a key event in the formation and progression of cancer. In at least some situations, these aberrant alterations occur early in the formation of malignancy and appear to be tumour specific. Multiple reports have suggested that measurement of the methylation status of the promoter regions of specific genes can aid early detection of cancer, determine prognosis and predict therapy responses. Promising DNA methylation biomarkers include the use of methylated GSTP1 for aiding the early diagnosis of prostate cancer, methylated PITX2 for predicting outcome in lymph node-negative breast cancer patients and methylated MGMT in predicting benefit from alkylating agents in patients with glioblastomas. However, prior to clinical utilisation, these findings require validation in prospective clinical studies. Furthermore, assays for measuring gene methylation need to be standardised, simplified and evaluated in external quality assurance programmes. It is concluded that methylated genes have the potential to provide a new generation of cancer biomarkers.

  11. Phase I/II study of azacitidine and capecitabine/oxaliplatin (CAPOX) in refractory CIMP-high metastatic colorectal cancer: evaluation of circulating methylated vimentin.

    Science.gov (United States)

    Overman, Michael J; Morris, Van; Moinova, Helen; Manyam, Ganiraju; Ensor, Joe; Lee, Michael S; Eng, Cathy; Kee, Bryan; Fogelman, David; Shroff, Rachna T; LaFramboise, Thomas; Mazard, Thibault; Feng, Tian; Hamilton, Stanley; Broom, Bradley; Lutterbaugh, James; Issa, Jean-Pierre; Markowitz, Sanford D; Kopetz, Scott

    2016-10-11

    Hypermethylation of promoter CpG islands (CIMP) has been strongly implicated in chemotherapy resistance and is implicated in the pathogenesis of a subset of colorectal cancers (CRCs) termed CIMP-high. This phase I/II study in CRC (phase II portion restricted to CIMP-high CRC), treated fluoropyrimidine/oxaliplatin refractory patients with azacitidine (75 mg/m2/day subcutaneously D1-5) and CAPOX (capecitibine and oxaliplatin) every three weeks. Twenty-six patients (pts) were enrolled in this study: 15 pts (12 treated at MTD) in phase I and 11 pts in phase II. No dose limiting toxicities were observed. A total of 14 pts were CIMP-high. No responses were seen. CIMP-high status did not correlate with efficacy endpoints [stable disease (SD) or progression-free survival (PFS)] or baseline vimentin methylation level. Changes in vimentin methylation over time did not correlate with efficacy outcomes. Baseline methylated vimentin correlated with tumor volume (PCIMP-high pts, but no objective responses. Serum methylated vimentin may be associated with benefit from a regimen including a hypomethylation agent, although this study is not able to separate a potential prognostic or predictive role for the biomarker.

  12. Genome-wide methylation analysis identified sexually dimorphic methylated regions in hybrid tilapia

    Science.gov (United States)

    Wan, Zi Yi; Xia, Jun Hong; Lin, Grace; Wang, Le; Lin, Valerie C. L.; Yue, Gen Hua

    2016-01-01

    Sexual dimorphism is an interesting biological phenomenon. Previous studies showed that DNA methylation might play a role in sexual dimorphism. However, the overall picture of the genome-wide methylation landscape in sexually dimorphic species remains unclear. We analyzed the DNA methylation landscape and transcriptome in hybrid tilapia (Oreochromis spp.) using whole genome bisulfite sequencing (WGBS) and RNA-sequencing (RNA-seq). We found 4,757 sexually dimorphic differentially methylated regions (DMRs), with significant clusters of DMRs located on chromosomal regions associated with sex determination. CpG methylation in promoter regions was negatively correlated with the gene expression level. MAPK/ERK pathway was upregulated in male tilapia. We also inferred active cis-regulatory regions (ACRs) in skeletal muscle tissues from WGBS datasets, revealing sexually dimorphic cis-regulatory regions. These results suggest that DNA methylation contribute to sex-specific phenotypes and serve as resources for further investigation to analyze the functions of these regions and their contributions towards sexual dimorphisms. PMID:27782217

  13. Disclosing bias in bisulfite assay: MethPrimers underestimate high DNA methylation.

    Directory of Open Access Journals (Sweden)

    Andrea Fuso

    Full Text Available Discordant results obtained in bisulfite assays using MethPrimers (PCR primers designed using MethPrimer software or assuming that non-CpGs cytosines are non methylated versus primers insensitive to cytosine methylation lead us to hypothesize a technical bias. We therefore used the two kinds of primers to study different experimental models and methylation statuses. We demonstrated that MethPrimers negatively select hypermethylated DNA sequences in the PCR step of the bisulfite assay, resulting in CpG methylation underestimation and non-CpG methylation masking, failing to evidence differential methylation statuses. We also describe the characteristics of "Methylation-Insensitive Primers" (MIPs, having degenerated bases (G/A to cope with the uncertain C/U conversion. As CpG and non-CpG DNA methylation patterns are largely variable depending on the species, developmental stage, tissue and cell type, a variable extent of the bias is expected. The more the methylome is methylated, the greater is the extent of the bias, with a prevalent effect of non-CpG methylation. These findings suggest a revision of several DNA methylation patterns so far documented and also point out the necessity of applying unbiased analyses to the increasing number of epigenomic studies.

  14. Identification of DNA methylation changes associated with human gastric cancer

    Directory of Open Access Journals (Sweden)

    Park Jung-Hoon

    2011-12-01

    Full Text Available Abstract Background Epigenetic alteration of gene expression is a common event in human cancer. DNA methylation is a well-known epigenetic process, but verifying the exact nature of epigenetic changes associated with cancer remains difficult. Methods We profiled the methylome of human gastric cancer tissue at 50-bp resolution using a methylated DNA enrichment technique (methylated CpG island recovery assay in combination with a genome analyzer and a new normalization algorithm. Results We were able to gain a comprehensive view of promoters with various CpG densities, including CpG Islands (CGIs, transcript bodies, and various repeat classes. We found that gastric cancer was associated with hypermethylation of 5' CGIs and the 5'-end of coding exons as well as hypomethylation of repeat elements, such as short interspersed nuclear elements and the composite element SVA. Hypermethylation of 5' CGIs was significantly correlated with downregulation of associated genes, such as those in the HOX and histone gene families. We also discovered long-range epigenetic silencing (LRES regions in gastric cancer tissue and identified several hypermethylated genes (MDM2, DYRK2, and LYZ within these regions. The methylation status of CGIs and gene annotation elements in metastatic lymph nodes was intermediate between normal and cancerous tissue, indicating that methylation of specific genes is gradually increased in cancerous tissue. Conclusions Our findings will provide valuable data for future analysis of CpG methylation patterns, useful markers for the diagnosis of stomach cancer, as well as a new analysis method for clinical epigenomics investigations.

  15. Analysis of DNA methylation in Arabidopsis thaliana based on methylation-sensitive AFLP markers.

    Science.gov (United States)

    Cervera, M T; Ruiz-García, L; Martínez-Zapater, J M

    2002-12-01

    AFLP analysis using restriction enzyme isoschizomers that differ in their sensitivity to methylation of their recognition sites has been used to analyse the methylation state of anonymous CCGG sequences in Arabidopsis thaliana. The technique was modified to improve the quality of fingerprints and to visualise larger numbers of scorable fragments. Sequencing of amplified fragments indicated that detection was generally associated with non-methylation of the cytosine to which the isoschizomer is sensitive. Comparison of EcoRI/ HpaII and EcoRI/ MspI patterns in different ecotypes revealed that 35-43% of CCGG sites were differentially digested by the isoschizomers. Interestingly, the pattern of digestion among different plants belonging to the same ecotype is highly conserved, with the rate of intra-ecotype methylation-sensitive polymorphisms being less than 1%. However, pairwise comparisons of methylation patterns between samples belonging to different ecotypes revealed differences in up to 34% of the methylation-sensitive polymorphisms. The lack of correlation between inter-ecotype similarity matrices based on methylation-insensitive or methylation-sensitive polymorphisms suggests that whatever the mechanisms regulating methylation may be, they are not related to nucleotide sequence variation.

  16. Comparison of methods for quantification of global DNA methylation in human cells and tissues.

    Directory of Open Access Journals (Sweden)

    Sofia Lisanti

    Full Text Available DNA methylation is a key epigenetic modification which, in mammals, occurs mainly at CpG dinucleotides. Most of the CpG methylation in the genome is found in repetitive regions, rich in dormant transposons and endogenous retroviruses. Global DNA hypomethylation, which is a common feature of several conditions such as ageing and cancer, can cause the undesirable activation of dormant repeat elements and lead to altered expression of associated genes. DNA hypomethylation can cause genomic instability and may contribute to mutations and chromosomal recombinations. Various approaches for quantification of global DNA methylation are widely used. Several of these approaches measure a surrogate for total genomic methyl cytosine and there is uncertainty about the comparability of these methods. Here we have applied 3 different approaches (luminometric methylation assay, pyrosequencing of the methylation status of the Alu repeat element and of the LINE1 repeat element for estimating global DNA methylation in the same human cell and tissue samples and have compared these estimates with the "gold standard" of methyl cytosine quantification by HPLC. Next to HPLC, the LINE1 approach shows the smallest variation between samples, followed by Alu. Pearson correlations and Bland-Altman analyses confirmed that global DNA methylation estimates obtained via the LINE1 approach corresponded best with HPLC-based measurements. Although, we did not find compelling evidence that the gold standard measurement by HPLC could be substituted with confidence by any of the surrogate assays for detecting global DNA methylation investigated here, the LINE1 assay seems likely to be an acceptable surrogate in many cases.

  17. [Methylation of selected tumor-supressor genes in benign and malignant ovarian tumors].

    Science.gov (United States)

    Cul'bová, M; Lasabová, Z; Stanclová, A; Tilandyová, P; Zúbor, P; Fiolka, R; Danko, J; Visnovský, J

    2011-09-01

    To evaluate the usefullness of examination of methylation status of selected tumor-supressor genes in early diagnosis of ovarian cancer. Prospective clinical study. Department of Gynecology and Obstetrics, Department of Molecular Biology, Jessenius Medical Faculty, Commenius University, Martin, Slovak Republic. In this study we analyzed hypermethylation of 5 genes RASSF1A, GSTP, E-cadherin, p16 and APC in ovarian tumor samples from 34 patients - 13 patients with epithelial ovarian cancer, 2 patients with border-line ovarian tumors, 12 patients with benign lesions of ovaries and 7 patients with healthy ovarian tissue. The methylation status of promoter region of tumor-supressor genes was determined by Methylation Specific Polymerase Chain Reaction (MSP) using a nested two-step approach with bisulfite modified DNA template and specific primers. Gene methylation analysis revealed hypermethylation of gene RASSF1A (46%) and GSTP (8%) only in malignant ovarian tissue samples. Ecad, p16 and APC genes were methylated both in maignant and benign tissue samples. Methylation positivity in observed genes was present independently to all clinical stages of ovarian cancer and to tumor grades. However, there was observed a trend of increased number and selective involvement of methylated genes with increasing disease stages. Furthermore, there was no association between positive methylation status and histological subtypes of ovarian carcinomas. RASSF1A and GSTP promoter methylation positivity is associated with ovarian cancer. The revealed gene-selective methylation positivity and the increased number of methylated genes with advancing disease stages could be considered as a useful molecular marker for early detection of ovarian cancer. However, there is need to find diagnostic approach of specifically and frequently methylated genes to determining a methylation phenotype for early detection of ovarian malignancies.

  18. DNA methylation modulates H19 and IGF2 expression in porcine female eye

    Directory of Open Access Journals (Sweden)

    Dongxu Wang

    2017-03-01

    Full Text Available Abstract The sexually dimorphic expression of H19/IGF2 is evolutionarily conserved. To investigate whether the expression of H19/IGF2 in the female porcine eye is sex-dependent, gene expression and methylation status were evaluated using quantitative real-time PCR (qPCR and bisulfite sequencing PCR (BSP. We hypothesized that H19/IGF2 might exhibit a different DNA methylation status in the female eye. In order to evaluate our hypothesis, parthenogenetic (PA cells were used for analysis by qPCR and BSP. Our results showed that H19 and IGF2 were over-expressed in the female eye compared with the male eye (3-fold and 2-fold, respectively. We observed a normal monoallelic methylation pattern for H19 differentially methylated regions (DMRs. Compared with H19 DMRs, IGF2 DMRs showed a different methylation pattern in the eye. Taken together, these results suggest that elevated expression of H19/IGF2 is caused by a specific chromatin structure that is regulated by the DNA methylation status of IGF2 DMRs in the female eye.

  19. DNA methylation modulates H19 and IGF2 expression in porcine female eye

    Science.gov (United States)

    Wang, Dongxu; Wang, Guodong; Yang, Hao; Liu, Haibo; Li, Cuie; Li, Xiaolan; Lin, Chao; Song, Yuning; Li, Zhanjun; Liu, Dianfeng

    2017-01-01

    Abstract The sexually dimorphic expression of H19/IGF2 is evolutionarily conserved. To investigate whether the expression of H19/IGF2 in the female porcine eye is sex-dependent, gene expression and methylation status were evaluated using quantitative real-time PCR (qPCR) and bisulfite sequencing PCR (BSP). We hypothesized that H19/IGF2 might exhibit a different DNA methylation status in the female eye. In order to evaluate our hypothesis, parthenogenetic (PA) cells were used for analysis by qPCR and BSP. Our results showed that H19 and IGF2 were over-expressed in the female eye compared with the male eye (3-fold and 2-fold, respectively). We observed a normal monoallelic methylation pattern for H19 differentially methylated regions (DMRs). Compared with H19 DMRs, IGF2 DMRs showed a different methylation pattern in the eye. Taken together, these results suggest that elevated expression of H19/IGF2 is caused by a specific chromatin structure that is regulated by the DNA methylation status of IGF2 DMRs in the female eye. PMID:28266684

  20. Modeling spatiotemporal dynamics of DNA methylation

    DEFF Research Database (Denmark)

    Lövkvist, Cecilia Elisabet

    into how epigenetic marks are distributed in the human genome. In the first part of the thesis, we investigate DNA methylation and maintenance of methylation patterns throughout cell division. We argue that collaborative models, those where the methylation of CpG sites depends on the methylation status...... into the game more explicitly in another type of model that speaks out the duality of the two aspects. Using statistical analysis of experimental data, this thesis further explores a link between DNA methylation and nucleosome occupancy. By comparing the patterns on promoters to regions with similar Cp...... division. The patterns of epigentic marks depend on enzymes that ensure their maintenance and introduction. Using theoretical models, this thesis proposes new mechanisms for how enzymes operate to maintain patterns of epigenetic marks. Through analysis of experimental data this work gives new insight...

  1. A DNA methylation microarray-based study identifies ERG as a gene commonly methylated in prostate cancer.

    Science.gov (United States)

    Schwartzman, Jacob; Mongoue-Tchokote, Solange; Gibbs, Angela; Gao, Lina; Corless, Christopher L; Jin, Jennifer; Zarour, Luai; Higano, Celestia; True, Lawrence D; Vessella, Robert L; Wilmot, Beth; Bottomly, Daniel; McWeeney, Shannon K; Bova, G Steven; Partin, Alan W; Mori, Motomi; Alumkal, Joshi

    2011-10-01

    DNA methylation of promoter regions is a common event in prostate cancer, one of the most common cancers in men worldwide. Because prior reports demonstrating that DNA methylation is important in prostate cancer studied a limited number of genes, we systematically quantified the DNA methylation status of 1505 CpG dinucleotides for 807 genes in 78 paraffin-embedded prostate cancer samples and three normal prostate samples. The ERG gene, commonly repressed in prostate cells in the absence of an oncogenic fusion to the TMPRSS2 gene, was one of the most commonly methylated genes, occurring in 74% of prostate cancer specimens. In an independent group of patient samples, we confirmed that ERG DNA methylation was common, occurring in 57% of specimens, and cancer-specific. The ERG promoter is marked by repressive chromatin marks mediated by polycomb proteins in both normal prostate cells and prostate cancer cells, which may explain ERG's predisposition to DNA methylation and the fact that tumors with ERG DNA methylation were more methylated, in general. These results demonstrate that bead arrays offer a high-throughput method to discover novel genes with promoter DNA methylation such as ERG, whose measurement may improve our ability to more accurately detect prostate cancer.

  2. Correlation among periodontal health status, maternal age and pre-term low birth weight.

    Science.gov (United States)

    Capasso, Francesca; Vozza, Iole; Capuccio, Veronica; Vestri, Anna Rita; Polimeni, Antonella; Ottolenghi, Livia

    2016-08-01

    To assess correlations between periodontal status, maternal age and adverse pregnancy outcomes, such as pre-term and low birth weight in a sample of pregnant women. Study population was represented by outpatient pregnant women, gestational age > 26 weeks. Medical history questionnaires were administered to all participants who underwent clinical evaluation; clinical obstetric outcome records were collected after delivery. A questionnaire was administered regarding personal information, socio-economic status, oral hygiene habits, and oral health conditions. A clinical oral examination was performed to collect Simplified Oral Hygiene Index (OHI-S) and Community Periodontal Index (CPI). Pregnancy outcome records included: delivery week, kind and causes of delivery, any relevant complications, and birth weight. Descriptive statistics were used to depict the data from the questionnaire while the relationship between delivery week, birth weight, maternal age and periodontal status was evaluated through multivariate tests of significance. 88 pregnant women were enrolled in the study. The results showed a statistically significant correlation (Pperiodontal disease and adverse pregnancy outcomes. No statistical correlation was found among pre-term and low birth weight, smoking, ethnicity and educational level of mothers. The results highlight the importance of including a routine oral and periodontal health examination in pregnant women older than 40 years of age. The correlation between periodontal status and adverse pregnancy outcomes in older mothers indicates the need for routine oral health examination and periodontal status assessment and care in pregnant women older than 40 years of age.

  3. [Correlation of psychoemotional status and adaptation to complete dentures].

    Science.gov (United States)

    Barkan, I Yu; Stafeev, A A; Repin, V S

    2015-01-01

    Patients with full adentia are characterized by the formation of specific psycho-emotional status. Rational psychotherapeutic support of these patients largely determines the efficiency of dental prosthetic treatment. At the same time, the definition of mental and emotional status is not included in the diagnostic examination protocol. Considering the above the purpose of the study was to evaluate mental and emotional status of patients receiving complete dentures. Prosthetic rehabilitation of 30 patients with complete teeth loss was performed and clinical evaluation and evaluation of mental and emotional status were carried out before and after treatment. Patients with negative experiences of prosthetics showed a higher level of personal and situational anxiety. There was correlation of adaptation to removable dentures and the patient's personality traits. It is determined that emotional instability during treatment tends to decrease affecting the timing of adaptation to complete dentures. It is noted that patients with repeated prosthetics have earlier recovery of coordination ability of the masticatory muscles.

  4. [Analysis of genomic DNA methylation level in radish under cadmium stress by methylation-sensitive amplified polymorphism technique].

    Science.gov (United States)

    Yang, Jin-Lan; Liu, Li-Wang; Gong, Yi-Qin; Huang, Dan-Qiong; Wang, Feng; He, Ling-Li

    2007-06-01

    The level of cytosine methylation induced by cadmium in radish (Raphanus sativus L.) genome was analysed using the technique of methylation-sensitive amplified polymorphism (MSAP). The MSAP ratios in radish seedling exposed to cadmium chloride at the concentration of 50, 250 and 500 mg/L were 37%, 43% and 51%, respectively, and the control was 34%; the full methylation levels (C(m)CGG in double strands) were at 23%, 25% and 27%, respectively, while the control was 22%. The level of increase in MSAP and full methylation indicated that de novo methylation occurred in some 5'-CCGG sites under Cd stress. There was significant positive correlation between increase of total DNA methylation level and CdCl(2) concentration. Four types of MSAP patterns: de novo methylation, de-methylation, atypical pattern and no changes of methylation pattern were identified among CdCl(2) treatments and the control. DNA methylation alteration in plants treated with CdCl(2) was mainly through de novo methylation.

  5. Usability of human Infinium MethylationEPIC BeadChip for mouse DNA methylation studies.

    Science.gov (United States)

    Needhamsen, Maria; Ewing, Ewoud; Lund, Harald; Gomez-Cabrero, David; Harris, Robert Adam; Kular, Lara; Jagodic, Maja

    2017-11-15

    The advent of array-based genome-wide DNA methylation methods has enabled quantitative measurement of single CpG methylation status at relatively low cost and sample input. Whereas the use of Infinium Human Methylation BeadChips has shown great utility in clinical studies, no equivalent tool is available for rodent animal samples. We examined the feasibility of using the new Infinium MethylationEPIC BeadChip for studying DNA methylation in mouse. In silico, we identified 19,420 EPIC probes (referred as mEPIC probes), which align with a unique best alignment score to the bisulfite converted reference mouse genome mm10. Further annotation revealed that 85% of mEPIC probes overlapped with mm10.refSeq genes at different genomic features including promoters (TSS1500 and TSS200), 1st exons, 5'UTRs, 3'UTRs, CpG islands, shores, shelves, open seas and FANTOM5 enhancers. Hybridization of mouse samples to Infinium Human MethylationEPIC BeadChips showed successful measurement of mEPIC probes and reproducibility between inter-array biological replicates. Finally, we demonstrated the utility of mEPIC probes for data exploration such as hierarchical clustering. Given the absence of cost and labor convenient genome-wide technologies in the murine system, our findings show that the Infinium MethylationEPIC BeadChip platform is suitable for investigation of the mouse methylome. Furthermore, we provide the "mEPICmanifest" with genomic features, available to users of Infinium Human MethylationEPIC arrays for mouse samples.

  6. LINE-1 methylation in visceral adipose tissue of severely obese individuals is associated with metabolic syndrome status and related phenotypes

    Directory of Open Access Journals (Sweden)

    Turcot Valérie

    2012-07-01

    Full Text Available Abstract Background Epigenetic mechanisms may be involved in the regulation of genes found to be differentially expressed in the visceral adipose tissue (VAT of severely obese subjects with (MetS+ versus without (MetS- metabolic syndrome (MetS. Long interspersed nuclear element 1 (LINE-1 elements DNA methylation levels (%meth in blood, a marker of global DNA methylation, have recently been associated with fasting glucose, blood lipids, heart diseases and stroke. Aim To test whether LINE-1%meth levels in VAT are associated with MetS phenotypes and whether they can predict MetS risk in severely obese individuals. Methods DNA was extracted from VAT of 34 men (MetS-: n = 14, MetS+: n = 20 and 152 premenopausal women (MetS-: n = 84; MetS+: n = 68 undergoing biliopancreatic diversion for the treatment of obesity. LINE-1%meth levels were assessed by pyrosequencing of sodium bisulfite-treated DNA. Results The mean LINE-1%meth in VAT was of 75.8% (SD = 3.0%. Multiple linear regression analyses revealed that LINE-1%meth was negatively associated with fasting glucose levels (β = -0.04; P = 0.03, diastolic blood pressure (β =  -0.65; P = 0.03 and MetS status (β = -0.04; P = 0.004 after adjustments for the effects of age, sex, waist circumference (except for MetS status and smoking. While dividing subjects into quartiles based on their LINE-1%meth (Q1 to Q4: lower %meth to higher %meth levels, greater risk were observed in the first (Q1: odds ratio (OR = 4.37, P = 0.004 and the second (Q2: OR = 4.76, P = 0.002 quartiles compared to Q4 (1.00 when adjusting for age, sex and smoking. Conclusions These results suggest that lower global DNA methylation, assessed by LINE-1 repetitive elements methylation analysis, would be associated with a greater risk for MetS in the presence of obesity.

  7. Correlation of pathologic features with CpG island methylator phenotype (CIMP) by quantitative DNA methylation analysis in colorectal carcinoma.

    Science.gov (United States)

    Ogino, Shuji; Odze, Robert D; Kawasaki, Takako; Brahmandam, Mohan; Kirkner, Gregory J; Laird, Peter W; Loda, Massimo; Fuchs, Charles S

    2006-09-01

    Extensive gene promoter methylation in colorectal carcinoma has been termed the CpG island methylator phenotype (CIMP). Previous studies on CIMP used primarily methylation-specific polymerase chain reaction (PCR), which, unfortunately, may detect low levels of methylation that has little or no biological significance. Utilizing quantitative real-time PCR (MethyLight), we measured DNA methylation in a panel of 5 CIMP-specific gene promoters (CACNA1G, CDKN2A (p16), CRABP1, MLH1, and NEUROG1) in 459 colorectal carcinomas obtained from 2 large prospective cohort studies. CIMP was defined as tumors that showed methylation in >or=4/5 promoters. CIMP was significantly associated with the presence of mucinous or signet ring cell morphology, marked Crohn's-like lymphoid reaction, tumor infiltrating lymphocytes, marked peritumoral lymphocytic reaction, tumor necrosis, tumor cell sheeting, and poor differentiation. All these features have previously been associated with microsatellite instability (MSI). Therefore, we divided the 459 colorectal carcinomas into 6 subtypes, namely, MSI-high (MSI-H)/CIMP, MSI-H/non-CIMP, MSI-low (MSI-L)/CIMP, MSI-L/non-CIMP, microsatellite stable/CIMP, and micro satellite sstable/non-CIMP. Compared with MSI-H/non-CIMP, MSI-H/CIMP was associated with marked tumor infiltrating lymphocytes, tumor necrosis, sheeting, and poor differentiation (all PCIMP, MSI-L/CIMP was associated with tumors that had CIMP. Both MSI and CIMP appear to play a role in the pathogenesis of specific morphologic patterns of colorectal carcinoma.

  8. The vaporization enthalpies and vapor pressures of fatty acid methyl esters C18, C21 to C23, and C25 to C29 by correlation - gas chromatography

    International Nuclear Information System (INIS)

    Chickos, James S.; Zhao Hui; Nichols, Gary

    2004-01-01

    Vapor pressures and vaporization enthalpies for methyl heptadecanoate and methyl heneicosanoate to methyl octacosanoate exclusive of methyl tricosanoate are evaluated as a function of temperature over the temperature range T = 298.15-450 K by correlation gas chromatography. The results are generated by an extrapolative process using literature values for methyl tetradecanoate to methyl eicosanoate as standards. Relationships for calculating vapor pressures of the title compounds from T = 298.15 to 450 K are provided. Experimental fusion enthalpies are also reported for the methyl esters from methyl hexadecanoate to methyl octacosanoate excluding methyl tridecanoate. Vaporization enthalpies and fusion enthalpies adjusted for temperature to T = 298.15 K are combined to provide sublimation enthalpies. The results are compared to available literature values. A rationale for the linear relationship observed between enthalpies of vaporization and enthalpies of transfer from solution to the vapor is also provided

  9. A methodological study of genome-wide DNA methylation analyses using matched archival formalin-fixed paraffin embedded and fresh frozen breast tumors.

    Science.gov (United States)

    Espinal, Allyson C; Wang, Dan; Yan, Li; Liu, Song; Tang, Li; Hu, Qiang; Morrison, Carl D; Ambrosone, Christine B; Higgins, Michael J; Sucheston-Campbell, Lara E

    2017-02-28

    DNA from archival formalin-fixed and paraffin embedded (FFPE) tissue is an invaluable resource for genome-wide methylation studies although concerns about poor quality may limit its use. In this study, we compared DNA methylation profiles of breast tumors using DNA from fresh-frozen (FF) tissues and three types of matched FFPE samples. For 9/10 patients, correlation and unsupervised clustering analysis revealed that the FF and FFPE samples were consistently correlated with each other and clustered into distinct subgroups. Greater than 84% of the top 100 loci previously shown to differentiate ER+ and ER- tumors in FF tissues were also FFPE DML. Weighted Correlation Gene Network Analyses (WCGNA) grouped the DML loci into 16 modules in FF tissue, with ~85% of the module membership preserved across tissue types. Restored FFPE and matched FF samples were profiled using the Illumina Infinium HumanMethylation450K platform. Methylation levels (β-values) across all loci and the top 100 loci previously shown to differentiate tumors by estrogen receptor status (ER+ or ER-) in a larger FF study, were compared between matched FF and FFPE samples using Pearson's correlation, hierarchical clustering and WCGNA. Positive predictive values and sensitivity levels for detecting differentially methylated loci (DML) in FF samples were calculated in an independent FFPE cohort. FFPE breast tumors samples show lower overall detection of DMLs versus FF, however FFPE and FF DMLs compare favorably. These results support the emerging consensus that the 450K platform can be employed to investigate epigenetics in large sets of archival FFPE tissues.

  10. DNA methylation of miRNA coding sequences putatively associated with childhood obesity.

    Science.gov (United States)

    Mansego, M L; Garcia-Lacarte, M; Milagro, F I; Marti, A; Martinez, J A

    2017-02-01

    Epigenetic mechanisms may be involved in obesity onset and its consequences. The aim of the present study was to evaluate whether DNA methylation status in microRNA (miRNA) coding regions is associated with childhood obesity. DNA isolated from white blood cells of 24 children (identification sample: 12 obese and 12 non-obese) from the Grupo Navarro de Obesidad Infantil study was hybridized in a 450 K methylation microarray. Several CpGs whose DNA methylation levels were statistically different between obese and non-obese were validated by MassArray® in 95 children (validation sample) from the same study. Microarray analysis identified 16 differentially methylated CpGs between both groups (6 hypermethylated and 10 hypomethylated). DNA methylation levels in miR-1203, miR-412 and miR-216A coding regions significantly correlated with body mass index standard deviation score (BMI-SDS) and explained up to 40% of the variation of BMI-SDS. The network analysis identified 19 well-defined obesity-relevant biological pathways from the KEGG database. MassArray® validation identified three regions located in or near miR-1203, miR-412 and miR-216A coding regions differentially methylated between obese and non-obese children. The current work identified three CpG sites located in coding regions of three miRNAs (miR-1203, miR-412 and miR-216A) that were differentially methylated between obese and non-obese children, suggesting a role of miRNA epigenetic regulation in childhood obesity. © 2016 World Obesity Federation.

  11. [Effect of total glucosides of peony on expression and DNA methylation status of ITGAL gene in CD4(+) T cells of systemic lupus erythematosus].

    Science.gov (United States)

    Zhao, Ming; Liang, Gongping; Luo, Shuangyan; Lu, Qianjin

    2012-05-01

    To investigate the effect of total glucosides of peony (TGP) on expression and DNA methylation status of ITGAL gene (CD11a) in CD4(+) T cells from patients with systemic lupus erythematosus (SLE). CD4(+) T cells were isolated by positive selection using CD4 beads. CD4(+) T cells were treated by TGP at 0, 62.5, 312.5 and 1562.5 mg/L for 48 h. The MTT method was used to assess cell viability; mRNA expression level was measured by realtime-PCR; protein level of CD11a was measured by flow cytometric analysis; DNA methylation status was assayed by bisulfite sequencing. No significant change in cell viability was found in CD4(+) T cells among the different concentration groups (P>0.05). Compared with control, the mRNA and protein levels of ITGAL were down-regulated significantly in SLE CD4(+) T cells treated with TGP (1562.5 mg/L) (PTGP (1562.5 mg/L) treated CD4(+) T cells compared with control group (PTGP can repress CD11a gene expression through enhancing DNA methylation of ITGAL promoter in CD4(+) T cells from patients with SLE. This observation represents a preliminary step in understanding the mechanism of TGP in SLE therapy.

  12. The application of methylation specific electrophoresis (MSE) to DNA methylation analysis of the 5' CpG island of mucin in cancer cells

    International Nuclear Information System (INIS)

    Yokoyama, Seiya; Yonezawa, Suguru; Kitamoto, Sho; Yamada, Norishige; Houjou, Izumi; Sugai, Tamotsu; Nakamura, Shin-ichi; Arisaka, Yoshifumi; Takaori, Kyoichi; Higashi, Michiyo

    2012-01-01

    Methylation of CpG sites in genomic DNA plays an important role in gene regulation and especially in gene silencing. We have reported mechanisms of epigenetic regulation for expression of mucins, which are markers of malignancy potential and early detection of human neoplasms. Epigenetic changes in promoter regions appear to be the first step in expression of mucins. Thus, detection of promoter methylation status is important for early diagnosis of cancer, monitoring of tumor behavior, and evaluating the response of tumors to targeted therapy. However, conventional analytical methods for DNA methylation require a large amount of DNA and have low sensitivity. Here, we report a modified version of the bisulfite-DGGE (denaturing gradient gel electrophoresis) using a nested PCR approach. We designated this method as methylation specific electrophoresis (MSE). The MSE method is comprised of the following steps: (a) bisulfite treatment of genomic DNA, (b) amplification of the target DNA by a nested PCR approach and (c) applying to DGGE. To examine whether the MSE method is able to analyze DNA methylation of mucin genes in various samples, we apply it to DNA obtained from state cell lines, ethanol-fixed colonic crypts and human pancreatic juices. The MSE method greatly decreases the amount of input DNA. The lower detection limit for distinguishing different methylation status is < 0.1% and the detectable minimum amount of DNA is 20 pg, which can be obtained from only a few cells. We also show that MSE can be used for analysis of challenging samples such as human isolated colonic crypts or human pancreatic juices, from which only a small amount of DNA can be extracted. The MSE method can provide a qualitative information of methylated sequence profile. The MSE method allows sensitive and specific analysis of the DNA methylation pattern of almost any block of multiple CpG sites. The MSE method can be applied to analysis of DNA methylation status in many different clinical

  13. Complementary vapor pressure data for 2-methyl-1-propanol and 3-methyl-1-butanol at a pressure range of (15 to 177) kPa

    Energy Technology Data Exchange (ETDEWEB)

    Bejarano, Arturo; Quezada, Nathalie [Departamento de Ingenieria Quimica y Ambiental, Universidad Tecnica Federico Santa Maria, Avda. Espana 1680, Valparaiso (Chile); Fuente, Juan C. de la [Departamento de Ingenieria Quimica y Ambiental, Universidad Tecnica Federico Santa Maria, Avda. Espana 1680, Valparaiso (Chile)], E-mail: juan.delafuente@usm.cl

    2009-09-15

    The vapor pressure of pure 2-methyl-1-propanol and 3-methyl-1-butanol, components called congeners that are present in aroma of wine, pisco, and other alcoholic beverages, were measured with a dynamic recirculation apparatus at a pressure range of (15 to 177) kPa with an estimated uncertainty <0.2%. The measurements were performed at temperature ranges of (337 to 392) K for 2-methyl-1-propanol and (358 to 422) K for 3-methyl-1-butanol. Data were correlated using a Wagner-type equation with standard deviations of 0.09 kPa for the vapor pressure of 2-methyl-1-propanol and 0.21 kPa for 3-methyl-1-butanol. The experimental data and correlation were compared with data selected from the literature.

  14. Promoter methylation of RASSF1A and DAPK and mutations of K-ras, p53, and EGFR in lung tumors from smokers and never-smokers

    International Nuclear Information System (INIS)

    Liu, Yang; Gao, Weimin; Siegfried, Jill M; Weissfeld, Joel L; Luketich, James D; Keohavong, Phouthone

    2007-01-01

    Epidemiological studies indicate that some characteristics of lung cancer among never-smokers significantly differ from those of smokers. Aberrant promoter methylation and mutations in some oncogenes and tumor suppressor genes are frequent in lung tumors from smokers but rare in those from never-smokers. In this study, we analyzed promoter methylation in the ras-association domain isoform A (RASSF1A) and the death-associated protein kinase (DAPK) genes in lung tumors from patients with primarily non-small cell lung cancer (NSCLC) from the Western Pennsylvania region. We compare the results with the smoking status of the patients and the mutation status of the K-ras, p53, and EGFR genes determined previously on these same lung tumors. Promoter methylation of the RASSF1A and DAPK genes was analyzed by using a modified two-stage methylation-specific PCR. Data on mutations of K-ras, p53, and EGFR were obtained from our previous studies. The RASSF1A gene promoter methylation was found in tumors from 46.7% (57/122) of the patients and was not significantly different between smokers and never-smokers, but was associated significantly in multiple variable analysis with tumor histology (p = 0.031) and marginally with tumor stage (p = 0.063). The DAPK gene promoter methylation frequency in these tumors was 32.8% (40/122) and did not differ according to the patients' smoking status, tumor histology, or tumor stage. Multivariate analysis adjusted for age, gender, smoking status, tumor histology and stage showed that the frequency of promoter methylation of the RASSF1A or DAPK genes did not correlate with the frequency of mutations of the K-ras, p53, and EGFR gene. Our results showed that RASSF1A and DAPK genes' promoter methylation occurred frequently in lung tumors, although the prevalence of this alteration in these genes was not associated with the smoking status of the patients or the occurrence of mutations in the K-ras, p53 and EGFR genes, suggesting each of

  15. The impact of endurance exercise on global and AMPK gene-specific DNA methylation

    Energy Technology Data Exchange (ETDEWEB)

    King-Himmelreich, Tanya S.; Schramm, Stefanie; Wolters, Miriam C.; Schmetzer, Julia; Möser, Christine V.; Knothe, Claudia [pharmazentrum frankfurt/ZAFES, Institut für Klinische Pharmakologie, Klinikum der Goethe-Universität Frankfurt, Theodor Stern Kai 7, 60590, Frankfurt am Main (Germany); Resch, Eduard [Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Project Group for Translational Medicine & Pharmacology (TMP), 60596, Frankfurt/Main (Germany); Peil, Johannes [Sports Clinic, Bad Nauheim, MCI GmbH, In der Aue 30-32, 61231, Bad Nauheim (Germany); Geisslinger, Gerd [pharmazentrum frankfurt/ZAFES, Institut für Klinische Pharmakologie, Klinikum der Goethe-Universität Frankfurt, Theodor Stern Kai 7, 60590, Frankfurt am Main (Germany); Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Project Group for Translational Medicine & Pharmacology (TMP), 60596, Frankfurt/Main (Germany); Niederberger, Ellen, E-mail: e.niederberger@em.uni-frankfurt.de [pharmazentrum frankfurt/ZAFES, Institut für Klinische Pharmakologie, Klinikum der Goethe-Universität Frankfurt, Theodor Stern Kai 7, 60590, Frankfurt am Main (Germany)

    2016-05-27

    Alterations in gene expression as a consequence of physical exercise are frequently described. The mechanism of these regulations might depend on epigenetic changes in global or gene-specific DNA methylation levels. The AMP-activated protein kinase (AMPK) plays a key role in maintenance of energy homeostasis and is activated by increases in the AMP/ATP ratio as occurring in skeletal muscles after sporting activity. To analyze whether exercise has an impact on the methylation status of the AMPK promoter, we determined the AMPK methylation status in human blood samples from patients before and after sporting activity in the context of rehabilitation as well as in skeletal muscles of trained and untrained mice. Further, we examined long interspersed nuclear element 1 (LINE-1) as indicator of global DNA methylation changes. Our results revealed that light sporting activity in mice and humans does not alter global DNA methylation but has an effect on methylation of specific CpG sites in the AMPKα2 gene. These regulations were associated with a reduced AMPKα2 mRNA and protein expression in muscle tissue, pointing at a contribution of the methylation status to AMPK expression. Taken together, these results suggest that exercise influences AMPKα2 gene methylation in human blood and eminently in the skeletal muscle of mice and therefore might repress AMPKα2 gene expression. -- Highlights: •AMPK gene methylation increases after moderate endurance exercise in humans and mice. •AMPKα mRNA and protein decrease after moderate endurance exercise in mice. •Global DNA methylation is not affected under the same conditions.

  16. The impact of endurance exercise on global and AMPK gene-specific DNA methylation

    International Nuclear Information System (INIS)

    King-Himmelreich, Tanya S.; Schramm, Stefanie; Wolters, Miriam C.; Schmetzer, Julia; Möser, Christine V.; Knothe, Claudia; Resch, Eduard; Peil, Johannes; Geisslinger, Gerd; Niederberger, Ellen

    2016-01-01

    Alterations in gene expression as a consequence of physical exercise are frequently described. The mechanism of these regulations might depend on epigenetic changes in global or gene-specific DNA methylation levels. The AMP-activated protein kinase (AMPK) plays a key role in maintenance of energy homeostasis and is activated by increases in the AMP/ATP ratio as occurring in skeletal muscles after sporting activity. To analyze whether exercise has an impact on the methylation status of the AMPK promoter, we determined the AMPK methylation status in human blood samples from patients before and after sporting activity in the context of rehabilitation as well as in skeletal muscles of trained and untrained mice. Further, we examined long interspersed nuclear element 1 (LINE-1) as indicator of global DNA methylation changes. Our results revealed that light sporting activity in mice and humans does not alter global DNA methylation but has an effect on methylation of specific CpG sites in the AMPKα2 gene. These regulations were associated with a reduced AMPKα2 mRNA and protein expression in muscle tissue, pointing at a contribution of the methylation status to AMPK expression. Taken together, these results suggest that exercise influences AMPKα2 gene methylation in human blood and eminently in the skeletal muscle of mice and therefore might repress AMPKα2 gene expression. -- Highlights: •AMPK gene methylation increases after moderate endurance exercise in humans and mice. •AMPKα mRNA and protein decrease after moderate endurance exercise in mice. •Global DNA methylation is not affected under the same conditions.

  17. DNA Methylation Adds Prognostic Value to Minimal Residual Disease Status in Pediatric T-Cell Acute Lymphoblastic Leukemia.

    Science.gov (United States)

    Borssén, Magnus; Haider, Zahra; Landfors, Mattias; Norén-Nyström, Ulrika; Schmiegelow, Kjeld; Åsberg, Ann E; Kanerva, Jukka; Madsen, Hans O; Marquart, Hanne; Heyman, Mats; Hultdin, Magnus; Roos, Göran; Forestier, Erik; Degerman, Sofie

    2016-07-01

    Despite increased knowledge about genetic aberrations in pediatric T-cell acute lymphoblastic leukemia (T-ALL), no clinically feasible treatment-stratifying marker exists at diagnosis. Instead patients are enrolled in intensive induction therapies with substantial side effects. In modern protocols, therapy response is monitored by minimal residual disease (MRD) analysis and used for postinduction risk group stratification. DNA methylation profiling is a candidate for subtype discrimination at diagnosis and we investigated its role as a prognostic marker in pediatric T-ALL. Sixty-five diagnostic T-ALL samples from Nordic pediatric patients treated according to the Nordic Society of Pediatric Hematology and Oncology ALL 2008 (NOPHO ALL 2008) protocol were analyzed by HumMeth450K genome wide DNA methylation arrays. Methylation status was analyzed in relation to clinical data and early T-cell precursor (ETP) phenotype. Two distinct CpG island methylator phenotype (CIMP) groups were identified. Patients with a CIMP-negative profile had an inferior response to treatment compared to CIMP-positive patients (3-year cumulative incidence of relapse (CIR3y ) rate: 29% vs. 6%, P = 0.01). Most importantly, CIMP classification at diagnosis allowed subgrouping of high-risk T-ALL patients (MRD ≥0.1% at day 29) into two groups with significant differences in outcome (CIR3y rates: CIMP negative 50% vs. CIMP positive 12%; P = 0.02). These groups did not differ regarding ETP phenotype, but the CIMP-negative group was younger (P = 0.02) and had higher white blood cell count at diagnosis (P = 0.004) compared with the CIMP-positive group. CIMP classification at diagnosis in combination with MRD during induction therapy is a strong candidate for further risk classification and could confer important information in treatment decision making. © 2016 Wiley Periodicals, Inc.

  18. Impact of IGF-1, IGF-1R, and IGFBP-3 promoter methylation on the risk and prognosis of esophageal carcinoma.

    Science.gov (United States)

    Ye, Peng; Qu, Chang-Fa; Hu, Xue-Lin

    2016-05-01

    The aim of this study is to investigate IGF-1, IGF-1R, and IGFBP-3 methylations in esophageal carcinoma (EC) patients and their relationship with the development and prognosis of EC. This study population consisted of 264 patients (case group) whom EC radical resection was performed and 283 healthy individuals (control group). Methylation-specific PCR (MSP) detected the methylation status of IGF-1, IGF-1R, and IGFBP-3 in the peripheral blood in both groups. The expressions of IGF-1, IGF-1R, and IGFBP-3 in EC and adjacent normal tissues were detected by immunohistochemistry (IHC). The methylation rates of IGF-1, IGF-1R, IGFBP3, and IGF-1 + IGF1R + IGFBP3 in the case group were higher than those in the control group (all P IGF-1, IGF-1R, IGFBP3, and IGF-1 + IGF1R + IGFBP3 IGF-1 among patients of different clinicopathological features (all P IGF-1 and IGF-1R in EC were significantly higher than those in adjacent normal tissues (both P IGF-1 and IGF1R gene promoter methylation was positively correlated with the positive expressions of IGF-1 (r = 0.139, P = 0.024) and IGF-1R (r = 0.135, P = 0.028), while the IGFBP3 methylation was negatively correlated with the positive expression of IGFBP3 (r = -0.133, P = 0.031). The positive expressions of IGF-1, IGF-1R, and IGFBP-3 were related to different clinicopathological features (all P IGF-1, IGF-1R, and IGF-1 + IGF1R + IGFBP3 ; expressions of IGF-1 and IGF-1R protein; infiltration depth; and lymph node metastasis (LNM) were independent factors of EC prognosis. Our study demonstrated that methylation of IGF-1, IGF1R, IGFBP3, and IGF-1 + IGF1R + IGFBP3 was closely linked with the occurrence of EC and patients' clinicopathological features. Besides, the methylation status of the target genes and the expressions of IGF-1 and IGF-1R protein were independent factors of EC prognosis, which could provide a direction for the prognosis and treatment of EC.

  19. Comparison of telomere length and insulin-like growth factor-binding protein 7 promoter methylation between breast cancer tissues and adjacent normal tissues in Turkish women.

    Science.gov (United States)

    Kaya, Zehra; Akkiprik, Mustafa; Karabulut, Sevgi; Peker, Irem; Gullu Amuran, Gokce; Ozmen, Tolga; Gulluoglu, Bahadır M; Kaya, Handan; Ozer, Ayse

    2017-09-01

    Both insulin-like growth factor-binding protein 7 (IGFBP7) and telomere length (TL) are associated with proliferation and senescence of human breast cancer. This study assessed the clinical significance of both TL and IGFBP7 methylation status in breast cancer tissues compared with adjacent normal tissues. We also investigated whether IGFBP7 methylation status could be affecting TL. Telomere length was measured by quantitative PCR to compare tumors with their adjacent normal tissues. The IGFBP7 promoter methylation status was evaluated by methylation-specific PCR and its expression levels were determined by western blotting. Telomeres were shorter in tumor tissues compared to controls (Pbreast cancer with invasive ductal carcinoma (IDC; n=72; P=.014) compared with other histological type (n=29), and TL in IDC with HER2 negative (n=53; P=.017) was higher than TL in IDC with HER2 positive (n=19). However, telomeres were shortened in advanced stages and growing tumors. IGFBP7 methylation was observed in 90% of tumor tissues and 59% of controls (P=.0002). Its frequency was significantly higher in IDC compared with invasive mixed carcinoma (IMC; P=.002) and it was not correlated either with protein expression or the other clinicopathological parameters. These results suggest that IGFBP7 promoter methylation and shorter TL in tumor compared with adjacent tissues may be predictive biomarkers for breast cancer. Telomere maintenance may be indicative of IDC and IDC with HER2 (-) of breast cancer. Further studies with larger number of cases are necessary to verify this association. © 2016 Wiley Periodicals, Inc.

  20. A genome-wide methylation study on obesity: differential variability and differential methylation.

    Science.gov (United States)

    Xu, Xiaojing; Su, Shaoyong; Barnes, Vernon A; De Miguel, Carmen; Pollock, Jennifer; Ownby, Dennis; Shi, Hidong; Zhu, Haidong; Snieder, Harold; Wang, Xiaoling

    2013-05-01

    Besides differential methylation, DNA methylation variation has recently been proposed and demonstrated to be a potential contributing factor to cancer risk. Here we aim to examine whether differential variability in methylation is also an important feature of obesity, a typical non-malignant common complex disease. We analyzed genome-wide methylation profiles of over 470,000 CpGs in peripheral blood samples from 48 obese and 48 lean African-American youth aged 14-20 y old. A substantial number of differentially variable CpG sites (DVCs), using statistics based on variances, as well as a substantial number of differentially methylated CpG sites (DMCs), using statistics based on means, were identified. Similar to the findings in cancers, DVCs generally exhibited an outlier structure and were more variable in cases than in controls. By randomly splitting the current sample into a discovery and validation set, we observed that both the DVCs and DMCs identified from the first set could independently predict obesity status in the second set. Furthermore, both the genes harboring DMCs and the genes harboring DVCs showed significant enrichment of genes identified by genome-wide association studies on obesity and related diseases, such as hypertension, dyslipidemia, type 2 diabetes and certain types of cancers, supporting their roles in the etiology and pathogenesis of obesity. We generalized the recent finding on methylation variability in cancer research to obesity and demonstrated that differential variability is also an important feature of obesity-related methylation changes. Future studies on the epigenetics of obesity will benefit from both statistics based on means and statistics based on variances.

  1. Predictive value of CHFR and MLH1 methylation in human gastric cancer.

    Science.gov (United States)

    Li, Yazhuo; Yang, Yunsheng; Lu, Youyong; Herman, James G; Brock, Malcolm V; Zhao, Po; Guo, Mingzhou

    2015-04-01

    Gastric carcinoma (GC) has one of the highest mortality rates of cancer diseases and has a high incidence rate in China. Palliative chemotherapy is the main treatment for advanced gastric cancer. It is necessary to compare the effectiveness and toxicities of different regimens. This study explores the possibility of methylation of DNA damage repair genes serving as a prognostic and chemo-sensitive marker in human gastric cancer. The methylation status of five DNA damage repair genes (CHFR, FANCF, MGMT, MLH1, and RASSF1A) was detected by nested methylation-specific PCR in 102 paraffin-embedded gastric cancer samples. Chi-square or Fisher's exact tests were used to evaluate the association of methylation status and clinic-pathological factors. The Kaplan-Meier method and Cox proportional hazards models were employed to analyze the association of methylation status and chemo-sensitivity. The results indicate that CHFR, MLH1, RASSF1A, MGMT, and FANCF were methylated in 34.3% (35/102), 21.6% (22/102), 12.7% (13/102), 9.8% (10/102), and 0% (0/102) of samples, respectively. No association was found between methylation of CHFR, MLH1, RASSF1A, MGMT, or FANCF with gender, age, tumor size, tumor differentiation, lymph node metastasis, and TNM stage. In docetaxel-treated gastric cancer patients, resistance to docetaxel was found in CHFR unmethylated patients by Cox proportional hazards model (HR 0.243, 95% CI, 0.069-0.859, p = 0.028), and overall survival is longer in the CHFR methylated group compared with the CHFR unmethylated group (log-rank, p = 0.036). In oxaliplatin-treated gastric cancer patients, resistance to oxaliplatin was found in MLH1 methylated patients (HR 2.988, 95% CI, 1.064-8.394, p = 0.038), and overall survival was longer in the MLH1 unmethylated group compared with the MLH1 methylated group (log-rank, p = 0.046). CHFR is frequently methylated in human gastric cancer, and CHFR methylation may serve as a docetaxel-sensitive marker. MLH1 methylation was

  2. Development of a multiplex methylation-specific PCR as candidate triage test for women with an HPV-positive cervical scrape

    International Nuclear Information System (INIS)

    Snellenberg, Suzanne; Strooper, Lise MA De; Hesselink, Albertus T; Meijer, Chris JLM; Snijders, Peter JF; Heideman, Daniëlle AM; Steenbergen, Renske DM

    2012-01-01

    Quantitative methylation-specific PCR (qMSP) analysis for determining the methylation status of (candidate) tumor suppressor genes has potential as objective and valuable test to triage high-risk human papillomavirus (hrHPV) positive women in cervical screening. Particularly combined methylation analysis of a panel of genes shows most promising clinical performance, with sensitivity levels that equal or exceed that of cytology. However, the wide application of such methylation marker panels is hampered by the lack of effective multiplex assays allowing simultaneous methylation detection of various targets in a single reaction. Here, we designed and analyzed a multiplex qMSP assay for three genes whose methylation was previously found to be informative for cervical (pre)cancer (i.e. CADM1, MAL and hsa-miR-124-2) as well as a reference gene β-actin. Based on our experience, we discuss the optimization of the parameters that provide a practical approach towards multiplex qMSP design. Primers and PCR reagents were optimized for multiplex qMSP purposes and the resulting assay was analytically validated on serial dilutions of methylated DNA in unmethylated DNA, and compared with singleplex counterparts on hrHPV-positive cervical scrapings. Upon optimization, including primer redesign and primer limiting assays, the multiplex qMSP showed the same analytical performance as the singleplex qMSPs. A strong correlation between the obtained normalized ratios of the singleplex and multiplex qMSPs on cervical scrapes was found for all three markers: CADM1 (R 2 =0.985), MAL (R 2 =0.986) and hsa-miR-124-2 (R 2 =0.944). Multiplex qMSP offers a promising approach for high-throughput diagnostic analysis of the methylation status of multiple genes, which after proper design and validation can be equally specific, sensitive and reproducible as its singleplex versions

  3. Trichloroethylene-Induced DNA Methylation Changes in Male F344 Rat Liver.

    Science.gov (United States)

    Jiang, Yan; Chen, Jiahong; Yue, Cong; Zhang, Hang; Chen, Tao

    2016-10-17

    Trichloroethylene (TCE), a common environmental contaminant, causes hepatocellular carcinoma in mice but not in rats. To understand the mechanisms of the species-specific hepatocarcinogenecity of TCE, we examined the methylation status of DNA in the liver of rats exposed to TCE at 0 or 1000 mg/kg b.w. for 5 days using MeDIP-chip, bisulfite sequencing, COBRA, and LC-MS/MS. The related mRNA expression levels were measured by qPCR. Although no global DNA methylation change was detected, 806 genes were hypermethylated and 186 genes were hypomethylated. The genes with hypermethylated DNA were enriched in endocytosis, MAPK, and cAMP signaling pathways. We further confirmed the hypermethylation of Uhrf2 DNA and the hypomethylation of Hadhb DNA, which were negatively correlated with their mRNA expression levels. The transcriptional levels of Jun, Ihh, and Tet2 were significantly downregulated, whereas Cdkn1a was overexpressed. No mRNA expression change was found for Mki67, Myc, Uhrf1, and Dnmt1. In conclusion, TCE-induced DNA methylation changes in rats appear to suppress instead of promote hepatocarcinogenesis, which might play a role in the species-specific hepatocarcinogenecity of TCE.

  4. Methylation in hepatocellular carcinoma

    Directory of Open Access Journals (Sweden)

    Regina M. Santella

    2007-02-01

    Full Text Available

    The development of HCC is a complex, multistep, multistage process. The molecular pathogenesis of HCC appears to involve multiple genetic aberrations in the molecular control of hepatocyte proliferation, differentiation and death and the maintenance of genomic integrity. This process is influenced by the cumulative activation and inactivation of oncogenes, tumor suppressor genes and other genes. p53, a tumor suppressor gene, is the most frequently mutated gene in human cancers. There is also a striking sequence specific binding and induction of mutations by AFB1 at codon 249 of p53 in HCC.

    Epigenetic alterations are also involved in cancer development and progression. Methylation of promoter CpG islands is associated with inhibition of transcriptional initiation and permanent silencing of downstream genes.

    It is now known that most important tumor suppressor genes are inactivated, not only by mutations and deletions but also by promoter methylation. Several studies indicated that p16, p15, RASSF1A, MGMT, and GSTP1 promoter hypermethylation are prevalent in HCC. In addition, geographic variation in the methylation status of tumor DNA indicates that environmental factors may influence the frequent and concordant degree of hypermethylation in multiple genes in HCC and that epigeneticenvironmental interactions may be involved in hepatocarcinogenesis. We have found significant relationships between promoter methylation and AFB1-DNA adducts confirming the impact of environmental exposures on gene methylation.

    DNA isolated from serum or plasma of cancer patients frequently contains the same genetic and

  5. Promoter de-methylation of cyclin D2 by sulforaphane in prostate cancer cells

    Directory of Open Access Journals (Sweden)

    Hsu Anna

    2011-10-01

    Full Text Available Abstract Sulforaphane (SFN, an isothiocyanate derived from cruciferous vegetables, induces potent anti-proliferative effects in prostate cancer cells. One mechanism that may contribute to the anti-proliferative effects of SFN is the modulation of epigenetic marks, such as inhibition of histone deacetylase (HDAC enzymes. However, the effects of SFN on other common epigenetic marks such as DNA methylation are understudied. Promoter hyper-methylation of cyclin D2, a major regulator of cell cycle, is correlated with prostate cancer progression, and restoration of cyclin D2 expression exerts anti-proliferative effects on LnCap prostate cancer cells. Our study aimed to investigate the effects of SFN on DNA methylation status of cyclin D2 promoter, and how alteration in promoter methylation impacts cyclin D2 gene expression in LnCap cells. We found that SFN significantly decreased the expression of DNA methyltransferases (DNMTs, especially DNMT1 and DNMT3b. Furthermore, SFN significantly decreased methylation in cyclin D2 promoter regions containing c-Myc and multiple Sp1 binding sites. Reduced methlyation of cyclin D2 promoter corresponded to an increase in cyclin D2 transcript levels, suggesting that SFN may de-repress methylation-silenced cyclin D2 by impacting epigenetic pathways. Our results demonstrated the ability of SFN to epigenetically modulate cyclin D2 expression, and provide novel insights into the mechanisms by which SFN may regulate gene expression as a prostate cancer chemopreventive agent.

  6. Preliminary individualized chemotherapy for malignant astrocytomas based on O6-methylguanine-deoxyribonucleic acid methyltransferase methylation analysis.

    Science.gov (United States)

    Watanabe, Takao; Katayama, Yoichi; Ogino, Akiyoshi; Ohta, Takashi; Yoshino, Atsuo; Fukushima, Takao

    2006-08-01

    O(6)-methylguanine-deoxyribonucleic acid methyltransferase gene (MGMT) methylation is apparently correlated with responsiveness to nitrosourea chemotherapy, suggesting this alkylating agent should be effective against MGMT-methylated tumors. MGMT appears not to be linked to platinum resistance, so platinum chemotherapy should be used for MGMT-unmethylated tumors. This study was a preliminary trial of individualized chemotherapy based on MGMT methylation status in a total of 20 patients with newly diagnosed malignant astrocytomas (9 anaplastic astrocytomas and 11 glioblastomas multiforme). The procarbazine, 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-2(2-chloroethyl)-3-nitrosourea, and vincristine (PAV) regimen was administered to seven patients with MGMT-methylated tumors, and the carboplatin and etoposide (CE) regimen was administered to 13 patients with MGMT-unmethylated tumors. Objective response to the PAV therapy was noted in all three patients with measurable residual tumor (2 complete responses and 1 partial response). Five of the seven patients continued to be disease-free after initiation of the PAV therapy. Objective response to the CE therapy was seen in only one of seven patients with measurable residual tumor (1 partial response). Three of the 13 patients were free from progression, whereas the remaining 10 patients showed early progression. The PAV regimen is effective against MGMT-methylated malignant astrocytomas, but the CE regimen is not useful at the given dose and schedule in MGMT-unmethylated tumors.

  7. Case–control study of HLA-G promoter methylation status, HPV infection and cervical neoplasia in Curitiba, Brazil: a pilot analysis

    International Nuclear Information System (INIS)

    Gillio-Tos, Anna; Carvalho, Newton S; Maestri, Carlos A; Lacerda, Hadriano M; Zugna, Daniela; Richiardi, Lorenzo; Merletti, Franco; Bicalho, Maria da Graça; Fiano, Valentina; Grasso, Chiara; Tarallo, Valentina; De Marco, Laura; Trevisan, Morena; Xavier, MarinaBarbaradeSousa; Slowik, Renata

    2012-01-01

    The causal association between persistent human papillomavirus (HPV) infection and cervical cancer has been established, but the mechanisms that favor HPV persistence in cervical cells are still unknown. The diminished capability of the immune system to control and resolve HPV infection is one of several hypotheses. The tolerogenic protein HLA-G has shown aberrant expression in a variety of cancers, which has been suggested as a mechanism for tumor escape from immunosurveillance. In the present study we evaluate the role of epigenetic modification (promoter de-methylation) of the HLA-G gene on susceptibility to HPV infection and development of high-grade cervical lesions. A case–control study was carried out in Curitiba, Brazil, between February and June 2010. A total of 789 women aged 15–47 years were recruited: 510 controls with normal cervical cytology, and 279 cases with histologically confirmed cervical intraepithelial neoplasia grade 2 (CIN2, N = 150) or grade 3 (CIN3, N = 129). All women were administered a questionnaire by interview, which collected information on demographic and lifestyle factors, and a cervical sample was collected. HPV DNA detection was performed by GP5+/GP6+ primer-mediated PCR. HPV-positive samples were genotyped by multiplex PCR. A pilot analysis of HLA-G promoter methylation was carried out in a subset of the study population (96 cases and 76 controls) by pyrosequencing. HLA-G methylation and HPV infection status of cases and controls were compared, and confounding factors were computed by t Student and non-parametric Wilcoxon tests. Comparison of HLA-G methylation between cases and controls was assessed by the Bonferroni correction. The association of HLA-G methylation with CIN2/3 was evaluated by logistic regression. HPV prevalence was 19.6% in controls and 94.3% in CIN2/3 cases. HPV16, 31, 33, 35 and 18 were the most prevalent types. Methylation analysis of seven CpGs in the HLA-G promoter did not reveal any spontaneous de-methylation

  8. Genetically contextual effects of smoking on genome wide DNA methylation.

    Science.gov (United States)

    Dogan, Meeshanthini V; Beach, Steven R H; Philibert, Robert A

    2017-09-01

    Smoking is the leading cause of death in the United States. It exerts its effects by increasing susceptibility to a variety of complex disorders among those who smoke, and if pregnant, to their unborn children. In prior efforts to understand the epigenetic mechanisms through which this increased vulnerability is conveyed, a number of investigators have conducted genome wide methylation analyses. Unfortunately, secondary to methodological limitations, these studies were unable to examine methylation in gene regions with significant amounts of genetic variation. Using genome wide genetic and epigenetic data from the Framingham Heart Study, we re-examined the relationship of smoking status to genome wide methylation status. When only methylation status is considered, smoking was significantly associated with differential methylation in 310 genes that map to a variety of biological process and cellular differentiation pathways. However, when SNP effects on the magnitude of smoking associated methylation changes are also considered, cis and trans-interaction effects were noted at a total of 266 and 4353 genes with no marked enrichment for any biological pathways. Furthermore, the SNP variation participating in the significant interaction effects is enriched for loci previously associated with complex medical illnesses. The enlarged scope of the methylome shown to be affected by smoking may better explicate the mediational pathways linking smoking with a myriad of smoking related complex syndromes. Additionally, these results strongly suggest that combined epigenetic and genetic data analyses may be critical for a more complete understanding of the relationship between environmental variables, such as smoking, and pathophysiological outcomes. © 2017 Wiley Periodicals, Inc.

  9. Evaluation of methylation pattern in promoter region of E-cadherin ...

    African Journals Online (AJOL)

    user

    2011-03-07

    Mar 7, 2011 ... promoter methylation in CDH1 gene inactivation in breast cancer, the CpG methylation status of E- ..... 5'CpG island of CDH1 in prostate, lung, liver, bladder, .... and estrogen receptor alpha from Sp1 sites to induce cell cycle.

  10. Alteration in Methylation Pattern of Retinoblastoma 1 Gene Promotor Region in Intestinal Metaplasia with or without Helicobacter pylori and Gastric Cancer Patients.

    Science.gov (United States)

    Boyacioglu, Seda Orenay; Kasap, Elmas; Yuceyar, Hakan; Korkmaz, Mehmet

    2016-01-01

    Helicobacter pylori, intestinal metaplasia (IM), and gene methylation play important roles in gastric carcinogenesis. However, the association among H. pylori infection, IM, gastric cancer (GC), and gene methylation is not fully understood. Cell cycle control involving retinoblastoma 1 (RB1) gene is one of the main regulatory pathways reported to be altered in gastric carcinogenesis. The purpose of this research is to assess the methylation status of RB1 gene in GC and IM with or without H. pylori infection, and to discuss the possible role of H. pylori-induced RB1 gene methylation in the mechanism of gastric carcinogenesis. The methylation profile of RB1 gene was analyzed by sodium bisulfite modification and methylation-specific PCR in GC (n = 24), IM patients with H. pylori positive (n = 20) and negative (n = 20), and control subjects (n = 20). According to methylation levels in RB1 gene; the high correlation values were detected between H. pylori positive-IM group and GC group, and between H. pylori positive-IM and H. pylori negative-IM groups (p gene. High methylation levels in RB1 gene in H. pylori positive individuals may suggest an elevated risk of gastric cancer occurrence.

  11. Modulation of DNA methylation levels sensitizes doxorubicin-resistant breast adenocarcinoma cells to radiation-induced apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Luzhna, Lidia [Department of Biological Sciences, University of Lethbridge, AB, Canada T1K 3M4 (Canada); Kovalchuk, Olga, E-mail: olga.kovalchuk@uleth.ca [Department of Biological Sciences, University of Lethbridge, AB, Canada T1K 3M4 (Canada)

    2010-02-05

    Chemoresistant tumors often fail to respond to other cytotoxic treatments such as radiation therapy. The mechanisms of chemo- and radiotherapy cross resistance are not fully understood and are believed to be epigenetic in nature. We hypothesize that MCF-7 cells and their doxorubicin-resistant variant MCF-7/DOX cells may exhibit different responses to ionizing radiation due to their dissimilar epigenetic status. Similar to previous studies, we found that MCF-7/DOX cells harbor much lower levels of global DNA methylation than MCF-7 cells. Furthermore, we found that MCF-7/DOX cells had lower background apoptosis levels and were less responsive to radiation than MCF-7 cells. Decreased radiation responsiveness correlated to significant global DNA hypomethylation in MCF-7/DOX cells. Here, for the first time, we show that the radiation resistance of MCF-7/DOX cells can be reversed by an epigenetic treatment - the application of methyl-donor SAM. SAM-mediated reversal of DNA methylation led to elevated radiation sensitivity in MCF-7/DOX cells. Contrarily, application of SAM on the radiation sensitive and higher methylated MCF-7 cells resulted in a decrease in their radiation responsiveness. This data suggests that a fine balance of DNA methylation is needed to insure proper radiation and drug responsiveness.

  12. Clinical Significance of IGFBP-3 Methylation in Patients with Early Stage Gastric Cancer

    Directory of Open Access Journals (Sweden)

    Seung Tae Kim

    2015-08-01

    Full Text Available BACKGROUND: IGFBP-3 is a multifunctional protein that inhibits growth and induces apoptosis of cancer cells. Hypermethylation of the promoter represses expression of the IGFBP-3 gene. We undertook this study to assess the impact of IGFBP-3 methylation on survival of early stage gastric cancer patients. METHODS: Of the 482 tissue samples from gastric cancer patients who underwent curative surgery, IGFBP-3 methylation was tested in 138 patients with stage IB/II gastric cancer. We also analyzed IGFBP-3 methylation in 26 gastric cancer cell lines. IGFBP-3 methylation was evaluated by methylation-specific polymerase chain reaction (MethyLight. Statistical analyses, all two-sided, were performed to investigate the prognostic effects of methylation status of the IGFBP-3 promoter on various clinical parameters. RESULTS: Hypermethylation of IGFBP-3 was observed in 26 (19% of the 138 stage IB/II gastric cancer patients. Clinicopathological factors such as age, Lauren classification, sex, tumor infiltration, lymph node metastasis, and histologic grade did not show a statistically significant association with the methylation status of the IGFBP-3 promoter. Patients with a hypermethylated IGFBP-3 promoter had similar 8-year disease-free survival compared with those without a hypermethylated IGFBP-3 promoter (73% vs 75%, P = .78. In subgroup analyses, females, but not males, seemed to have poorer prognosis for DFS and OS in the subset of patients with IGFBP-3 methylation as compared with those without IGFBP-3 methylation (8-year DFS: 55.6% vs 71.6%, P = .3694 and 8-year overall survival: 55.6% vs 68.4%, P = .491, respectively even with no statistical significance. CONCLUSIONS: The status of IGFBP-3 methylation as measured by methylation-specific polymerase chain reaction proposed the modest role for predicting survival in specific subgroups of patients with early-stage gastric cancer who undergo curative surgery. However, this needs further investigation.

  13. Integrated DNA methylation and copy-number profiling identify three clinically and biologically relevant groups of anaplastic glioma.

    Science.gov (United States)

    Wiestler, Benedikt; Capper, David; Sill, Martin; Jones, David T W; Hovestadt, Volker; Sturm, Dominik; Koelsche, Christian; Bertoni, Anna; Schweizer, Leonille; Korshunov, Andrey; Weiß, Elisa K; Schliesser, Maximilian G; Radbruch, Alexander; Herold-Mende, Christel; Roth, Patrick; Unterberg, Andreas; Hartmann, Christian; Pietsch, Torsten; Reifenberger, Guido; Lichter, Peter; Radlwimmer, Bernhard; Platten, Michael; Pfister, Stefan M; von Deimling, Andreas; Weller, Michael; Wick, Wolfgang

    2014-10-01

    The outcome of patients with anaplastic gliomas varies considerably. Whether a molecular classification of anaplastic gliomas based on large-scale genomic or epigenomic analyses is superior to histopathology for reflecting distinct biological groups, predicting outcomes and guiding therapy decisions has yet to be determined. Epigenome-wide DNA methylation analysis, using a platform which also allows the detection of copy-number aberrations, was performed in a cohort of 228 patients with anaplastic gliomas (astrocytomas, oligoastrocytomas, and oligodendrogliomas), including 115 patients of the NOA-04 trial. We further compared these tumors with a group of 55 glioblastomas. Unsupervised clustering of DNA methylation patterns revealed two main groups correlated with IDH status: CpG island methylator phenotype (CIMP) positive (77.5 %) or negative (22.5 %). CIMP(pos) (IDH mutant) tumors showed a further separation based on copy-number status of chromosome arms 1p and 19q. CIMP(neg) (IDH wild type) tumors showed hallmark copy-number alterations of glioblastomas, and clustered together with CIMP(neg) glioblastomas without forming separate groups based on WHO grade. Notably, there was no molecular evidence for a distinct biological entity representing anaplastic oligoastrocytoma. Tumor classification based on CIMP and 1p/19q status was significantly associated with survival, allowing a better prediction of outcome than the current histopathological classification: patients with CIMP(pos) tumors with 1p/19q codeletion (CIMP-codel) had the best prognosis, followed by patients with CIMP(pos) tumors but intact 1p/19q status (CIMP-non-codel). Patients with CIMP(neg) anaplastic gliomas (GBM-like) had the worst prognosis. Collectively, our data suggest that anaplastic gliomas can be grouped by IDH and 1p/19q status into three molecular groups that show clear links to underlying biology and a significant association with clinical outcome in a prospective trial cohort.

  14. Experimental vapor pressures (from 1 Pa to 100 kPa) of six saturated Fatty Acid Methyl Esters (FAMEs): Methyl hexanoate, methyl octanoate, methyl decanoate, methyl dodecanoate, methyl tetradecanoate and methyl hexadecanoate

    International Nuclear Information System (INIS)

    Sahraoui, Lakhdar; Khimeche, Kamel; Dahmani, Abdallah; Mokbel, Ilham; Jose, Jacques

    2016-01-01

    Highlight: • Vapor-liquid equilibria, Enthalpy of Vaporization, saturated Fatty Acid Methyl Ester. - Abstract: Vapor pressures of six saturated Fatty Acid Methyl Esters (FAMEs), methyl hexanoate (or methyl caproate), methyl octanoate (or methyl caprylate), Methyl decanoate (or methyl caprate), methyl dodecanoate (or methyl laurate), methyl tetradecanoate (or methyl myristate), and methyl hexadecanoate (or methyl palmitate) were measured from 1 Pa to 100 kPa and at temperature range between 262 and 453 K using a static apparatus. The experimental data (P-T) were compared with the available literature data.

  15. Examination of DNA methylation status of the ELOVL2 marker may be useful for human age prediction in forensic science.

    Science.gov (United States)

    Zbieć-Piekarska, Renata; Spólnicka, Magdalena; Kupiec, Tomasz; Makowska, Żanetta; Spas, Anna; Parys-Proszek, Agnieszka; Kucharczyk, Krzysztof; Płoski, Rafał; Branicki, Wojciech

    2015-01-01

    Age estimation in forensic investigations may complement the prediction of externally visible characteristics and the inference of biogeographical ancestry, thus allowing a better description of an unknown individual. Multiple CpG sites that show linear correlation between age and degree of DNA methylation have been identified in the human genome, providing a selection of candidates for age prediction. In this study, we optimized an assay based on bisulfite conversion and pyrosequencing of 7 CpG sites located in the ELOVL2 gene. Examination of 303 blood samples collected from individuals aged 2-75 years allowed selection of the most informative site, explaining 83% of variation in age. The final linear regression model included two CpG sites in ELOVL2 and enabled age prediction with R(2)=0.859, prediction error=6.85 and mean absolute deviation MAD=5.03. Examination of a testing set of 124 blood samples (MAD=5.75) showed that 68.5% of samples were correctly predicted, assuming that chronological and predicted ages matched ± 7 years. It was found that the ELOVL2 methylation status in bloodstains had not changed significantly after 4 weeks of storage in room temperature conditions. Analysis of 45 bloodstains deposited on tissue paper after 5, 10 and 15 years of storage in room conditions indicated that although a gradual decrease of positive PCR results was observed, the general age prediction success rate remained similar and equaled 60-78%. The obtained results show that the ELOVL2 locus provides a very good source of information about human chronological age based on analysis of blood, including bloodstains, and it may constitute a powerful and reliable predictor in future forensic age estimation models. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  16. Exploring the Link between Nucleosome Occupancy and DNA Methylation

    Directory of Open Access Journals (Sweden)

    Cecilia Lövkvist

    2018-01-01

    Full Text Available Near promoters, both nucleosomes and CpG sites form characteristic spatial patterns. Previously, nucleosome depleted regions were observed upstream of transcription start sites and nucleosome occupancy was reported to correlate both with CpG density and the level of CpG methylation. Several studies imply a causal link where CpG methylation might induce nucleosome formation, whereas others argue the opposite, i.e., that nucleosome occupancy might influence CpG methylation. Correlations are indeed evident between nucleosomes, CpG density and CpG methylation—at least near promoter sites. It is however less established whether there is an immediate causal relation between nucleosome occupancy and the presence of CpG sites—or if nucleosome occupancy could be influenced by other factors. In this work, we test for such causality in human genomes by analyzing the three quantities both near and away from promoter sites. For data from the human genome we compare promoter regions with given CpG densities with genomic regions without promoters but of similar CpG densities. We find the observed correlation between nucleosome occupancy and CpG density, respectively CpG methylation, to be specific to promoter regions. In other regions along the genome nucleosome occupancy is statistically independent of the positioning of CpGs or their methylation levels. Anti-correlation between CpG density and methylation level is however similarly strong in both regions. On promoters, nucleosome occupancy is more strongly affected by the level of gene expression than CpG density or CpG methylation—calling into question any direct causal relation between nucleosome occupancy and CpG organization. Rather, our results suggest that for organisms with cytosine methylation nucleosome occupancy might be primarily linked to gene expression, with no strong impact on methylation.

  17. Detection and discrimination of maintenance and de novo CpG methylation events using MethylBreak.

    Science.gov (United States)

    Hsu, William; Mercado, Augustus T; Hsiao, George; Yeh, Jui-Ming; Chen, Chung-Yung

    2017-05-15

    Understanding the principles governing the establishment and maintenance activities of DNA methyltransferases (DNMTs) can help in the development of predictive biomarkers associated with genetic disorders and diseases. A detection system was developed that distinguishes and quantifies methylation events using methylation-sensitive endonucleases and molecular beacon technology. MethylBreak (MB) is a 22-mer oligonucleotide with one hemimethylated and two unmethylated CpG sites, which are also recognition sites for Sau96I and SacII, and is attached to a fluorophore and a quencher. Maintenance methylation was quantified by fluorescence emission due to the digestion of SacII when the hemimethylated CpG site is methylated, which inhibits Sau96I cleavage. The signal difference between SacII digestion of both MB substrate and maintenance methylated MB corresponds to de novo methylation event. Our technology successfully discriminated and measured both methylation activities at different concentrations of MB and achieved a high correlation coefficient of R 2 =0.997. Additionally, MB was effectively applied to normal and cancer cell lines and in the analysis of enzymatic kinetics and RNA inhibition of recombinant human DNMT1. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Clinicopathologic Risk Factor Distributions for MLH1 Promoter Region Methylation in CIMP-Positive Tumors.

    Science.gov (United States)

    Levine, A Joan; Phipps, Amanda I; Baron, John A; Buchanan, Daniel D; Ahnen, Dennis J; Cohen, Stacey A; Lindor, Noralane M; Newcomb, Polly A; Rosty, Christophe; Haile, Robert W; Laird, Peter W; Weisenberger, Daniel J

    2016-01-01

    The CpG island methylator phenotype (CIMP) is a major molecular pathway in colorectal cancer. Approximately 25% to 60% of CIMP tumors are microsatellite unstable (MSI-H) due to DNA hypermethylation of the MLH1 gene promoter. Our aim was to determine if the distributions of clinicopathologic factors in CIMP-positive tumors with MLH1 DNA methylation differed from those in CIMP-positive tumors without DNA methylation of MLH1. We assessed the associations between age, sex, tumor-site, MSI status BRAF and KRAS mutations, and family colorectal cancer history with MLH1 methylation status in a large population-based sample of CIMP-positive colorectal cancers defined by a 5-marker panel using unconditional logistic regression to assess the odds of MLH1 methylation by study variables. Subjects with CIMP-positive tumors without MLH1 methylation were significantly younger, more likely to be male, and more likely to have distal colon or rectal primaries and the MSI-L phenotype. CIMP-positive MLH1-unmethylated tumors were significantly less likely than CIMP-positive MLH1-methylated tumors to harbor a BRAF V600E mutation and significantly more likely to harbor a KRAS mutation. MLH1 methylation was associated with significantly better overall survival (HR, 0.50; 95% confidence interval, 0.31-0.82). These data suggest that MLH1 methylation in CIMP-positive tumors is not a completely random event and implies that there are environmental or genetic determinants that modify the probability that MLH1 will become methylated during CIMP pathogenesis. MLH1 DNA methylation status should be taken into account in etiologic studies. ©2015 American Association for Cancer Research.

  19. Clinicopathological risk factor distributions for MLH1 promoter region methylation in CIMP positive tumors

    Science.gov (United States)

    Levine, A. Joan; Phipps, Amanda I.; Baron, John A.; Buchanan, Daniel D.; Ahnen, Dennis J.; Cohen, Stacey A.; Lindor, Noralane M.; Newcomb, Polly A.; Rosty, Christophe; Haile, Robert W.; Laird, Peter W.; Weisenberger, Daniel J.

    2015-01-01

    Background The CpG Island Methylator Phenotype (CIMP) is a major molecular pathway in colorectal cancer (CRC). Approximately 25% to 60% of CIMP tumors are microsatellite unstable (MSI-H) due to DNA hypermethylation of the MLH1 gene promoter. Our aim was to determine if the distributions of clinicopathologic factors in CIMP-positive tumors with MLH1 DNA methylation differed from those in CIMP-positive tumors without DNA methylation of MLH1. Methods We assessed the associations between age, sex, tumor-site, MSI status BRAF and KRAS mutations and family CRC history with MLH1 methylation status in a large population-based sample of CIMP-positive CRCs defined by a 5-marker panel using unconditional logistic regression to assess the odds of MLH1 methylation by study variables. Results Subjects with CIMP-positive tumors without MLH1 methylation were significantly younger, more likely to be male, more likely to have distal colon or rectal primaries and the MSI-L phenotype. CIMP-positive MLH1-unmethylated tumors were significantly less likely than CIMP-positive MLH1-methylated tumors to harbor a BRAF V600E mutation and significantly more likely to harbor a KRAS mutation. MLH1 methylation was associated with significantly better overall survival (HR=0.50; 95% Confidence Interval (0.31, 0.82)). Conclusions These data suggest that MLH1 methylation in CIMP-positive tumors is not a completely random event and implies that there are environmental or genetic determinants that modify the probability that MLH1 will become methylated during CIMP pathogenesis. Impact MLH1 DNA methylation status should be taken into account in etiologic studies. PMID:26512054

  20. Prediction of methyl-side Chain Dynamics in Proteins

    International Nuclear Information System (INIS)

    Ming Dengming; Brueschweiler, Rafael

    2004-01-01

    A simple analytical model is presented for the prediction of methyl-side chain dynamics in comparison with S 2 order parameters obtained by NMR relaxation spectroscopy. The model, which is an extension of the local contact model for backbone order parameter prediction, uses a static 3D protein structure as input. It expresses the methyl-group S 2 order parameters as a function of local contacts of the methyl carbon with respect to the neighboring atoms in combination with the number of consecutive mobile dihedral angles between the methyl group and the protein backbone. For six out of seven proteins the prediction results are good when compared with experimentally determined methyl-group S 2 values with an average correlation coefficient r-bar=0.65±0.14. For the unusually rigid cytochrome c 2 no significant correlation between prediction and experiment is found. The presented model provides independent support for the reliability of current side-chain relaxation methods along with their interpretation by the model-free formalism

  1. Methylation-associated down-regulation of RASSF1A and up-regulation of RASSF1C in pancreatic endocrine tumors

    International Nuclear Information System (INIS)

    Malpeli, Giorgio; Amato, Eliana; Dandrea, Mario; Fumagalli, Caterina; Debattisti, Valentina; Boninsegna, Letizia; Pelosi, Giuseppe; Falconi, Massimo; Scarpa, Aldo

    2011-01-01

    RASSF1A gene silencing by DNA methylation has been suggested as a major event in pancreatic endocrine tumor (PET) but RASSF1A expression has never been studied. The RASSF1 locus contains two CpG islands (A and C) and generates seven transcripts (RASSF1A-RASSF1G) by differential promoter usage and alternative splicing. We studied 20 primary PETs, their matched normal pancreas and three PET cell lines for the (i) methylation status of the RASSF1 CpG islands using methylation-specific PCR and pyrosequencing and (ii) expression of RASSF1 isoforms by quantitative RT-PCR in 13 cases. CpG island A methylation was evaluated by methylation-specific PCR (MSP) and by quantitative methylation-specific PCR (qMSP); pyrosequencing was applied to quantify the methylation of 51 CpGs also encompassing those explored by MSP and qMSP approaches. MSP detected methylation in 16/20 (80%) PETs and 13/20 (65%) normal pancreas. At qMSP, 11/20 PETs (55%) and 9/20 (45%) normals were methylated in at least 20% of RASSF1A alleles. Pyrosequencing showed variable distribution and levels of methylation within and among samples, with PETs having average methylation higher than normals in 15/20 (75%) cases (P = 0.01). The evaluation of mRNA expression of RASSF1 variants showed that: i) RASSF1A was always expressed in PET and normal tissues, but it was, on average, expressed 6.8 times less in PET (P = 0.003); ii) RASSF1A methylation inversely correlated with its expression; iii) RASSF1 isoforms were rarely found, except for RASSF1B that was always expressed and RASSF1C whose expression was 11.4 times higher in PET than in normal tissue (P = 0.001). A correlation between RASSF1A expression and gene methylation was found in two of the three PET cell lines, which also showed a significant increase in RASSF1A expression upon demethylating treatment. RASSF1A gene methylation in PET is higher than normal pancreas in no more than 75% of cases and as such it cannot be considered a marker for this neoplasm

  2. DNA methylation changes in Mexican children exposed to arsenic from two historic mining areas in San Luis potosí.

    Science.gov (United States)

    Alegría-Torres, Jorge Alejandro; Carrizales-Yánez, Leticia; Díaz-Barriga, Fernando; Rosso-Camacho, Fernando; Motta, Valeria; Tarantini, Letizia; Bollati, Valentina

    2016-12-01

    Arsenic is a carcinogen and epimutagen that threatens the health of exposed populations worldwide. In this study, we examined the methylation status of Alu and long interspersed nucleotide elements (LINE-1) and their association with levels of urinary arsenic in 84 Mexican children between 6 and 12 years old from two historic mining areas in the State of San Luis Potosí, Mexico. Urinary arsenic levels were determined by atomic absorption spectrophotometry and DNA methylation analysis was performed in peripheral blood leukocytes by bisulfite-pyrosequencing. The geometric mean of urinary arsenic was 26.44 µg/g Cr (range 1.93-139.35). No significant differences in urinary arsenic or methylation patterns due to gender were observed. A positive correlation was found between urinary arsenic and the mean percentage of methylated cytosines in Alu sequences (Spearman correlation coefficient r = 0.532, P < 0.001), and a trend of LINE-1 hypomethylation was also observed (Spearman correlation coefficient r = -0.232, P = 0.038) after adjustment for sex and age. A linear regression model showed an association with log-normalized urinary arsenic for Alu (β = 1.05, 95% CI: 0.67; 1.43, P < 0.001) and LINE-1 (β = -0.703, 95% CI: -1.36; -0.38, P = 0.038). Despite the low-level arsenic exposure, a subtle epigenetic imbalance measured as DNA methylation was detected in the leukocytes of Mexican children living in two historic mining areas. Environ. Mol. Mutagen. 57:717-723, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  3. The altered promoter methylation of oxytocin receptor gene in autism.

    Science.gov (United States)

    Elagoz Yuksel, Mine; Yuceturk, Betul; Karatas, Omer Faruk; Ozen, Mustafa; Dogangun, Burak

    Autism spectrum disorder (ASD) is one of the lifelong existing disorders. Abnormal methylation status of gene promoters of oxytonergic system has been implicated as among the etiologic factors of ASDs. We, therefore, investigated the methylation frequency of oxytocin receptor gene (OXTR) promoter from peripheral blood samples of children with autistic features. Our sample includes 66 children in total (22-94 months); 27 children with ASDs according to the DSM-IV-TR and the Childhood Autism Rating Scale (CARS) and 39 children who do not have any autistic like symptoms as the healthy control group. We investigated the DNA methylation status of OXTR promoter by methylation specific enzymatic digestion of genomic DNA and polymerase chain reaction. A significant relationship has been found between ASDs and healthy controls for the reduction of methylation frequency of the regions MT1 and MT3 of OXTR. We could not find any association in the methylation frequency of MT2 and MT4 regions of OXTR. Although our findings indicate high frequency of OXTR promoter hypomethylation in ASDs, there is need for independent replication of the results for a bigger sample set. We expect that future studies with the inclusion of larger, more homogeneous samples will attempt to disentangle the causes of ASDs.

  4. Indices of methylation in sperm DNA from fertile men differ between distinct geographical regions

    DEFF Research Database (Denmark)

    Consales, C; Leter, G; Bonde, Jens Peter

    2014-01-01

    STUDY QUESTION: Which are the main determinants, if any, of sperm DNA methylation levels? SUMMARY ANSWER: Geographical region resulted associated with the sperm methylation status assessed on genome-wide repetitive sequences. WHAT IS KNOWN ALREADY: DNA methylation level, assessed on repetitive se...

  5. Methylated Host Cell Gene Promoters and Human Papillomavirus Type 16 and 18 Predicting Cervical Lesions and Cancer.

    Directory of Open Access Journals (Sweden)

    Nina Milutin Gašperov

    Full Text Available Change in the host and/or human papillomavirus (HPV DNA methylation profile is probably one of the main factors responsible for the malignant progression of cervical lesions to cancer. To investigate those changes we studied 173 cervical samples with different grades of cervical lesion, from normal to cervical cancer. The methylation status of nine cellular gene promoters, CCNA1, CDH1, C13ORF18, DAPK1, HIC1, RARβ2, hTERT1, hTERT2 and TWIST1, was investigated by Methylation Specific Polymerase Chain Reaction (MSP. The methylation of HPV18 L1-gene was also investigated by MSP, while the methylated cytosines within four regions, L1, 5'LCR, enhancer, and promoter of the HPV16 genome covering 19 CpG sites were evaluated by bisulfite sequencing. Statistically significant methylation biomarkers distinguishing between cervical precursor lesions from normal cervix were primarily C13ORF18 and secondly CCNA1, and those distinguishing cervical cancer from normal or cervical precursor lesions were CCNA1, C13ORF18, hTERT1, hTERT2 and TWIST1. In addition, the methylation analysis of individual CpG sites of the HPV16 genome in different sample groups, notably the 7455 and 7694 sites, proved to be more important than the overall methylation frequency. The majority of HPV18 positive samples contained both methylated and unmethylated L1 gene, and samples with L1-gene methylated forms alone had better prognosis when correlated with the host cell gene promoters' methylation profiles. In conclusion, both cellular and viral methylation biomarkers should be used for monitoring cervical lesion progression to prevent invasive cervical cancer.

  6. Possible Role of GADD45γ Methylation in Diffuse Large B-Cell Lymphoma: Does It Affect the Progression and Tissue Involvement?

    Directory of Open Access Journals (Sweden)

    İkbal Cansu Barış

    2015-12-01

    Full Text Available INTRODUCTION: Diffuse large B-cell lymphoma (DLBCL is the most common type of non-Hodgkin lymphoma among adults and is characterized by heterogeneous clinical, immunophenotypic, and genetic features. Different mechanisms deregulating cell cycle and apoptosis play a role in the pathogenesis of DLBCL. Growth arrest DNA damage-inducible 45 (GADD45γ is an important gene family involved in these mechanisms. The aims of this study are to determine the frequency of GADD45γ methylation, to evaluate the correlation between GADD45γ methylation and protein expression, and to investigate the relation between methylation status and clinicopathologic parameters in DLBCL tissues and reactive lymphoid node tissues from patients with reactive lymphoid hyperplasia. METHODS: Thirty-six tissue samples of DLBCL and 40 nonmalignant reactive lymphoid node tissues were analyzed in this study. Methylation-sensitive high-resolution melting analysis was used for the determination of GADD45γ methylation status. The GADD45γ protein expression was determined by immunohistochemistry. RESULTS: GADD45γ methylation was frequent (50.0% in DLBCL. It was also significantly higher in advanced-stage tumors compared with early-stage (p=0.041. In contrast, unmethylated GADD45γ was associated with nodal involvement as the primary anatomical site (p=0.040. DISCUSSION AND CONCLUSION: The results of this study show that, in contrast to solid tumors, the frequency of GADD45γ methylation is higher and this epigenetic alteration of GADD45γ may be associated with progression in DLBCL. In addition, nodal involvement is more likely to be present in patients with unmethylated GADD45γ.

  7. Search for methylation-sensitive amplification polymorphisms associated with the mantled variant phenotype in oil palm (Elaeis guineensis Jacq).

    Science.gov (United States)

    Jaligot, E; Beulé, T; Baurens, F-C; Billotte, N; Rival, A

    2004-02-01

    The methylation-sensitive amplification polymorphism (MSAP) technique has been employed on somatic embryo-derived oil palms (Elaeis guineensis Jacq.) to identify methylation polymorphisms correlated with the "mantled" somaclonal variation. The variant phenotype displays an unstable feminization of male organs in both male and female flowers. Using MSAP, the methylation status of CCGG sites was compared in three normal versus three mantled regenerants sampled in clonal populations obtained through somatic embryogenesis from four genotypically distinct mother palms. Overall, 64 selective primer combinations were used and they have amplified 23 markers exhibiting a differential methylation pattern between the two phenotypes. Our results indicate that CCGG sites are poorly affected by the considerable decrease in global DNA methylation that has been previously associated with the mantled phenotype. Each of the 23 markers isolated in the present study could discriminate between the two phenotypes only when they were from the same genetic origin. This result hampers at the moment the direct use of MSAP markers for the early detection of variants, even though valuable information on putative target sequences will be obtained from a further characterization of these polymorphic markers.

  8. Socio-Economic Status And Birth-Order As Correlates Of Women ...

    African Journals Online (AJOL)

    This study investigated socio-economic status and birth-order as correlates of women spiritual help-seeking behavior. Five hundred women help-seekers were sampled from 10 spiritual houses within Ibadan metropolis. Their age ranged between 17-70 years. Fifty percent (50 %,) i.e. 250 of the total sample were singles; ...

  9. Correlation between Ahlbäck radiographic classification and anterior cruciate ligament status in primary knee arthrosis

    Directory of Open Access Journals (Sweden)

    Glaucus Cajaty Martins

    Full Text Available ABSTRACT OBJECTIVE: To correlate the Ahlbäck radiographic classification with the anterior cruciate ligament (ACL status in knee arthritis patients. METHODS: The study evaluated 89 knees of patients who underwent total knee arthroplasty due to primary osteoarthritis: 16 male and 69 females, with mean age 69.79 years (53-87 years. Osteoarthritis was classified radiographically by the Ahlbäck radiographic classification into five grades. The ACL was classified in the surgery as present or absent. The correlation of ACL status and Ahlbäck classification was assessed, as well as those of ACL status and the parameters age, gender, and tibiofemoral angulation (varus-valgus. RESULTS: In cases of varus knees, there was a correlation between grades I to III and ACL presence in 41/47 (86.7% cases and between grades IV and V and ACL absence in 15/17 (88.2% cases (p < 0.0001. In valgus knees, no statistically significant correlation was observed between the ACL status and the Ahlbäck classification. In the present study, absence of the ACL was more common in men (9/17; 52% than in women (19/72; 26%. CONCLUSION: In cases of medial osteoarthritis, the Ahlbäck radiographic classification is a useful parameter to predict ACL status (presence or absence. In gonarthritis in genu valgum, ACL status was not predicted by Ahlbäck's classification.

  10. Methylated genes as new cancer biomarkers

    DEFF Research Database (Denmark)

    Brunner, Nils; Duffy, M.J; Napieralski, R.

    2009-01-01

    Aberrant hypermethylation of promoter regions in specific genes is a key event in the formation and progression of cancer. In at least some situations, these aberrant alterations occur early in the formation of malignancy and appear to be tumour specific. Multiple reports have suggested that meas......Aberrant hypermethylation of promoter regions in specific genes is a key event in the formation and progression of cancer. In at least some situations, these aberrant alterations occur early in the formation of malignancy and appear to be tumour specific. Multiple reports have suggested...... that measurement of the methylation status of the promoter regions of specific genes can aid early detection of cancer, determine prognosis and predict therapy responses. Promising DNA methylation biomarkers include the use of methylated GSTP1 for aiding the early diagnosis of prostate cancer, methylated PITX2...... for predicting outcome in lymph node-negative breast cancer patients and methylated MGMT in predicting benefit from alkylating agents in patients with glioblastomas. However, prior to clinical utilisation, these findings require validation in prospective clinical studies. Furthermore, assays for measuring gene...

  11. Effect of dietary antioxidants, training, and performance correlates on antioxidant status in competitive rowers.

    Science.gov (United States)

    Braakhuis, Andrea J; Hopkins, Will G; Lowe, Timothy E

    2013-09-01

    The beneficial effects of exercise and a healthy diet are well documented in the general population but poorly understood in elite athletes. Previous research in subelite athletes suggests that regular training and an antioxidant-rich diet enhance antioxidant defenses but not performance. To investigate whether habitual diet and/or exercise (training status or performance) affect antioxidant status in elite athletes. Antioxidant blood biomarkers were assessed before and after a 30-min ergometer time trial in 28 male and 34 female rowers. The antioxidant blood biomarkers included ascorbic acid, uric acid, total antioxidant capacity (TAC), erythrocyte- superoxide dismutase, glutathione peroxidase (GPx), and catalase. Rowers completed a 7-d food diary and an antioxidant-intake questionnaire. Effects of diet, training, and performance on resting biomarkers were assessed with Pearson correlations, and their effect on exercise-induced changes in blood biomarkers was assessed by a method of standardization. With the exception of GPx, there were small to moderate increases with exercise for all markers. Blood resting TAC had a small correlation with total antioxidant intake (correlation .29; 90% confidence limits, ±.27), and the exercise-induced change in TAC had a trivial to small association with dietary antioxidant intake from vitamin C (standardized effect .19; ±.22), vegetables (.20; ±.23), and vitamin A (.25; ±.27). Most other dietary intakes had trivial associations with antioxidant biomarkers. Years of training had a small inverse correlation with TAC (-.32; ±.19) and a small association with the exercise-induced change in TAC (.27; ±.24). Training status correlates more strongly with antioxidant status than diet does.

  12. Correlation Analysis of Freeway Traffic Status and Crashes with Nevada Data.

    Science.gov (United States)

    2017-11-11

    This project is to study the correlation between freeway traffic status and crash risks with the historical freeway ITS data and related crash data in Nevada. With the comprehensive review of previous research results, the Center for Advanced Transpo...

  13. Promoter methylation-associated loss of ID4 expression is a marker of tumour recurrence in human breast cancer

    International Nuclear Information System (INIS)

    Noetzel, Erik; Veeck, Jürgen; Niederacher, Dieter; Galm, Oliver; Horn, Felicitas; Hartmann, Arndt; Knüchel, Ruth; Dahl, Edgar

    2008-01-01

    Inhibitor of DNA binding/Inhibitor of differentiation 4 (ID4) is a critical factor for cell proliferation and differentiation in normal vertebrate development. ID4 has regulative functions for differentiation and growth of the developing brain. The role of ID1, ID2 and ID3 are expected to be oncogenic due to their overexpression in pancreatic cancer and colorectal adenocarcinomas, respectively. Aside from these findings, loss of ID3 expression was demonstrated in ovarian cancer. The aim of the present study was to reveal the factual role of ID4 in carcinogenesis in more detail, since its role for the pathogenesis of human breast cancer has been discussed controversially, assigning both oncogenic and tumour suppressive functions. ID4 promoter methylation, ID4 mRNA expression and ID4 protein expression were analysed in primary human breast cancer specimens using methylation-specific PCR (MSP) (n=170), semiquantitative realtime RT-PCR (n=46) and immunhistochemistry (n=3), respectively. In order to demonstrate a functional association of ID4 promoter methylation with its gene silencing, we performed DNA demethylation analysis with four human breast cell lines using MSP and semiquantitative realtime RT-PCR. In addition, we performed correlations of ID4 promoter methylation with ID4 mRNA and ID4 protein expression in matched samples of breast tumour and corresponding normal tissue. We carried out statistical analyses in order to find correlations between ID4 promoter methylation and clinicopathological parameters. Frequent ID4 promoter methylation was observed in primary breast cancer samples (69%, 117/170). We found a tight correlation (P<0.0001) between ID4 promoter methylation and loss of ID4 expression in primary breast cancer 3 specimens. Demethylating treatment with breast cancer cell lines was associated with clear ID4 mRNA re-expression. Tumours with ID4 promoter methylation showed distinct loss of ID4 expression on both transcription and protein level

  14. COX-2 gene expression in colon cancer tissue related to regulating factors and promoter methylation status

    International Nuclear Information System (INIS)

    Asting, Annika Gustafsson; Carén, Helena; Andersson, Marianne; Lönnroth, Christina; Lagerstedt, Kristina; Lundholm, Kent

    2011-01-01

    Increased cyclooxygenase activity promotes progression of colorectal cancer, but the mechanisms behind COX-2 induction remain elusive. This study was therefore aimed to define external cell signaling and transcription factors relating to high COX-2 expression in colon cancer tissue. Tumor and normal colon tissue were collected at primary curative operation in 48 unselected patients. COX-2 expression in tumor and normal colon tissue was quantified including microarray analyses on tumor mRNA accounting for high and low tumor COX-2 expression. Cross hybridization was performed between tumor and normal colon tissue. Methylation status of up-stream COX-2 promoter region was evaluated. Tumors with high COX-2 expression displayed large differences in gene expression compared to normal colon. Numerous genes with altered expression appeared in tumors of high COX-2 expression compared to tumors of low COX-2. COX-2 expression in normal colon was increased in patients with tumors of high COX-2 compared to normal colon from patients with tumors of low COX-2. IL1β, IL6 and iNOS transcripts were up-regulated among external cell signaling factors; nine transcription factors (ATF3, C/EBP, c-Fos, Fos-B, JDP2, JunB, c-Maf, NF-κB, TCF4) showed increased expression and 5 (AP-2, CBP, Elk-1, p53, PEA3) were decreased in tumors with high COX-2. The promoter region of COX-2 gene did not show consistent methylation in tumor or normal colon tissue. Transcription and external cell signaling factors are altered as covariates to COX-2 expression in colon cancer tissue, but DNA methylation of the COX-2 promoter region was not a significant factor behind COX-2 expression in tumor and normal colon tissue

  15. COX-2 gene expression in colon cancer tissue related to regulating factors and promoter methylation status

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    Lagerstedt Kristina

    2011-06-01

    Full Text Available Abstract Background Increased cyclooxygenase activity promotes progression of colorectal cancer, but the mechanisms behind COX-2 induction remain elusive. This study was therefore aimed to define external cell signaling and transcription factors relating to high COX-2 expression in colon cancer tissue. Method Tumor and normal colon tissue were collected at primary curative operation in 48 unselected patients. COX-2 expression in tumor and normal colon tissue was quantified including microarray analyses on tumor mRNA accounting for high and low tumor COX-2 expression. Cross hybridization was performed between tumor and normal colon tissue. Methylation status of up-stream COX-2 promoter region was evaluated. Results Tumors with high COX-2 expression displayed large differences in gene expression compared to normal colon. Numerous genes with altered expression appeared in tumors of high COX-2 expression compared to tumors of low COX-2. COX-2 expression in normal colon was increased in patients with tumors of high COX-2 compared to normal colon from patients with tumors of low COX-2. IL1β, IL6 and iNOS transcripts were up-regulated among external cell signaling factors; nine transcription factors (ATF3, C/EBP, c-Fos, Fos-B, JDP2, JunB, c-Maf, NF-κB, TCF4 showed increased expression and 5 (AP-2, CBP, Elk-1, p53, PEA3 were decreased in tumors with high COX-2. The promoter region of COX-2 gene did not show consistent methylation in tumor or normal colon tissue. Conclusions Transcription and external cell signaling factors are altered as covariates to COX-2 expression in colon cancer tissue, but DNA methylation of the COX-2 promoter region was not a significant factor behind COX-2 expression in tumor and normal colon tissue.

  16. O6-Methylguanine-DNA methyltransferase status in neuroendocrine tumours: prognostic relevance and association with response to alkylating agents.

    Science.gov (United States)

    Walter, T; van Brakel, B; Vercherat, C; Hervieu, V; Forestier, J; Chayvialle, J-A; Molin, Y; Lombard-Bohas, C; Joly, M-O; Scoazec, J-Y

    2015-02-03

    O(6)-Methylguanine-DNA methyltransferase (MGMT) loss of expression has been suggested to be predictive of response to temozolomide in neuroendocrine tumours (NETs), but so far, only limited data are available. We evaluated the prognostic and predictive value of MGMT status, assessed by two molecular methods and immunohistochemistry, in a large series of NETs of different origins. A total of 107 patients, including 53 treated by alkylants (temozolomide, dacarbazine or streptozotocin), were retrospectively studied. In each case, we used methyl-specific PCR (MS-PCR) and pyrosequencing for evaluation of promoter methylation and immunohistochemistry for evaluation of protein status. MGMT promoter methylation was detected in 12 out of 99 (12%) interpretable cases by MS-PCR and in 24 out of 99 (24%) by pyrosequencing. O(6)-Methylguanine-DNA methyltransferase loss of expression was observed in 29 out of 89 (33%) interpretable cases. Status of MGMT was not correlated with overall survival (OS) from diagnosis. Progression-free survival and OS from first alkylant use (temozolomide, dacarbazine and streptozotocin) were higher in patients with MGMT protein loss (respectively, 20.2 vs 7.6 months, Palkylant-based chemotherapy in NETs.

  17. DNA methylation-based variation between human populations.

    Science.gov (United States)

    Kader, Farzeen; Ghai, Meenu

    2017-02-01

    Several studies have proved that DNA methylation affects regulation of gene expression and development. Epigenome-wide studies have reported variation in methylation patterns between populations, including Caucasians, non-Caucasians (Blacks), Hispanics, Arabs, and numerous populations of the African continent. Not only has DNA methylation differences shown to impact externally visible characteristics, but is also a potential biomarker for underlying racial health disparities between human populations. Ethnicity-related methylation differences set their mark during early embryonic development. Genetic variations, such as single-nucleotide polymorphisms and environmental factors, such as age, dietary folate, socioeconomic status, and smoking, impacts DNA methylation levels, which reciprocally impacts expression of phenotypes. Studies show that it is necessary to address these external influences when attempting to differentiate between populations since the relative impacts of these factors on the human methylome remain uncertain. The present review summarises several reported attempts to establish the contribution of differential DNA methylation to natural human variation, and shows that DNA methylation could represent new opportunities for risk stratification and prevention of several diseases amongst populations world-wide. Variation of methylation patterns between human populations is an exciting prospect which inspires further valuable research to apply the concept in routine medical and forensic casework. However, trans-generational inheritance needs to be quantified to decipher the proportion of variation contributed by DNA methylation. The future holds thorough evaluation of the epigenome to understand quantification, heritability, and the effect of DNA methylation on phenotypes. In addition, methylation profiling of the same ethnic groups across geographical locations will shed light on conserved methylation differences in populations.

  18. Methyl cation affinities of neutral and anionic maingroup-element hydrides: trends across the periodic table and correlation with proton affinities.

    Science.gov (United States)

    Mulder, R Joshua; Guerra, Célia Fonseca; Bickelhaupt, F Matthias

    2010-07-22

    We have computed the methyl cation affinities in the gas phase of archetypal anionic and neutral bases across the periodic table using ZORA-relativistic density functional theory (DFT) at BP86/QZ4P//BP86/TZ2P. The main purpose of this work is to provide the methyl cation affinities (and corresponding entropies) at 298 K of all anionic (XH(n-1)(-)) and neutral bases (XH(n)) constituted by maingroup-element hydrides of groups 14-17 and the noble gases (i.e., group 18) along the periods 2-6. The cation affinity of the bases decreases from H(+) to CH(3)(+). To understand this trend, we have carried out quantitative bond energy decomposition analyses (EDA). Quantitative correlations are established between the MCA and PA values.

  19. Correlation between apparent diffusion coefficients and HER2 status in gastric cancers: pilot study

    International Nuclear Information System (INIS)

    He, Jian; Shi, Hua; Zhou, Zhuping; Chen, Jun; Guan, Wenxian; Wang, Hao; Yu, Haiping; Liu, Song; Zhou, Zhengyang; Yang, Xiaofeng; Liu, Tian

    2015-01-01

    To evaluate whether apparent diffusion coefficient (ADC) value of gastric cancer obtained from diffusion weighted imaging (DWI) correlates with the HER2 status. Forty-five patients, who had been diagnosed with gastric cancer through biopsy, were enrolled in this IRB-approved study. Each patient underwent a DWI (b values: 0 and 1,000 sec/mm 2 ) prior to surgery (curative gastrectomy or palliative resection). Postoperative microscopic findings, HER2 status by immunohistochemical analysis and fluorescence in situ hybridization (FISH) were obtained. HER2 status was compared among gastric cancers with various histopathological features using the chi square test. The ADC values of gastric cancers with positive and negative HER2 were compared using the student t test. A weak yet significant correlation was observed between the mean ADC values and HER2 status (r = 0.312, P = 0.037) and scores (r = 0.419, P = 0.004). The mean ADC value of HER2-positive gastric cancers was significantly higher than those of HER2-negative tumors (1.211 vs. 0.984 mm 2 /s, P = 0.020). The minimal ADC value of HER2-positive gastric cancers was significantly higher than those of HER2-negative tumors (1.105 vs. 0.905 × 10 −3 mm 2 /s, P = 0.036). In this pilot study, we have demonstrated that the ADC values of gastric cancer correlate with the HER2 status. Future research is warranted to see if DWI can predict HER2 status and help in tailoring therapy for gastric cancer

  20. DNA Methylation at the DAT Promoter and Risk for Psychopathology: Intergenerational Transmission between School-Age Youths and Their Parents in a Community Sample

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    Silvia Cimino

    2018-01-01

    Full Text Available BackgroundThe effect of gene polymorphisms and promoter methylation, associated with maladaptive developmental outcomes, vary depending on environmental factors (e.g., parental psychopathology. Most studies have focused on 0- to 5-year-old children, adolescents, or adults, whereas there is dearth of research on school-age youths and pre-adolescents.MethodsIn a sample of 21 families recruited at schools, we addressed parents’ psychopathological symptoms (through SCL-90-R; offspring emotional–behavioral functioning (through CBCL-6–18; dopamine transporter gene (DAT1 for epigenetic status of the 5′-untranslated region (UTR and for genotype, i.e., variable number of tandem repeats polymorphism at the 3′-UTR. Possible associations were explored between bio-genetic and psychological characteristics within the same individual and between triplets of children, mothers, and fathers.ResultsDAT methylation of CpG at positions M1, M6, and M7 in mothers was correlated with maternal (phobic anxiety, whereas in fathers’ position M6 was related to paternal depression, anxiety, hostility, psychoticism, and higher Global Severity Index (GSI. No significant correlations were found between maternal and offspring DAT methylation. Significant correlations were found between fathers’ methylation at CpG M1 and children’s methylation at CpG M6. Linear regressions showed that mothers and fathers’ GSI predicted children’s methylation at CpG sites M2, M3, and M6, whereas fathers’ GSI predicted children’s methylation at CpG sites, particularly M1, M2, and M6. Moreover, offspring methylation of DAT at CpG M2 predicted somatic complaint, internalizing and attention problems; methylation of DAT at CpG M6 predicted withdraw.ConclusionThis study may have important clinical implication for the prevention and treatment of emotional–behavioral difficulties in children, as it adds to previous knowledge about the role of genetic and environmental factors in

  1. Correlation of neuter status and expression of heritable disorders

    OpenAIRE

    Belanger, Janelle M.; Bellumori, Thomas P.; Bannasch, Danika L.; Famula, Thomas R.; Oberbauer, Anita M.

    2017-01-01

    Background Gonadectomy, or neutering, is a very common surgery for dogs having many positive effects on behavior, health, and longevity. There are also certain risks associated with neutering including the development of orthopedic conditions, cognitive decline, and a predisposition to some neoplasias. This study was designed specifically to identify if a correlation exists between neuter status and inherited conditions in a large aggregate cohort of dogs representing many different breeds. R...

  2. Obesity-related DNA methylation at imprinted genes in human sperm: Results from the TIEGER study.

    Science.gov (United States)

    Soubry, Adelheid; Guo, Lisa; Huang, Zhiqing; Hoyo, Cathrine; Romanus, Stephanie; Price, Thomas; Murphy, Susan K

    2016-01-01

    Epigenetic reprogramming in mammalian gametes resets methylation marks that regulate monoallelic expression of imprinted genes. In males, this involves erasure of the maternal methylation marks and establishment of paternal-specific methylation to appropriately guide normal development. The degree to which exogenous factors influence the fidelity of methylation reprogramming is unknown. We previously found an association between paternal obesity and altered DNA methylation in umbilical cord blood, suggesting that the father's endocrine, nutritional, or lifestyle status could potentiate intergenerational heritable epigenetic abnormalities. In these analyses, we examine the relationship between male overweight/obesity and DNA methylation status of imprinted gene regulatory regions in the gametes. Linear regression models were used to compare sperm DNA methylation percentages, quantified by bisulfite pyrosequencing, at 12 differentially methylated regions (DMRs) from 23 overweight/obese and 44 normal weight men. Our study population included 69 volunteers from The Influence of the Environment on Gametic Epigenetic Reprogramming (TIEGER) study, based in NC, USA. After adjusting for age and fertility patient status, semen from overweight or obese men had significantly lower methylation percentages at the MEG3 (β = -1.99; SE = 0.84; p = 0.02), NDN (β = -1.10; SE = 0.47; p = 0.02), SNRPN (β = -0.65; SE = 0.27; p = 0.02), and SGCE/PEG10 (β = -2.5; SE = 1.01; p = 0.01) DMRs. Our data further suggest a slight increase in DNA methylation at the MEG3-IG DMR (β = +1.22; SE = 0.59; p = 0.04) and H19 DMR (β = +1.37; SE = 0.62; p = 0.03) in sperm of overweight/obese men. Our data support that male overweight/obesity status is traceable in the sperm epigenome. Further research is needed to understand the effect of such changes and the point of origin of DNA methylation differences between lean and

  3. Divergence of gene body DNA methylation and evolution of plant duplicate genes.

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    Jun Wang

    Full Text Available It has been shown that gene body DNA methylation is associated with gene expression. However, whether and how deviation of gene body DNA methylation between duplicate genes can influence their divergence remains largely unexplored. Here, we aim to elucidate the potential role of gene body DNA methylation in the fate of duplicate genes. We identified paralogous gene pairs from Arabidopsis and rice (Oryza sativa ssp. japonica genomes and reprocessed their single-base resolution methylome data. We show that methylation in paralogous genes nonlinearly correlates with several gene properties including exon number/gene length, expression level and mutation rate. Further, we demonstrated that divergence of methylation level and pattern in paralogs indeed positively correlate with their sequence and expression divergences. This result held even after controlling for other confounding factors known to influence the divergence of paralogs. We observed that methylation level divergence might be more relevant to the expression divergence of paralogs than methylation pattern divergence. Finally, we explored the mechanisms that might give rise to the divergence of gene body methylation in paralogs. We found that exonic methylation divergence more closely correlates with expression divergence than intronic methylation divergence. We show that genomic environments (e.g., flanked by transposable elements and repetitive sequences of paralogs generated by various duplication mechanisms are associated with the methylation divergence of paralogs. Overall, our results suggest that the changes in gene body DNA methylation could provide another avenue for duplicate genes to develop differential expression patterns and undergo different evolutionary fates in plant genomes.

  4. Are clinicopathological features of colorectal cancers with methylation in half of CpG island methylator phenotype panel markers different from those of CpG island methylator phenotype-high colorectal cancers?

    Science.gov (United States)

    Bae, Jeong Mo; Rhee, Ye-Young; Kim, Kyung Ju; Wen, Xianyu; Song, Young Seok; Cho, Nam-Yun; Kim, Jung Ho; Kang, Gyeong Hoon

    2016-01-01

    CpG island methylator phenotype (CIMP)-high (CIMP-H) colorectal cancer (CRC) is defined when a tumor shows methylation at greater than or equal to 60% of CIMP panel markers. Although CRCs with methylation at 50% of panel markers are classified as CIMP-low/CIMP-0 tumors, little is known regarding the clinicopathological and molecular features of CRCs with methylation at 4/8 panel markers (4/8 methylated markers) and whether they are akin to CIMP-H or CIMP-low/CIMP-0 CRCs in terms of their clinicopathological or molecular features. A total of 1164 cases of surgically resected CRC were analyzed for their methylation status in 8 CIMP panel markers, and the frequencies of various clinicopathological and molecular features were compared between CRCs with 0/8, 1/8 to 3/8, 4/8, and 5/8 to 8/8 methylated markers. CRCs with 4/8 methylated markers were closer to CRCs with 5/8 to 8/8 methylated markers in terms of sex distribution, mucin production, serration, nodal metastasis, CK7 expression, CK20 loss, and CDX2 loss frequencies and overall survival rate. CRCs with methylation at 4/8 markers were closer to CRCs with 1/8 to 3/8 methylated markers in terms of less frequent right colon location and poor differentiation. CRCs with 4/8 methylated markers showed the shortest overall survival time compared with CRCs with 0/8, 1/8 to 3/8, 4/8, or 5/8 to 8/8 methylated markers. In terms of clinicopathological and molecular features, CRCs with 4/8 methylated markers appeared to be closer to CIMP-H than to CIMP-low/CIMP-0 and would thus be better classified as CIMP-H if the CRCs require classification into either CIMP-H or CIMP-low/CIMP-0. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. CpG methylation of APC promoter 1A in sporadic and familial breast cancer patients.

    Science.gov (United States)

    Debouki-Joudi, Saoussen; Trifa, Fatma; Khabir, Abdelmajid; Sellami-Boudawara, Tahia; Frikha, Mounir; Daoud, Jamel; Mokdad-Gargouri, Raja

    2017-01-01

    Tumour suppressor gene (TSG) silencing through promoter hypermethylation plays an important role in cancer initiation. The aim of this study was to assess the extent of methylation of APC gene promoter in 91 sporadic and 44 familial cases of Tunisian patients with breast cancer (BC) in. The frequency of APC promoter methylation is somewhat similar for sporadic and familial breast cancer cases, (52.1%, and 54.5% respectively). For sporadic breast cancer patients, there was a significant correlation of APC promoter hypermethylation with TNM stage (p = 0.024) and 3-year survival (p = 0.025). Regarding the hormonal status (HR), we found significant association between negativity to PR and unmethylated APC (p= 0.005) while ER and Her2/neu are not correlated. Moreover, unmethylated APC promoter is more frequent in tumours expressing at least one out the 3 proteins compared to triple negative cases (p= 0.053). On the other hand, aberrant methylation of APC was associated with tumour size (p = 0.036), lymph node (p = 0.028), distant metastasis (p = 0.031), and 3-year survival (p = 0.046) in the group of patients with familial breast cancer. Moreover, patients with sporadic breast cancer displaying the unmethylated profile have a significant prolonged overall survival compared to those with the methylated pattern of APC promoter (p log rank = 0.008). Epigenetic change at the CpG islands in the APC promoter was associated with the silence of its transcript and the loss of protein expression suggesting that this event is the main mechanism regulating the APC expression in breast cancer. In conclusion, our data showed that the loss of APC through aberrant methylation is associated with the aggressive behavior of both sporadic and familial breast cancer in Tunisian patients.

  6. Evaluating genome-wide DNA methylation changes in mice by Methylation Specific Digital Karyotyping

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    Maruoka Shuichiro

    2008-12-01

    Full Text Available Abstract Background The study of genome-wide DNA methylation changes has become more accessible with the development of various array-based technologies though when studying species other than human the choice of applications are limited and not always within reach. In this study, we adapted and tested the applicability of Methylation Specific Digital Karyotyping (MSDK, a non-array based method, for the prospective analysis of epigenetic changes after perinatal nutritional modifications in a mouse model of allergic airway disease. MSDK is a sequenced based method that allows a comprehensive and unbiased methylation profiling. The method generates 21 base pairs long sequence tags derived from specific locations in the genome. The resulting tag frequencies determine in a quantitative manner the methylation level of the corresponding loci. Results Genomic DNA from whole lung was isolated and subjected to MSDK analysis using the methylation-sensitive enzyme Not I as the mapping enzyme and Nla III as the fragmenting enzyme. In a pair wise comparison of the generated mouse MSDK libraries we identified 158 loci that are significantly differentially methylated (P-value = 0.05 after perinatal dietary changes in our mouse model. Quantitative methylation specific PCR and sequence analysis of bisulfate modified genomic DNA confirmed changes in methylation at specific loci. Differences in genomic MSDK tag counts for a selected set of genes, correlated well with changes in transcription levels as measured by real-time PCR. Furthermore serial analysis of gene expression profiling demonstrated a dramatic difference in expressed transcripts in mice exposed to perinatal nutritional changes. Conclusion The genome-wide methylation survey applied in this study allowed for an unbiased methylation profiling revealing subtle changes in DNA methylation in mice maternally exposed to dietary changes in methyl-donor content. The MSDK method is applicable for mouse models

  7. MicroRNA-137 promoter methylation in oral lichen planus and oral squamous cell carcinoma

    DEFF Research Database (Denmark)

    Dang, Jun; Bian, Yong-qian; Sun, Jian-yong

    2013-01-01

    and patients with oral squamous cell carcinoma (OSCC). A total of 20 OLP and 12 patients with OSCC as well as 10 healthy subjects were subjected to miR-137 promoter methylation analysis using methylation-specific PCR (MSP). To address the malignancy prediction potential from miR-137 promoter methylation status...

  8. Promoter DNA methylation pattern identifies prognostic subgroups in childhood T-cell acute lymphoblastic leukemia.

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    Magnus Borssén

    Full Text Available BACKGROUND: Treatment of pediatric T-cell acute lymphoblastic leukemia (T-ALL has improved, but there is a considerable fraction of patients experiencing a poor outcome. There is a need for better prognostic markers and aberrant DNA methylation is a candidate in other malignancies, but its potential prognostic significance in T-ALL is hitherto undecided. DESIGN AND METHODS: Genome wide promoter DNA methylation analysis was performed in pediatric T-ALL samples (n = 43 using arrays covering >27000 CpG sites. Clinical outcome was evaluated in relation to methylation status and compared with a contemporary T-ALL group not tested for methylation (n = 32. RESULTS: Based on CpG island methylator phenotype (CIMP, T-ALL samples were subgrouped as CIMP+ (high methylation and CIMP- (low methylation. CIMP- T-ALL patients had significantly worse overall and event free survival (p = 0.02 and p = 0.001, respectively compared to CIMP+ cases. CIMP status was an independent factor for survival in multivariate analysis including age, gender and white blood cell count. Analysis of differently methylated genes in the CIMP subgroups showed an overrepresentation of transcription factors, ligands and polycomb target genes. CONCLUSIONS: We identified global promoter methylation profiling as being of relevance for subgrouping and prognostication of pediatric T-ALL.

  9. The correlation between pulmonary fibrosis and methylation of peripheral Smad3 in cases of pigeon breeder's lung in a Chinese Uygur population.

    Science.gov (United States)

    Wu, Chao; Ding, Wei; Li, Qifeng; Wang, Wenyi; Deng, Mingqin; Jin, Rong; Pang, Baosen; Yang, Xiaohong

    2017-06-27

    Smad3 is a key protein in the transforming growth factor-beta (TGF-β)/Smad signaling pathway, which is involved in fibrosis in many organs. We investigated the relationship between Smad3 gene methylation and pulmonary fibrosis in pigeon breeder's lung (PBL). Twenty Uygur PBL patients with pulmonary fibrosis in Kashi between October 2015 and March 2016 were enrolled. Twenty PBL-free pigeon breeders and 20 healthy non-pigeon breeders enrolled during the same period constituted the negative and normal control groups, respectively. Participants' data and peripheral blood samples were collected, and three Smad3 CpG loci were examined. Distributions of CpG_2 and CpG_4 methylation rates did not differ across groups, whereas distributions of CpG_3 methylation rates were significantly different among the three groups. The CpG_3 methylation rate was significantly lower in the patient group than in the negative control group. Smad3 mRNA expression was significantly higher in the patient group than in the negative control group but did not differ between the two control groups. TGF-βlevels were significantly higher in the patient group than in either control group (both Ppulmonary fibrosis in Uygur PBL patients via increased Smad3 mRNA expression. Smad3 methylation, Smad3 mRNA expression and TGF-β level were correlated with the number of pigeons bred by patients.

  10. Bisulfite sequencing reveals that Aspergillus flavus holds a hollow in DNA methylation.

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    Si-Yang Liu

    Full Text Available Aspergillus flavus first gained scientific attention for its production of aflatoxin. The underlying regulation of aflatoxin biosynthesis has been serving as a theoretical model for biosynthesis of other microbial secondary metabolites. Nevertheless, for several decades, the DNA methylation status, one of the important epigenomic modifications involved in gene regulation, in A. flavus remains to be controversial. Here, we applied bisulfite sequencing in conjunction with a biological replicate strategy to investigate the DNA methylation profiling of A. flavus genome. Both the bisulfite sequencing data and the methylome comparisons with other fungi confirm that the DNA methylation level of this fungus is negligible. Further investigation into the DNA methyltransferase of Aspergillus uncovers its close relationship with RID-like enzymes as well as its divergence with the methyltransferase of species with validated DNA methylation. The lack of repeat contents of the A. flavus' genome and the high RIP-index of the small amount of remanent repeat potentially support our speculation that DNA methylation may be absent in A. flavus or that it may possess de novo DNA methylation which occurs very transiently during the obscure sexual stage of this fungal species. This work contributes to our understanding on the DNA methylation status of A. flavus, as well as reinforces our views on the DNA methylation in fungal species. In addition, our strategy of applying bisulfite sequencing to DNA methylation detection in species with low DNA methylation may serve as a reference for later scientific investigations in other hypomethylated species.

  11. DNA Methylation Alterations at 5'-CCGG Sites in the Interspecific and Intraspecific Hybridizations Derived from Brassica rapa and B. napus.

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    Wanshan Xiong

    Full Text Available DNA methylation is an important regulatory mechanism for gene expression that involved in the biological processes of development and differentiation in plants. To investigate the association of DNA methylation with heterosis in Brassica, a set of intraspecific hybrids in Brassica rapa and B. napus and interspecific hybrids between B. rapa and B. napus, together with parental lines, were used to monitor alterations in cytosine methylation at 5'-CCGG sites in seedlings and buds by methylation-sensitive amplification polymorphism analysis. The methylation status of approximately a quarter of the methylation sites changed between seedlings and buds. These alterations were related closely to the genomic structure and heterozygous status among accessions. The methylation status in the majority of DNA methylation sites detected in hybrids was the same as that in at least one of the parental lines in both seedlings and buds. However, the association between patterns of cytosine methylation and heterosis varied among different traits and between tissues in hybrids of Brassica, although a few methylation loci were associated with heterosis. Our data suggest that changes in DNA methylation at 5'-CCGG sites are not associated simply with heterosis in the interspecific and intraspecific hybridizations derived from B. rapa and B. napus.

  12. Initial estimation of correlation between estrogen receptor status and histopathology, and also some selected prognostic factors in breast cancer patients

    International Nuclear Information System (INIS)

    Cwikla, J.; Badowski, J.; Shafie, D.; Gugala, K.; Koziorowski, M.

    1996-01-01

    The goal of this study was to assess the correlation between estrogen receptor (ER) status and histopathology findings, likewise to assess some selected prognostic factors in patients with breast cancer. The study was carried out on 126 patients with breast cancer. ER concentration was estimated by the standard biochemical assay (DCC-dextran-coated charcoal assay). The correlation between established risk factors like: lymph node status; age menopausal status and ER status were analysed.The ER yielded in 61% positive results. The mean value of ER in invasive ductal carcinoma was 43.9 fmol/mg protein and the mean value of ER in invasive lobular carcinoma 51.4 fmol/mg protein. The significant statistics negative correlation between ER status of pre-menopausal patients with ductal breast carcinoma and regional lymph nodes involvement was found. There was no difference between ER status and histological type of the cancer. No correlation was found between ER status and age of patients. (author)

  13. Quantitative Methylation Profiles for Multiple Tumor Suppressor Gene Promoters in Salivary Gland Tumors

    Science.gov (United States)

    Durr, Megan L.; Mydlarz, Wojciech K.; Shao, Chunbo; Zahurak, Marianna L.; Chuang, Alice Y.; Hoque, Mohammad O.; Westra, William H.; Liegeois, Nanette J.; Califano, Joseph A.; Sidransky, David; Ha, Patrick K.

    2010-01-01

    Background Methylation profiling of tumor suppressor gene (TSGs) promoters is quickly becoming a powerful diagnostic tool for the early detection, prognosis, and even prediction of clinical response to treatment. Few studies address this in salivary gland tumors (SGTs); hence the promoter methylation profile of various TSGs was quantitatively assessed in primary SGT tissue to determine if tumor-specific alterations could be detected. Methodology DNA isolated from 78 tumor and 17 normal parotid gland specimens was assayed for promoter methylation status of 19 TSGs by fluorescence-based, quantitative methylation-specific PCR (qMSP). The data were utilized in a binary fashion as well as quantitatively (using a methylation quotient) allowing for better profiling and interpretation of results. Principal Findings The average number of methylation events across the studied genes was highest in salivary duct carcinoma (SDC), with a methylation value of 9.6, compared to the normal 4.5 (ptrend for increasing methylation in APC, Mint 1, PGP9.5, RAR-β, and Timp3. Conclusions/Significance Screening promoter methylation profiles in SGTs showed considerable heterogeneity. The methylation status of certain markers was surprisingly high in even normal salivary tissue, confirming the need for such controls. Several TSGs were found to be associated with malignant SGTs, especially SDC. Further study is needed to evaluate the potential use of these associations in the detection, prognosis, and therapeutic outcome of these rare tumors. PMID:20520817

  14. In vivo estradiol-dependent dephosphorylation of the repressor MDBP-2-H1 correlates with the loss of in vitro preferential binding to methylated DNA.

    Science.gov (United States)

    Bruhat, A; Jost, J P

    1995-01-01

    We have previously shown that estradiol treatment of roosters resulted in a rapid loss of binding activity of the repressor MDBP-2-H1 (a member of the histone H1 family) to methylated DNA that was not due to a decrease in MDBP-2-H1 concentration. Here we demonstrate that MDBP-2-H1 from rooster liver nuclear extracts is a phosphoprotein. Phosphoamino acid analysis reveals that the phosphorylation occurs exclusively on serine residues. Two-dimensional gel electrophoresis and tryptic phosphopeptide analysis show that MDBP-2-H1 is phosphorylated at several sites. Treatment of roosters with estradiol triggers a dephosphorylation of at least two sites in the protein. Phosphatase treatment of purified rooster MDBP-2-H1 combined with gel mobility shift assay indicates that phosphorylation of MDBP-2-H1 is essential for the binding to methylated DNA and that the dephosphorylation can occur on the protein bound to methylated DNA causing its release from DNA. Thus, these results suggest that in vivo modification of the phosphorylation status of MDBP-2-H1 caused by estradiol treatment may be a key step for the down regulation of its binding to methylated DNA. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:7731964

  15. Aberrant Methylation-Mediated Suppression of APAF1 in Myelodysplastic Syndrome.

    Science.gov (United States)

    Zaker, Farhad; Nasiri, Nahid; Amirizadeh, Naser; Razavi, Seyed Mohsen; Yaghmaie, Marjan; Teimoori-Toolabi, Ladan; Maleki, Ali; Bakhshayesh, Masoumeh

    2017-04-01

    Background: Myelodysplastic syndromes (MDSs) include a diverse group of clonal bone marrow disorders characterized by ineffective hematopoiesis and pancytopenia. It was found that down regulation of APAF1, a putative tumor suppressor gene (TSG), leads to resistance to chemotherapy and disease development in some cancers. In this study, we investigated the relation of APAF1 methylation status with its expression and clinicopathological factors in myelodysplastic syndrome (MDS) patients. Materials and Methods: Methylation Sensitive-High Resolution Melting Curve Analysis (MS-HRM) was employed in studying the methylation of CpG islands in the APAF1promoter region in MDS. Gene expression was analyzed by using real time RT-PCR. Results: 42.6% of patient samples were methylated in promoter region of APAF1analyzed, while methylation of the gene was not seen in controls (P<0.05). Methylation of APAF1was significantly associated with the suppression of its mRNA expression (P=0.00). The methylation status of APAF1in advanced-stage MDS patients (80%) was significantly higher than that of the early-stage MDS patients (28.2%) (P=0.001). The difference in frequency of hypermethylatedAPAF1 gene was significant between good (37.5%) and poor (85.71%) cytogenetic risk groups (P=0.043). In addition, a higher frequency of APAF1hypermethylation was observed in higher-risk MDS group (69.2%) compared to lower-risk MDS group (34.14%) (P=0.026). Conclusion: Our study indicated that APAF1hypermethylation in MDS was associated to high-risk disease classified according to the IPSS, WHO and cytogenetic risk.

  16. Regulation of homocysteine metabolism and methylation in human and mouse tissues

    Science.gov (United States)

    Chen, Natalie C.; Yang, Fan; Capecci, Louis M.; Gu, Ziyu; Schafer, Andrew I.; Durante, William; Yang, Xiao-Feng; Wang, Hong

    2010-01-01

    Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Homocysteine (Hcy) metabolism involves multiple enzymes; however, tissue Hcy metabolism and its relevance to methylation remain unknown. Here, we established gene expression profiles of 8 Hcy metabolic and 12 methylation enzymes in 20 human and 19 mouse tissues through bioinformatic analysis using expression sequence tag clone counts in tissue cDNA libraries. We analyzed correlations between gene expression, Hcy, S-adenosylhomocysteine (SAH), and S-adenosylmethionine (SAM) levels, and SAM/SAH ratios in mouse tissues. Hcy metabolic and methylation enzymes were classified into two types. The expression of Type 1 enzymes positively correlated with tissue Hcy and SAH levels. These include cystathionine β-synthase, cystathionine-γ-lyase, paraxonase 1, 5,10-methylenetetrahydrofolate reductase, betaine:homocysteine methyltransferase, methionine adenosyltransferase, phosphatidylethanolamine N-methyltransferases and glycine N-methyltransferase. Type 2 enzyme expressions correlate with neither tissue Hcy nor SAH levels. These include SAH hydrolase, methionyl-tRNA synthase, 5-methyltetrahydrofolate:Hcy methyltransferase, S-adenosylmethionine decarboxylase, DNA methyltransferase 1/3a, isoprenylcysteine carboxyl methyltransferases, and histone-lysine N-methyltransferase. SAH is the only Hcy metabolite significantly correlated with Hcy levels and methylation enzyme expression. We established equations expressing combined effects of methylation enzymes on tissue SAH, SAM, and SAM/SAH ratios. Our study is the first to provide panoramic tissue gene expression profiles and mathematical models of tissue methylation regulation.—Chen, N. C., Yang, F., Capecci, L. M., Gu, Z., Schafer, A. I., Durante, W., Yang, X.-F., Wang, H. Regulation of homocysteine metabolism and methylation in human and mouse tissues. PMID:20305127

  17. The DNA methylation status alteration of two steroidogenic genes in gonads of rare minnow after bisphenol A exposure.

    Science.gov (United States)

    Zhang, Ting; Liu, Yan; Chen, Hong; Gao, Jiancao; Zhang, Yingying; Yuan, Cong; Wang, Zaizhao

    2017-08-01

    Both cytochrome P450c17 (CYP17A1) and P-450 side chain cleavage (CYP11A1) play important roles in steroid biosynthesis. According to our previous studies, bisphenol A (BPA) could regulate the mRNA expression of cyp17a1 and cyp11a1 in rare minnow Gobiocypris rarus. However, the potential mechanism of the regulation is barely understood. In the present study, aiming to explore how BPA affects the mRNA expression of cyp17a1 and cyp11a1 in testes and ovaries of G. rarus, we firstly cloned 340-bp fragment of 5' flanking region of cyp11a1 and then detected the methylation level of CpG loci involved in 5' flanking of cyp11a1 and cyp17a1 and their mRNA expression levels. Results showed that exposure to BPA significantly increased serum estradiol (E2) and 11-ketotesterone (11-KT) concentrations. Ovarian mRNA expression of cyp17a1 and cyp11a1 were significantly decreased after BPA exposure 7- for and 14-days. However, transcriptions of testicular cyp17a1 and cyp11a1 were significantly increased and decreased respectively after BPA treatment for 14days. The DNA methylation levels of cyp17a1 were decreased in ovaries on day 7 and increased in ovaries and decreased in testes respectively on day 14. The methylation levels of cyp11a1 were increased in ovaries on day 7 and both ovaries and testes on day 14. There were a significant correlation between DNA methylation at specific CpG loci and cyp17a1 and cyp11a1 genes transcription levels. In conclusion, the CpG loci methylation in 5' flanking region appears to involve in the regulation of mRNA expression of cyp17a1 and cyp11a1 mediated by BPA. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Oxidative Stress and DNA Methylation in Prostate Cancer

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    Krishna Vanaja Donkena

    2010-01-01

    Full Text Available The protective effects of fruits, vegetables, and other foods on prostate cancer may be due to their antioxidant properties. An imbalance in the oxidative stress/antioxidant status is observed in prostate cancer patients. Genome oxidative damage in prostate cancer patients is associated with higher lipid peroxidation and lower antioxidant levels. Oxygen radicals are associated with different steps of carcinogenesis, including structural DNA damage, epigenetic changes, and protein and lipid alterations. Epigenetics affects genetic regulation, cellular differentiation, embryology, aging, cancer, and other diseases. DNA methylation is perhaps the most extensively studied epigenetic modification, which plays an important role in the regulation of gene expression and chromatin architecture, in association with histone modification and other chromatin-associated proteins. This review will provide a broad overview of the interplay of oxidative stress and DNA methylation, DNA methylation changes in regulation of gene expression, lifestyle changes for prostate cancer prevention, DNA methylation as biomarkers for prostate cancer, methods for detection of methylation, and clinical application of DNA methylation inhibitors for epigenetic therapy.

  19. Spatial control of protein phosphatase 2A (de)methylation

    International Nuclear Information System (INIS)

    Longin, Sari; Zwaenepoel, Karen; Martens, Ellen; Louis, Justin V.; Rondelez, Evelien; Goris, Jozef; Janssens, Veerle

    2008-01-01

    Reversible methylation of the protein phosphatase 2A catalytic subunit (PP2A C ) is an important regulatory mechanism playing a crucial role in the selective recruitment of regulatory B subunits. Here, we investigated the subcellular localization of leucine carboxyl methyltransferase (LCMT1) and protein phosphatase methylesterase (PME-1), the two enzymes catalyzing this process. The results show that PME-1 is predominantly localized in the nucleus and harbors a functional nuclear localization signal, whereas LCMT1 is underrepresented in the nucleus and mainly localizes to the cytoplasm, Golgi region and late endosomes. Indirect immunofluorescence with methylation-sensitive anti-PP2A C antibodies revealed a good correlation with the methylation status of PP2A C , demethylated PP2A C being substantially nuclear. Throughout mitosis, demethylated PP2A C is associated with the mitotic spindle and during cytokinesis with the cleavage furrow. Overexpression of PME-1, but not of an inactive mutant, results in increased demethylation of PP2A C in the nucleus, whereas overexpression of a cytoplasmic PME-1 mutant lacking the NLS results in increased demethylation in the cytoplasm-in all cases, however, without any obvious functional consequences. PME-1 associates with an inactive PP2A population, regardless of its esterase activity or localization. We propose that stabilization of this inactive, nuclear PP2A pool is a major in vivo function of PME-1

  20. FAMILY’S ECONOMIC LEVEL AND CULTURE CORRELATE WITH NUTRITIONAL STATUS OF CHILDREN UNDER FIVE YEARS

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    Abdul Muhith

    2017-01-01

    Full Text Available Introduction: Nutrition is an important thing for human life. Variety in family’s economic level and culture have effect on family’s eating habit. Family with higher economic status have big opportunity to met under fi ve year’s nutrition. Cultural diversity on each family has an impact on the difference of raw food selection, processing methods, and presentation of food. The purpose of this study was to determine the correlation between family’s economic level and culture with nutritional status of children under fi ve year. Method: Research design was observational analytic with cross sectional approach. The population were mother and their children under fi ve years at Desa Jatigono Kunir, Kabupaten Lumajang. Sampel were 184 respondents, taken by using cluster sampling. Independent variables were family’s economic level and culture. Dependent variable was nutritional status of children under fi ve years. Data were collected by using questionnaire and observational sheet. Then, data were analyzed by using Spearman Rho Test with α<0.05. Result: The results showed that 140 (76.1% respondents have low economic level, 105 (57.1% respondents have negative culture in children’s nutrition, and 89 (48% respondents have good nutritional status. The result of Spearman-rho test showed that family’s economic level (p=0.000 and culture (0.019 have correlated with nutritional status of children under five years. Discussion: It can be concluded that family’s economic level and culture have correlated with nutritional status of children under fi ve years. Nurses should develop health education and counseling to improve family’s knowledge about nutrition, so children will have good nutritional status. Keywords: economic level, family’s culture, nutritional status, children under five years

  1. Correlates of AUDIT Risk Status for Male and Female College Students

    Science.gov (United States)

    DeMartini, Kelly S.; Carey, Kate B.

    2009-01-01

    Objective: The current study identified gender-specific correlates of hazardous drinker status as defined by the AUDIT. Participants: A total of 462 college student volunteers completed the study in 2006. The sample was predominantly Caucasian (75%) and female (55%). Methods: Participants completed a survey assessing demographics, alcohol use…

  2. The Correlation of Parenting and Socioeconomic Status Towards English Learning Readiness of Children

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    Andi Ummul Khair

    2014-08-01

    Full Text Available This research is about the correlation of Parenting and Socioeconomic Status (SES towards English Learning Readiness (ELR of children. This study was aimed to find out the correlation of parenting quality and socioeconomic status towards English learning readiness of children. This research applied quantitative research, the case conducts in correlational research which describes an existing condition. The population of this research was students from all elementary school in Kecamatan Tamalate Makassar where English is tought at second grade. The representation of the population in this research is the 2nd year students of six elementary schools in Kecamatan Tamalate academic year of 2012/2013 who have collected the two questionnaires which is distributed to them and filled out by their parents. Total number of the sample is 105 students chosen from the questionnaires which is collected and has filled properly by parents. The data were obtained by using two kinds of instruments, those are questionnaires of parenting and socioeconomic status which have tested the validity in a number of students and data of the ELR of children got from student’s English achievement in school. Those data were analyzed by using path analysis of Amos 20.0. The researcher concludes that (1 the correlation of parenting with ELR indicates the higher quality of parenting they have the higher children gain ELR, on the contrary the less quality of parenting they have the less children gain ELR, (2 SES has almost none indication to have correlation with ELR, (3 The higher SES the better parenting do and the lower SES the worst parenting do.

  3. Expression and promoter DNA methylation of MLH1 in colorectal cancer and lung cancer.

    Science.gov (United States)

    Ma, Yunxia; Chen, Yuan; Petersen, Iver

    2017-04-01

    Aberrant DNA methylation is a common molecular feature in human cancer. The aims of this study were to analyze the methylation status of MLH1, one of the DNA mismatch repair (MMR) genes, in human colorectal and lung cancer and to evaluate its clinical relevance. The expression of MLH1 was analyzed in 8 colorectal cancer (CRC) and 8 lung cancer cell lines by real-time RT-PCR and western blotting. The MLH1 protein expression was evaluated by immunohistochemistry on tissue microarrays including 121 primary CRC and 90 lung cancer patient samples. In cancer cell lines, the methylation status of MLH1 promoter and exon 2 was investigated by bisulfite sequencing (BS). Methylation-specific-PCR (MSP) was used to evaluate methylation status of MLH1. The expression of MLH1 mRNA was detected in 8 CRC cell lines as well as normal colonic fibroblast cells CCD-33Co. At protein levels, MLH1 was lost in one CRC cell line HCT-116 and normal cells CCD-33Co. No methylation was found in the promoter and exon 2 of MLH1 in CRC cell lines. MLH1 was expressed in 8 lung cancer cell lines at both mRNA and protein levels. Compared to cancer cells, normal bronchial epithelial cells (HBEC) had lower expression of MLH1 protein. In primary CRC, 54.5% of cases exhibited positive staining, while 47.8% of lung tumors were positive for MLH1 protein. MSP analysis showed that 58 out of 92 (63.0%) CRC and 41 out of 73 (56.2%) lung cancer exhibited MLH1 methylation. In CRC, the MLH1 methylation was significantly associated with tumor invasion in veins (P=0.012). However, no significant links were found between MLH1 expression and promoter methylation in both tumor entities. MLH1 methylation is a frequent molecular event in CRC and lung cancer patients. In CRC, methylation of MLH1 could be linked to vascular invasiveness. Copyright © 2017 Elsevier GmbH. All rights reserved.

  4. DNA methylation map in circulating leukocytes mirrors subcutaneous adipose tissue methylation pattern: a genome-wide analysis from non-obese and obese patients

    Science.gov (United States)

    Crujeiras, A. B.; Diaz-Lagares, A.; Sandoval, J.; Milagro, F. I.; Navas-Carretero, S.; Carreira, M. C.; Gomez, A.; Hervas, D.; Monteiro, M. P.; Casanueva, F. F.; Esteller, M.; Martinez, J. A.

    2017-01-01

    The characterization of the epigenetic changes within the obesity-related adipose tissue will provide new insights to understand this metabolic disorder, but adipose tissue is not easy to sample in population-based studies. We aimed to evaluate the capacity of circulating leukocytes to reflect the adipose tissue-specific DNA methylation status of obesity susceptibility. DNA samples isolated from subcutaneous adipose tissue and circulating leukocytes were hybridized in the Infinium HumanMethylation 450 BeadChip. Data were compared between samples from obese (n = 45) and non-obese (n = 8–10) patients by Wilcoxon-rank test, unadjusted for cell type distributions. A global hypomethylation of the differentially methylated CpG sites (DMCpGs) was observed in the obese subcutaneous adipose tissue and leukocytes. The overlap analysis yielded a number of genes mapped by the common DMCpGs that were identified to reflect the obesity state in the leukocytes. Specifically, the methylation levels of FGFRL1, NCAPH2, PNKD and SMAD3 exhibited excellent and statistically significant efficiencies in the discrimination of obesity from non-obesity status (AUC > 0.80; p obesity-related adipose tissue pathogenesis through peripheral blood analysis, an easily accessible and minimally invasive biological material instead of adipose tissue. PMID:28211912

  5. What do unicellular organisms teach us about DNA methylation?

    Science.gov (United States)

    Harony, Hala; Ankri, Serge

    2008-05-01

    DNA methylation is an epigenetic hallmark that has been studied intensively in mammals and plants. However, knowledge of this phenomenon in unicellular organisms is scanty. Examining epigenetic regulation, and more specifically DNA methylation, in these organisms represents a unique opportunity to better understand their biology. The determination of their methylation status is often complicated by the presence of several differentiation stages in their life cycle. This article focuses on some recent advances that have revealed the unexpected nature of the epigenetic determinants present in protozoa. The role of the enigmatic DNA methyltransferase Dnmt2 in unicellular organisms is discussed.

  6. Differential methylation at the RELN gene promoter in temporal cortex from autistic and typically developing post-puberal subjects.

    Science.gov (United States)

    Lintas, Carla; Sacco, Roberto; Persico, Antonio M

    2016-01-01

    Reelin plays a pivotal role in neurodevelopment and in post-natal synaptic plasticity and has been implicated in the pathogenesis of autism spectrum disorder (ASD). The reelin (RELN) gene expression is significantly decreased in ASD, both in the brain and peripherally. Methylation at the RELN gene promoter is largely triggered at puberty, and hypermethylation has been found in post-mortem brains of schizophrenic and bipolar patients. In this study, we assessed RELN gene methylation status in post-mortem temporocortical tissue samples (BA41/42 or 22) of six pairs of post-puberal individuals with ASD and typically developing subjects, matched for sex (male:female, M:F = 5:1), age, and post-mortem interval. ASD patients display a significantly higher number of methylated CpG islands and heavier methylation in the 5' region of the RELN gene promoter, spanning from -458 to -223 bp, whereas controls have more methylated CpG positions and greater extent of methylation at the 3' promoter region, spanning from -222 to +1 bp. The most upstream promoter region (-458 to -364 bp) is methylated only in ASD brains, while the most downstream region (-131 to +1 bp) is methylated exclusively in control brains. Within this general framework, three different methylation patterns are discernible, each correlated with different extents of reduction in reelin gene expression among ASD individuals compared to controls. The methylation pattern is different in ASD and control post-mortem brains. ASD-specific CpG positions, located in the most upstream gene promoter region, may exert a functional role potentially conferring ASD risk by blunting RELN gene expression.

  7. Role of a redox-based methylation switch in mRNA life cycle ( pre- & post- transcriptional maturation and protein turnover : Implications in neurological disorders

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    MALAV SUCHIN TRIVEDI

    2012-06-01

    Full Text Available Homeostatic synaptic scaling in response to neuronal stimulus or activation, as well as due to changes in cellular niche, is an important phenomenon for memory consolidation, retrieval, and other similar cognitive functions. Neurological disorders and cognitive disabilities in autism, Rett syndrome, schizophrenia, dementia etc., are strongly correlated to alterations in protein expression (both synaptic and cytoplasmic. This correlation suggests that efficient temporal regulation of synaptic protein expression is important for synaptic plasticity. In addition, equilibrium between mRNA processing, protein translation and protein turnover is a critical sensor/trigger for recording synaptic information, normal cognition and behavior. Thus a regulatory switch, controlling the lifespan, maturation and processing of mRNA, might influence cognition and adaptive behavior. Here, we propose a two part novel hypothesis that methylation might act as this suggested coordinating switch to critically regulate mRNA maturation at 1.The pre-transcription level, by regulating precursor-RNA (pre-RNA processing into mRNA, via other non-coding RNAs and their influence on splicing phenomenon, and 2. the post-transcription level by modulating the regulatory functions of ribonucleoproteins (RNP and RNA binding proteins (RNABP in mRNA translation, dendritic translocation as well as protein synthesis and synaptic turnover. DNA methylation changes are well recognized and highly correlated to gene expression levels as well as, learning and memory; however, RNA methylation changes are recently characterized and yet their functional implications are not established. This review article provides some insight on the intriguing consequences of changes in methylation levels on mRNA life-cycle. We also suggest that, since methylation is under the control of glutathione antioxidant levels, the redox status of neurons might be the central regulatory switch for methylation

  8. Epigenetic status of H19/IGF2 and SNRPN imprinted genes in aborted and successfully derived embryonic stem cell lines in non-human primates

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    Florence Wianny

    2016-05-01

    Full Text Available The imprinted genes of primate embryonic stem cells (ESCs often show altered DNA methylation. It is unknown whether these alterations emerge while deriving the ESCs. Here we studied the methylation patterns of two differentially methylated regions (DMRs, SNRPN and H19/IGF2 DMRs, during the derivation of monkey ESCs. We show that the SNRPN DMR is characteristically methylated at maternal alleles, whereas the H19/IGF2 DMR is globally highly methylated, with unusual methylation on the maternal alleles. These methylation patterns remain stable from the early stages of ESC derivation to late passages of monkey ESCs and following differentiation. Importantly, the methylation status of H19/IGF2 DMR and the expression levels of IGF2, H19, and DNMT3B mRNAs in early embryo-derived cells were correlated with their capacity to generate genuine ESC lines. Thus, we propose that these markers could be useful to predict the outcomes of establishing an ESC line in primates.

  9. Gene methylation profiles of normal mucosa, and benign and malignant colorectal tumors identify early onset markers

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    Vatn Morten

    2008-12-01

    Full Text Available Abstract Background Multiple epigenetic and genetic changes have been reported in colorectal tumors, but few of these have clinical impact. This study aims to pinpoint epigenetic markers that can discriminate between non-malignant and malignant tissue from the large bowel, i.e. markers with diagnostic potential. The methylation status of eleven genes (ADAMTS1, CDKN2A, CRABP1, HOXA9, MAL, MGMT, MLH1, NR3C1, PTEN, RUNX3, and SCGB3A1 was determined in 154 tissue samples including normal mucosa, adenomas, and carcinomas of the colorectum. The gene-specific and widespread methylation status among the carcinomas was related to patient gender and age, and microsatellite instability status. Possible CIMP tumors were identified by comparing the methylation profile with microsatellite instability (MSI, BRAF-, KRAS-, and TP53 mutation status. Results The mean number of methylated genes per sample was 0.4 in normal colon mucosa from tumor-free individuals, 1.2 in mucosa from cancerous bowels, 2.2 in adenomas, and 3.9 in carcinomas. Widespread methylation was found in both adenomas and carcinomas. The promoters of ADAMTS1, MAL, and MGMT were frequently methylated in benign samples as well as in malignant tumors, independent of microsatellite instability. In contrast, normal mucosa samples taken from bowels without tumor were rarely methylated for the same genes. Hypermethylated CRABP1, MLH1, NR3C1, RUNX3, and SCGB3A1 were shown to be identifiers of carcinomas with microsatellite instability. In agreement with the CIMP concept, MSI and mutated BRAF were associated with samples harboring hypermethylation of several target genes. Conclusion Methylated ADAMTS1, MGMT, and MAL are suitable as markers for early tumor detection.

  10. BDNF Methylation and Maternal Brain Activity in a Violence-Related Sample.

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    Dominik A Moser

    Full Text Available It is known that increased circulating glucocorticoids in the wake of excessive, chronic, repetitive stress increases anxiety and impairs Brain-Derived Neurotrophic Factor (BDNF signaling. Recent studies of BDNF gene methylation in relation to maternal care have linked high BDNF methylation levels in the blood of adults to lower quality of received maternal care measured via self-report. Yet the specific mechanisms by which these phenomena occur remain to be established. The present study examines the link between methylation of the BDNF gene promoter region and patterns of neural activity that are associated with maternal response to stressful versus non-stressful child stimuli within a sample that includes mothers with interpersonal violence-related PTSD (IPV-PTSD. 46 mothers underwent fMRI. The contrast of neural activity when watching children-including their own-was then correlated to BDNF methylation. Consistent with the existing literature, the present study found that maternal BDNF methylation was associated with higher levels of maternal anxiety and greater childhood exposure to domestic violence. fMRI results showed a positive correlation of BDNF methylation with maternal brain activity in the anterior cingulate (ACC, and ventromedial prefrontal cortex (vmPFC, regions generally credited with a regulatory function toward brain areas that are generating emotions. Furthermore we found a negative correlation of BDNF methylation with the activity of the right hippocampus. Since our stimuli focus on stressful parenting conditions, these data suggest that the correlation between vmPFC/ACC activity and BDNF methylation may be linked to mothers who are at a disadvantage with respect to emotion regulation when facing stressful parenting situations. Overall, this study provides evidence that epigenetic signatures of stress-related genes can be linked to functional brain regions regulating parenting stress, thus advancing our understanding of

  11. The expression status of TRX, AR, and cyclin D1 correlates with clinicopathological characteristics and ER status in breast cancer.

    Science.gov (United States)

    Huang, Weisun; Nie, Weiwei; Zhang, Wenwen; Wang, Yanru; Zhu, Aiyu; Guan, Xiaoxiang

    2016-01-01

    The ER signaling pathway plays a critical role in breast cancer. ER signaling pathway-related proteins, such as TRX, AR, and cyclin D1, may have an important function in breast cancer. However, the ways that they influence breast cancer development and progression are still unclear. A total of 101 Chinese female patients diagnosed with invasive ductal breast adenocarcinoma were retrospectively enrolled in the study. The expression levels of TRX, AR, and cyclin D1 were detected by immunohistochemistry and analyzed via correlation with clinicopathological characteristics and the expression status of ER, PR, and HER2. The expression status of TRX, AR, and cyclin D1 was not associated with the patient's age, menopausal status, tumor size, or histological differentiation (P>0.05), but was positively correlated with ER and PR (PTRX-positive patients were also HER2-positive (P=0.003). Of AR- or cyclin D1-positive patients, most had relatively earlier I-II tumor stage (P=0.005 and P=0.047, respectively) and no metastatic lymph node involvement (P=0.008 and P=0.005, respectively). TRX was found to be positively correlated with ER and PR expression, whereas it was negatively correlated with HER2 expression. In addition, we found that the positive expression of AR and cyclin D1 was correlated with lower TNM stage and fewer metastatic lymph nodes, and it was more common in ER-positive breast cancer than in the basal-like subtype. This may indicate that AR and cyclin D1 are good predictive and prognostic factors and closely interact with ER signaling pathway. Further studies will be necessary to investigate the response and clinical outcomes of treatment targeting TRX, AR, and cyclin D1.

  12. Methylation-sensitive linking libraries enhance gene-enriched sequencing of complex genomes and map DNA methylation domains

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    Bharti Arvind K

    2008-12-01

    Full Text Available Abstract Background Many plant genomes are resistant to whole-genome assembly due to an abundance of repetitive sequence, leading to the development of gene-rich sequencing techniques. Two such techniques are hypomethylated partial restriction (HMPR and methylation spanning linker libraries (MSLL. These libraries differ from other gene-rich datasets in having larger insert sizes, and the MSLL clones are designed to provide reads localized to "epigenetic boundaries" where methylation begins or ends. Results A large-scale study in maize generated 40,299 HMPR sequences and 80,723 MSLL sequences, including MSLL clones exceeding 100 kb. The paired end reads of MSLL and HMPR clones were shown to be effective in linking existing gene-rich sequences into scaffolds. In addition, it was shown that the MSLL clones can be used for anchoring these scaffolds to a BAC-based physical map. The MSLL end reads effectively identified epigenetic boundaries, as indicated by their preferential alignment to regions upstream and downstream from annotated genes. The ability to precisely map long stretches of fully methylated DNA sequence is a unique outcome of MSLL analysis, and was also shown to provide evidence for errors in gene identification. MSLL clones were observed to be significantly more repeat-rich in their interiors than in their end reads, confirming the correlation between methylation and retroelement content. Both MSLL and HMPR reads were found to be substantially gene-enriched, with the SalI MSLL libraries being the most highly enriched (31% align to an EST contig, while the HMPR clones exhibited exceptional depletion of repetitive DNA (to ~11%. These two techniques were compared with other gene-enrichment methods, and shown to be complementary. Conclusion MSLL technology provides an unparalleled approach for mapping the epigenetic status of repetitive blocks and for identifying sequences mis-identified as genes. Although the types and natures of

  13. Global DNA methylation and oxidative stress biomarkers in workers exposed to metal oxide nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Liou, Saou-Hsing; Wu, Wei-Te; Liao, Hui-Yi [National Institute of Environmental Health Sciences, National Health Research Institutes, Zhunan, Miaoli County, Taiwan (China); Chen, Chao-Yu; Tsai, Cheng-Yen; Jung, Wei-Ting [Department of Chemistry, Fu Jen Catholic University, New Taipei City, Taiwan (China); Lee, Hui-Ling, E-mail: huilinglee3573@gmail.com [Department of Chemistry, Fu Jen Catholic University, New Taipei City, Taiwan (China)

    2017-06-05

    Highlights: • Global methylation and oxidative DNA damage levels in nanomaterial handling workers were assessed. • 8-isoprostane in exhaled breath condensate of workers exposed to nanoparticles was higher. • 8-OHdG was negatively correlated with global methylation. • Exposure to metal oxide nanoparticles may lead to global methylation and DNA oxidative damage. - Abstract: This is the first study to assess global methylation, oxidative DNA damage, and lipid peroxidation in workers with occupational exposure to metal oxide nanomaterials (NMs). Urinary and white blood cell (WBC) 8-hydroxydeoxyguanosine (8-OHdG), and exhaled breath condensate (EBC) 8-isoprostane were measured as oxidative stress biomarkers. WBC global methylation was measured as an epigenetic alteration. Exposure to TiO{sub 2}, SiO{sub 2,} and indium tin oxide (ITO) resulted in significantly higher oxidative biomarkers such as urinary 8-OHdG and EBC 8-isoprostane. However, significantly higher WBC 8-OHdG and lower global methylation were only observed in ITO handling workers. Significant positive correlations were noted between WBC and urinary 8-OHdG (Spearman correlation r = 0.256, p = 0.003). Furthermore, a significant negative correlation was found between WBC 8-OHdG and global methylation (r = −0.272, p = 0.002). These results suggest that exposure to metal oxide NMs may lead to global methylation, DNA oxidative damage, and lipid peroxidation.

  14. Correlations between disease-specific and generic health status questionnaires in patients with advanced COPD: a one-year observational study

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    Wilke Sarah

    2012-08-01

    Full Text Available Abstract Background Longitudinal studies analyzing the correlations between disease-specific and generic health status questionnaires at different time points in patients with advanced COPD are lacking. The aim of this study was to determine whether and to what extent a disease-specific health status questionnaire (Saint George’s Respiratory Questionnaire, SGRQ correlates with generic health status questionnaires (EuroQol-5-Dimensions, EQ-5D; Assessment of Quality of Life instrument, AQoL; Medical Outcomes Study 36-Item Short-Form Health Survey, SF-36 at four different time points in patients with advanced COPD; and to determine the correlation between the changes in these questionnaires during one-year follow-up. Methods Demographic and clinical characteristics were assessed in 105 outpatients with advanced COPD at baseline. Disease-specific health status (SGRQ and generic health status (EQ-5D, AQoL, SF-36 were assessed at baseline, four, eight, and 12 months. Correlations were determined between SGRQ and EQ-5D, AQoL, and SF-36 scores and changes in these scores. Agreement in direction of change was assessed. Results Eighty-four patients (80% completed one-year follow-up and were included for analysis. SGRQ total score and EQ-5D index score, AQoL total score and SF-36 Physical Component Summary measure (SF-36 PCS score were moderately to strongly correlated. The correlation of the changes between the SGRQ total score and EQ-5D index score, AQoL total score, SF-36 PCS, and SF-36 Mental Component Summary measure (SF-36 MCS score were weak or absent. The direction of changes in SGRQ total scores agreed slightly with the direction of changes in EQ-5D index score, AQoL total score, and SF-36 PCS score. Conclusions At four, eight and 12 months after baseline, SGRQ total scores and EQ-5D index scores, AQoL total scores and SF-36 PCS scores were moderately to strongly correlated, while SGRQ total scores were weakly correlated with SF-36 MCS scores

  15. Genome-wide signatures of differential DNA methylation in pediatric acute lymphoblastic leukemia

    DEFF Research Database (Denmark)

    Nordlund, Jessica; Bäcklin, Christofer L; Wahlberg, Per

    2013-01-01

    BACKGROUND: Although aberrant DNA methylation has been observed previously in acute lymphoblastic leukemia (ALL), the patterns of differential methylation have not been comprehensively determined in all subtypes of ALL on a genome-wide scale. The relationship between DNA methylation, cytogenetic...... background, drug resistance and relapse in ALL is poorly understood. RESULTS: We surveyed the DNA methylation levels of 435,941 CpG sites in samples from 764 children at diagnosis of ALL and from 27 children at relapse. This survey uncovered four characteristic methylation signatures. First, compared...... cells at relapse, compared with matched samples at diagnosis. Analysis of relapse-free survival identified CpG sites with subtype-specific differential methylation that divided the patients into different risk groups, depending on their methylation status. CONCLUSIONS: Our results suggest an important...

  16. Regulation of DNA methylation on EEF1D and RPL8 expression in cattle.

    Science.gov (United States)

    Liu, Xuan; Yang, Jie; Zhang, Qin; Jiang, Li

    2017-10-01

    Dynamic changes to the epigenome play a critical role in a variety of biology processes and complex traits. Many important candidate genes have been identified through our previous genome wide association study (GWAS) on milk production traits in dairy cattle. However, the underlying mechanism of candidate genes have not yet been clearly understood. In this study, we analyzed the methylation variation of the candidate genes, EEF1D and RPL8, which were identified to be strongly associated with milk production traits in dairy cattle in our previous studies, and its effect on protein and mRNA expression. We compared DNA methylation profiles and gene expression levels of EEF1D and RPL8 in five different tissues (heart, liver, mammary gland, ovary and muscle) of three cows. Both genes showed the highest expression level in mammary gland. For RPL8, there was no difference in the DNA methylation pattern in the five tissues, suggesting no effect of DNA methylation on gene expression. For EEF1D, the DNA methylation levels of its first CpG island differed in the five tissues and were negatively correlated with the gene expression levels. To further investigate the function of DNA methylation on the expression of EEF1D, we collected blood samples of three cows at early stage of lactation and in dry period and analyzed its expression and the methylation status of the first CpG island in blood. As a result, the mRNA expression of EEF1D in the dry period was higher than that at the early stage of lactation, while the DNA methylation level in the dry period was lower than that at the early stage of lactation. Our result suggests that the DNA methylation of EEF1D plays an important role in the spatial and temporal regulation of its expression and possibly have an effect on the milk production traits.

  17. Methyl-Deficient Diets and Risks of Breast Cancer Among African-American Women: A Case-Control Study by Methylation Status of the ER Gene

    National Research Council Canada - National Science Library

    Zhu, Kangmin

    2001-01-01

    This is the final report of our case-control study testing the hypothesis that methyl-deficient diets are more likely to be related to breast cancer with methylated CpG islands of the estrogen-receptor (ER) gene...

  18. Skeletal Muscle PGC1α -1 Nucleosome Position and -260 nt DNA Methylation Determine Exercise Response and Prevent Ectopic Lipid Accumulation in Men.

    Science.gov (United States)

    Bajpeyi, Sudip; Covington, Jeffrey D; Taylor, Erin M; Stewart, Laura K; Galgani, Jose E; Henagan, Tara M

    2017-07-01

    Endurance exercise has been shown to improve lipid oxidation and increase mitochondrial content in skeletal muscle, two features that have shown dependence on increased expression of the peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α). It is also hypothesized that exercise-related alterations in PGC1α expression occur through epigenetic regulation of nucleosome positioning in association with differential DNA methylation status within the PGC1α promoter. In this study, we show that when primary human myotubes from obese patients with type 2 diabetes are exposed to lipolytic stimulus (palmitate, forskolin, inomycin) in vitro, nucleosome occupancy surrounding the -260 nucleotide (nt) region, a known regulatory DNA methylation site, is reduced. This finding is reproduced in vivo in the vastus lateralis from 11 healthy males after a single, long endurance exercise bout in which participants expended 650 kcal. Additionally, we show a significant positive correlation between fold change of PGC1α messenger RNA expression and -1 nucleosome repositioning away from the -260 nt methylation site in skeletal muscle tissue following exercise. Finally, we found that when exercise participants are divided into high and low responders based on the -260 nt methylation status, the -1 nucleosome is repositioned away from the regulatory -260 nt methylation site in high responders, those exhibiting a significant decrease in -260 nt methylation, but not in low responders. Additionally, high but not low responders showed a significant decrease in intramyocellular lipid content after exercise. These findings suggest a potential target for epigenetic modification of the PGC1α promoter to stimulate the therapeutic effects of endurance exercise in skeletal muscle. Copyright © 2017 Endocrine Society.

  19. Vascular measurements correlate with estrogen receptor status

    International Nuclear Information System (INIS)

    Lloyd, Mark C; Alfarouk, Khalid O; Verduzco, Daniel; Bui, Marilyn M; Gillies, Robert J; Ibrahim, Muntaser E; Brown, Joel S; Gatenby, Robert A

    2014-01-01

    Breast carcinoma can be classified as either Estrogen Receptor (ER) positive or negative by immunohistochemical phenotyping, although ER expression may vary from 1 to 100% of malignant cells within an ER + tumor. This is similar to genetic variability observed in other tumor types and is generally viewed as a consequence of intratumoral evolution driven by random genetic mutations. Here we view cellular evolution within tumors as a classical Darwinian system in which variations in molecular properties represent predictable adaptations to spatially heterogeneous environmental selection forces. We hypothesize that ER expression is a successful adaptive strategy only if estrogen is present in the microenvironment. Since the dominant source of estrogen is blood flow, we hypothesized that, in general, intratumoral regions with higher blood flow would contain larger numbers of ER + cells when compared to areas of low blood flow and in turn necrosis. This study used digital pathology whole slide image acquisition and advanced image analysis algorithms. We examined the spatial distribution of ER + and ER- cells, vascular density, vessel area, and tissue necrosis within histological sections of 24 breast cancer specimens. These data were correlated with the patients ER status and molecular pathology report findings. ANOVA analyses revealed a strong correlation between vascular area and ER expression and between high fractional necrosis and absent ER expression (R 2 = 39%; p < 0.003 and R 2 = 46%; p < 0.001), respectively). ER expression did not correlate with tumor grade or size. We conclude that ER expression can be understood as a Darwinian process and linked to variations in estrogen delivery by temporal and spatial heterogeneity in blood flow. This correlation suggests strategies to promote intratumoral blood flow or a cyclic introduction of estrogen in the treatment schedule could be explored as a counter-intuitive approach to increase the efficacy of anti

  20. Different diagnostic values of imaging parameters to predict pseudoprogression in glioblastoma subgroups stratified by MGMT promoter methylation

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Ra Gyoung [Catholic Kwandong University International St. Mary' s Hospital, Department of Radiology, Catholic Kwandong University College of Medicine, Incheon (Korea, Republic of); Kim, Ho Sung; Shim, Woo Hyun; Kim, Sang Joon [University of Ulsan College of Medicine, Asan Medical Center, Department of Radiology and Research Institute of Radiology, Seoul (Korea, Republic of); Paik, Wooyul [Dankook Unversity Hospital, Department of Radiology, Cheonan-si, Chungcheongnam-do (Korea, Republic of); Kim, Jeong Hoon [University of Ulsan College of Medicine, Asan Medical Center, Department of Neurosurgery, Seoul (Korea, Republic of)

    2017-01-15

    The aim of this study was to determine whether diffusion and perfusion imaging parameters demonstrate different diagnostic values for predicting pseudoprogression between glioblastoma subgroups stratified by O{sup 6}-mythylguanine-DNA methyltransferase (MGMT) promoter methylation status. We enrolled seventy-five glioblastoma patients that had presented with enlarged contrast-enhanced lesions on magnetic resonance imaging (MRI) one month after completing concurrent chemoradiotherapy and undergoing MGMT promoter methylation testing. The imaging parameters included 10 or 90 % histogram cutoffs of apparent diffusion coefficient (ADC10), normalized cerebral blood volume (nCBV90), and initial area under the time signal-intensity curve (IAUC90). The results of the areas under the receiver operating characteristic curve (AUCs) with cross-validation were compared between MGMT methylation and unmethylation groups. MR imaging parameters demonstrated a trend toward higher accuracy in the MGMT promoter methylation group than in the unmethylation group (cross-validated AUCs = 0.70-0.95 and 0.56-0.87, respectively). The combination of MGMT methylation status with imaging parameters improved the AUCs from 0.70 to 0.75-0.90 for both readers in comparison with MGMT methylation status alone. The probability of pseudoprogression was highest (95.7 %) when nCBV90 was below 4.02 in the MGMT promoter methylation group. MR imaging parameters could be stronger predictors of pseudoprogression in glioblastoma patients with the methylated MGMT promoter than in patients with the unmethylated MGMT promoter. (orig.)

  1. Methyl-Analyzer--whole genome DNA methylation profiling.

    Science.gov (United States)

    Xin, Yurong; Ge, Yongchao; Haghighi, Fatemeh G

    2011-08-15

    Methyl-Analyzer is a python package that analyzes genome-wide DNA methylation data produced by the Methyl-MAPS (methylation mapping analysis by paired-end sequencing) method. Methyl-MAPS is an enzymatic-based method that uses both methylation-sensitive and -dependent enzymes covering >80% of CpG dinucleotides within mammalian genomes. It combines enzymatic-based approaches with high-throughput next-generation sequencing technology to provide whole genome DNA methylation profiles. Methyl-Analyzer processes and integrates sequencing reads from methylated and unmethylated compartments and estimates CpG methylation probabilities at single base resolution. Methyl-Analyzer is available at http://github.com/epigenomics/methylmaps. Sample dataset is available for download at http://epigenomicspub.columbia.edu/methylanalyzer_data.html. fgh3@columbia.edu Supplementary data are available at Bioinformatics online.

  2. Promoter methylation of APC and RAR-β genes as prognostic markers in non-small cell lung cancer (NSCLC).

    Science.gov (United States)

    Feng, Hongxiang; Zhang, Zhenrong; Qing, Xin; Wang, Xiaowei; Liang, Chaoyang; Liu, Deruo

    2016-02-01

    Aberrant promoter hypermethylations of tumor suppressor genes are promising markers for lung cancer diagnosis and prognosis. The purpose of this study was to determine methylation status at APC and RAR-β promoters in primary NSCLC, and whether they have any relationship with survival. APC and RAR-β promoter methylation status were determined in 41 NSCLC patients using methylation specific PCR. APC promoter methylation was detectable in 9 (22.0%) tumor samples and 6 (14.6%) corresponding non-tumor samples (P=0.391). RAR-β promoter methylation was detectable in 13 (31.7%) tumor samples and 4 (9.8%) corresponding non-tumor samples (P=0.049) in the NSCLC patients. APC promoter methylation was found to be associated with T stage (P=0.046) and nodal status (P=0.019) in non-tumor samples, and with smoking (P=0.004) in tumor samples. RAR-β promoter methylation was found associated with age (P=0.031) in non-tumor samples and with primary tumor site in tumor samples. Patients with APC promoter methylation in tumor samples showed significantly longer survival than patients without it (Log-rank P=0.014). In a multivariate analysis of prognostic factors, APC methylation in tumor samples was an independent prognostic factor (P=0.012), as were N1 positive lymph node number (P=0.025) and N2 positive lymph node number (P=0.06). Our study shows that RAR-β methylation detected in lung tissue may be used as a predictive marker for NSCLC diagnosis and that APC methylation in tumor sample may be a useful marker for superior survival in NSCLC patients. Copyright © 2015. Published by Elsevier Inc.

  3. Genome-wide Differences in DNA Methylation Changes in Two Contrasting Rice Genotypes in Response to Drought Conditions

    Directory of Open Access Journals (Sweden)

    Wensheng Wang

    2016-11-01

    Full Text Available Differences in drought stress tolerance within diverse rice genotypes have been attributed to genetic diversity and epigenetic alterations. DNA methylation is an important epigenetic modification that influences diverse biological processes, but its effects on rice drought stress tolerance are poorly understood. In this study, methylated DNA immunoprecipitation sequencing and an Affymetrix GeneChip rice genome array were used to profile the DNA methylation patterns and transcriptomes of the drought-tolerant introgression line DK151 and its drought-sensitive recurrent parent IR64 under drought and control conditions. The introgression of donor genomic DNA induced genome-wide DNA methylation changes in DK151 plants. A total of 1190 differentially methylated regions (DMRs were detected between the two genotypes under normal growth conditions, and the DMR-associated genes in DK151 plants were mainly related to stress response, programmed cell death, and nutrient reservoir activity, which are implicated to constitutive drought stress tolerance. A comparison of the DNA methylation changes in the two genotypes under drought conditions indicated that DK151 plants have a more stable methylome, with only 92 drought-induced DMRs, than IR64 plants with 506 DMRs. Gene ontology analyses of the DMR-associated genes in drought-stressed plants revealed that changes to the DNA methylation status of genotype-specific genes are associated with the epigenetic regulation of drought stress responses. Transcriptome analysis further helped to identify a set of 12 and 23 DMR-associated genes that were differentially expressed in DK151 and IR64, respectively, under drought stress compared with respective controls. Correlation analysis indicated that DNA methylation has various effects on gene expression, implying that it affects gene expression directly or indirectly through diverse regulatory pathways. Our results indicate that drought-induced alterations to DNA

  4. Expression and methylation of BDNF in the human brain in schizophrenia.

    Science.gov (United States)

    Cheah, Sern-Yih; McLeay, Robert; Wockner, Leesa F; Lawford, Bruce R; Young, Ross McD; Morris, Charles P; Voisey, Joanne

    2017-08-01

    To examine the combined effect of the BDNF Val66Met (rs6265) polymorphism and BDNF DNA methylation on transcriptional regulation of the BDNF gene. DNA methylation profiles were generated for CpG sites proximal to Val66Met, within BDNF promoter I and exon V for prefrontal cortex samples from 25 schizophrenia and 25 control subjects. Val66Met genotypes and BDNF mRNA expression data were generated by transcriptome sequencing. Expression, methylation and genotype data were correlated and examined for association with schizophrenia. There was 43% more of the BDNF V-VIII-IX transcript in schizophrenia samples. BDNF mRNA expression and DNA methylation of seven CpG sites were not associated with schizophrenia after accounting for age and PMI effects. BDNF mRNA expression and DNA methylation were not altered by Val66Met after accounting for age and PMI effects. DNA methylation of one CpG site had a marginally significant positive correlation with mRNA expression in schizophrenia subjects. Schizophrenia risk was not associated with differential BDNF mRNA expression and DNA methylation. A larger age-matched cohort with comprehensive clinical history is required to accurately identify the effects of genotype, mRNA expression and DNA methylation on schizophrenia risk.

  5. Links between DNA methylation and nucleosome occupancy in the human genome.

    Science.gov (United States)

    Collings, Clayton K; Anderson, John N

    2017-01-01

    DNA methylation is an epigenetic modification that is enriched in heterochromatin but depleted at active promoters and enhancers. However, the debate on whether or not DNA methylation is a reliable indicator of high nucleosome occupancy has not been settled. For example, the methylation levels of DNA flanking CTCF sites are higher in linker DNA than in nucleosomal DNA, while other studies have shown that the nucleosome core is the preferred site of methylation. In this study, we make progress toward understanding these conflicting phenomena by implementing a bioinformatics approach that combines MNase-seq and NOMe-seq data and by comprehensively profiling DNA methylation and nucleosome occupancy throughout the human genome. The results demonstrated that increasing methylated CpG density is correlated with nucleosome occupancy in the total genome and within nearly all subgenomic regions. Features with elevated methylated CpG density such as exons, SINE-Alu sequences, H3K36-trimethylated peaks, and methylated CpG islands are among the highest nucleosome occupied elements in the genome, while some of the lowest occupancies are displayed by unmethylated CpG islands and unmethylated transcription factor binding sites. Additionally, outside of CpG islands, the density of CpGs within nucleosomes was shown to be important for the nucleosomal location of DNA methylation with low CpG frequencies favoring linker methylation and high CpG frequencies favoring core particle methylation. Prominent exceptions to the correlations between methylated CpG density and nucleosome occupancy include CpG islands marked by H3K27me3 and CpG-poor heterochromatin marked by H3K9me3, and these modifications, along with DNA methylation, distinguish the major silencing mechanisms of the human epigenome. Thus, the relationship between DNA methylation and nucleosome occupancy is influenced by the density of methylated CpG dinucleotides and by other epigenomic components in chromatin.

  6. Molecular Classification and Correlates in Colorectal Cancer

    OpenAIRE

    Ogino, Shuji; Goel, Ajay

    2008-01-01

    Molecular classification of colorectal cancer is evolving. As our understanding of colorectal carcinogenesis improves, we are incorporating new knowledge into the classification system. In particular, global genomic status [microsatellite instability (MSI) status and chromosomal instability (CIN) status] and epigenomic status [CpG island methylator phenotype (CIMP) status] play a significant role in determining clinical, pathological and biological characteristics of colorectal cancer. In thi...

  7. The Fine LINE: Methylation Drawing the Cancer Landscape

    Directory of Open Access Journals (Sweden)

    Isabelle R. Miousse

    2015-01-01

    Full Text Available LINE-1 (L1 is the most abundant mammalian transposable element that comprises nearly 20% of the genome, and nearly half of the mammalian genome has stemmed from L1-mediated mobilization. Expression and retrotransposition of L1 are suppressed by complex mechanisms, where the key role belongs to DNA methylation. Alterations in L1 methylation may lead to aberrant expression of L1 and have been described in numerous diseases. Accumulating evidence clearly indicates that loss of global DNA methylation observed in cancer development and progression is tightly associated with hypomethylation of L1 elements. Significant progress achieved in the last several years suggests that such parameters as L1 methylation status can be potentially utilized as clinical biomarkers for determination of the disease stage and in predicting the disease-free survival in cancer patients. In this paper, we summarize the current knowledge on L1 methylation, with specific emphasis given to success and challenges on the way of introduction of L1 into clinical practice.

  8. The dynamic changes of DNA methylation and histone modifications of salt responsive transcription factor genes in soybean.

    Directory of Open Access Journals (Sweden)

    Yuguang Song

    Full Text Available Epigenetic modification contributes to the regulation of gene expression and plant development under salinity stress. Here we describe the identification of 49 soybean transcription factors by microarray analysis as being inducible by salinity stress. A semi-quantitative RT-PCR-based expression assay confirmed the salinity stress inducibility of 45 of these 49 transcription factors, and showed that ten of them were up-regulated when seedlings were exposed to the demethylation agent 5-aza-2-deoxycytidine. Salinity stress was shown to affect the methylation status of four of these ten transcription factors (one MYB, one b-ZIP and two AP2/DREB family members using a combination of bisulfite sequencing and DNA methylation-sensitive DNA gel blot analysis. ChIP analysis indicated that the activation of three of the four DNA methylated transcription factors was correlated with an increased level of histone H3K4 trimethylation and H3K9 acetylation, and/or a reduced level of H3K9 demethylation in various parts of the promoter or coding regions. Our results suggest a critical role for some transcription factors' activation/repression by DNA methylation and/or histone modifications in soybean tolerance to salinity stress.

  9. Methylation associated inactivation of RASSF1A and its synergistic effect with activated K-Ras in nasopharyngeal carcinoma

    Directory of Open Access Journals (Sweden)

    Yu Jing

    2009-12-01

    Full Text Available Abstract Background Epigenetic silencing of tumor suppressor genes associated with promoter methylation is considered to be a hallmark of oncogenesis. RASSF1A is a candidate tumor suppressor gene which was found to be inactivated in many human cancers. Although we have had a prelimilary cognition about the function of RASSF1A, the exact mechanisms about how RASSF1A functions in human cancers were largely unknown. Moreover, the effect of mutated K-Ras gene on the function of RASSF1A is lacking. The aim of this study was to investigate the expression profile and methylation status of RASSF1A gene, and to explore its concrete mechanisms as a tumor suppressor gene in Nasopharyngeal Carcinoma. Methods We examined the expression profile and methylation status of RASSF1A in two NPC cell lines, 38 primary nasopharyngeal carcinoma and 14 normal nasopharyngeal epithelia using RT-PCR and methylated specific PCR(MSP respectively. 5-aza-dC was then added to confirm the correlation between hypermethylation status and inactivation of RASSF1A. The NPC cell line CNE-2 was transfected with exogenous pcDNA3.1(+/RASSF1A plasmid in the presence or absence of mutated K-Ras by liposome-mediated gene transfer method. Flow cytometry was used to examine the effect of RASSF1A on cell cycle modulation and apoptosis. Meanwhile, trypan blue dye exclusion assays was used to detect the effect of RASSF1A transfection alone and the co-transfection of RASSF1A and K-Ras on cell proliferation. Results Promoter methylation of RASSF1A could be detected in 71.05% (27/38 of NPC samples, but not in normal nasopharyngeal epithelia. RASSF1A expression in NPC primary tumors was lower than that in normal nasopharyngeal epithelial (p p p p Conclusion Expression of RASSF1A is down-regulated in NPC due to the hypermethylation of promoter. Exogenous expression of RASSF1A is able to induce growth inhibition effect and apoptosis in tumor cell lines, and this effect could be enhanced by activated

  10. Donor age and C1orf132/MIR29B2C determine age-related methylation signature of blood after allogeneic hematopoietic stem cell transplantation.

    Science.gov (United States)

    Spólnicka, Magdalena; Piekarska, Renata Zbieć; Jaskuła, Emilia; Basak, Grzegorz W; Jacewicz, Renata; Pięta, Agnieszka; Makowska, Żanetta; Jedrzejczyk, Maciej; Wierzbowska, Agnieszka; Pluta, Agnieszka; Robak, Tadeusz; Berent, Jarosław; Branicki, Wojciech; Jędrzejczak, Wiesław; Lange, Andrzej; Płoski, Rafał

    2016-01-01

    Our recent study demonstrated that DNA methylation status in a set of CpGs located in ELOVL2, C1orf132, TRIM59, KLF14, and FHL2 can accurately predict calendar age in blood. In the present work, we used these markers to evaluate the effect of allogeneic hematopoietic stem cell transplantation (HSCT) on the age-related methylation signature of human blood. DNA methylation in 32 CpGs was investigated in 16 donor-recipient pairs using pyrosequencing. DNA was isolated from the whole blood collected from recipients 27-360 days (mean 126) after HSCT and from the donors shortly before the HSCT. It was found that in the recipients, the predicted age did not correlate with their calendar age but was correlated with the calendar age (r = 0.94, p = 4 × 10(-8)) and predicted age (r = 0.97, p = 5 × 10(-10)) of a respective donor. Despite this strong correlation, the predicted age of a recipient was consistently lower than the predicted age of a donor by 3.7 years (p = 7.8 × 10(-4)). This shift was caused by hypermethylation of the C1orf132 CpGs, for C1orf132 CpG_1. Intriguingly, the recipient-donor methylation difference correlated with calendar age of the donor (r = 0.76, p = 6 × 10(-4)). This finding could not trivially be explained by shifts of the major cellular factions of blood. We confirm the single previous report that after HSCT, the age of the donor is the major determinant of age-specific methylation signature in recipient's blood. A novel finding is the unique methylation dynamics of C1orf132 which encodes MIR29B2C implicated in the self-renewing of hematopoietic stem cells. This observation suggests that C1orf132 could influence graft function after HSCT.

  11. Using a medium-throughput comet assay to evaluate the global DNA methylation status of single cells

    Science.gov (United States)

    Lewies, Angélique; Van Dyk, Etresia; Wentzel, Johannes F.; Pretorius, Pieter J.

    2014-01-01

    The comet assay is a simple and cost effective technique, commonly used to analyze and quantify DNA damage in individual cells. The versatility of the comet assay allows introduction of various modifications to the basic technique. The difference in the methylation sensitivity of the isoschizomeric restriction enzymes HpaII and MspI are used to demonstrate the ability of the comet assay to measure the global DNA methylation level of individual cells when using cell cultures. In the experiments described here, a medium-throughput comet assay and methylation sensitive comet assay are combined to produce a methylation sensitive medium-throughput comet assay to measure changes in the global DNA methylation pattern in individual cells under various growth conditions. PMID:25071840

  12. Regulation and function of DNA methylation in plants and animals

    KAUST Repository

    He, Xinjian

    2011-02-15

    DNA methylation is an important epigenetic mark involved in diverse biological processes. In plants, DNA methylation can be established through the RNA-directed DNA methylation pathway, an RNA interference pathway for transcriptional gene silencing (TGS), which requires 24-nt small interfering RNAs. In mammals, de novo DNA methylation occurs primarily at two developmental stages: during early embryogenesis and during gametogenesis. While it is not clear whether establishment of DNA methylation patterns in mammals involves RNA interference in general, de novo DNA methylation and suppression of transposons in germ cells require 24-32-nt piwi-interacting small RNAs. DNA methylation status is dynamically regulated by DNA methylation and demethylation reactions. In plants, active DNA demethylation relies on the repressor of silencing 1 family of bifunctional DNA glycosylases, which remove the 5-methylcytosine base and then cleave the DNA backbone at the abasic site, initiating a base excision repair (BER) pathway. In animals, multiple mechanisms of active DNA demethylation have been proposed, including a deaminase- and DNA glycosylase-initiated BER pathway. New information concerning the effects of various histone modifications on the establishment and maintenance of DNA methylation has broadened our understanding of the regulation of DNA methylation. The function of DNA methylation in plants and animals is also discussed in this review. © 2011 IBCB, SIBS, CAS All rights reserved.

  13. High fructose consumption induces DNA methylation at PPARα and CPT1A promoter regions in the rat liver

    Energy Technology Data Exchange (ETDEWEB)

    Ohashi, Koji [Department of Clinical Biochemistry, Fujita Health University School of Health Sciences, Toyoake (Japan); Munetsuna, Eiji [Department of Biochemistry, Fujita Health University School of Medicine, Toyoake (Japan); Yamada, Hiroya, E-mail: hyamada@fujita-hu.ac.jp [Department of Hygiene, Fujita Health University School of Medicine, Toyoake (Japan); Ando, Yoshitaka [Department of Joint Research Laboratory of Clinical Medicine, Fujita Health University Hospital, Toyoake (Japan); Yamazaki, Mirai; Taromaru, Nao; Nagura, Ayuri; Ishikawa, Hiroaki [Department of Clinical Biochemistry, Fujita Health University School of Health Sciences, Toyoake (Japan); Suzuki, Koji [Department of Public Health, Fujita Health University School of Health Sciences, Toyoake (Japan); Teradaira, Ryoji [Department of Clinical Biochemistry, Fujita Health University School of Health Sciences, Toyoake (Japan); Hashimoto, Shuji [Department of Hygiene, Fujita Health University School of Medicine, Toyoake (Japan)

    2015-12-04

    DNA methylation status is affected by environmental factors, including nutrition. Fructose consumption is considered a risk factor for the conditions that make up metabolic syndrome such as dyslipidemia. However, the pathogenetic mechanism by which fructose consumption leads to metabolic syndrome is unclear. Based on observations that epigenetic modifications are closely related to induction of metabolic syndrome, we hypothesized that fructose-induced metabolic syndrome is caused by epigenetic alterations. Male SD rats were designated to receive water or 20% fructose solution for 14 weeks. mRNA levels for peroxisome proliferator-activated receptor alpha (PPARα) and carnitine palmitoyltransferase 1A (CPT1A) was analyzed using Real-time PCR. Restriction digestion and real-time PCR (qAMP) was used for the analysis of DNA methylation status. Hepatic lipid accumulation was also observed by fructose intake. Fructose feeding also significantly decreased mRNA levels for PPARα and CPT1A. qAMP analysis demonstrated the hypermethylation of promoter regions of PPARα and CTP1A genes. Fructose-mediated attenuated gene expression may be mediated by alterations of DNA methylation status, and pathogenesis of metabolic syndrome induced by fructose relates to DNA methylation status. - Highlights: • No general consensus has been reached regarding the molecular mechanisms of the pathogenesis of fructose-induced diseases. • Significant increase in hepatic total methylation level was observed after fructose-supplemented feeding. • Fructose feeding significantly decreased mRNA levels for PPARα and CPT1A. • qAMP analysis demonstrated the hypermethylation of promoter regions of PPARα and CTP1A genes. • Fructose-mediated attenuated gene expression may be mediated by alterations of DNA methylation status in rat liver.

  14. High fructose consumption induces DNA methylation at PPARα and CPT1A promoter regions in the rat liver

    International Nuclear Information System (INIS)

    Ohashi, Koji; Munetsuna, Eiji; Yamada, Hiroya; Ando, Yoshitaka; Yamazaki, Mirai; Taromaru, Nao; Nagura, Ayuri; Ishikawa, Hiroaki; Suzuki, Koji; Teradaira, Ryoji; Hashimoto, Shuji

    2015-01-01

    DNA methylation status is affected by environmental factors, including nutrition. Fructose consumption is considered a risk factor for the conditions that make up metabolic syndrome such as dyslipidemia. However, the pathogenetic mechanism by which fructose consumption leads to metabolic syndrome is unclear. Based on observations that epigenetic modifications are closely related to induction of metabolic syndrome, we hypothesized that fructose-induced metabolic syndrome is caused by epigenetic alterations. Male SD rats were designated to receive water or 20% fructose solution for 14 weeks. mRNA levels for peroxisome proliferator-activated receptor alpha (PPARα) and carnitine palmitoyltransferase 1A (CPT1A) was analyzed using Real-time PCR. Restriction digestion and real-time PCR (qAMP) was used for the analysis of DNA methylation status. Hepatic lipid accumulation was also observed by fructose intake. Fructose feeding also significantly decreased mRNA levels for PPARα and CPT1A. qAMP analysis demonstrated the hypermethylation of promoter regions of PPARα and CTP1A genes. Fructose-mediated attenuated gene expression may be mediated by alterations of DNA methylation status, and pathogenesis of metabolic syndrome induced by fructose relates to DNA methylation status. - Highlights: • No general consensus has been reached regarding the molecular mechanisms of the pathogenesis of fructose-induced diseases. • Significant increase in hepatic total methylation level was observed after fructose-supplemented feeding. • Fructose feeding significantly decreased mRNA levels for PPARα and CPT1A. • qAMP analysis demonstrated the hypermethylation of promoter regions of PPARα and CTP1A genes. • Fructose-mediated attenuated gene expression may be mediated by alterations of DNA methylation status in rat liver.

  15. DNA damage, homology-directed repair, and DNA methylation.

    Directory of Open Access Journals (Sweden)

    Concetta Cuozzo

    2007-07-01

    Full Text Available To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP genes (DR-GFP. A total of 2%-4% of the cells generated a functional GFP by homology-directed repair (HR and gene conversion. However, approximately 50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2'-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments.

  16. Colorectal Cancer "Methylator Phenotype": Fact or Artifact?

    Directory of Open Access Journals (Sweden)

    Charles Anacleto

    2005-04-01

    Full Text Available It has been proposed that human colorectal tumors can be classified into two groups: one in which methylation is rare, and another with methylation of several loci associated with a "CpG island methylated phenotype (CIMP," characterized by preferential proximal location in the colon, but otherwise poorly defined. There is considerable overlap between this putative methylator phenotype and the well-known mutator phenotype associated with microsatellite instability (MSI. We have examined hypermethylation of the promoter region of five genes (DAPK, MGMT, hMLH1, p16INK4a, and p14ARF in 106 primary colorectal cancers. A graph depicting the frequency of methylated loci in the series of tumors showed a continuous, monotonically decreasing distribution quite different from the previously claimed discontinuity. We observed a significant association between the presence of three or more methylated loci and the proximal location of the tumors. However, if we remove from analysis the tumors with hMLH1 methylation or those with MSI, the significance vanishes, suggesting that the association between multiple methylations and proximal location was indirect due to the correlation with MSI. Thus, our data do not support the independent existence of the so-called methylator phenotype and suggest that it rather may represent a statistical artifact caused by confounding of associations.

  17. MGMT and MLH1 methylation in Helicobacter pylori-infected children and adults.

    Science.gov (United States)

    Alvarez, Marisa C; Santos, Juliana C; Maniezzo, Nathália; Ladeira, Marcelo S; da Silva, Artur L C; Scaletsky, Isabel C A; Pedrazzoli, José; Ribeiro, Marcelo L

    2013-05-28

    To evaluate the association between Helicobacter pylori (H. pylori) infection and MLH1 and MGMT methylation and its relationship with microsatellite instability (MSI). The methylation status of the MLH1 and MGMT promoter region was analysed by methylation specific methylation-polymerase chain reaction (MSP-PCR) in gastric biopsy samples from uninfected or H. pylori-infected children (n = 50), from adults with chronic gastritis (n = 97) and from adults with gastric cancer (n = 92). MLH1 and MGMT mRNA expression were measured by real-time PCR and normalised to a constitutive gene (β actin). MSI analysis was performed by screening MSI markers at 4 loci (Bat-25, Bat-26, D17S250 and D2S123) with PCR; PCR products were analysed by single strand conformation polymorphism followed by silver staining. Statistical analyses were performed with either the χ(2) test with Yates continuity correction or Fisher's exact test, and statistical significance for expression analysis was assessed using an unpaired Student's t-test. Methylation was not detected in the promoter regions of MLH1 and MGMT in gastric biopsy samples from children, regardless of H. pylori infection status. The MGMT promoter was methylated in 51% of chronic gastritis adult patients and was associated with H. pylori infection (P MLH1 methylation frequencies among H. pylori-infected and non-infected chronic gastritis adult patients were 13% and 7%, respectively. We observed methylation of the MLH1 promoter (39%) and increased MSI levels (68%) in samples from gastric cancer patients in comparison to samples from H. pylori-infected adult chronic gastritis patients (P MLH1 and MGMT mRNA were significantly reduced in chronic gastritis samples that were also hypermethylated (P MLH1 methylation did not occur in earlier-stage H. pylori infections and thus might depend on the duration of infection.

  18. Overexpression of Human-Derived DNMT3A Induced Intergenerational Inheritance of Active DNA Methylation Changes in Rat Sperm

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    Xiaoguo Zheng

    2017-12-01

    Full Text Available DNA methylation is the major focus of studies on paternal epigenetic inheritance in mammals, but most previous studies about inheritable DNA methylation changes are passively induced by environmental factors. However, it is unclear whether the active changes mediated by variations in DNA methyltransferase activity are heritable. Here, we established human-derived DNMT3A (hDNMT3A transgenic rats to study the effect of hDNMT3A overexpression on the DNA methylation pattern of rat sperm and to investigate whether this actively altered DNA methylation status is inheritable. Our results revealed that hDNMT3A was overexpressed in the testis of transgenic rats and induced genome-wide alterations in the DNA methylation pattern of rat sperm. Among 5438 reliable loci identified with 64 primer-pair combinations using a methylation-sensitive amplification polymorphism method, 28.01% showed altered amplified band types. Among these amplicons altered loci, 68.42% showed an altered DNA methylation status in the offspring of transgenic rats compared with wild-type rats. Further analysis based on loci which had identical DNA methylation status in all three biological replicates revealed that overexpression of hDNMT3A in paternal testis induced hypermethylation in sperm of both genotype-negative and genotype-positive offspring. Among the differentially methylated loci, 34.26% occurred in both positive and negative offspring of transgenic rats, indicating intergenerational inheritance of active DNA methylation changes in the absence of hDNM3A transmission. Furthermore, 75.07% of the inheritable loci were hyper-methylated while the remaining were hypomethylated. Distribution analysis revealed that the DNA methylation variations mainly occurred in introns and intergenic regions. Functional analysis revealed that genes related to differentially methylated loci were involved in a wide range of functions. Finally, this study demonstrated that active DNA methylation

  19. CpG Methylation Analysis—Current Status of Clinical Assays and Potential Applications in Molecular Diagnostics

    Science.gov (United States)

    Sepulveda, Antonia R.; Jones, Dan; Ogino, Shuji; Samowitz, Wade; Gulley, Margaret L.; Edwards, Robin; Levenson, Victor; Pratt, Victoria M.; Yang, Bin; Nafa, Khedoudja; Yan, Liying; Vitazka, Patrick

    2009-01-01

    Methylation of CpG islands in gene promoter regions is a major molecular mechanism of gene silencing and underlies both cancer development and progression. In molecular oncology, testing for the CpG methylation of tissue DNA has emerged as a clinically useful tool for tumor detection, outcome prediction, and treatment selection, as well as for assessing the efficacy of treatment with the use of demethylating agents and monitoring for tumor recurrence. In addition, because CpG methylation occurs early in pre-neoplastic tissues, methylation tests may be useful as markers of cancer risk in patients with either infectious or inflammatory conditions. The Methylation Working Group of the Clinical Practice Committee of the Association of Molecular Pathology has reviewed the current state of clinical testing in this area. We report here our summary of both the advantages and disadvantages of various methods, as well as the needs for standardization and reporting. We then conclude by summarizing the most promising areas for future clinical testing in cancer molecular diagnostics. PMID:19541921

  20. Osteoponin Promoter Controlled by DNA Methylation: Aberrant Methylation in Cloned Porcine Genome

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    Chih-Jie Shen

    2014-01-01

    Full Text Available Cloned animals usually exhibited many defects in physical characteristics or aberrant epigenetic reprogramming, especially in some important organ development. Osteoponin (OPN is an extracellular-matrix protein involved in heart and bone development and diseases. In this study, we investigated the correlation between OPN mRNA and its promoter methylation changes by the 5-aza-dc treatment in fibroblast cell and promoter assay. Aberrant methylation of porcine OPN was frequently found in different tissues of somatic nuclear transferred cloning pigs, and bisulfite sequence data suggested that the OPN promoter region −2615 to −2239 nucleotides (nt may be a crucial regulation DNA element. In pig ear fibroblast cell culture study, the demethylation of OPN promoter was found in dose-dependent response of 5-aza-dc treatment and followed the OPN mRNA reexpression. In cloned pig study, discrepant expression pattern was identified in several cloned pig tissues, especially in brain, heart, and ear. Promoter assay data revealed that four methylated CpG sites presenting in the −2615 to −2239 nt region cause significant downregulation of OPN promoter activity. These data suggested that methylation in the OPN promoter plays a crucial role in the regulation of OPN expression that we found in cloned pigs genome.

  1. Endogenous isoflavone methylation correlates with the in vitro rooting phases of Spartium junceum L. (Leguminosae).

    Science.gov (United States)

    Clematis, Francesca; Viglione, Serena; Beruto, Margherita; Lanzotti, Virginia; Dolci, Paola; Poncet, Christine; Curir, Paolo

    2014-09-01

    Spartium junceum L. (Leguminosae) is a perennial shrub, native to the Mediterranean region in southern Europe, widespread in all the Italian regions and, as a leguminous species, it has a high isoflavone content. An in vitro culture protocol was developed for this species starting from stem nodal sections of in vivo plants, and isoflavone components of the in vitro cultured tissues were studied by means of High Performance Liquid Chromatography (HPLC) analytical techniques. Two main isoflavones were detected in the S. junceum tissues during the in vitro propagation phases: Genistein (4',5,7-Trihydroxyisoflavone), already reported in this species, and its methylated form 4',5,7-Trimethoxyisoflavone, detected for the first time in this plant species (0.750 ± 0.02 mg g(-1) dry tissue). The presence of both of these compounds in S. junceum tissues was consistently detected during the in vitro multiplication phase. The absence of the methylated form within plant tissues in the early phases of the in vitro adventitious root formation was correlated with its negative effect displayed on root induction and initiation phases, while its presence in the final "root manifestation" phase influenced positively the rooting process. The unmethylated form, although detectable in tissues in the precocious rooting phases, was no longer present in the final rooting phase. Its effect on rooting, however, proved always to be beneficial. Copyright © 2014 Elsevier GmbH. All rights reserved.

  2. Microarray-based DNA methylation study of Ewing's sarcoma of the bone.

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    Park, Hye-Rim; Jung, Woon-Won; Kim, Hyun-Sook; Park, Yong-Koo

    2014-10-01

    Alterations in DNA methylation patterns are a hallmark of malignancy. However, the majority of epigenetic studies of Ewing's sarcoma have focused on the analysis of only a few candidate genes. Comprehensive studies are thus lacking and are required. The aim of the present study was to identify novel methylation markers in Ewing's sarcoma using microarray analysis. The current study reports the microarray-based DNA methylation study of 1,505 CpG sites of 807 cancer-related genes from 69 Ewing's sarcoma samples. The Illumina GoldenGate Methylation Cancer Panel I microarray was used, and with the appropriate controls (n=14), a total of 92 hypermethylated genes were identified in the Ewing's sarcoma samples. The majority of the hypermethylated genes were associated with cell adhesion, cell regulation, development and signal transduction. The overall methylation mean values were compared between patients who survived and those that did not. The overall methylation mean was significantly higher in the patients who did not survive (0.25±0.03) than in those who did (0.22±0.05) (P=0.0322). However, the overall methylation mean was not found to significantly correlate with age, gender or tumor location. GDF10 , OSM , APC and HOXA11 were the most significant differentially-methylated genes, however, their methylation levels were not found to significantly correlate with the survival rate. The DNA methylation profile of Ewing's sarcoma was characterized and 92 genes that were significantly hypermethylated were detected. A trend towards a more aggressive behavior was identified in the methylated group. The results of this study indicated that methylation may be significant in the development of Ewing's sarcoma.

  3. Differential clinicopathological features in microsatellite instability-positive colorectal cancers depending on CIMP status.

    Science.gov (United States)

    Bae, Jeong Mo; Kim, Mi Jung; Kim, Jung Ho; Koh, Jae Moon; Cho, Nam-Yun; Kim, Tae-You; Kang, Gyeong Hoon

    2011-07-01

    Microsatellite instability-positive (MSI+) colorectal cancers (CRCs) are divided into CpG island methylator phenotype-positive (CIMP+) and CpG island methylator phenotype-negative (CIMP-) tumors. The repertoire of inactivated genes in CIMP+/MSI+ CRCs overlaps with but is likely to differ from that of CIMP-/MSI+ CRCs. Because epigenotypic differences are likely to be manifested as phenotypic differences, CIMP+/MSI+ CRCs are expected to differ from CIMP-/MSI+ CRCs in some clinicopathological features. This study aimed to characterize both common and different features between the two subtypes. A total of 72 MSI+ CRCs were analyzed for their methylation status in eight CIMP panel markers using MethyLight assay. CIMP+/MSI+ and CIMP-/MSI+ CRCs were compared regarding clinicopathologic features and mutation in KRAS/BRAF. An independent set of MSI+ CRCs (n = 97) was analyzed for their relationship of CIMP+ status with clinical outcome. Eighteen cases (25%) were CIMP+, and this CIMP+ subtype was highly correlated with older age (P CIMP-/MSI+ CRCs (18.5%, P = 0.057). CIMP+/MSI+ CRCs were closely associated with poor differentiation, medullary appearance, signet ring cell appearance, and acinar-form appearance, whereas the CIMP-/MSI+ subtype was closely associated with intraglandular eosinophilic mucin and stratified nuclei (all P values CIMP+/MSI+ CRCs showed worse overall survival than patients with CIMP-/MSI+ CRCs. Our results demonstrate heterogeneity in the clinicopathological features of MSI+ CRCs depending on CIMP status. The observation that CIMP+ and CIMP- subtypes showed different clinical behaviors may provide a clue for establishing subtype-specific therapeutic strategies for these two subtypes.

  4. Use of MSAP markers to analyse the effects of salt stress on DNA methylation in rapeseed (Brassica napus var. oleifera.

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    Gianpiero Marconi

    Full Text Available Excessive soil salinity is a major ecological and agronomical problem, the adverse effects of which are becoming a serious issue in regions where saline water is used for irrigation. Plants can employ regulatory strategies, such as DNA methylation, to enable relatively rapid adaptation to new conditions. In this regard, cytosine methylation might play an integral role in the regulation of gene expression at both the transcriptional and post-transcriptional levels. Rapeseed, which is the most important oilseed crop in Europe, is classified as being tolerant of salinity, although cultivars can vary substantially in their levels of tolerance. In this study, the Methylation Sensitive Amplified Polymorphism (MSAP approach was used to assess the extent of cytosine methylation under salinity stress in salinity-tolerant (Exagone and salinity-sensitive (Toccata rapeseed cultivars. Our data show that salinity affected the level of DNA methylation. In particular methylation decreased in Exagone and increased in Toccata. Nineteen DNA fragments showing polymorphisms related to differences in methylation were sequenced. In particular, two of these were highly similar to genes involved in stress responses (Lacerata and trehalose-6-phosphatase synthase S4 and were chosen to further characterization. Bisulfite sequencing and quantitative RT-PCR analysis of selected MSAP loci showed that cytosine methylation changes under salinity as well as gene expression varied. In particular, our data show that salinity stress influences the expression of the two stress-related genes. Moreover, we quantified the level of trehalose in Exagone shoots and found that it was correlated to TPS4 expression and, therefore, to DNA methylation. In conclusion, we found that salinity could induce genome-wide changes in DNA methylation status, and that these changes, when averaged across different genotypes and developmental stages, accounted for 16.8% of the total site

  5. Analysis of DNA methylation of perennial ryegrass under drought using the methylation-sensitive amplification polymorphism (MSAP) technique.

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    Tang, Xiao-Mei; Tao, Xiang; Wang, Yan; Ma, Dong-Wei; Li, Dan; Yang, Hong; Ma, Xin-Rong

    2014-12-01

    Perennial ryegrass (Lolium perenne), an excellent grass for forage and turf, is widespread in temperate regions. Drought is an important factor that limits its growth, distribution, and yield. DNA methylation affects gene expression and plays an important role in adaptation to adverse environments. In this study, the DNA methylation changes in perennial ryegrass under drought stress were assessed using methylation-sensitive amplified polymorphism (MSAP). After 15 days of drought stress treatment, the plant height was less than half of the control, and the leaves were smaller and darker. Genome-wide, a total of 652 CCGG sites were detected by MSAP. The total methylation level was 57.67 and 47.39 % in the control and drought treatment, respectively, indicating a decrease of 10.28 % due to drought exposure. Fifteen differentially displayed DNA fragments in MSAP profiles were cloned for sequencing analysis. The results showed that most of the genes involved in stress responses. The relative expression levels revealed that three demethylated fragments were up-regulated. The expression of a predicted retrotransposon increased significantly, changing from hypermethylation to non-methylation. Although the extent of methylation in two other genes decreased, the sites of methylation remained, and the expression increased only slightly. All of these results suggested that drought stress decreased the total DNA methylation level in perennial ryegrass and demethylation up-regulated related gene expressions and that the extent of methylation was negatively correlated with expression. Overall, the induced epigenetic changes in genome probably are an important regulatory mechanism for acclimating perennial ryegrass to drought and possibly other environmental stresses.

  6. Hepatocellular carcinoma displays distinct DNA methylation signatures with potential as clinical predictors.

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    Hector Hernandez-Vargas

    Full Text Available BACKGROUND: Hepatocellular carcinoma (HCC is characterized by late detection and fast progression, and it is believed that epigenetic disruption may be the cause of its molecular and clinicopathological heterogeneity. A better understanding of the global deregulation of methylation states and how they correlate with disease progression will aid in the design of strategies for earlier detection and better therapeutic decisions. METHODS AND FINDINGS: We characterized the changes in promoter methylation in a series of 30 HCC tumors and their respective surrounding tissue and identified methylation signatures associated with major risk factors and clinical correlates. A wide panel of cancer-related gene promoters was analyzed using Illumina bead array technology, and CpG sites were then selected according to their ability to classify clinicopathological parameters. An independent series of HCC tumors and matched surrounding tissue was used for validation of the signatures. We were able to develop and validate a signature of methylation in HCC. This signature distinguished HCC from surrounding tissue and from other tumor types, and was independent of risk factors. However, aberrant methylation of an independent subset of promoters was associated with tumor progression and etiological risk factors (HBV or HCV infection and alcohol consumption. Interestingly, distinct methylation of an independent panel of gene promoters was strongly correlated with survival after cancer therapy. CONCLUSION: Our study shows that HCC tumors exhibit specific DNA methylation signatures associated with major risk factors and tumor progression stage, with potential clinical applications in diagnosis and prognosis.

  7. What is popular? Distinguishing bullying and aggression as status correlates within specific peer normative contexts

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    Diego Palacios

    2016-01-01

    Full Text Available Abstract This study tested social status correlates of aggression and bullying and how these are influenced by peer groups’ normative beliefs about aggression and prosocial behavior among 1165 fourth, fifth and sixth graders in Chile. Associations between aggression and popularity (positive and social preference (negative were confirmed, whereas bullying was negatively associated with both dimensions. Normative beliefs about aggression and prosocial behavior were assessed at the group level, while social status was assessed at the classroom level through peer nominations. Hierarchical Linear Analyses showed that in groups with a higher value associated with aggression, classmates rated aggressive peers as less popular but also less disliked. The status correlates of bullying remained unaffected by peer normative beliefs. The discussion focuses on the social function of aggression as compared to the social sanction associated with bullying, and on the specificity of these associations at different layers of the social ecology.

  8. Methylation sensitive-sequence related amplified polymorphism (MS ...

    African Journals Online (AJOL)

    DR NJ TONUKARI

    2011-04-25

    Apr 25, 2011 ... Sequence-related amplified polymorphism (SRAP) is a simple but an efficient gene amplification marker system for both .... Each polymorphic band reflecting different methylation status at the ... After boiling for 5 min in the water, the .... CpG dinucleotides in the open reading frame of a testicular germ cell-.

  9. O6-methylguanine-DNA methyltransferase activity is associated with response to alkylating agent therapy and with MGMT promoter methylation in glioblastoma and anaplastic glioma

    Science.gov (United States)

    Bobola, Michael S.; Alnoor, Mohammad; Chen, John Y.-S.; Kolstoe, Douglas D.; Silbergeld, Daniel L.; Rostomily, Robert C.; Blank, A.; Chamberlain, Marc C.; Silber, John R.

    2014-01-01

    Background CpG methylation in the O6-methylguanine-DNA methyltransferase (MGMT) promoter is associated with better outcome following alkylating agent chemotherapy in glioblastoma (GBM) and anaplastic glioma (AG). To what extent improved response reflects low or absent MGMT activity in glioma tissue has not been unequivocally assessed. This information is central to developing anti-resistance therapies. Methods We examined the relationship of MGMT activity in 91 GBMs and 84 AGs with progression-free survival (PFS) following alkylator therapy and with promoter methylation status determined by methylation-specific PCR (MSP). Results Cox regression analysis revealed that GBMs with high activity had a significantly greater risk for progression in dichotomous (P ≤ 0.001) and continuous (P ≤ 0.003) models, an association observed for different alkylator regimens, including concurrent chemo-radiation with temozolomide. Analysis of MGMT promoter methylation status in 47 of the GBMs revealed that methylated tumors had significantly lower activity (P ≤ 0.005) and longer PFS (P ≤ 0.036) compared to unmethylated tumors, despite overlapping activities. PFS was also significantly greater in methylated vs. unmethylated GBMs with comparable activity (P ≤ 0.005), and among unmethylated tumors with less than median activity (P ≤ 0.026), suggesting that mechanisms in addition to MGMT promote alkylator resistance. Similar associations of MGMT activity with PFS and promoter methylation status were observed for AGs. Conclusions Our results provide strong support for the hypotheses that MGMT activity promotes alkylator resistance and reflects promoter methylation status in malignant gliomas. General significance MGMT activity is an attractive target for anti-resistance therapy regardless of methylation status. PMID:25558448

  10. Zinc sulfate contributes to promote telomere length extension via increasing telomerase gene expression, telomerase activity and change in the TERT gene promoter CpG island methylation status of human adipose-derived mesenchymal stem cells.

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    Raheleh Farahzadi

    Full Text Available The use of mesenchymal stem cells (MSCs for cell therapy and regenerative medicine has received widespread attention over the past few years, but their application can be complicated by factors such as reduction in proliferation potential, the senescent tendency of the MSCs upon expansion and their age-dependent decline in number and function. It was shown that all the mentioned features were accompanied by a reduction in telomerase activity and telomere shortening. Furthermore, the role of epigenetic changes in aging, especially changes in promoter methylation, was reported. In this study, MSCs were isolated from the adipose tissue with enzymatic digestion. In addition, immunocytochemistry staining and flow cytometric analysis were performed to investigate the cell-surface markers. In addition, alizarin red-S, sudan III, toluidine blue, and cresyl violet staining were performed to evaluate the multi-lineage differentiation of hADSCs. In order to improve the effective application of MSCs, these cells were treated with 1.5 × 10-8 and 2.99 × 10-10 M of ZnSO4 for 48 hours. The length of the absolute telomere, human telomerase reverse transcriptase (hTERT gene expression, telomerase activity, the investigation of methylation status of the hTERT gene promoter and the percentage of senescent cells were analyzed with quantitative real-time PCR, PCR-ELISA TRAP assay, methylation specific PCR (MSP, and beta-galactosidase (SA-β-gal staining, respectively. The results showed that the telomere length, the hTERT gene expression, and the telomerase activity had significantly increased. In addition, the percentage of senescent cells had significantly decreased and changes in the methylation status of the CpG islands in the hTERT promoter region under treatment with ZnSO4 were seen. In conclusion, it seems that ZnSO4 as a proper antioxidant could improve the aging-related features due to lengthening of the telomeres, increasing the telomerase gene expression

  11. Epigenetic Variation in Monozygotic Twins: A Genome-Wide Analysis of DNA Methylation in Buccal Cells

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    Jenny van Dongen

    2014-05-01

    Full Text Available DNA methylation is one of the most extensively studied epigenetic marks in humans. Yet, it is largely unknown what causes variation in DNA methylation between individuals. The comparison of DNA methylation profiles of monozygotic (MZ twins offers a unique experimental design to examine the extent to which such variation is related to individual-specific environmental influences and stochastic events or to familial factors (DNA sequence and shared environment. We measured genome-wide DNA methylation in buccal samples from ten MZ pairs (age 8–19 using the Illumina 450k array and examined twin correlations for methylation level at 420,921 CpGs after QC. After selecting CpGs showing the most variation in the methylation level between subjects, the mean genome-wide correlation (rho was 0.54. The correlation was higher, on average, for CpGs within CpG islands (CGIs, compared to CGI shores, shelves and non-CGI regions, particularly at hypomethylated CpGs. This finding suggests that individual-specific environmental and stochastic influences account for more variation in DNA methylation in CpG-poor regions. Our findings also indicate that it is worthwhile to examine heritable and shared environmental influences on buccal DNA methylation in larger studies that also include dizygotic twins.

  12. Methylation of the RASSF1A, RARβ2, and SEMA3B genes in epithelial breast and ovarian tumors, and in patients with polyneoplasia

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    T. P. Kazubskaya

    2012-01-01

    Full Text Available The methylation status of the tumor suppressor genes RASSF1A, RARβ2, and SEMA3B was studied in the samples of cancer and its histologically normal tissue of the breast and ovaries. The high rate of abnormal methylation of the CpG islet in the RASSF1A, RARβ2, and SEMA3B genes was found in the tumors of the breast (78% (32/41, 46% (26/56, and 35% (22/65, respectively and ovaries 73% (33/45, 30% (15/50, and 50% (25/51. Hypermethylation in the CpG islets belonging to the RASSF1A and RARβ2 genes was first ascertained in 90% of the patients with polyneoplasms involving the breast and ovaries. Abnormal methylation of the promotor region of the RASSF1A gene was shown to be detectable in preclinical-stage and anaplasia-degree breast and ovarian cancer. There was a correlation of the rate of methylation in the promoter regions of the RARβ2 and SEMA3B genes with clinical-stage and anaplasia-degree breast and ovarian cancer. Analysis of gene methylation in biological fluids provides considerable opportunity to use methylation of DNA as a marker in clinical practice.

  13. Down-regulation of p21 (CDKN1A/CIP1) is inversely associated with microsatellite instability and CpG island methylator phenotype (CIMP) in colorectal cancer.

    Science.gov (United States)

    Ogino, S; Kawasaki, T; Kirkner, G J; Ogawa, A; Dorfman, I; Loda, M; Fuchs, C S

    2006-10-01

    p21 (CDKN1A/CIP1/WAF1), one of the cyclin-dependent kinase inhibitors, plays a key role in regulating the cell cycle and is transcriptionally regulated by p53. Down-regulation of p21 is caused by TP53 mutations in colorectal cancer. CpG island methylator phenotype (CIMP) appears to be a distinct subtype of colorectal cancer with concordant methylation of multiple gene promoters and is associated with a high degree of microsatellite instability (MSI-H) and BRAF mutations. However, no study to date has evaluated the relationship between p21 expression and CIMP in colorectal cancer. The purpose of this study was to examine the inter-relationships between p21, p53, CIMP, MSI and KRAS/BRAF status in colorectal cancer. We utilized 737 relatively unbiased samples of colorectal cancers from two large prospective cohort studies. Using quantitative real-time PCR (MethyLight), we measured DNA methylation in five CIMP-specific gene promoters [CACNA1G, CDKN2A (p16/INK4A), CRABP1, MLH1 and NEUROG1]. CIMP-high (>or=4/5 methylated promoters) was diagnosed in 118 (16%) of the 737 tumours. We also assessed expression of p21 and p53 by immunohistochemistry. Among the 737 tumours, 371 (50%) showed p21 loss. Both p21 loss and p53 positivity were inversely associated with CIMP-high, MSI-H and BRAF mutations. The associations of p21 with these molecular features were still present after tumours were stratified by p53 status. In contrast, the associations of p53 positivity with the molecular features were no longer present after tumours were stratified by p21 status. When CIMP-high and non-CIMP-high tumours were stratified by MSI or KRAS/BRAF status, CIMP-high and MSI-H (but not BRAF mutations) were still inversely associated with p21 loss. In conclusion, down-regulation of p21 is inversely correlated with CIMP-high and MSI-H in colorectal cancer, independent of TP53 and BRAF status.

  14. Parvovirus b19 DNA CpG dinucleotide methylation and epigenetic regulation of viral expression.

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    Francesca Bonvicini

    Full Text Available CpG DNA methylation is one of the main epigenetic modifications playing a role in the control of gene expression. For DNA viruses whose genome has the ability to integrate in the host genome or to maintain as a latent episome, a correlation has been found between the extent of DNA methylation and viral quiescence. No information is available for Parvovirus B19, a human pathogenic virus, which is capable of both lytic and persistent infections. Within Parvovirus B19 genome, the inverted terminal regions display all the characteristic signatures of a genomic CpG island; therefore we hypothesised a role of CpG dinucleotide methylation in the regulation of viral genome expression.The analysis of CpG dinucleotide methylation of Parvovirus B19 DNA was carried out by an aptly designed quantitative real-time PCR assay on bisulfite-modified DNA. The effects of CpG methylation on the regulation of viral genome expression were first investigated by transfection of either unmethylated or in vitro methylated viral DNA in a model cell line, showing that methylation of viral DNA was correlated to lower expression levels of the viral genome. Then, in the course of in vitro infections in different cellular environments, it was observed that absence of viral expression and genome replication were both correlated to increasing levels of CpG methylation of viral DNA. Finally, the presence of CpG methylation was documented in viral DNA present in bioptic samples, indicating the occurrence and a possible role of this epigenetic modification in the course of natural infections.The presence of an epigenetic level of regulation of viral genome expression, possibly correlated to the silencing of the viral genome and contributing to the maintenance of the virus in tissues, can be relevant to the balance and outcome of the different types of infection associated to Parvovirus B19.

  15. Parvovirus B19 DNA CpG Dinucleotide Methylation and Epigenetic Regulation of Viral Expression

    Science.gov (United States)

    Bonvicini, Francesca; Manaresi, Elisabetta; Di Furio, Francesca; De Falco, Luisa; Gallinella, Giorgio

    2012-01-01

    CpG DNA methylation is one of the main epigenetic modifications playing a role in the control of gene expression. For DNA viruses whose genome has the ability to integrate in the host genome or to maintain as a latent episome, a correlation has been found between the extent of DNA methylation and viral quiescence. No information is available for Parvovirus B19, a human pathogenic virus, which is capable of both lytic and persistent infections. Within Parvovirus B19 genome, the inverted terminal regions display all the characteristic signatures of a genomic CpG island; therefore we hypothesised a role of CpG dinucleotide methylation in the regulation of viral genome expression. The analysis of CpG dinucleotide methylation of Parvovirus B19 DNA was carried out by an aptly designed quantitative real-time PCR assay on bisulfite-modified DNA. The effects of CpG methylation on the regulation of viral genome expression were first investigated by transfection of either unmethylated or in vitro methylated viral DNA in a model cell line, showing that methylation of viral DNA was correlated to lower expression levels of the viral genome. Then, in the course of in vitro infections in different cellular environments, it was observed that absence of viral expression and genome replication were both correlated to increasing levels of CpG methylation of viral DNA. Finally, the presence of CpG methylation was documented in viral DNA present in bioptic samples, indicating the occurrence and a possible role of this epigenetic modification in the course of natural infections. The presence of an epigenetic level of regulation of viral genome expression, possibly correlated to the silencing of the viral genome and contributing to the maintenance of the virus in tissues, can be relevant to the balance and outcome of the different types of infection associated to Parvovirus B19. PMID:22413013

  16. Detection of Methylated Circulating DNA as Noninvasive Biomarkers for Breast Cancer Diagnosis

    Science.gov (United States)

    Cheuk, Isabella Wai Yin; Shin, Vivian Yvonne

    2017-01-01

    Internationally, breast cancer is the most common female cancer, and is induced by a combination of environmental, genetic, and epigenetic risk factors. Despite the advancement of imaging techniques, invasive sampling of breast epithelial cells is the only definitive diagnostic procedure for patients with breast cancer. To date, molecular biomarkers with high sensitivity and specificity for the screening and early detection of breast cancer are lacking. Recent evidence suggests that the detection of methylated circulating cell-free DNA in the peripheral blood of patients with cancer may be a promising quantitative and noninvasive method for cancer diagnosis. Methylation detection based on a multi-gene panel, rather than on the methylation status of a single gene, may be used to increase the sensitivity and specificity of breast cancer screening. In this review, the results of 14 relevant studies, investigating the efficacy of cell-free DNA methylation screening for breast cancer diagnosis, have been summarized. The genetic risk factors for breast cancer, the methods used for breast cancer detection, and the techniques and limitations related to the detection of cell-free DNA methylation status, have also been reviewed and discussed. From this review, we conclude that the analysis of peripheral blood or other samples to detect differentially methylated cell-free DNA is a promising technique for use in clinical settings, and may improve the sensitivity of screening for both, early detection and disease relapse, and thus improve the future prognosis of patients with breast cancer. PMID:28382090

  17. Folate, colorectal cancer and the involvement of DNA methylation.

    Science.gov (United States)

    Williams, Elizabeth A

    2012-11-01

    Diet is a major factor in the aetiology of colorectal cancer (CRC). Epidemiological evidence suggests that folate confers a modest protection against CRC risk. However, the relationship is complex, and evidence from human intervention trials and animal studies suggests that a high-dose of folic acid supplementation may enhance the risk of colorectal carcinogenesis in certain circumstances. The molecular mechanisms underlying the apparent dual modulatory effect of folate on colorectal carcinogenesis are not fully understood. Folate is central to C1 metabolism and is needed for both DNA synthesis and DNA methylation, providing plausible biological mechanisms through which folate could modulate cancer risk. Aberrant DNA methylation is an early event in colorectal carcinogenesis and is typically associated with the transcriptional silencing of tumour suppressor genes. Folate is required for the production of S-adenosyl methionine, which serves as a methyl donor for DNA methylation events; thereby folate availability is proposed to modulate DNA methylation status. The evidence for an effect of folate on DNA methylation in the human colon is limited, but a modulation of DNA methylation in response to folate has been demonstrated. More research is required to clarify the optimum intake of folate for CRC prevention and to elucidate the effect of folate availability on DNA methylation and the associated impact on CRC biology.

  18. Lgr5 Methylation in Cancer Stem Cell Differentiation and Prognosis-Prediction in Colorectal Cancer.

    Directory of Open Access Journals (Sweden)

    Shasha Su

    Full Text Available Leucine-rich-repeat-containing G-protein-coupled receptor 5 (lgr5 is a candidate marker for colorectal cancer stem cells (CSC. In the current study, we investigated the methylation status within thelgr5 promoter and evaluated its relationship with CSC differentiation, prognosis for colorectal cancer, and its clinicopathological features.The methylation status within Lgr5 promoter was detected with a methylation-specific PCR in six colorectal cancer cell lines as well as 169 primary colorectal tumor tissues. Differentiation of CSC was examined with immunofluorescence and immunocytochemistry. Down-regulation of lgr5 was achieved with gene-specific siRNA. The associations between lgr5 methylation and the clinicopathological features as well as survival of patients were analyzed with statistical methods.The lgr5 promoter was methylated to different degrees for the six colorectal cell lines examined, with complete methylation observed in HCT116 cells in which the lgr5 expression was partially recovered following DAC treatment. The stem-cell sphere formation from HCT116 cells was accompanied by increasing methylation within the lgr5 promoter and decreasing expression of lgr5. Knocking down lgr5 by siRNA also led to stem-cell spheres formation. Among primary colorectal tumors, 40% (67/169 were positive for lgr5 methylation, while none of the normal colon tissues were positive for lgr5 methylation. Furthermore, lgr5 methylation significantly associated with higher tumor grade, and negative distant metastasis (p < 0.05, as well as better prognosis (p = 0.001 in patients with colorectal cancer.Our data suggests that lgr5 methylation, through the regulation of lgr5 expression and colorectal CSC differentiation, may constitute a novel prognostic marker for colorectal cancer patients.

  19. Reference Materials for Calibration of Analytical Biases in Quantification of DNA Methylation.

    Science.gov (United States)

    Yu, Hannah; Hahn, Yoonsoo; Yang, Inchul

    2015-01-01

    Most contemporary methods for the quantification of DNA methylation employ bisulfite conversion and PCR amplification. However, many reports have indicated that bisulfite-mediated PCR methodologies can result in inaccurate measurements of DNA methylation owing to amplification biases. To calibrate analytical biases in quantification of gene methylation, especially those that arise during PCR, we utilized reference materials that represent exact bisulfite-converted sequences with 0% and 100% methylation status of specific genes. After determining relative quantities using qPCR, pairs of plasmids were gravimetrically mixed to generate working standards with predefined DNA methylation levels at 10% intervals in terms of mole fractions. The working standards were used as controls to optimize the experimental conditions and also as calibration standards in melting-based and sequencing-based analyses of DNA methylation. Use of the reference materials enabled precise characterization and proper calibration of various biases during PCR and subsequent methylation measurement processes, resulting in accurate measurements.

  20. Integrated analysis of epigenomic and genomic changes by DNA methylation dependent mechanisms provides potential novel biomarkers for prostate cancer.

    Science.gov (United States)

    White-Al Habeeb, Nicole M A; Ho, Linh T; Olkhov-Mitsel, Ekaterina; Kron, Ken; Pethe, Vaijayanti; Lehman, Melanie; Jovanovic, Lidija; Fleshner, Neil; van der Kwast, Theodorus; Nelson, Colleen C; Bapat, Bharati

    2014-09-15

    Epigenetic silencing mediated by CpG methylation is a common feature of many cancers. Characterizing aberrant DNA methylation changes associated with tumor progression may identify potential prognostic markers for prostate cancer (PCa). We treated two PCa cell lines, 22Rv1 and DU-145 with the demethylating agent 5-Aza 2'-deoxycitidine (DAC) and global methylation status was analyzed by performing methylation-sensitive restriction enzyme based differential methylation hybridization strategy followed by genome-wide CpG methylation array profiling. In addition, we examined gene expression changes using a custom microarray. Gene Set Enrichment Analysis (GSEA) identified the most significantly dysregulated pathways. In addition, we assessed methylation status of candidate genes that showed reduced CpG methylation and increased gene expression after DAC treatment, in Gleason score (GS) 8 vs. GS6 patients using three independent cohorts of patients; the publically available The Cancer Genome Atlas (TCGA) dataset, and two separate patient cohorts. Our analysis, by integrating methylation and gene expression in PCa cell lines, combined with patient tumor data, identified novel potential biomarkers for PCa patients. These markers may help elucidate the pathogenesis of PCa and represent potential prognostic markers for PCa patients.

  1. Validation of methylation-sensitive high-resolution melting (MS-HRM) for the detection of stool DNA methylation in colorectal neoplasms.

    Science.gov (United States)

    Xiao, Zhujun; Li, Bingsheng; Wang, Guozhen; Zhu, Weisi; Wang, Zhongqiu; Lin, Jinfeng; Xu, Angao; Wang, Xinying

    2014-04-20

    Methylation-sensitive high-resolution melting (MS-HRM) is a new technique for assaying DNA methylation, but its feasibility for assaying stool in patients with colorectal cancer (CRC) is unknown. First, the MS-HRM and methylation-specific PCR (MSP) detection limits were tested. Second, the methylation statuses of SFRP2 and VIM were analyzed in stool samples by MS-HRM, and in matching tumor and normal colon tissues via bisulfite sequencing PCR (BSP). Third, a case-control study evaluated the diagnostic sensitivity and specificity of MS-HRM relative to results obtained with MSP and the fecal immunochemical test (FIT). Finally, the linearity and reproducibility of MS-HRM were assessed. The detection limits of MS-HRM and MSP were 1% and 5%, respectively. The diagnostic sensitivities of MS-HRM (87.3%, 55/63) in stool and BSP in matching tumor tissue (92.1%, 58/63) were highly consistent (κ=0.744). The MS-HRM assay detected 92.5% (37/40) methylation in CRCs, 94.4% (34/36) in advanced adenomas, and 8.8% (5/57) in normal controls. The results of MS-HRM analysis were stable and reliable and showed fairly good linearity for both SFRP2 (PHRM shows potential for CRC screening. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Fragile X mental retardation 1 (FMR1) intron 1 methylation in blood predicts verbal cognitive impairment in female carriers of expanded FMR1 alleles: evidence from a pilot study.

    Science.gov (United States)

    Godler, David E; Slater, Howard R; Bui, Quang M; Storey, Elsdon; Ono, Michele Y; Gehling, Freya; Inaba, Yoshimi; Francis, David; Hopper, John L; Kinsella, Glynda; Amor, David J; Hagerman, Randi J; Loesch, Danuta Z

    2012-03-01

    Cognitive status in females with mutations in the FMR1 (fragile X mental retardation 1) gene is highly variable. A biomarker would be of value for predicting which individuals were liable to develop cognitive impairment and could benefit from early intervention. A detailed analysis of CpG sites bridging exon 1 and intron 1 of FMR1, known as fragile X-related epigenetic element 2 (FREE2), suggests that a simple blood test could identify these individuals. Study participants included 74 control females (Wechsler intelligence quotient (IQ) tests. We used MALDI-TOF mass spectrometry to determine the methylation status of FREE2 CpG sites that best identified low-functioning (IQ 200 CGG repeats), compared the results with those for Southern blot FMR1 activation ratios, and related these assessments to the level of production of the FMR1 protein product in blood. A methylation analysis of intron 1 CpG sites 10-12 showed the highest diagnostic sensitivity (100%) and specificity (98%) of all the molecular measures tested for detecting females with a standardized verbal IQ of <70 among the study participants. In the group consisting of only FM females, methylation of these sites was significantly correlated with full-scale IQ, verbal IQ, and performance IQ. Several verbal subtest scores showed strong correlation with the methylation of these sites (P = 1.2 × 10(-5)) after adjustment for multiple measures. The data suggest that hypermethylation of the FMR1 intron 1 sites in blood is predictive of cognitive impairment in FM females, with implications for improved fragile X syndrome diagnostics in young children and screening of the newborn population.

  3. DNA methylation perturbations in genes involved in polyunsaturated Fatty Acid biosynthesis associated with depression and suicide risk.

    Science.gov (United States)

    Haghighi, Fatemeh; Galfalvy, Hanga; Chen, Sean; Huang, Yung-Yu; Cooper, Thomas B; Burke, Ainsley K; Oquendo, Maria A; Mann, J John; Sublette, M Elizabeth

    2015-01-01

    Polyunsaturated fatty acid (PUFA) status has been associated with neuropsychiatric disorders, including depression and risk of suicide. Long-chain PUFAs (LC-PUFAs) are obtained in the diet or produced by sequential desaturation and elongation of shorter-chain precursor fatty acids linoleic acid (LA, 18:2n-6) and α-linolenic acid (ALA, 18:3n-3). We compared DNA methylation patterns in genes involved in LC-PUFA biosynthesis in major depressive disorder (MDD) with (n = 22) and without (n = 39) history of suicide attempt, and age- and sex-matched healthy volunteers (n = 59). Plasma levels of selected PUFAs along the LC-PUFA biosynthesis pathway were determined by transesterification and gas chromatography. CpG methylation levels for the main human LC-PUFA biosynthetic genes, fatty acid desaturases 1 (Fads1) and 2 (Fads2), and elongation of very long-chain fatty acids protein 5 (Elovl5), were assayed by bisulfite pyrosequencing. Associations between PUFA levels and diagnosis or suicide attempt status did not survive correction for multiple testing. However, MDD diagnosis and suicide attempts were significantly associated with DNA methylation in Elovl5 gene regulatory regions. Also the relative roles of PUFA levels and DNA methylation with respect to diagnostic and suicide attempt status were determined by least absolute shrinkage and selection operator logistic regression analyses. We found that PUFA associations with suicide attempt status were explained by effects of Elovl5 DNA methylation within the regulatory regions. The observed link between plasma PUFA levels, DNA methylation, and suicide risk may have implications for modulation of disease-associated epigenetic marks by nutritional intervention.

  4. DNA methylation perturbations in genes involved in polyunsaturated fatty acid biosynthesis associated with depression and suicide risk

    Directory of Open Access Journals (Sweden)

    Fatemeh eHaghighi

    2015-04-01

    Full Text Available Polyunsaturated fatty acid (PUFA status has been associated with neuropsychiatric disorders, including depression and risk of suicide. Long-chain PUFAs (LC-PUFAs are obtained in the diet or produced by sequential desaturation and elongation of shorter-chain precursor fatty acids linoleic acid (LA, 18:2n-6 and α-linolenic acid (ALA, 18:3n-3. We compared DNA methylation patterns in genes involved in LC-PUFA biosynthesis in major depressive disorder (MDD with (n=22 and without (n=39 history of suicide attempt, and age- and sex-matched healthy volunteers (n=59. Plasma levels of selected PUFAs along the LC-PUFA biosynthesis pathway were determined by transesterification and gas chromatography. CpG methylation levels for the main human LC-PUFA biosynthetic genes, fatty acid desaturases 1 (Fads1 and 2 (Fads2, and elongation of very long chain fatty acids protein 5 (Elovl5, were assayed by bisulfite pyrosequencing. Associations between PUFA levels and diagnosis or suicide attempt status did not survive correction for multiple testing. However, MDD diagnosis and suicide attempts were significantly associated with DNA methylation in Elovl5 gene regulatory regions. Also the relative roles of PUFA levels and DNA methylation with respect to diagnostic and suicide attempt status were determined by least absolute shrinkage and selection operator (LASSO logistic regression analyses. We found that PUFA associations with suicide attempt status were explained by effects of Elovl5 DNA methylation within the regulatory regions. The observed link between plasma PUFA levels, DNA methylation, and suicide risk may have implications for modulation of disease-associated epigenetic marks by nutritional intervention.

  5. Effect of Exposure to 900 MHz GSM Mobile Phone Radiofrequency Radiation on Estrogen Receptor Methylation Status in Colon Cells of Male Sprague Dawley Rats

    Science.gov (United States)

    Mokarram, P.; Sheikhi, M.; Mortazavi, S.M.J.; Saeb, S.; Shokrpour, N.

    2017-01-01

    Background: Over the past several years, the rapidly increasing use of mobile phones has raised global concerns about the biological effects of exposure to radiofrequency (RF) radiation. Numerous studies have shown that exposure to electromagnetic fields (EMFs) can be associated with effects on the nervous, endocrine, immune, cardiovascular, hematopoietic and ocular systems. In spite of genetic diversity, the onset and progression of cancer can be controlled by epigenetic mechanisms such as gene promoter methylation. There are extensive studies on the epigenetic changes of the tumor suppressor genes as well as the identification of methylation biomarkers in colorectal cancer. Some studies have revealed that genetic changes can be induced by exposure to RF radiation. However, whether or not RF radiation is capable of inducing epigenetic alteration has not been clarified yet. To date, no study has been conducted on the effect of radiation on epigenetic alterations in colorectal cancer (CRC). Several studies have also shown that methylation of estrogen receptor α (ERα), MYOD, MGMT, SFRP2 and P16 play an important role in CRC. It can be hypothesized that RF exposure can be a reason for the high incidence of CRC in Iran. This study aimed to investigate whether epigenetic pattern of ERα is susceptible to RF radiation and if RF radiation can induce radioadaptive response as epigenetic changes after receiving the challenge dose (γ-ray). Material and Method: 40 male Sprague-Dawley rats were divided into 4 equal groups (Group I: exposure to RF radiation of a GSM cell phone for 4 hours and sacrificed after 24 hours; Group II: RF exposure for 4 hours, exposure to Co-60 gamma radiation (3 Gy) after 24 hours and sacrificed after 72 hrs; Group III: only 3Gy gamma radiation; Group 4: control group). DNA from colon tissues was extracted to evaluate the methylation status by methylation specific PCR. Results: Our finding showed that exposure to GSM cell phone RF radiation was

  6. Effect of Exposure to 900 MHz GSM Mobile Phone Radiofrequency Radiation on Estrogen Receptor Methylation Status in Colon Cells of Male Sprague Dawley Rats

    Directory of Open Access Journals (Sweden)

    Mokarram P.

    2017-03-01

    Full Text Available Background: Over the past several years, the rapidly increasing use of mobile phones has raised global concerns about the biological effects of exposure to radiofrequency (RF radiation. Numerous studies have shown that exposure to electromagnetic fields (EMFs can be associated with effects on the nervous, endocrine, immune, cardiovascular, hematopoietic and ocular systems. In spite of genetic diversity, the onset and progression of cancer can be controlled by epigenetic mechanisms such as gene promoter methylation. There are extensive studies on the epigenetic changes of the tumor suppressor genes as well as the identification of methylation biomarkers in colorectal cancer. Some studies have revealed that genetic changes can be induced by exposure to RF radiation. However, whether or not RF radiation is capable of inducing epigenetic alteration has not been clarified yet. To date, no study has been conducted on the effect of radiation on epigenetic alterations in colorectal cancer (CRC. Several studies have also shown that methylation of estrogen receptor α (ERα, MYOD, MGMT, SFRP2 and P16 play an important role in CRC. It can be hypothesized that RF exposure can be a reason for the high incidence of CRC in Iran. This study aimed to investigate whether epigenetic pattern of ERα is susceptible to RF radiation and if RF radiation can induce radioadaptive response as epigenetic changes after receiving the challenge dose (γ-ray. Material and Method: 40 male Sprague-Dawley rats were divided into 4 equal groups (Group I: exposure to RF radiation of a GSM cell phone for 4 hours and sacrificed after 24 hours; Group II: RF exposure for 4 hours, exposure to Co-60 gamma radiation (3 Gy after 24 hours and sacrificed after 72 hrs; Group III: only 3Gy gamma radiation; Group 4: control group. DNA from colon tissues was extracted to evaluate the methylation status by methylation specific PCR. Results: Our finding showed that exposure to GSM cell phone RF

  7. Aberrant gene methylation in non-neoplastic mucosa as a predictive marker of ulcerative colitis-associated CRC.

    Science.gov (United States)

    Scarpa, Marco; Scarpa, Melania; Castagliuolo, Ignazio; Erroi, Francesca; Kotsafti, Andromachi; Basato, Silvia; Brun, Paola; D'Incà, Renata; Rugge, Massimo; Angriman, Imerio; Castoro, Carlo

    2016-03-01

    BACKGROUND PROMOTER: hypermethylation plays a major role in cancer through transcriptional silencing of critical genes. The aim of our study is to evaluate the methylation status of these genes in the colonic mucosa without dysplasia or adenocarcinoma at the different steps of sporadic and UC-related carcinogenesis and to investigate the possible role of genomic methylation as a marker of CRC. The expression of Dnmts 1 and 3A was significantly increased in UC-related carcinogenesis compared to non inflammatory colorectal carcinogenesis. In non-neoplastic colonic mucosa, the number of methylated genes resulted significantly higher in patients with CRC and in those with UC-related CRC compared to the HC and UC patients and patients with dysplastic lesion of the colon. The number of methylated genes in non-neoplastic colonic mucosa predicted the presence of CRC with good accuracy either in non inflammatory and inflammatory related CRC. Colonic mucosal samples were collected from healthy subjects (HC) (n = 30) and from patients with ulcerative colitis (UC) (n = 29), UC and dysplasia (n = 14), UC and cancer (n = 10), dysplastic adenoma (n = 14), and colon adenocarcinoma (n = 10). DNA methyltransferases-1, -3a, -3b, mRNA expression were quantified by real time qRT-PCR. The methylation status of CDH13, APC, MLH1, MGMT1 and RUNX3 gene promoters was assessed by methylation-specific PCR. Methylation status of APC, CDH13, MGMT, MLH1 and RUNX3 in the non-neoplastic mucosa may be used as a marker of CRC: these preliminary results could allow for the adjustment of a patient's surveillance interval and to select UC patients who should undergo intensive surveillance.

  8. Methylation-sensitive amplification polymorphism in date palms (Phoenix dactylifera L.) and their off-shoots.

    Science.gov (United States)

    Fang, J-G; Chao, C T

    2007-07-01

    DNA methylation plays an important role in the regulation of gene expression in eukaryotes. In this study, the extent and patterns of DNA methylation were assessed in date palm mother-plants and their off-shoots using the methylation-sensitive amplified polymorphism (MSAP) technique. Three types of bands were generated using 12 pairs of primers. Type I were present in both ECOR I + HPA II and ECOR I + MSP I lanes; type II were present in ECOR I + HPA II lanes, but not in ECOR I + MSP I lanes; and type III bands were present in ECOR I + MSP I lanes, but not in ECOR I + HPA II lanes. The total numbers of these three types of bands were 782, 55, and 34, respectively. Among these three types of bands, the polymorphic bands were, respectively, 37, 10, and 0. The distribution of polymorphic bands among mother-plants and off-shoots suggests the methylation variation was present in both the mother-plants and off-shoots. Forty- four out of these 47 polymorphic bands show clear difference between mother-plant and off-shoots: 38 were present only in off-shoots and 6 in both mother-plants and off-shoots. Compared to methylation status in mother-plants, the methylation variation during off-shoot growth of date palm can be characterized as a process involving primarily de-methylation. Hypomethylation of DNA in off-shoots, compared with mother-plants, reflects the marked expression of this molecular feature, which may be related to gene expression during off-shoot development. The methylation or de-methylation status of specific loci in the mother-plants and their off-shoots were probably random events.

  9. Social Status Correlates of Reporting Racial Discrimination and Gender Discrimination among Racially Diverse Women

    Science.gov (United States)

    Ro, Annie E.; Choi, Kyung-Hee

    2009-01-01

    The growing body of research on discrimination and health indicates a deleterious effect of discrimination on various health outcomes. However, less is known about the sociodemographic correlates of reporting racial discrimination and gender discrimination among racially diverse women. We examined the associations of social status characteristics with lifetime experiences of racial discrimination and gender discrimination using a racially-diverse sample of 754 women attending family planning clinics in Northern California (11.4% African American, 16.8% Latina, 10.1% Asian and 61.7% Caucasian). A multivariate analysis revealed that race, financial difficulty and marital status were significantly correlated with higher reports of racial discrimination, while race, education, financial difficulty and nativity were significantly correlated with gender discrimination scores. Our findings suggest that the social patterning of perceiving racial discrimination is somewhat different from that of gender discrimination. This has implications in the realm of discrimination research and applied interventions, as different forms of discrimination may have unique covariates that should be accounted for in research analysis or program design. PMID:19485231

  10. Social status correlates of reporting gender discrimination and racial discrimination among racially diverse women.

    Science.gov (United States)

    Ro, Annie E; Choi, Kyung-Hee

    2009-01-01

    The growing body of research on discrimination and health indicates a deleterious effect of discrimination on various health outcomes. However, less is known about the sociodemographic correlates of reporting racial discrimination and gender discrimination among racially diverse women. We examined the associations of social status characteristics with lifetime experiences of racial discrimination and gender discrimination using a racially-diverse sample of 754 women attending family planning clinics in North California (11.4% African American, 16.8% Latina, 10.1% Asian and 61.7% Caucasian). A multivariate analysis revealed that race, financial difficulty and marital status were significantly correlated with higher reports of racial discrimination, while race, education, financial difficulty and nativity were significantly correlated with gender discrimination scores. Our findings suggest that the social patterning of perceiving racial discrimination is somewhat different from that of gender discrimination. This has implications in the realm of discrimination research and applied interventions, as different forms of discrimination may have unique covariates that should be accounted for in research analysis or program design.

  11. Digital PCR quantification of MGMT methylation refines prediction of clinical benefit from alkylating agents in glioblastoma and metastatic colorectal cancer.

    Science.gov (United States)

    Barault, L; Amatu, A; Bleeker, F E; Moutinho, C; Falcomatà, C; Fiano, V; Cassingena, A; Siravegna, G; Milione, M; Cassoni, P; De Braud, F; Rudà, R; Soffietti, R; Venesio, T; Bardelli, A; Wesseling, P; de Witt Hamer, P; Pietrantonio, F; Siena, S; Esteller, M; Sartore-Bianchi, A; Di Nicolantonio, F

    2015-09-01

    O(6)-methyl-guanine-methyl-transferase (MGMT) silencing by promoter methylation may identify cancer patients responding to the alkylating agents dacarbazine or temozolomide. We evaluated the prognostic and predictive value of MGMT methylation testing both in tumor and cell-free circulating DNA (cfDNA) from plasma samples using an ultra-sensitive two-step digital PCR technique (methyl-BEAMing). Results were compared with two established techniques, methylation-specific PCR (MSP) and Bs-pyrosequencing. Thresholds for MGMT methylated status for each technique were established in a training set of 98 glioblastoma (GBM) patients. The prognostic and the predictive value of MGMT methylated status was validated in a second cohort of 66 GBM patients treated with temozolomide in which methyl-BEAMing displayed a better specificity than the other techniques. Cutoff values of MGMT methylation specific for metastatic colorectal cancer (mCRC) tissue samples were established in a cohort of 60 patients treated with dacarbazine. In mCRC, both quantitative assays methyl-BEAMing and Bs-pyrosequencing outperformed MSP, providing better prediction of treatment response and improvement in progression-free survival (PFS) (P alkylating agents. © The Author 2015. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  12. DNA Methylation of Regulatory Regions of Imprinted Genes at Birth and Its Relation to Infant Temperament

    Directory of Open Access Journals (Sweden)

    Bernard F. Fuemmeler

    2016-01-01

    Full Text Available BACKGROUND DNA methylation of the differentially methylated regions (DMRs of imprinted genes is relevant to neurodevelopment. METHODS DNA methylation status of the DMRs of nine imprinted genes in umbilical cord blood leukocytes was analyzed in relation to infant behaviors and temperament (n = 158. RESULTS MEG3 DMR levels were positively associated with internalizing ( β = 0.15, P = 0.044 and surgency ( β = 0.19, P = 0.018 behaviors, after adjusting for birth weight, gender, gestational age at birth, maternal age at delivery, race/ethnicity, education level, smoking status, parity, and a history of anxiety or depression. Higher methylation levels at the intergenic MEG3-IG methylation regions were associated with surgency ( β = 0.28, P = 0.0003 and PEG3 was positively related to externalizing ( β = 0.20, P = 0.01 and negative affectivity ( β = 0.18, P = 0.02. CONCLUSION While the small sample size limits inference, these pilot data support gene-specific associations between epigenetic differences in regulatory regions of imprinted domains at birth and later infant temperament.

  13. Analysis of RET promoter CpG island methylation using methylation-specific PCR (MSP), pyrosequencing, and methylation-sensitive high-resolution melting (MS-HRM): impact on stage II colon cancer patient outcome.

    Science.gov (United States)

    Draht, Muriel X G; Smits, Kim M; Jooste, Valérie; Tournier, Benjamin; Vervoort, Martijn; Ramaekers, Chantal; Chapusot, Caroline; Weijenberg, Matty P; van Engeland, Manon; Melotte, Veerle

    2016-01-01

    Already since the 1990s, promoter CpG island methylation markers have been considered promising diagnostic, prognostic, and predictive cancer biomarkers. However, so far, only a limited number of DNA methylation markers have been introduced into clinical practice. One reason why the vast majority of methylation markers do not translate into clinical applications is lack of independent validation of methylation markers, often caused by differences in methylation analysis techniques. We recently described RET promoter CpG island methylation as a potential prognostic marker in stage II colorectal cancer (CRC) patients of two independent series. In the current study, we analyzed the RET promoter CpG island methylation of 241 stage II colon cancer patients by direct methylation-specific PCR (MSP), nested-MSP, pyrosequencing, and methylation-sensitive high-resolution melting (MS-HRM). All primers were designed as close as possible to the same genomic region. In order to investigate the effect of different DNA methylation assays on patient outcome, we assessed the clinical sensitivity and specificity as well as the association of RET methylation with overall survival for three and five years of follow-up. Using direct-MSP and nested-MSP, 12.0 % (25/209) and 29.6 % (71/240) of the patients showed RET promoter CpG island methylation. Methylation frequencies detected by pyrosequencing were related to the threshold for positivity that defined RET methylation. Methylation frequencies obtained by pyrosequencing (threshold for positivity at 20 %) and MS-HRM were 13.3 % (32/240) and 13.8 % (33/239), respectively. The pyrosequencing threshold for positivity of 20 % showed the best correlation with MS-HRM and direct-MSP results. Nested-MSP detected RET promoter CpG island methylation in deceased patients with a higher sensitivity (33.1 %) compared to direct-MSP (10.7 %), pyrosequencing (14.4 %), and MS-HRM (15.4 %). While RET methylation frequencies detected by nested

  14. The Main Correlations of the Hungarian’s Health Status and Food Consumption

    Directory of Open Access Journals (Sweden)

    Bakos Izabella Mária

    2016-11-01

    Full Text Available It is a general socio-political objective of the mid- and long term food industry development strategy of Hungary to promote healthy food production and consumption. The realization of the strategy of the domestic food industry increasingly promotes healthy eating, for example consuming natural, domestic, fresh ingredients, prepared foods, in order to improve the overall health of the population (EFS, 2014-2020. Our study presents the regional tendencies of staple food consumption in Hungarian regions and the changes in indicators reflecting the health status of the population. Furthermore, our hypothesis states that there is a statistically provable correlation between the annual food consumption of Hungarian households per capita and the health status, on regional level.

  15. Trend analysis of the correlation of amino acid plasma profile with glycemic status in Saudi diabetic patients

    Directory of Open Access Journals (Sweden)

    Fahad A. Al-Abbasi

    2012-10-01

    Full Text Available The role of amino acids in diabetes mellitus and its metabolic traits have been suggested previously; however, studied to a very limited scale in the Saudi patient population. Patients diagnosed with diabetes mellitus were included in the current clinical study. Sample was representative and in accordance with the national population distribution. Blood samples were drawn and assayed for glucose, total cholesterol, triglyceride, high density lipoprotein and low density lipoprotein. General biochemical markers, such as alkaline phosphatase (ALP, creatinine kinase (CK, aspartate transaminase (AST, alanine transaminase (ALT and blood urea nitrogen (BUN were assessed. Serum amino acids of different categories (essential, semi-essential and metabolic indicator amino acids were assessed. Correlation co-efficient between each amino acid and serum glucose level was calculated. The current study showed positive correlation between amino acid level and glucose serum concentration in male while it showed negative correlation in female Saudi diabetic patients. Male patients had significantly higher methionine concentration parallel to their glycemic status. Metabolic indicator amino acids significantly changed in concordance with the glycemic status of female patients more than in male patients. In conclusion, serum amino acid is positively correlated with glycemic status in Saudi male diabetic patients while negatively correlated in female patients. Yet, further study would be recommended to utilize serum amino acid profile as surrogate parameter for the metabolic complications of diabetes mellitus.

  16. A relative quantitative Methylation-Sensitive Amplified Polymorphism (MSAP) method for the analysis of abiotic stress

    OpenAIRE

    Bednarek, Piotr T.; Or?owska, Renata; Niedziela, Agnieszka

    2017-01-01

    Background We present a new methylation-sensitive amplified polymorphism (MSAP) approach for the evaluation of relative quantitative characteristics such as demethylation, de novo methylation, and preservation of methylation status of CCGG sequences, which are recognized by the isoschizomers HpaII and MspI. We applied the technique to analyze aluminum (Al)-tolerant and non-tolerant control and Al-stressed inbred triticale lines. The approach is based on detailed analysis of events affecting H...

  17. Genome-wide DNA methylation profiling identifies ALDH1A3 promoter methylation as a prognostic predictor in G-CIMP- primary glioblastoma.

    Science.gov (United States)

    Zhang, Wei; Yan, Wei; You, Gan; Bao, Zhaoshi; Wang, Yongzhi; Liu, Yanwei; You, Yongping; Jiang, Tao

    2013-01-01

    To date, the aberrations in the DNA methylation patterns that are associated with different prognoses of G-CIMP- primary GBMs remain to be elucidated. Here, DNA methylation profiling of primary GBM tissues from 13 long-term survivors (LTS; overall survival ⩾18months) and 20 short-term survivors (STS; overall survival ⩽9months) was performed. Then G-CIMP+ samples were excluded. The differentially expressed CpG loci were identified between residual 18 STS and 9 LTS G-CIMP- samples. Methylation levels of 11 CpG loci (10genes) were statistically significantly lower, and 43 CpG loci (40genes) were statistically significantly higher in the tumor tissues of LTS than those of STS G-CIMP- samples (PCIMP- samples, 3 CpG loci localized in the promoter of ALDH1A3. Furthermore, using an independent validation cohort containing 37 primary GBM samples without IDH1 mutation and MGMT promoter methylation, the hypermethylation status of ALDH1A3 promoter predicted a better prognosis with an accompanied low expression of ALDH1A3 protein. Taken together, our results defined prognosis-related methylation signatures systematically for the first time in G-CIMP- primary GBMs. ALDH1A3 promoter methylation conferred a favorable prognosis in G-CIMP- primary GBMs. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  18. DNA Methylation in Peripheral Blood Cells of Pigs Cloned by Somatic Cell Nuclear Transfer

    DEFF Research Database (Denmark)

    Gao, Fei; Li, Shengting; Lin, Lin

    2011-01-01

    To date, the genome-wide DNA methylation status of cloned pigs has not been investigated. Due to the relatively low success rate of pig cloning by somatic cell nuclear transfer, a better understanding of the epigenetic reprogramming and the global methylation patterns associated with development...... in cloned pigs is required. In this study we applied methylation-specific digital karyotyping tag sequencing by Solexa technology and investigated the genome-wide DNA methylation profiles of peripheral blood cells in cloned pigs with normal phenotypes in comparison with their naturally bred controls....... In the result, we found that globally there was no significant difference of DNA methylation patterns between the two groups. Locus-specifically, some genes involved in embryonic development presented a generally increased level of methylation. Our findings suggest that in cloned pigs with normal phenotypes...

  19. Analysis of DNA methylation related to rice adult plant resistance to bacterial blight based on methylation-sensitive AFLP (MSAP) analysis.

    Science.gov (United States)

    Sha, A H; Lin, X H; Huang, J B; Zhang, D P

    2005-07-01

    DNA methylation is known to play an important role in the regulation of gene expression in eukaryotes. The rice cultivar Wase Aikoku 3 becomes resistant to the blight pathogen Xanthomonas oryzae pv. oryzae at the adult stage. Using methylation-sensitive amplified polymorphism (MSAP) analysis, we compared the patterns of cytosine methylation in seedlings and adult plants of the rice cultivar Wase Aikoku 3 that had been inoculated with the pathogen Xanthomonas oryzae pv. oryzae, subjected to mock inoculation or left untreated. In all, 2000 DNA fragments, each representing a recognition site cleaved by either or both of two isoschizomers, were amplified using 60 pairs of selective primers. A total of 380 sites were found to be methylated. Of these, 45 showed differential cytosine methylation among the seedlings and adult plants subjected to different treatments, and overall levels of methylation were higher in adult plants than in seedlings. All polymorphic fragments were sequenced, and six showed homology to genes that code for products of known function. Northern analysis of three fragments indicated that their expression varied with methylation pattern, with hypermethylation being correlated with repression of transcription, as expected. The results suggest that significant differences in cytosine methylation exist between seedlings and adult plants, and that hypermethylation or hypomethylation of specific genes may be involved in the development of adult plant resistance (APR) in rice plants.

  20. Correlation of human papillomavirus status with apparent diffusion coefficient of diffusion-weighted MRI in head and neck squamous cell carcinomas.

    Science.gov (United States)

    Driessen, Juliette P; van Bemmel, Alexander J M; van Kempen, Pauline M W; Janssen, Luuk M; Terhaard, Chris H J; Pameijer, Frank A; Willems, Stefan M; Stegeman, Inge; Grolman, Wilko; Philippens, Marielle E P

    2016-04-01

    Identification of prognostic patient characteristics in head and neck squamous cell carcinoma (HNSCC) is of great importance. Human papillomavirus (HPV)-positive HNSCCs have favorable response to (chemo)radiotherapy. Apparent diffusion coefficient, derived from diffusion-weighted MRI, has also shown to predict treatment response. The purpose of this study was to evaluate the correlation between HPV status and apparent diffusion coefficient. Seventy-three patients with histologically proven HNSCC were retrospectively analyzed. Mean pretreatment apparent diffusion coefficient was calculated by delineation of total tumor volume on diffusion-weighted MRI. HPV status was analyzed and correlated to apparent diffusion coefficient. Six HNSCCs were HPV-positive. HPV-positive HNSCC showed significantly lower apparent diffusion coefficient compared to HPV-negative. This correlation was independent of other patient characteristics. In HNSCC, positive HPV status correlates with low mean apparent diffusion coefficient. The favorable prognostic value of low pretreatment apparent diffusion coefficient might be partially attributed to patients with a positive HPV status. © 2015 Wiley Periodicals, Inc. Head Neck 38: E613-E618, 2016. © 2015 Wiley Periodicals, Inc.

  1. Infraspecific DNA methylation polymorphism in cotton (Gossypium hirsutum L.).

    Science.gov (United States)

    Keyte, Anna L; Percifield, Ryan; Liu, Bao; Wendel, Jonathan F

    2006-01-01

    Cytosine methylation is important in the epigenetic regulation of gene expression and development in plants and has been implicated in silencing duplicate genes after polyploid formation in several plant groups. Relatively little information exists, however, on levels and patterns of methylation polymorphism (MP) at homologous loci within species. Here we explored the levels and patterns of methylation-polymorphism diversity at CCGG sites within allotetraploid cotton, Gossypium hirsutum, using a methylation-sensitive amplified fragment length polymorphism screen and a selected set of 20 G. hirsutum accessions for which we have information on genetic polymorphism levels and relationships. Methylation and MP exist at high levels within G. hirsutum: of 150 HpaII/MspI sites surveyed, 48 were methylated at the inner cytosine (32%) and 32 of these were polymorphic (67%). Both these values are higher than comparable measures of genetic diversity using restriction fragment length polymorphisms. The high percentage of methylation-polymorphic sites and potential relationship to gene expression underscore the potential significance of MP within and among populations. We speculate that biased correlation of methylation-polymorphic sites and genes in cotton may be a consequence of polyploidy and the attendant doubling of all genes.

  2. Mechanisms of Breast Cancer in Shift Workers: DNA Methylation in Five Core Circadian Genes in Nurses Working Night Shifts.

    Science.gov (United States)

    Samulin Erdem, Johanna; Skare, Øivind; Petersen-Øverleir, Marte; Notø, Heidi Ødegaard; Lie, Jenny-Anne S; Reszka, Edyta; Pepłońska, Beata; Zienolddiny, Shanbeh

    2017-01-01

    Shift work has been suggested to be associated with breast cancer risk, and circadian disruption in shift workers is hypothesized as one of the mechanisms of increased cancer risk. There is, however, insufficient molecular evidence supporting this hypothesis. Using the quantitative methodology of pyrosequencing, epigenetic changes in 5-methyl cytosine (5mC) in five circadian genes CLOCK , BMAL1 , CRY1, PER1 and PER2 in female nurses working night shift work (278 breast cancer cases, 280 controls) were analyzed. In breast cancer cases, a medium exposure to night work was associated with increased methylation levels of the CLOCK (p=0.050), BMAL1 (p=0.001) and CRY1 (p=0.040) genes, compared with controls. Within the cases, analysis of the effects of shift work on the methylation patterns showed that methylation of CRY1 was lower in those who had worked night shift and had a high exposure (p=0.006) compared with cases that had worked only days. For cases with a medium exposure to night work, an increase in BMAL1 (p=0.003) and PER1 (p=0.035) methylation was observed compared with day working (unexposed) cases. The methylation levels of the five core circadian genes were also analyzed in relation to the estrogen and progesterone receptors status of the tumors in the cases, and no correlations were observed. Furthermore, nineteen polymorphisms in the five circadian genes were assessed for their effects on the methylation levels of the respective genes, but no associations were found. In summary, our data suggest that epigenetic regulation of CLOCK , BMAL1, CRY1 and PER1 may contribute to breast cancer in shift workers.

  3. Combined Analysis of COX-2 and p53 Expressions Reveals Synergistic Inverse Correlations with Microsatellite Instability and CpG Island Methylator Phenotype in Colorectal Cancer

    Directory of Open Access Journals (Sweden)

    Shuji Ogino

    2006-06-01

    Full Text Available Cyclooxygenase-2 (COX-2 overexpression and mutations of p53 (a known COX-2 regulator are inversely associated with microsatellite instability—high (MSI-H and CpG island methylator phenotype (CIMP, characterized by extensive promoter methylation, is associated with MSI-H. However, no studies have comprehensively examined interrelations between COX-2, p53, MSI, and CIMP. Using MethyLight, we measured DNA methylation in five CIMP-specific gene promoters [CACNA1G, CDKN2A (p16/INK4A, CRABP1, MLH1, and NEUROG1] in relatively unbiased samples of 751 colorectal cancer cases obtained from two large prospective cohorts; 115 (15% tumors were CIMP-high (≥ 4 of 5 methylated promoters, 251 (33% were CIMP-low (1 to 3 methylated promoters, and the remaining 385 (51% were CIMP-0 (no methylated promoters. CIMP-high tumors were much less frequent in COX-2+/p53+ tumors (4.6% than in COX-2+/p53- tumors (19%; P < .0001, COX-2-/p53+ tumors (17%; P = .04, and COX-2-/p53- tumors (28%; P < .0001. In addition, COX-2+/p53+ tumors were significantly less common in MSI-H CIMP-high tumors (9.7% than in non-MSI-H CIMP-low/CIMP-0 tumors (44–47%; P < .0001. In conclusion, COX-2 and p53 alterations were synergistically inversely correlated with both MSI-H and CIMP-high. Our data suggest that a combined analysis of COX-2 and p53 may be more useful for the molecular classification of colorectal cancer than either COX-2 or p53 analysis alone.

  4. The association between personal sun exposure, serum vitamin D and global methylation in human lymphocytes in a population of healthy adults in South Australia

    Energy Technology Data Exchange (ETDEWEB)

    Nair-Shalliker, Visalini, E-mail: visalinin@nswcc.org.au [Cancer Research Division, Cancer Council New South Wales (Australia); Dhillon, Varinderpal [CSIRO Food and Nutritional Sciences (Australia); Clements, Mark [Department of Medical Epidemiology and Biostatistics, Karolinska Institutet (Sweden); Armstrong, Bruce K. [Sydney School of Public Health, University of Sydney (Australia); Fenech, Michael [CSIRO Food and Nutritional Sciences (Australia)

    2014-07-15

    Highlights: • Solar UV exposure is positively correlated with LINE 1 hypomethylation. • This was observed in human peripheral blood lymphocytes. • There was no evident effect modification by serum vitamin D (25OHD) levels. • This was observed in a population of healthy adults from South Australia. - Abstract: Background: There is a positive association between solar UV exposure and micronucleus frequency in peripheral blood lymphocytes (PBL) and this association may be stronger when serum vitamin D (25(OH)D) levels are insufficient (<50 nmol/L). Micronucleus formation can result from global hypomethylation of DNA repeat sequences. The aim of this analysis was to evaluate the relationship between solar UV exposure and methylation pattern in LINE-1 repetitive elements in PBL DNA and to see if serum 25(OH)D levels modify it. Method: Personal solar UV exposure was estimated from hours of outdoor exposure over 6 weeks recalled at the time of blood collection in 208 male and female participants living in South Australia. Methylation in LINE-1 repetitive elements was assessed in PBL using pyrosequencing. Results: Methylation in LINE-1 decreased with increasing solar UV exposure (% decrease = 0.5% per doubling of sUV; 95%CI: −0.7 to −0.2 p{sub value} = 0.00003). Although there was no correlation between LINE-1 methylation and micronucleus frequency, there was a 4.3% increase (95%CI: 0.6–8.1 p-value = 0.02) in nucleoplasmic bridges and a 4.3% increase in necrosis (CI: 1.9–6.8 p-value = 0.0005) for every 1% increase in LINE-1 methylation. Serum 25(OH)D was not associated with DNA methylation; or did it modify the association of solar UV with DNA methylation. Conclusion: Exposure to solar UV radiation may reduce DNA methylation in circulating lymphocytes. This association does not appear to be influenced or mediated by vitamin D status.

  5. The DNA methylation status of MyoD and IGF-I genes are correlated with muscle growth during different developmental stages of Japanese flounder (Paralichthys olivaceus).

    Science.gov (United States)

    Huang, Yajuan; Wen, Haishen; Zhang, Meizhao; Hu, Nan; Si, Yufeng; Li, Siping; He, Feng

    2018-05-01

    Many genes related to muscle growth modulate myoblast proliferation and differentiation and promote muscle hypertrophy. MyoD is a myogenic determinant that contributes to myoblast determination, and insulin-like growth factor 1 (IGF-I) interacts with MyoD to regulate muscle hypertrophy and muscle mass. In this study, we aimed to assess DNA methylation and mRNA expression patterns of MyoD and IGF-I during different developmental stages of Japanese flounder, and to examine the relationship between MyoD and IGF-I gene. DNA and RNA were extracted from muscles, and DNA methylation of MyoD and IGF-I promoter and exons was detected by bisulfite sequencing. The relative expression of MyoD and IGF-I was measured by quantitative polymerase chain reaction. IGF-I was measured by radioimmunoassay. Interestingly, the lowest expression of MyoD and IGF-I emerged at larva stage, and the mRNA expression was negatively associated with methylation. We hypothesized that many skeletal muscle were required to complete metamorphosis; thus, the expression levels of MyoD and IGF-I genes increased from larva stage and then decreased. The relative expression levels of MyoD and IGF-I exhibited similar patterns, suggesting that MyoD and IGF-I regulated muscle growth through combined effects. Changes in the concentrations of IGF-I hormone were similar to those of IGF-I gene expression. Our results the mechanism through which MyoD and IGF-I regulate muscle development and demonstrated that MyoD interacted with IGF-I to regulate muscle growth during different developmental stages. Copyright © 2018 Elsevier Inc. All rights reserved.

  6. Absolute quantification of DNA methylation using microfluidic chip-based digital PCR.

    Science.gov (United States)

    Wu, Zhenhua; Bai, Yanan; Cheng, Zule; Liu, Fangming; Wang, Ping; Yang, Dawei; Li, Gang; Jin, Qinghui; Mao, Hongju; Zhao, Jianlong

    2017-10-15

    Hypermethylation of CpG islands in the promoter region of many tumor suppressor genes downregulates their expression and in a result promotes tumorigenesis. Therefore, detection of DNA methylation status is a convenient diagnostic tool for cancer detection. Here, we reported a novel method for the integrative detection of methylation by the microfluidic chip-based digital PCR. This method relies on methylation-sensitive restriction enzyme HpaII, which cleaves the unmethylated DNA strands while keeping the methylated ones intact. After HpaII treatment, the DNA methylation level is determined quantitatively by the microfluidic chip-based digital PCR with the lower limit of detection equal to 0.52%. To validate the applicability of this method, promoter methylation of two tumor suppressor genes (PCDHGB6 and HOXA9) was tested in 10 samples of early stage lung adenocarcinoma and their adjacent non-tumorous tissues. The consistency was observed in the analysis of these samples using our method and a conventional bisulfite pyrosequencing. Combining high sensitivity and low cost, the microfluidic chip-based digital PCR method might provide a promising alternative for the detection of DNA methylation and early diagnosis of epigenetics-related diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Reduced Histone H3 Lysine 9 Methylation Contributes to the Pathogenesis of Latent Autoimmune Diabetes in Adults via Regulation of SUV39H2 and KDM4C

    Directory of Open Access Journals (Sweden)

    Xi-yu Liu

    2017-01-01

    Full Text Available Aims. Latent autoimmune diabetes in adults (LADA is an autoimmune disease of which the mechanism is not clear. Emerging evidence suggests that histone methylation contributes to autoimmunity. Methods. Blood CD4+ T lymphocytes from 26 LADA patients and 26 healthy controls were isolated to detect histone H3 lysine 4 and H3 lysine 9 methylation status. Results. Reduced global H3 lysine 9 methylation was observed in LADA patients’ CD4+ T lymphocytes, compared to healthy controls (P < 0.05. H3 lysine 4 methylation was not statistically different. The reduced H3 lysine 9 methylation was associated with GADA titer but not correlated with glycosylated hemoglobin (HbA1c. When the LADA patient group was divided into those with complication and those without, relatively reduced global H3 lysine 9 methylation was observed in LADA patients with complication (P < 0.05. The expression of histone methyltransferase SUV39H2 for H3 lysine 9 methylation was downregulated in LADA patients, and the expression of histone demethylase KDM4C which made H3 lysine 9 demethylation was upregulated. Conclusion. The reduction of histone H3 lysine 9 methylation which may due to the downregulation of methyltransferase SUV39H2 and the upregulation of demethylase KDM4C was found in CD4+ T lymphocytes of LADA patients.

  8. Genome-wide DNA methylation sequencing reveals miR-663a is a novel epimutation candidate in CIMP-high endometrial cancer

    OpenAIRE

    Yanokura, Megumi; Banno, Kouji; Adachi, Masataka; Aoki, Daisuke; Abe, Kuniya

    2017-01-01

    Aberrant DNA methylation is widely observed in many cancers. Concurrent DNA methylation of multiple genes occurs in endometrial cancer and is referred to as the CpG island methylator phenotype (CIMP). However, the features and causes of CIMP-positive endometrial cancer are not well understood. To investigate DNA methylation features characteristic to CIMP-positive endometrial cancer, we first classified samples from 25 patients with endometrial cancer based on the methylation status of three ...

  9. Hyper-Methylated Loci Persisting from Sessile Serrated Polyps to Serrated Cancers.

    Science.gov (United States)

    Andrew, Angeline S; Baron, John A; Butterly, Lynn F; Suriawinata, Arief A; Tsongalis, Gregory J; Robinson, Christina M; Amos, Christopher I

    2017-03-02

    Although serrated polyps were historically considered to pose little risk, it is now understood that progression down the serrated pathway could account for as many as 15%-35% of colorectal cancers. The sessile serrated adenoma/polyp (SSA/P) is the most prevalent pre-invasive serrated lesion. Our objective was to identify the CpG loci that are persistently hyper-methylated during serrated carcinogenesis, from the early SSA/P lesion through the later cancer phases of neoplasia development. We queried the loci hyper-methylated in serrated cancers within our rightsided SSA/Ps from the New Hampshire Colonoscopy Registry, using the Illumina Infinium Human Methylation 450 k panel to comprehensively assess the DNA methylation status. We identified CpG loci and regions consistently hyper-methylated throughout the serrated carcinogenesis spectrum, in both our SSA/P specimens and in serrated cancers. Hyper-methylated CpG loci included the known the tumor suppressor gene RET (p = 5.72 x 10-10), as well as loci in differentially methylated regions for GSG1L, MIR4493, NTNG1, MCIDAS, ZNF568, and RERG. The hyper-methylated loci that we identified help characterize the biology of SSA/P development, and could be useful as therapeutic targets, or for future identification of patients who may benefit from shorter surveillance intervals.

  10. Restoration of CpG Methylation in The Egf Promoter Region during Rat Liver Regeneration

    Science.gov (United States)

    Deming, Li; Ziwei, Li; Xueqiang, Guo; Cunshuan, Xu

    2015-01-01

    Epidermal growth factor (EGF) is an important factor for healing after tissue damage in diverse experimental models. It plays an important role in liver regeneration (LR). The objective of this experiment is to investigate the methylation variation of 10 CpG sites in the Egf promoter region and their relevance to Egf expression during rat liver regenera- tion. As a follow up of our previous study, rat liver tissue was collected after rat 2/3 partial hepatectomy (PH) during the re-organization phase (from days 14 to days 28). Liver DNA was extracted and modified by sodium bisulfate. The methylation status of 10 CpG sites in Egf promoter region was determined using bisulfite sequencing polymerase chain reaction (PCR), as BSP method. The results showed that 3 (sites 3, 4 and 9) out of 10 CpG sites have strikingly methylation changes during the re-organization phase compared to the regeneration phase (from 2 hours to 168 hours, P=0.002, 0.048 and 0.018, respectively). Our results showed that methylation modification of CpGs in the Egf promoter region could be restored to the status before PH operation and changes of methylation didn’t affect Egf mRNA expression during the re-organization phase. PMID:26464832

  11. A CpG island methylator phenotype of colorectal cancer that is contiguous with conventional adenomas, but not serrated polyps.

    Science.gov (United States)

    Hokazono, Koji; Ueki, Takashi; Nagayoshi, Kinuko; Nishioka, Yasunobu; Hatae, Tatsunobu; Koga, Yutaka; Hirahashi, Minako; Oda, Yoshinao; Tanaka, Masao

    2014-11-01

    A subset of colorectal cancers (CRCs) harbor the CpG island methylator phenotype (CIMP), with concurrent multiple promoter hypermethylation of tumor-related genes. A serrated pathway in which CIMP is developed from serrated polyps is proposed. The present study characterized CIMP and morphologically examined precursor lesions of CIMP. In total, 104 CRCs treated between January 1996 and December 2004 were examined. Aberrant promoter methylation of 15 cancer-related genes was analyzed. CIMP status was classified according to the number of methylated genes and was correlated with the clinicopathological features, including the concomitant polyps in and around the tumors. The frequency of aberrant methylation in each CRC showed a bimodal pattern, and the CRCs were classified as CIMP-high (CIMP-H), CIMP-low (CIMP-L) and CIMP-negative (CIMP-N). CIMP-H was associated with aberrant methylation of MLH1 (P=0.005) and with an improved recurrence-free survival (RFS) rate following curative resection compared with CIMP-L/N (five-year RFS rate, 93.8 vs. 67.1%; P=0.044), while CIMP-N tumors were associated with frequent distant metastases at diagnosis (P=0.023). No concomitant serrated lesions were present in the tumors, whereas conventional adenoma was contiguous with 11 (10.6%) of 104 CRCs, including four CIMP-H CRCs. CIMP-H was classified in CRCs by a novel CIMP marker panel and the presence of concomitant tumors revealed that certain CIMP-H CRCs may have arisen from conventional adenomas.

  12. Methylation-sensitive amplification polymorphism analysis of fat and muscle tissues in pigs.

    Science.gov (United States)

    Ma, J D; Li, M Z; Zhou, S L; Zhou, C W; Li, X W

    2012-09-26

    DNA methylation may be involved in regulating the expression of protein-coding genes, resulting in different fat and muscle phenotypes. Using a methylation-sensitive amplified polymorphism approach, we obtained 7423 bands by selective amplification of genomic DNA from six different fat depots and two heterogeneous muscle types from Duroc/Landrace/Yorkshire cross-bred pigs. The degrees of DNA methylation, determined by the percentages of hemi- and fully methylated sites relative to the total number of CCGG sites, were similar in male and female pigs for each specific tissue [χ(2) test; P (two-tailed) > 0.05]. Gender bias was therefore ignored. There were significant differences in the degree of DNA methylation among the eight tissue types [χ(2) test; P(total) (two-tailed) = 0.009]. However, similar degrees of methylation were observed among the six fat depots [χ(2) test; P(fat) (two-tailed) = 0.24 > 0.05]and between the two muscle types [χ(2) test; P(muscle) (two-tailed) = 0.76 > 0.05]. We conclude that the degree of DNA methylation differs between porcine fat and muscle tissue, but that the methylation status of a particular tissue type is similar, despite being deposited at different body sites.

  13. Non-Steroidal Anti-Inflammatory Drug Use and Genomic DNA Methylation in Blood.

    Directory of Open Access Journals (Sweden)

    Lauren E Wilson

    Full Text Available Non-steroidal anti-inflammatory drug (NSAID use is associated with decreased risk of some cancers. NSAID use modulates the epigenetic profile of normal colonic epithelium and may reduce risk of colon cancer through this pathway; however, the effect of NSAID use on the DNA methylation profile of other tissues including whole blood has not yet been examined.Using the Sister Study cohort, we examined the association between NSAID usage and whole genome methylation patterns in blood DNA. Blood DNA methylation status across 27,589 CpG sites was evaluated for 871 women using the Illumina Infinium HumanMethylation27 Beadchip, and in a non-overlapping replication sample of 187 women at 485,512 CpG sites using the Infinium HumanMethylation450 Beadchip. We identified a number of CpG sites that were differentially methylated in regular, long-term users of NSAIDs in the discovery group, but none of these sites were statistically significant in our replication group.We found no replicable methylation differences in blood related to NSAID usage. If NSAID use does effect blood DNA methylation patterns, differences are likely small.

  14. ROLE OF DNA METHYLATION AS A DIAGNOSTIC BIOMARKER OF SPORADIC BREAST CANCER

    Directory of Open Access Journals (Sweden)

    Wirsma Arif Harahap

    2017-02-01

    Full Text Available The initiation and progression of breast cancer have been recognized for many years to be secondary to the accumulation of genetic mutations which lead to aberrant cellular function. Genetic mutations, either inherited or sporadic, may result in the activation of oncogenes and the inactivation of tumor suppressor genes. The more recent discovery that reversible alterations in histone proteins and deoxyribonucleic acid (DNA can also lead to tumorigenesis has introduced a novel term to the field of cancer research: epigenetics.  Epigenetics refers to the study of heritable changes in gene regulation that do not involve a change in the DNA sequence. The most often studied in epigenetics of breast cancer is DNA methylation. That a promoter methylation result in transcription blockade supports the notion that cellular inhibition takes place. Compared to normal tissues, hypermethylation occurs from double to triple in cancerous ones. DNA methylation plays a crucial role in oncogenesis and is one of the hallmarks of cancer. Detection of aberrantly methylated CpG islands in promoter region of several genes in DNA sample derived from nipple aspirates, serum, or cancer tissue associated with down regulation of expression or loss of function of these genes has been associated with early stages of breast cancer, where  hypermethylation of CpG island points to poorer prognosis in breast cancer.  DNA methylation has been identified as signature for TNBC. Methylation of BRCA1 gene is frequently demonstrated in young, estrogen receptor-negative breast cancer patients. Methylation of specific genes is known to differ across race and socioeconomic status. BRCA1 methylation in premenopausal women with sporadic breast cancer in West Sumatra region has been higher than in Western women. DNA methylation may be used to enhance current breast cancer classification. There is such a distinction between methylation and gene expression profiles of breast cancer that not

  15. The tumour suppressor SOX11 is associated with improved survival among high grade epithelial ovarian cancers and is regulated by reversible promoter methylation

    International Nuclear Information System (INIS)

    Sernbo, Sandra; Gustavsson, Elin; Brennan, Donal J; Gallagher, William M; Rexhepaj, Elton; Rydnert, Frida; Jirström, Karin; Borrebaeck, Carl AK; Ek, Sara

    2011-01-01

    The neural transcription factor SOX11 has been described as a prognostic marker in epithelial ovarian cancers (EOC), however its role in individual histological subtypes and tumour grade requires further clarification. Furthermore, methylation-dependent silencing of SOX11 has been reported for B cell lymphomas and indicates that epigenetic drugs may be used to re-express this tumour suppressor, but information on SOX11 promoter methylation in EOC is still lacking. SOX11 expression and clinicopathological data was compared using χ 2 test in a cohort of 154 cases of primary invasive EOC. Kaplan-Meier analysis and the log rank test were applied to evaluate ovarian cancer-specific survival (OCSS) and overall survival (OS) in strata, according to SOX11 expression. Also, the methylation status of the SOX11 promoter was determined by sodium bisulfite sequencing and methylation specific PCR (MSP). Furthermore, the effect of ectopic overexpression of SOX11 on proliferation was studied through [3H]-thymidine incorporation. SOX11 expression was associated with an improved survival of patients with high grade EOC, although not independent of stage. Further analyses of EOC cell lines showed that SOX11 mRNA and protein were expressed in two of five cell lines, correlating with promoter methylation status. Demethylation was successfully performed using 5'-Aza-2'deoxycytidine (5-Aza-dC) resulting in SOX11 mRNA and protein expression in a previously negative EOC cell line. Furthermore, overexpression of SOX11 in EOC cell lines confirmed the growth regulatory role of SOX11. SOX11 is a functionally associated protein in EOC with prognostic value for high-grade tumours. Re-expression of SOX11 in EOC indicates a potential use of epigenetic drugs to affect cellular growth in SOX11-negative tumours

  16. The tumour suppressor SOX11 is associated with improved survival among high grade epithelial ovarian cancers and is regulated by reversible promoter methylation

    LENUS (Irish Health Repository)

    Sernbo, Sandra

    2011-09-24

    Abstract Background The neural transcription factor SOX11 has been described as a prognostic marker in epithelial ovarian cancers (EOC), however its role in individual histological subtypes and tumour grade requires further clarification. Furthermore, methylation-dependent silencing of SOX11 has been reported for B cell lymphomas and indicates that epigenetic drugs may be used to re-express this tumour suppressor, but information on SOX11 promoter methylation in EOC is still lacking. Methods SOX11 expression and clinicopathological data was compared using χ2 test in a cohort of 154 cases of primary invasive EOC. Kaplan-Meier analysis and the log rank test were applied to evaluate ovarian cancer-specific survival (OCSS) and overall survival (OS) in strata, according to SOX11 expression. Also, the methylation status of the SOX11 promoter was determined by sodium bisulfite sequencing and methylation specific PCR (MSP). Furthermore, the effect of ectopic overexpression of SOX11 on proliferation was studied through [3H]-thymidine incorporation. Results SOX11 expression was associated with an improved survival of patients with high grade EOC, although not independent of stage. Further analyses of EOC cell lines showed that SOX11 mRNA and protein were expressed in two of five cell lines, correlating with promoter methylation status. Demethylation was successfully performed using 5\\'-Aza-2\\'deoxycytidine (5-Aza-dC) resulting in SOX11 mRNA and protein expression in a previously negative EOC cell line. Furthermore, overexpression of SOX11 in EOC cell lines confirmed the growth regulatory role of SOX11. Conclusions SOX11 is a functionally associated protein in EOC with prognostic value for high-grade tumours. Re-expression of SOX11 in EOC indicates a potential use of epigenetic drugs to affect cellular growth in SOX11-negative tumours.

  17. Parental Socio-Economic Status as Correlate of Child Labour in Ile-Ife, Nigeria

    Science.gov (United States)

    Elegbeleye, O. S.; Olasupo, M. O.

    2012-01-01

    This study investigated the relationship between parental socio-economic status and child labour practices in Ile-Ife, Nigeria. The study employed survey method to gather data from 200 parents which constituted the study population. Pearson Product Moment Correlation and t-test statistics were used for the data analyses. The outcome of the study…

  18. Frequent silencing of RASSF1A by DNA methylation in thymic neuroendocrine tumours.

    Science.gov (United States)

    Kajiura, Koichiro; Takizawa, Hiromitsu; Morimoto, Yuki; Masuda, Kiyoshi; Tsuboi, Mitsuhiro; Kishibuchi, Reina; Wusiman, Nuliamina; Sawada, Toru; Kawakita, Naoya; Toba, Hiroaki; Yoshida, Mitsuteru; Kawakami, Yukikiyo; Naruto, Takuya; Imoto, Issei; Tangoku, Akira; Kondo, Kazuya

    2017-09-01

    Aberrant methylation of promoter CpG islands (CGIs) of tumour suppressor genes is a common epigenetic mechanism underlying cancer pathogenesis. The methylation patterns of thymic tumours have not been studied in detail since such tumours are rare. Herein, we sought to identify genes that could serve as epigenetic targets for thymic neuroendocrine tumour (NET) therapy. Genome-wide screening for aberrantly methylated CGIs was performed in three NET samples, seven thymic carcinoma (TC) samples, and eight type-B3 thymoma samples. The methylation status of thymic epithelial tumours (TETs) samples was validated by pyrosequencing in a larger cohort. The expression status was analysed by quantitative polymerase chain reaction (PCR) and immunohistochemistry. We identified a CGI on a novel gene, RASSF1A, which was strongly hypermethylated in NET, but not in thymic carcinoma or B3 thymoma. RASSF1A was identified as a candidate gene statistically and bibliographically, as it showed frequent CGI hypermethylation in NET by genome-wide screening. Pyrosequencing confirmed significant hypermethylation of a RASSF1A CGI in NET. Low-grade NET tissue was more strongly methylated than high-grade NET. Quantitative PCR and immunohistochemical staining revealed that RASSF1A mRNA and protein expression levels were negatively regulated by DNA methylation. RASSF1A is a tumour suppressor gene epigenetically dysregulated in NET. Aberrant methylation of RASSF1A has been reported in various tumours, but this is the first report of RASSF1A hypermethylation in TETs. RASSF1A may represent an epigenetic therapeutic target in thymic NET. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Variation in the DNA methylation pattern of expressed and nonexpressed genes in chicken.

    Science.gov (United States)

    Cooper, D N; Errington, L H; Clayton, R M

    1983-01-01

    Using methyl-sensitive and -insensitive restriction enzymes, Hpa II and Msp I, the methylation status of various chicken genes was examined in different tissues and developmental stages. Tissue-specific differences in methylation were found for the delta-crystallin, beta-tubulin, G3PDH, rDNA, and actin genes but not for the histone genes. Developmental decreases in methylation were noted for the delta-crystallin and actin genes in chicken kidney between embryo and adult. Since most of the sequences examined were housekeeping genes, transcriptional differences are apparently not a necessary accompaniment to changes in DNA methylation at the CpG sites examined. The only exception is sperm DNA where the delta-crystallin, beta-tubulin, and actin genes are highly methylated and almost certainly not transcribed. However the G3PDH genes are no more highly methylated in sperm than in other somatic tissues. Many sequences homologous to the rDNA and histone probes used are unmethylated in all tissues examined including sperm, but a methylated rDNA subfraction is more heavily methylated in sperm than in other tissues. We speculate as to the significance of these differences in sperm DNA methylation in the light of possible requirements for early gene activation and the probable deleterious mutagenic effects of heavy methylation within coding sequences.

  20. Comprehensive analyses of imprinted differentially methylated regions reveal epigenetic and genetic characteristics in hepatoblastoma

    International Nuclear Information System (INIS)

    Rumbajan, Janette Mareska; Aoki, Shigehisa; Kohashi, Kenichi; Oda, Yoshinao; Hata, Kenichiro; Saji, Tsutomu; Taguchi, Tomoaki; Tajiri, Tatsuro; Soejima, Hidenobu; Joh, Keiichiro; Maeda, Toshiyuki; Souzaki, Ryota; Mitsui, Kazumasa; Higashimoto, Ken; Nakabayashi, Kazuhiko; Yatsuki, Hitomi; Nishioka, Kenichi; Harada, Ryoko

    2013-01-01

    Aberrant methylation at imprinted differentially methylated regions (DMRs) in human 11p15.5 has been reported in many tumors including hepatoblastoma. However, the methylation status of imprinted DMRs in imprinted loci scattered through the human genome has not been analyzed yet in any tumors. The methylation statuses of 33 imprinted DMRs were analyzed in 12 hepatoblastomas and adjacent normal liver tissue by MALDI-TOF MS and pyrosequencing. Uniparental disomy (UPD) and copy number abnormalities were investigated with DNA polymorphisms. Among 33 DMRs analyzed, 18 showed aberrant methylation in at least 1 tumor. There was large deviation in the incidence of aberrant methylation among the DMRs. KvDMR1 and IGF2-DMR0 were the most frequently hypomethylated DMRs. INPP5Fv2-DMR and RB1-DMR were hypermethylated with high frequencies. Hypomethylation was observed at certain DMRs not only in tumors but also in a small number of adjacent histologically normal liver tissue, whereas hypermethylation was observed only in tumor samples. The methylation levels of long interspersed nuclear element-1 (LINE-1) did not show large differences between tumor tissue and normal liver controls. Chromosomal abnormalities were also found in some tumors. 11p15.5 and 20q13.3 loci showed the frequent occurrence of both genetic and epigenetic alterations. Our analyses revealed tumor-specific aberrant hypermethylation at some imprinted DMRs in 12 hepatoblastomas with additional suggestion for the possibility of hypomethylation prior to tumor development. Some loci showed both genetic and epigenetic alterations with high frequencies. These findings will aid in understanding the development of hepatoblastoma

  1. Molecular characterization of HOXC8 gene and methylation status analysis of its exon 1 associated with the length of cashmere fiber in Liaoning cashmere goat.

    Science.gov (United States)

    Bai, Wen L; Wang, Jiao J; Yin, Rong H; Dang, Yun L; Wang, Ze Y; Zhu, Yu B; Cong, Yu Y; Deng, Liang; Guo, Dan; Wang, Shi Q; Yang, Shu H; Xue, Hui L

    2017-02-01

    Homeobox protein Hox-C8 (HOXC8) is a member of Hox family. It is expressed in the dermal papilla of the skin and is thought to be associated with the hair inductive capacity of dermal papilla cells. In the present study, we isolated and characterized a full-length open reading frame of HOXC8 cDNA from the skin tissue of Liaoning cashmere goat, as well as, established a phylogenetic relationship of goat HOXC8 with that of other species. Also, we investigated the effect of methylation status of HOXC8 exon 1 at anagen secondary hair follicle on the cashmere fiber traits in Liaoning cashmere goat. The sequence analysis indicated that the obtained cDNA was 1134-bp in length containing a complete ORF of 729-bp. It encoded a peptide of 242 amino acid residues in length. The structural analysis indicated that goat HOXC8 contained a typical homeobox domain. The phylogenetic analysis revealed that Capra hircus HOXC8 had a closer genetic relationship with that of Ovis aries, followed by Bos Taurus and Bubalus bubalis. The methylation analysis suggested that the methylation degree of HOXC8 exon 1 in anagen secondary hair follicle might be involved in regulating the growth of cashmere fiber in Liaoning cashmere goat. Our results provide new evidence for understanding the molecular structural and evolutionary characteristics of HOXC8 in Liaoning cashmere goat, as well as, for further insight into the role of methylation degree of HOXC8 exon 1 regulates the growth of cashmere fiber in goat.

  2. Genome-wide DNA methylation maps in follicular lymphoma cells determined by methylation-enriched bisulfite sequencing.

    Directory of Open Access Journals (Sweden)

    Jeong-Hyeon Choi

    Full Text Available BACKGROUND: Follicular lymphoma (FL is a form of non-Hodgkin's lymphoma (NHL that arises from germinal center (GC B-cells. Despite the significant advances in immunotherapy, FL is still not curable. Beyond transcriptional profiling and genomics datasets, there currently is no epigenome-scale dataset or integrative biology approach that can adequately model this disease and therefore identify novel mechanisms and targets for successful prevention and treatment of FL. METHODOLOGY/PRINCIPAL FINDINGS: We performed methylation-enriched genome-wide bisulfite sequencing of FL cells and normal CD19(+ B-cells using 454 sequencing technology. The methylated DNA fragments were enriched with methyl-binding proteins, treated with bisulfite, and sequenced using the Roche-454 GS FLX sequencer. The total number of bases covered in the human genome was 18.2 and 49.3 million including 726,003 and 1.3 million CpGs in FL and CD19(+ B-cells, respectively. 11,971 and 7,882 methylated regions of interest (MRIs were identified respectively. The genome-wide distribution of these MRIs displayed significant differences between FL and normal B-cells. A reverse trend in the distribution of MRIs between the promoter and the gene body was observed in FL and CD19(+ B-cells. The MRIs identified in FL cells also correlated well with transcriptomic data and ChIP-on-Chip analyses of genome-wide histone modifications such as tri-methyl-H3K27, and tri-methyl-H3K4, indicating a concerted epigenetic alteration in FL cells. CONCLUSIONS/SIGNIFICANCE: This study is the first to provide a large scale and comprehensive analysis of the DNA methylation sequence composition and distribution in the FL epigenome. These integrated approaches have led to the discovery of novel and frequent targets of aberrant epigenetic alterations. The genome-wide bisulfite sequencing approach developed here can be a useful tool for profiling DNA methylation in clinical samples.

  3. MGMT promoter methylation determined by HRM in comparison to MSP and pyrosequencing for predicting high-grade glioma response.

    Science.gov (United States)

    Switzeny, Olivier J; Christmann, Markus; Renovanz, Mirjam; Giese, Alf; Sommer, Clemens; Kaina, Bernd

    2016-01-01

    The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) causes resistance of cancer cells to alkylating agents and, therefore, is a well-established predictive marker for high-grade gliomas that are routinely treated with alkylating drugs. Since MGMT is highly epigenetically regulated, the MGMT promoter methylation status is taken as an indicator of MGMT silencing, predicting the outcome of glioma therapy. MGMT promoter methylation is usually determined by methylation specific PCR (MSP), which is a labor intensive and error-prone method often used semi-quantitatively. Searching for alternatives, we used closed-tube high resolution melt (HRM) analysis, which is a quantitative method, and compared it with MSP and pyrosequencing regarding its predictive value. We analyzed glioblastoma cell lines with known MGMT activity and formalin-fixed samples from IDH1 wild-type high-grade glioma patients (WHO grade III/IV) treated with radiation and temozolomide by HRM, MSP, and pyrosequencing. The data were compared as to progression-free survival (PFS) and overall survival (OS) of patients exhibiting the methylated and unmethylated MGMT status. A promoter methylation cut-off level relevant for PFS and OS was determined. In a multivariate Cox regression model, methylation of MGMT promoter of high-grade gliomas analyzed by HRM, but not MSP, was found to be an independent predictive marker for OS. Univariate Kaplan-Meier analyses revealed for PFS and OS a significant and better discrimination between methylated and unmethylated tumors when quantitative HRM was used instead of MSP. Compared to MSP and pyrosequencing, the HRM method is simple, cost effective, highly accurate and fast. HRM is at least equivalent to pyrosequencing in quantifying the methylation level. It is superior in predicting PFS and OS of high-grade glioma patients compared to MSP and, therefore, can be recommended being used routinely for determination of the MGMT status of gliomas.

  4. eMethylsorb: electrochemical quantification of DNA methylation at CpG resolution using DNA-gold affinity interactions.

    Science.gov (United States)

    Sina, Abu Ali Ibn; Howell, Sidney; Carrascosa, Laura G; Rauf, Sakandar; Shiddiky, Muhammad J A; Trau, Matt

    2014-11-07

    We report a simple electrochemical method referred to as "eMethylsorb" for the detection of DNA methylation. The method relies on the base dependent affinity interaction of DNA with gold. The methylation status of DNA is quantified by monitoring the electrochemical current as a function of the relative adsorption level of bisulphite treated DNA samples onto a bare gold electrode. This method can successfully distinguish methylated and unmethylated epigenotypes at single CpG resolution.

  5. Determination of epigenetic inheritance, genetic inheritance, and estimation of genome DNA methylation in a full-sib family of Cupressus sempervirens L.

    Science.gov (United States)

    Avramidou, Evangelia V; Doulis, Andreas G; Aravanopoulos, Filippos A

    2015-05-15

    Genetic inheritance and epigenetic inheritance are significant determinants of plant evolution, adaptation and plasticity. We studied inheritance of restriction site polymorphisms by the f-AFLP method and epigenetic DNA cytosine methylation inheritance by the f-MSAP technique. The study involved parents and 190 progeny of a Cupressus sempervirens L. full-sib family. Results from AFLP genetic data revealed that 71.8% of the fragments studied are under Mendelian genetic control, whereas faithful Mendelian inheritance for the MSAP fragments was low (4.29%). Further, MSAP fragment analysis showed that total methylation presented a mean of 28.2%, which was higher than the midparent value, while maternal inheritance was higher (5.65%) than paternal (3.01%). Interestingly de novo methylation in the progeny was high (19.65%) compared to parental methylation. Genetic and epigenetic distances for parents and offspring were not correlated (R(2)=0.0005). Furthermore, we studied correlation of total relative methylation and CG methylation with growth (height, diameter). We found CG/CNG methylation (N: A, C, T) to be positively correlated with height and diameter, while total relative methylation and CG methylation were positively correlated with height. Results are discussed in light of further research needed and of their potential application in breeding. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Correlation between HPV status at T and N sites of oropharyngeal squamous cell carcinomas

    DEFF Research Database (Denmark)

    Josiassen, Michael Vallop; Charabi, Birgitte; Lajer, Christel Braemer

    2017-01-01

    Objectives: Human papilloma virus (HPV) is known to be associated with oropharyngeal squamous cell carcinomas (OPSCC) and may potentially play a vital role in tumor metastasis. The purpose of this study was to correlate HPV status of cervical lymph node metastases with their respective primary...

  7. MECP2 promoter methylation and X chromosome inactivation in autism.

    Science.gov (United States)

    Nagarajan, Raman P; Patzel, Katherine A; Martin, Michelle; Yasui, Dag H; Swanberg, Susan E; Hertz-Picciotto, Irva; Hansen, Robin L; Van de Water, Judy; Pessah, Isaac N; Jiang, Ruby; Robinson, Wendy P; LaSalle, Janine M

    2008-06-01

    Epigenetic mechanisms have been proposed to play a role in the etiology of autism. This hypothesis is supported by the discovery of increased MECP2 promoter methylation associated with decreased MeCP2 protein expression in autism male brain. To further understand the influence of female X chromosome inactivation (XCI) and neighboring methylation patterns on aberrant MECP2 promoter methylation in autism, multiple methylation analyses were peformed on brain and blood samples from individuals with autism. Bisulfite sequencing analyses of a region 0.6 kb upstream of MECP2 in brain DNA samples revealed an abrupt transition from a highly methylated region in both sexes to a region unmethylated in males and subject to XCI in females. Chromatin immunoprecipitation analysis demonstrated that the CCTC-binding factor (CTCF) bound to this transition region in neuronal cells, consistent with a chromatin boundary at the methylation transition. Male autism brain DNA samples displayed a slight increase in methylation in this transition region, suggesting a possible aberrant spreading of methylation into the MECP2 promoter in autism males across this boundary element. In addition, autistic female brain DNA samples showed evidence for aberrant MECP2 promoter methylation as an increase in the number of bisulfite sequenced clones with undefined XCI status for MECP2 but not androgen receptor (AR). To further investigate the specificity of MECP2 methylation alterations in autism, blood DNA samples from females and mothers of males with autism were also examined for XCI skewing at AR, but no significant increase in XCI skewing was observed compared to controls. These results suggest that the aberrant MECP2 methylation in autism brain DNA samples is due to locus-specific rather than global X chromosome methylation changes.

  8. Stratified randomization controls better for batch effects in 450K methylation analysis: A cautionary tale

    Directory of Open Access Journals (Sweden)

    Olive D. Buhule

    2014-10-01

    Full Text Available Background: Batch effects in DNA methylation microarray experiments can lead to spurious results if not properly handled during the plating of samples. Methods: Two pilot studies examining the association of DNA methylation patterns across the genome with obesity in Samoan men were investigated for chip- and row-specific batch effects. For each study, the DNA of 46 obese men and 46 lean men were assayed using Illumina's Infinium HumanMethylation450 BeadChip. In the first study (Sample One, samples from obese and lean subjects were examined on separate chips. In the second study (Sample Two, the samples were balanced on the chips by lean/obese status, age group, and census region. We used methylumi, watermelon, and limma R packages, as well as ComBat, to analyze the data. Principal component analysis and linear regression were respectively employed to identify the top principal components and to test for their association with the batches and lean/obese status. To identify differentially methylated positions (DMPs between obese and lean males at each locus, we used a moderated t-test.Results: Chip effects were effectively removed from Sample Two but not Sample One. In addition, dramatic differences were observed between the two sets of DMP results. After removing'' batch effects with ComBat, Sample One had 94,191 probes differentially methylated at a q-value threshold of 0.05 while Sample Two had zero differentially methylated probes. The disparate results from Sample One and Sample Two likely arise due to the confounding of lean/obese status with chip and row batch effects.Conclusion: Even the best possible statistical adjustments for batch effects may not completely remove them. Proper study design is vital for guarding against spurious findings due to such effects.

  9. DNA methylation-based subtype prediction for pediatric acute lymphoblastic leukemia

    DEFF Research Database (Denmark)

    Nordlund, Jessica; Bäcklin, Christofer L; Zachariadis, Vasilios

    2015-01-01

    BACKGROUND: We present a method that utilizes DNA methylation profiling for prediction of the cytogenetic subtypes of acute lymphoblastic leukemia (ALL) cells from pediatric ALL patients. The primary aim of our study was to improve risk stratification of ALL patients into treatment groups using DNA...... in cytogenetically undefined ALL patient groups and could be implemented as a complementary method for diagnosis of ALL. The results of our study provide clues to the origin and development of leukemic transformation. The methylation status of the CpG sites constituting the classifiers also highlight relevant...

  10. [The Role of 5-Aza-CdR on Methylation of Promoter in RASSF1A Gene in Endometrial Carcinoma].

    Science.gov (United States)

    Huang, Li-ping; Chen, Chen; Wang, Xue-ping; Liu, Hui

    2015-05-01

    To explore the effect of demethylating drug 5-Aza-2'-deoxycytidine (5-Aza-CdR) on methtylation status of the Ras-association domain familylA gene (RASSF1A) in human endometrial carcinoma. Randomly'assign the human endometrial carcinoma cell line HEC-1-B into groups and use demethylating drug 5-Aza-CdR of different concentration to treat them. Then Methylation-specific polymerase chain reaction (MSP), real-time PCR, Western blot, TUNEL technology were used to analyze methylation status of RASSF1A promoter CpG islands, RASSF1A mRNA expression, RASSF1A protein expression and apoptosis of HEC-1-B cell. High DNA methylation in RASSF1A gene promoter region, low RASSF1A mRNA level and protein expression and out of control of human endometrial carcinoma cell HEC-1-B apoptosis were observed. 5-Aza-CdR of different concentration could reverse RASSF1A gene's methylation status, recover the expression of mRNA and protein, and control the growth of HEC-1-B by inducing apoptosis. Aberrant methylation of RASSF1A in endometrial cancer as a therapeutic target, demethylating agent 5-Aza-CdR could be an effective way of gene therapy.

  11. Integration of CpG-free DNA induces de novo methylation of CpG islands in pluripotent stem cells

    KAUST Repository

    Takahashi, Yuta; Wu, Jun; Suzuki, Keiichiro; Martinez-Redondo, Paloma; Li, Mo; Liao, Hsin-Kai; Wu, Min-Zu; Herná ndez-Bení tez, Reyna; Hishida, Tomoaki; Shokhirev, Maxim Nikolaievich; Esteban, Concepcion Rodriguez; Sancho-Martinez, Ignacio; Belmonte, Juan Carlos Izpisua

    2017-01-01

    that insertion of CpG-free DNA into targeted CGIs induces de novo methylation of the entire CGI in human pluripotent stem cells (PSCs). The methylation status is stably maintained even after CpG-free DNA removal, extensive passaging, and differentiation

  12. Theoretical spectroscopic characterization at low temperatures of S-methyl thioformate and O-methyl thioformate

    International Nuclear Information System (INIS)

    Senent, M. L.; Puzzarini, C.; Hochlaf, M.; Domínguez-Gómez, R.; Carvajal, M.

    2014-01-01

    Highly correlated ab initio methods are employed to determine spectroscopic properties at low temperatures of two S-analogs of methyl formate: S-methyl thioformate CH 3 -S-CHO (MSCHO) and O-methyl thioformate CH 3 -O-CHS (MOCHS). Both species are detectable and they are expected to play an important role in Astrochemistry. Molecular properties are compared with those of the O-analog, methyl formate. Both isomers present two conformers cis and trans. cis-CH 3 -S-CHO represents the most stable structure lying 4372.2 cm −1 below cis-CH 3 -O-CHS. The energy difference between the cis and trans forms is drastically lower for MSCHO (1134 cm −1 ) than for MOCHS (1963.6 cm −1 ). Harmonic and anharmonic fundamentals and the corresponding intensities, as well as the rotational constants for the ground vibrational and first excited torsional states and the centrifugal distortions constants, are provided. Low torsional energy levels have been obtained by solving variationally a two dimensional Hamiltonian expressed in terms of the two torsional degrees of freedom. The corresponding 2D potential energy surfaces have been computed at the CCSD(T)/aug-cc-pVTZ level of theory. The methyl torsional barriers V 3 (cis) are determined to be 139.7 cm −1 (CH 3 -S-CHO) and 670.4 cm −1 (CH 3 -O-CHS). The A/E splitting of ground torsional state has been estimated to be 0.438 cm −1 for CH 3 -S-CHO and negligible for CH 3 -O-CHS

  13. Methylation profiling in individuals with Russell-Silver syndrome.

    Science.gov (United States)

    Peñaherrera, Maria S; Weindler, Susanne; Van Allen, Margot I; Yong, Siu-Li; Metzger, Daniel L; McGillivray, Barbara; Boerkoel, Cornelius; Langlois, Sylvie; Robinson, Wendy P

    2010-02-01

    Russell-Silver syndrome (RSS) is a heterogeneous disorder associated with pre- and post-natal growth restriction and relative macrocephaly. Involvement of imprinted genes on both chromosome 7 and 11p15.5 has been reported. To further characterize the role of epimutations in RSS we evaluated the methylation status at both 11p15.5 imprinting control regions (ICRs): ICR1 associated with H19/IGF2 expression and ICR2 (KvDMR1) associated with CDKN1C expression in a series of 35 patients with RSS. We also evaluated methylation at the promoter regions of other imprinted genes involved in growth such as PLAGL1 (6q24), GCE (7q21), and PEG10 (7q21) in this series of 35 patients with RSS. Thirteen of the 35 patient samples, but none of 22 controls, showed methylation levels at ICR1 that were more than 2 SD below the mean for controls. Three RSS patients were highly methylated at the SCGE promoter, all of which were diagnosed with upd(7)mat. To identify further potential global methylation changes in RSS patients, a subset of 22 patients were evaluated at 1505 CpG sites by the Illumina GoldenGate methylation array. Among the few CpG sites displaying a significant difference between RSS patients and controls, was a CpG associated with the H19 promoter. No other sites associated with known imprinted genes were identified as abnormally methylated in RSS patients by this approach. While the association of hypomethylation of the H19/IGF2 ICR1 is clear, the continuous distribution of methylation values among the patients and controls complicates the establishment of clear cut-offs for clinical diagnosis. Copyright 2010 Wiley-Liss, Inc.

  14. Integrated analysis of dynamic FET PET/CT parameters, histology, and methylation profiling of 44 gliomas.

    Science.gov (United States)

    Röhrich, Manuel; Huang, Kristin; Schrimpf, Daniel; Albert, Nathalie L; Hielscher, Thomas; von Deimling, Andreas; Schüller, Ulrich; Dimitrakopoulou-Strauss, Antonia; Haberkorn, Uwe

    2018-05-07

    Dynamic 18 F-FET PET/CT is a powerful tool for the diagnosis of gliomas. 18 F-FET PET time-activity curves (TAC) allow differentiation between histological low-grade gliomas (LGG) and high-grade gliomas (HGG). Molecular methods such as epigenetic profiling are of rising importance for glioma grading and subclassification. Here, we analysed dynamic 18 F-FET PET data, and the histological and epigenetic features of 44 gliomas. Dynamic 18 F-FET PET was performed in 44 patients with newly diagnosed, untreated glioma: 10 WHO grade II glioma, 13 WHO grade III glioma and 21 glioblastoma (GBM). All patients underwent stereotactic biopsy or tumour resection after 18 F-FET PET imaging. As well as histological analysis of tissue samples, DNA was subjected to epigenetic analysis using the Illumina 850 K methylation array. TACs, standardized uptake values corrected for background uptake in healthy tissue (SUVmax/BG), time to peak (TTP) and kinetic modelling parameters were correlated with histological diagnoses and with epigenetic signatures. Multivariate analyses were performed to evaluate the diagnostic accuracy of 18 F-FET PET in relation to the tumour groups identified by histological and methylation-based analysis. Epigenetic profiling led to substantial tumour reclassification, with six grade II/III gliomas reclassified as GBM. Overlap of HGG-typical TACs and LGG-typical TACs was dramatically reduced when tumours were clustered on the basis of their methylation profile. SUVmax/BG values of GBM were higher than those of LGGs following both histological diagnosis and methylation-based diagnosis. The differences in TTP between GBMs and grade II/III gliomas were greater following methylation-based diagnosis than following histological diagnosis. Kinetic modeling showed that relative K1 and fractal dimension (FD) values significantly differed in histology- and methylation-based GBM and grade II/III glioma between those diagnosed histologically and those diagnosed by

  15. Promoter methylation of Wnt-antagonists in polypoid and nonpolypoid colorectal adenomas

    International Nuclear Information System (INIS)

    Voorham, Quirinus JM; Mulder, Chris JJ; Engeland, Manon van; Meijer, Gerrit A; Steenbergen, Renske DM; Carvalho, Beatriz; Janssen, Jerry; Tijssen, Marianne; Snellenberg, Suzanne; Mongera, Sandra; Grieken, Nicole CT van; Grabsch, Heike; Kliment, Martin; Rembacken, Bjorn J

    2013-01-01

    Nonpolypoid adenomas are a subgroup of colorectal adenomas that have been associated with a more aggressive clinical behaviour compared to their polypoid counterparts. A substantial proportion of nonpolypoid and polypoid adenomas lack APC mutations, APC methylation or chromosomal loss of the APC locus on chromosome 5q, suggesting the involvement of other Wnt-pathway genes. The present study investigated promoter methylation of several Wnt-pathway antagonists in both nonpolypoid and polypoid adenomas. Quantitative methylation-specific PCR (qMSP) was used to evaluate methylation of four Wnt-antagonists, SFRP2, WIF-1, DKK3 and SOX17 in 18 normal colorectal mucosa samples, 9 colorectal cancer cell lines, 18 carcinomas, 44 nonpolypoid and 44 polypoid adenomas. Results were integrated with previously obtained data on APC mutation, methylation and chromosome 5q status from the same samples. Increased methylation of all genes was found in the majority of cell lines, adenomas and carcinomas compared to normal controls. WIF-1 and DKK3 showed a significantly lower level of methylation in nonpolypoid compared to polypoid adenomas (p < 0.01). Combining both adenoma types, a positive trend between APC mutation and both WIF-1 and DKK3 methylation was observed (p < 0.05). Methylation of Wnt-pathway antagonists represents an additional mechanism of constitutive Wnt-pathway activation in colorectal adenomas. Current results further substantiate the existence of partially alternative Wnt-pathway disruption mechanisms in nonpolypoid compared to polypoid adenomas, in line with previous observations

  16. Influence of rice straw amendment on mercury methylation and nitrification in paddy soils

    International Nuclear Information System (INIS)

    Liu, Yu-Rong; Dong, Ji-Xin; Han, Li-Li; Zheng, Yuan-Ming; He, Ji-Zheng

    2016-01-01

    Currently, rice straw return in place of burning is becoming more intensive in China than observed previously. However, little is known on the effect of returned rice straw on mercury (Hg) methylation and microbial activity in contaminated paddy fields. Here, we conduct a microcosm experiment to evaluate the effect of rice straw amendment on the Hg methylation and potential nitrification in two paddy soils with distinct Hg levels. Our results show that amended rice straw enhanced Hg methylation for relatively high Hg content soil, but not for low Hg soil, spiking the same additional fresh Hg. methylmercury (MeHg) concentration was significantly correlated to the dissolved organic carbon (DOC) content and relative abundance of dominant microbes associated with Hg methylation. Similarly, amended rice straw was found to only enhance the potential nitrification rate in soil with relatively high Hg content. These findings provide evidence that amended rice straw differentially modulates Hg methylation and nitrification in Hg contaminated soils possibly resulting from different characteristics in the soil microbial community. This highlights that caution should be taken when returning rice straw to contaminated paddy fields, as this practice may increase the risk of more MeHg production. Main finding: Rice straw amendment enhanced both Hg methylation and nitrification potential in the relatively high, but not low, Hg soil. - Highlights: • Rice straw enhanced Hg methylation in relatively high Hg content paddy soils. • Microbial community directly correlated to the Hg methylation. • Mercury methylation in soils depend on Hg bioavailability and microbial activities. • Hg input affects microbial community associated with decomposition of rice straw.

  17. SNP rs16906252C>T is an expression and methylation quantitative trait locus associated with an increased risk of developing MGMT-methylated colorectal cancer

    Science.gov (United States)

    Kuroiwa-Trzmielina, Joice; Wang, Fan; Rapkins, Robert W.; Ward, Robyn L.; Buchanan, Daniel D.; Win, Aung Ko; Clendenning, Mark; Rosty, Christophe; Southey, Melissa C.; Winship, Ingrid M.; Hopper, John L.; Jenkins, Mark A.; Olivier, Jake; Hawkins, Nicholas J.; Hitchins, Megan P.

    2016-01-01

    Purpose Methylation of the MGMT promoter is the major cause of O6-methylguanine methyltransferase deficiency in cancer and has been associated with the T variant of the promoter-enhancer SNP rs16906252C>T. We sought evidence for an association between the rs16906252C>T genotype and increased risk of developing a subtype of colorectal cancer (CRC) featuring MGMT methylation, mediated by genotype-dependent epigenetic silencing within normal tissues. Experimental design By applying a molecular pathological epidemiology case-control study design, associations between rs16906252C>T and risk for CRC overall, and CRC stratified by MGMT methylation status, were estimated using multinomial logistic regression in two independent retrospective series of CRC cases and controls. The test sample comprised 1054 CRC cases and 451 controls from Sydney, Australia. The validation sample comprised 612 CRC cases and 245 controls from the Australasian Colon Cancer Family Registry (ACCFR). To determine if rs16906252C>T was linked to a constitutively altered epigenetic state, quantitative allelic expression and methylation analyses were performed in normal tissues. Results An association between rs16906252C>T and increased risk of developing MGMT-methylated CRC in the Sydney sample was observed (OR 3.3; 95%CI=2.0–5.3; PT represents an expression and methylation quantitative trait locus. Conclusions We provide evidence that rs16906252C>T is associated with elevated risk for MGMT-methylated CRC, likely mediated by constitutive epigenetic repression of the T allele. PMID:27267851

  18. DNA methylation regulates expression of VEGF-C, and S-adenosylmethionine is effective for VEGF-C methylation and for inhibiting cancer growth

    Energy Technology Data Exchange (ETDEWEB)

    Da, M.X. [Department of Surgical Oncology, Gansu Provincial Hospital, Lanzhou (China); Zhang, Y.B. [Department of Surgery, Ningxia Medical University, Yinchuan (China); Yao, J.B. [Department of Surgical Oncology, Gansu Provincial Hospital, Lanzhou (China); Duan, Y.X. [Department of Surgery, Ningxia Medical University, Yinchuan (China)

    2014-09-30

    DNA hypomethylation may activate oncogene transcription, thus promoting carcinogenesis and tumor development. S-adenosylmethionine (SAM) is a methyl donor in numerous methylation reactions and acts as an inhibitor of intracellular demethylase activity, which results in hypermethylation of DNA. The main objectives of this study were to determine whether DNA hypomethylation correlated with vascular endothelial growth factor-C (VEGF-C) expression, and the effect of SAM on VEGF-C methylation and gastric cancer growth inhibition. VEGF-C expression was assayed by Western blotting and RT-qPCR in gastric cancer cells, and by immunohistochemistry in tumor xenografts. VEGF-C methylation was assayed by bisulfite DNA sequencing. The effect of SAM on cell apoptosis was assayed by flow cytometry analyses and its effect on cancer growth was assessed in nude mice. The VEGF-C promoters of MGC-803, BGC-823, and SGC-7901 gastric cancer cells, which normally express VEGF-C, were nearly unmethylated. After SAM treatment, the VEGF-C promoters in these cells were highly methylated and VEGF-C expression was downregulated. SAM also significantly inhibited tumor growth in vitro and in vivo. DNA methylation regulates expression of VEGF-C. SAM can effectively induce VEGF-C methylation, reduce the expression of VEGF-C, and inhibit tumor growth. SAM has potential as a drug therapy to silence oncogenes and block the progression of gastric cancer.

  19. Somatic mutations, allele loss, and DNA methylation of the Cub and Sushi Multiple Domains 1 (CSMD1 gene reveals association with early age of diagnosis in colorectal cancer patients.

    Directory of Open Access Journals (Sweden)

    Austin Y Shull

    Full Text Available The Cub and Sushi Multiple Domains 1 (CSMD1 gene, located on the short arm of chromosome 8, codes for a type I transmembrane protein whose function is currently unknown. CSMD1 expression is frequently lost in many epithelial cancers. Our goal was to characterize the relationships between CSMD1 somatic mutations, allele imbalance, DNA methylation, and the clinical characteristics in colorectal cancer patients.We sequenced the CSMD1 coding regions in 54 colorectal tumors using the 454FLX pyrosequencing platform to interrogate 72 amplicons covering the entire coding sequence. We used heterozygous SNP allele ratios at multiple CSMD1 loci to determine allelic balance and infer loss of heterozygosity. Finally, we performed methylation-specific PCR on 76 colorectal tumors to determine DNA methylation status for CSMD1 and known methylation targets ALX4, RUNX3, NEUROG1, and CDKN2A.Using 454FLX sequencing and confirming with Sanger sequencing, 16 CSMD1 somatic mutations were identified in 6 of the 54 colorectal tumors (11%. The nonsynonymous to synonymous mutation ratio of the 16 somatic mutations was 15:1, a ratio significantly higher than the expected 2:1 ratio (p = 0.014. This ratio indicates a presence of positive selection for mutations in the CSMD1 protein sequence. CSMD1 allelic imbalance was present in 19 of 37 informative cases (56%. Patients with allelic imbalance and CSMD1 mutations were significantly younger (average age, 41 years than those without somatic mutations (average age, 68 years. The majority of tumors were methylated at one or more CpG loci within the CSMD1 coding sequence, and CSMD1 methylation significantly correlated with two known methylation targets ALX4 and RUNX3. C:G>T:A substitutions were significantly overrepresented (47%, suggesting extensive cytosine methylation predisposing to somatic mutations.Deep amplicon sequencing and methylation-specific PCR reveal that CSMD1 alterations can correlate with earlier clinical

  20. Concordant association validates MGMT methylation and protein expression as favorable prognostic factors in glioma patients on alkylating chemotherapy (Temozolomide).

    Science.gov (United States)

    Pandith, Arshad A; Qasim, Iqbal; Zahoor, Wani; Shah, Parveen; Bhat, Abdul R; Sanadhya, Dheera; Shah, Zafar A; Naikoo, Niyaz A

    2018-04-30

    O 6 -methylguanine-DNA methyltransferase (MGMT) promoter methylation and its subsequent loss of protein expression has been identified to have a variable impact on clinical outcome of glioma patients indicated for chemotherapy with alkylating agents (Temozolomide). This study investigated methylation status of MGMT gene along with in situ protein expression in malignant glioma patients of different histological types to evaluate the associated clinical outcome vis-a-vis use of alkylating drugs and radiotherapy. Sixty three cases of glioma were evaluated for MGMT promoter methylation by methylation-specific PCR (MS-PCR) and protein expression by immunostaining (IHC). Methylation status of MGMT and loss of protein expression showed a very high concordant association with better survival and progression free survival (PFS) (p < 0.0001). Multivariate Cox regression analysis showed both MGMT methylation and loss of protein as significant independent prognostic factors in glioma patients with respect to lower Hazard Ratio (HR) for better OS and PFS) [p < 0.05]. Interestingly concordant MGMT methylation and lack of protein showed better response in TMZ therapy treated patient subgroups with HR of 2.02 and 0.76 (p < 0.05). We found the merits of prognostication of MGMT parameters, methylation as well as loss of its protein as predictive factors for favorable outcome in terms of better survival for TMZ therapy.

  1. Whole-genome methylation caller designed for methyl- DNA ...

    African Journals Online (AJOL)

    etchie

    2013-02-20

    Feb 20, 2013 ... Our method uses a single-CpG-resolution, whole-genome methylation ... Key words: Methyl-DNA immunoprecipitation, next-generation sequencing, ...... methylation is prevalent in embryonic stem cells andmaybe mediated.

  2. Involvement of aberrant DNA methylation on reduced expression of lysophosphatidic acid receptor-1 gene in rat tumor cell lines

    International Nuclear Information System (INIS)

    Tsujiuchi, Toshifumi; Shimizu, Kyoko; Onishi, Mariko; Sugata, Eriko; Fujii, Hiromasa; Mori, Toshio; Honoki, Kanya; Fukushima, Nobuyuki

    2006-01-01

    Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation, migration, and protects cells from apoptosis. It interacts with specific G protein-coupled transmembrane receptors. Recently, it has been reported that alterations of LPA receptor expression might be important in the malignant transformation of tumor cells. Therefore, to assess an involvement of DNA methylation in reduced expression of the LPA receptor-1 (lpa1) gene, we investigated the expression of the lpa1 gene and its DNA methylation patterns in rat tumor cell lines. Both rat brain-derived neuroblastoma B103 and liver-derived hepatoma RH7777 cells used in this study indicated no expression of lpa1. For the analysis of methylation status, bisulfite sequencing was performed with B103 and RH7777 cells, comparing with other lpa1 expressed cells and normal tissues of brain and liver. The lpa1 expressed cells and tissues were all unmethylated in this region of lpa1. In contrast, both B103 and RH7777 cells were highly methylated, correlating with reduced expression of the lpa1. Treatment with 5-aza 2'-deoxycytidine induced expression of lpa1 gene in B103 and RH7777 cells after 24 h. In RH7777 cells treated with 5-aza 2'-deoxycytidine, stress fiber formation was also observed in response to LPA in RH7777 cells, but not in untreated RH7777 cells. These results suggest that aberrant DNA methylation of the lpa1 gene may be involved in its reduced expression in rat tumor cells

  3. PRmePRed: A protein arginine methylation prediction tool.

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    Pawan Kumar

    Full Text Available Protein methylation is an important Post-Translational Modification (PTMs of proteins. Arginine methylation carries out and regulates several important biological functions, including gene regulation and signal transduction. Experimental identification of arginine methylation site is a daunting task as it is costly as well as time and labour intensive. Hence reliable prediction tools play an important task in rapid screening and identification of possible methylation sites in proteomes. Our preliminary assessment using the available prediction methods on collected data yielded unimpressive results. This motivated us to perform a comprehensive data analysis and appraisal of features relevant in the context of biological significance, that led to the development of a prediction tool PRmePRed with better performance. The PRmePRed perform reasonably well with an accuracy of 84.10%, 82.38% sensitivity, 83.77% specificity, and Matthew's correlation coefficient of 66.20% in 10-fold cross-validation. PRmePRed is freely available at http://bioinfo.icgeb.res.in/PRmePRed/.

  4. Methylation alterations are not a major cause of PTTG1 missregulation

    International Nuclear Information System (INIS)

    Hidalgo, Manuel; Royo, Jose Luis; Galan, Jose Jorge; Sáez, Carmen; Ferrero, Eduardo; Castilla, Carolina; Ramirez-Lorca, Reposo; Pelaez, Pablo; Ruiz, Agustin; Japón, Miguel A

    2008-01-01

    On its physiological cellular context, PTTG1 controls sister chromatid segregation during mitosis. Within its crosstalk to the cellular arrest machinery, relies a checkpoint of integrity for which gained the over name of securin. PTTG1 was found to promote malignant transformation in 3T3 fibroblasts, and further found to be overexpressed in different tumor types. More recently, PTTG1 has been also related to different processes such as DNA repair and found to trans-activate different cellular pathways involving c-myc, bax or p53, among others. PTTG1 over-expression has been correlated to a worse prognosis in thyroid, lung, colorectal cancer patients, and it can not be excluded that this effect may also occur in other tumor types. Despite the clinical relevance and the increasing molecular characterization of PTTG1, the reason for its up-regulation remains unclear. We analysed PTTG1 differential expression in PC-3, DU-145 and LNCaP tumor cell lines, cultured in the presence of the methyl-transferase inhibitor 5-Aza-2'-deoxycytidine. We also tested whether the CpG island mapping PTTG1 proximal promoter evidenced a differential methylation pattern in differentiated thyroid cancer biopsies concordant to their PTTG1 immunohistochemistry status. Finally, we performed whole-genome LOH studies using Affymetix 50 K microarray technology and FRET analysis to search for allelic imbalances comprising the PTTG1 locus. Our data suggest that neither methylation alterations nor LOH are involved in PTTG1 over-expression. These data, together with those previously reported, point towards a post-transcriptional level of missregulation associated to PTTG1 over-expression

  5. Patterns of DNMT1 Promoter Methylation in Patients with Acute Lymphoblastic Leukemia.

    Science.gov (United States)

    Rahmani, Tirdad; Azad, Mehdi; Chahardouli, Bahram; Nasiri, Hajar; Vatanmakanian, Mousa; Kaviani, Saeid

    2017-07-01

    Background: Acute lymphoblastic leukemia (ALL) is a clonal malignant disorder characterized by an uncontrolled proliferation of immature T or B lymphocytes. Extensive studies have shown that the epigenetic changes, especially modified DNA methylation patterns in the regulatory regions through the DNA methyltransferase (DNMTs), play an important role in the development of genetic disorders and abnormal growth and maturation capacity of leukemic stem cells (LSCs).The aim of this study was to evaluate the changes in DNMT1 promoter methylation and its expression pattern in patients with ALL. Materials and Methods: In this experimental study, methylation specific PCR (MSP) was used to assess the methylation status of DNMT1 promoter regions in samples collected from ALL patients (n=45) and healthy control subjects. According to this method, un-methylated cytosine nucleotides are converted to uracil by sodium bisulfite and the proliferation of methylated and un-methylated regions are performed using specific primers for target sequences. Results: None of the patients with B and T-ALL showed methylated promoter regions of the DNMT1 gene, while the methylation pattern of both pre-B ALL patients and the control group showed a relative promoter methylation. Conclusion: Analysis of promoter methylation patterns in various subgroups of ALL has revealed the importance of DNMT1 in the regulation of gene expression. Likewise, extensive data have also highlighted the methylation-based mechanisms exerted by DNAM1 as one of the main participants regulating gene expression in B-ALL and T-ALL patients. Investigation of the overall DNA methylation pattern offers significant improvements in the prediction of disease prognosis and treatment response.

  6. Theoretical spectroscopic characterization at low temperatures of S-methyl thioformate and O-methyl thioformate

    Energy Technology Data Exchange (ETDEWEB)

    Senent, M. L., E-mail: senent@iem.cfmac.csic.es [Departamento de Química y Física Teóricas, Instituto de Estructura de la Materia, IEM-C.S.I.C., Serrano 121, Madrid 28006 (Spain); Puzzarini, C., E-mail: cristina.puzzarini@unibo.it [Dipartimento di Chimica G. Ciamician, Università di Bologna, Via F. Selmi 2, I-40126 Bologna (Italy); Hochlaf, M., E-mail: hochlaf@univ-mlv.fr [Laboratoire de Modélisation et Simulation Multi Echelle, Université Paris-Est, MSME UMR 8208 CNRS, 5 boulevard Descartes, 77454 Marne-la-Vallée (France); Domínguez-Gómez, R., E-mail: rosa.dominguez@upm.es [Departamento de Ingeniería Civil, Cátedra de Química, E.U.I.T. Obras Públicas, Universidad Politécnica de Madrid, Madrid (Spain); Carvajal, M., E-mail: miguel.carvajal@dfa.uhu.es [Departamento de Física Aplicada, Facultad de Ciencias Experimentales, Unidad Asociada IEM-CSIC-U.Huelva, Universidad de Huelva, 21071 Huelva (Spain)

    2014-09-14

    Highly correlated ab initio methods are employed to determine spectroscopic properties at low temperatures of two S-analogs of methyl formate: S-methyl thioformate CH{sub 3}-S-CHO (MSCHO) and O-methyl thioformate CH{sub 3}-O-CHS (MOCHS). Both species are detectable and they are expected to play an important role in Astrochemistry. Molecular properties are compared with those of the O-analog, methyl formate. Both isomers present two conformers cis and trans. cis-CH{sub 3}-S-CHO represents the most stable structure lying 4372.2 cm{sup −1} below cis-CH{sub 3}-O-CHS. The energy difference between the cis and trans forms is drastically lower for MSCHO (1134 cm{sup −1}) than for MOCHS (1963.6 cm{sup −1}). Harmonic and anharmonic fundamentals and the corresponding intensities, as well as the rotational constants for the ground vibrational and first excited torsional states and the centrifugal distortions constants, are provided. Low torsional energy levels have been obtained by solving variationally a two dimensional Hamiltonian expressed in terms of the two torsional degrees of freedom. The corresponding 2D potential energy surfaces have been computed at the CCSD(T)/aug-cc-pVTZ level of theory. The methyl torsional barriers V{sub 3}(cis) are determined to be 139.7 cm{sup −1} (CH{sub 3}-S-CHO) and 670.4 cm{sup −1} (CH{sub 3}-O-CHS). The A/E splitting of ground torsional state has been estimated to be 0.438 cm{sup −1} for CH{sub 3}-S-CHO and negligible for CH{sub 3}-O-CHS.

  7. Methylation of Promoter Regions of Genes of the Human Intrauterine Renin Angiotensin System and Their Expression

    Directory of Open Access Journals (Sweden)

    Shane D. Sykes

    2015-01-01

    Full Text Available The intrauterine renin angiotensin system (RAS is implicated in placentation and labour onset. Here we investigate whether promoter methylation of RAS genes changes with gestation or labour and if it affects gene expression. Early gestation amnion and placenta were studied, as were term amnion, decidua, and placenta collected before labour (at elective caesarean section or after spontaneous labour and delivery. The expression and degree of methylation of the prorenin receptor (ATP6AP2, angiotensin converting enzyme (ACE, angiotensin II type 1 receptor (AGTR1, and two proteases that can activate prorenin (kallikrein, KLK1, and cathepsin D, CTSD were measured by qPCR and a DNA methylation array. There was no effect of gestation or labour on the methylation of RAS genes and CTSD. Amnion and decidua displayed strong correlations between the percent hypermethylation of RAS genes and CTSD, suggestive of global methylation. There were no correlations between the degree of methylation and mRNA abundance of any genes studied. KLK1 was the most methylated gene and the proportion of hypermethylated KLK1 alleles was lower in placenta than decidua. The presence of intermediate methylated alleles of KLK1 in early gestation placenta and in amnion after labour suggests that KLK1 methylation is uniquely dynamic in these tissues.

  8. Epigenetic Diversity of Clonal White Poplar (Populus alba L. Populations: Could Methylation Support the Success of Vegetative Reproduction Strategy?

    Directory of Open Access Journals (Sweden)

    Francesco Guarino

    Full Text Available The widespread poplar populations of Sardinia are vegetatively propagated and live in different natural environments forming large monoclonal stands. The main goals of the present study were: i to investigate/measure the epigenetic diversity of the poplar populations by determining their DNA methylation status; ii to assess if and how methylation status influences population clustering; iii to shed light on the changes that occur in the epigenome of ramets of the same poplar clone. To these purposes, 83 white poplar trees were sampled at different locations on the island of Sardinia. Methylation sensitive amplified polymorphism analysis was carried out on the genomic DNA extracted from leaves at the same juvenile stage. The study showed that the genetic biodiversity of poplars is quite limited but it is counterbalanced by epigenetic inter-population molecular variability. The comparison between MspI and HpaII DNA fragmentation profiles revealed that environmental conditions strongly influence hemi-methylation of the inner cytosine. The variable epigenetic status of Sardinian white poplars revealed a decreased number of population clusters. Landscape genetics analyses clearly demonstrated that ramets of the same clone were differentially methylated in relation to their geographic position. Therefore, our data support the notion that studies on plant biodiversity should no longer be restricted to genetic aspects, especially in the case of vegetatively propagated plant species.

  9. Identification of endometrial cancer methylation features using combined methylation analysis methods.

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    Michael P Trimarchi

    Full Text Available DNA methylation is a stable epigenetic mark that is frequently altered in tumors. DNA methylation features are attractive biomarkers for disease states given the stability of DNA methylation in living cells and in biologic specimens typically available for analysis. Widespread accumulation of methylation in regulatory elements in some cancers (specifically the CpG island methylator phenotype, CIMP can play an important role in tumorigenesis. High resolution assessment of CIMP for the entire genome, however, remains cost prohibitive and requires quantities of DNA not available for many tissue samples of interest. Genome-wide scans of methylation have been undertaken for large numbers of tumors, and higher resolution analyses for a limited number of cancer specimens. Methods for analyzing such large datasets and integrating findings from different studies continue to evolve. An approach for comparison of findings from a genome-wide assessment of the methylated component of tumor DNA and more widely applied methylation scans was developed.Methylomes for 76 primary endometrial cancer and 12 normal endometrial samples were generated using methylated fragment capture and second generation sequencing, MethylCap-seq. Publically available Infinium HumanMethylation 450 data from The Cancer Genome Atlas (TCGA were compared to MethylCap-seq data.Analysis of methylation in promoter CpG islands (CGIs identified a subset of tumors with a methylator phenotype. We used a two-stage approach to develop a 13-region methylation signature associated with a "hypermethylator state." High level methylation for the 13-region methylation signatures was associated with mismatch repair deficiency, high mutation rate, and low somatic copy number alteration in the TCGA test set. In addition, the signature devised showed good agreement with previously described methylation clusters devised by TCGA.We identified a methylation signature for a "hypermethylator phenotype" in

  10. Heterogeneity in white blood cells has potential to confound DNA methylation measurements.

    Directory of Open Access Journals (Sweden)

    Bjorn T Adalsteinsson

    Full Text Available Epigenetic studies are commonly conducted on DNA from tissue samples. However, tissues are ensembles of cells that may each have their own epigenetic profile, and therefore inter-individual cellular heterogeneity may compromise these studies. Here, we explore the potential for such confounding on DNA methylation measurement outcomes when using DNA from whole blood. DNA methylation was measured using pyrosequencing-based methodology in whole blood (n = 50-179 and in two white blood cell fractions (n = 20, isolated using density gradient centrifugation, in four CGIs (CpG Islands located in genes HHEX (10 CpG sites assayed, KCNJ11 (8 CpGs, KCNQ1 (4 CpGs and PM20D1 (7 CpGs. Cellular heterogeneity (variation in proportional white blood cell counts of neutrophils, lymphocytes, monocytes, eosinophils and basophils, counted by an automated cell counter explained up to 40% (p<0.0001 of the inter-individual variation in whole blood DNA methylation levels in the HHEX CGI, but not a significant proportion of the variation in the other three CGIs tested. DNA methylation levels in the two cell fractions, polymorphonuclear and mononuclear cells, differed significantly in the HHEX CGI; specifically the average absolute difference ranged between 3.4-15.7 percentage points per CpG site. In the other three CGIs tested, methylation levels in the two fractions did not differ significantly, and/or the difference was more moderate. In the examined CGIs, methylation levels were highly correlated between cell fractions. In summary, our analysis detects region-specific differential DNA methylation between white blood cell subtypes, which can confound the outcome of whole blood DNA methylation measurements. Finally, by demonstrating the high correlation between methylation levels in cell fractions, our results suggest a possibility to use a proportional number of a single white blood cell type to correct for this confounding effect in analyses.

  11. Size ratio correlates with intracranial aneurysm rupture status: a prospective study.

    Science.gov (United States)

    Rahman, Maryam; Smietana, Janel; Hauck, Erik; Hoh, Brian; Hopkins, Nick; Siddiqui, Adnan; Levy, Elad I; Meng, Hui; Mocco, J

    2010-05-01

    The prediction of intracranial aneurysm (IA) rupture risk has generated significant controversy. The findings of the International Study of Unruptured Intracranial Aneurysms (ISUIA) that small anterior circulation aneurysms (IAs are small. These discrepancies have led to the search for better aneurysm parameters to predict rupture. We previously reported that size ratio (SR), IA size divided by parent vessel diameter, correlated strongly with IA rupture status (ruptured versus unruptured). These data were all collected retrospectively off 3-dimensional angiographic images. Therefore, we performed a blinded prospective collection and evaluation of SR data from 2-dimensional angiographic images for a consecutive series of patients with ruptured and unruptured IAs. We prospectively enrolled 40 consecutive patients presenting to a single institution with either ruptured IA or for first-time evaluation of an incidental IA. Blinded technologists acquired all measurements from 2-dimensional angiographic images. Aneurysm rupture status, location, IA maximum size, and parent vessel diameter were documented. The SR was calculated by dividing the aneurysm size (mm) by the average parent vessel size (mm). A 2-tailed Mann-Whitney test was performed to assess statistical significance between ruptured and unruptured groups. Fisher exact test was used to compare medical comorbidities between the ruptured and unruptured groups. Significant differences between the 2 groups were subsequently tested with logistic regression. SE and probability values are reported. Forty consecutive patients with 24 unruptured and 16 ruptured aneurysms met the inclusion criteria. No significant differences were found in age, gender, smoking status, or medical comorbidities between ruptured and unruptured groups. The average maximum size of the unruptured IAs (6.18 + or - 0.60 mm) was significantly smaller compared with the ruptured IAs (7.91 + or - 0.47 mm; P=0.03), and the unruptured group had

  12. Maternal exposure to anti-androgenic compounds, vinclozolin, flutamide and procymidone, has no effects on spermatogenesis and DNA methylation in male rats of subsequent generations

    International Nuclear Information System (INIS)

    Inawaka, Kunifumi; Kawabe, Mayumi; Takahashi, Satoru; Doi, Yuko; Tomigahara, Yoshitaka; Tarui, Hirokazu; Abe, Jun; Kawamura, Satoshi; Shirai, Tomoyuki

    2009-01-01

    To verify whether anti-androgens cause transgenerational effects on spermatogenesis and DNA methylation in rats, gravid Crl:CD(SD) female rats (4 or 5/group, gestational day (GD) 0 = day sperm detected) were intraperitoneally treated with anti-androgenic compounds, such as vinclozolin (100 mg/kg/day), procymidone (100 mg/kg/day), or flutamide (10 mg/kg/day), from GD 8 to GD 15. Testes were collected from F1 male pups at postnatal day (PND) 6 for DNA methylation analysis of the region (210 bp including 7 CpG sites) within the lysophospholipase gene by bisulfite DNA sequencing method. F0 and F1 males underwent the sperm analysis (count, motility and morphology), followed by DNA methylation analysis of the sperm. Remaining F1 males were cohabited with untreated-females to obtain F2 male pups for subsequent DNA methylation analysis of the testes at PND 6. These analyses showed no effects on spermatogenesis and fertility in F1 males of any treatment group. DNA methylation status in testes (F1 and F2 pups at PND 6) or sperms (F1 males at 13 weeks old) of the treatment groups were comparable to the control at all observation points, although DNA methylation rates in testes were slightly lower than those in sperm. In F0 males, no abnormalities in the spermatogenesis, fertility and DNA methylation status of sperm were observed. No transgenerational abnormalities of spermatogenesis and DNA methylation status caused by anti-androgenic compounds were observed.

  13. Maternal exposure to anti-androgenic compounds, vinclozolin, flutamide and procymidone, has no effects on spermatogenesis and DNA methylation in male rats of subsequent generations.

    Science.gov (United States)

    Inawaka, Kunifumi; Kawabe, Mayumi; Takahashi, Satoru; Doi, Yuko; Tomigahara, Yoshitaka; Tarui, Hirokazu; Abe, Jun; Kawamura, Satoshi; Shirai, Tomoyuki

    2009-06-01

    To verify whether anti-androgens cause transgenerational effects on spermatogenesis and DNA methylation in rats, gravid Crl:CD(SD) female rats (4 or 5/group, gestational day (GD) 0=day sperm detected) were intraperitoneally treated with anti-androgenic compounds, such as vinclozolin (100 mg/kg/day), procymidone (100 mg/kg/day), or flutamide (10 mg/kg/day), from GD 8 to GD 15. Testes were collected from F1 male pups at postnatal day (PND) 6 for DNA methylation analysis of the region (210 bp including 7 CpG sites) within the lysophospholipase gene by bisulfite DNA sequencing method. F0 and F1 males underwent the sperm analysis (count, motility and morphology), followed by DNA methylation analysis of the sperm. Remaining F1 males were cohabited with untreated-females to obtain F2 male pups for subsequent DNA methylation analysis of the testes at PND 6. These analyses showed no effects on spermatogenesis and fertility in F1 males of any treatment group. DNA methylation status in testes (F1 and F2 pups at PND 6) or sperms (F1 males at 13 weeks old) of the treatment groups were comparable to the control at all observation points, although DNA methylation rates in testes were slightly lower than those in sperm. In F0 males, no abnormalities in the spermatogenesis, fertility and DNA methylation status of sperm were observed. No transgenerational abnormalities of spermatogenesis and DNA methylation status caused by anti-androgenic compounds were observed.

  14. Tumor-associated endothelial cells display GSTP1 and RARβ2 promoter methylation in human prostate cancer

    Directory of Open Access Journals (Sweden)

    Pohida Thomas J

    2006-03-01

    Full Text Available Abstract Background A functional blood supply is essential for tumor growth and proliferation. However, the mechanism of blood vessel recruitment to the tumor is still poorly understood. Ideally, a thorough molecular assessment of blood vessel cells would be critical in our comprehension of this process. Yet, to date, there is little known about the molecular makeup of the endothelial cells of tumor-associated blood vessels, due in part to the difficulty of isolating a pure population of endothelial cells from the heterogeneous tissue environment. Methods Here we describe the use of a recently developed technique, Expression Microdissection, to isolate endothelial cells from the tumor microenvironment. The methylation status of the dissected samples was evaluated for GSTP1 and RARβ2 promoters via the QMS-PCR method. Results Comparing GSTP1 and RARβ2 promoter methylation data, we show that 100% and 88% methylation is detected, respectively, in the tumor areas, both in epithelium and endothelium. Little to no methylation is observed in non-tumor tissue areas. Conclusion We applied an accurate microdissection technique to isolate endothelial cells from tissues, enabling DNA analysis such as promoter methylation status. The observations suggest that epigenetic alterations may play a role in determining the phenotype of tumor-associated vasculature.

  15. Isobaric (vapour + liquid) equilibrium for N-methyl-2-pyrrolidone with branched alcohols

    International Nuclear Information System (INIS)

    Gnanakumari, P.; Venkatesu, P.; Hsieh, C.-T.; Rao, M.V. Prabhakara; Lee, M.-J.; Lin, Ho-mu

    2009-01-01

    The (vapour + liquid) equilibrium (VLE) and boiling temperature measurements have been determined at 95.3 kPa as a function of composition for the binary liquid mixtures of N-methyl-2-pyrrolidone (NMP) with branched alcohols using a Swietoslawski-ebulliometer. The branched alcohols include 2-propanol, 2-butanol, 2-methyl-l- propanol, 2-methyl-2-propanol, and 3-methyl-l-butanol. The experimental temperature-composition (T-x) results were used to estimate Wilson parameters and then used to calculate the equilibrium vapour compositions and the excess Gibbs free energy at T = 298.15 K. The experimental temperature-composition (T, x) results were correlated with the Wilson, the NRTL and the UNIQUAC models. The experimental results are interpreted in terms of intermolecular interactions between constituent molecules

  16. Oxidative stress and repetitive element methylation changes in artisanal gold miners occupationally exposed to mercury

    Directory of Open Access Journals (Sweden)

    Diana M. Narváez

    2017-09-01

    Full Text Available Mercury (Hg exposure is a public health concern due to its persistence in the environment and its high toxicity. Such toxicity has been associated with the generation of oxidative stress in occupationally exposed subjects, such as artisanal gold miners. In this study, we characterize occupational exposure to Hg by measuring blood, urine and hair levels, and investigate oxidative stress and DNA methylation associated with gold mining. To do this, samples from 53 miners and 36 controls were assessed. We show higher levels of oxidative stress marker 8-OHdG in the miners. Differences in LINE1 and Alu(Yb8 DNA methylation between gold miners and control group are present in peripheral blood leukocytes. LINE1 methylation is positively correlated with 8-OHdG levels, while XRCC1 and LINE1 methylation are positively correlated with Hg levels. These results suggest an effect of Hg on oxidative stress and DNA methylation in gold miners that may have an impact on miners’ health.

  17. Diethylstilbestrol (DES)-stimulated hormonal toxicity is mediated by ERα alteration of target gene methylation patterns and epigenetic modifiers (DNMT3A, MBD2, and HDAC2) in the mouse seminal vesicle.

    Science.gov (United States)

    Li, Yin; Hamilton, Katherine J; Lai, Anne Y; Burns, Katherine A; Li, Leping; Wade, Paul A; Korach, Kenneth S

    2014-03-01

    Diethylstilbestrol (DES) is a synthetic estrogen associated with adverse effects on reproductive organs. DES-induced toxicity of the mouse seminal vesicle (SV) is mediated by estrogen receptor α (ERα), which alters expression of seminal vesicle secretory protein IV (Svs4) and lactoferrin (Ltf) genes. We examined a role for nuclear receptor activity in association with DNA methylation and altered gene expression. We used the neonatal DES exposure mouse model to examine DNA methylation patterns via bisulfite conversion sequencing in SVs of wild-type (WT) and ERα-knockout (αERKO) mice. The DNA methylation status at four specific CpGs (-160, -237, -306, and -367) in the Svs4 gene promoter changed during mouse development from methylated to unmethylated, and DES prevented this change at 10 weeks of age in WT SV. At two specific CpGs (-449 and -459) of the Ltf gene promoter, DES altered the methylation status from methylated to unmethylated. Alterations in DNA methylation of Svs4 and Ltf were not observed in αERKO SVs, suggesting that changes of methylation status at these CpGs are ERα dependent. The methylation status was associated with the level of gene expression. In addition, gene expression of three epigenetic modifiers-DNMT3A, MBD2, and HDAC2-increased in the SV of DES-exposed WT mice. DES-induced hormonal toxicity resulted from altered gene expression of Svs4 and Ltf associated with changes in DNA methylation that were mediated by ERα. Alterations in gene expression of DNMT3A, MBD2, and HDAC2 in DES-exposed male mice may be involved in mediating the changes in methylation status in the SV. Li Y, Hamilton KJ, Lai AY, Burns KA, Li L, Wade PA, Korach KS. 2014. Diethylstilbestrol (DES)-stimulated hormonal toxicity is mediated by ERα alteration of target gene methylation patterns and epigenetic modifiers (DNMT3A, MBD2, and HDAC2) in the mouse seminal vesicle. Environ Health Perspect 122:262-268; http://dx.doi.org/10.1289/ehp.1307351.

  18. Meta-analysis of the association between APC promoter methylation and colorectal cancer.

    Science.gov (United States)

    Ding, Zhenyu; Jiang, Tong; Piao, Ying; Han, Tao; Han, Yaling; Xie, Xiaodong

    2015-01-01

    Previous studies investigating the association between adenomatous polyposis coli (APC) gene promoter methylation and colorectal cancer (CRC) have yielded conflicting results. The aim of this study was to comprehensively evaluate the potential application of the detection of APC promoter methylation to the prevention and treatment of CRC. PubMed, Embase, and MEDLINE (results updated to October 2014) were searched for relevant studies. The effect size was defined as the weighted odds ratio (OR), which was calculated using either the fixed-effects or random-effects model. Prespecified subgroup and sensitivity analyses were conducted to evaluate potential heterogeneity among the included studies. Nineteen studies comprising 2,426 participants were selected for our meta-analysis. The pooled results of nine studies comprising a total of 740 subjects indicated that APC promoter methylation was significantly associated with CRC risk (pooled OR 5.53; 95% confidence interval [CI] 3.50-8.76; PAPC promoter methylation and the presence of CRC metastasis, and the pooled OR was 0.80 (95% CI 0.44-1.46; P=0.47). A meta-analysis conducted with four studies with a total of 467 patients found no significant correlation between APC promoter methylation and the presence of colorectal adenoma (pooled OR 1.85; 95% CI 0.67-5.10; P=0.23). No significant correlation between APC promoter methylation and patients' Dukes' stage, TNM stage, differentiation grade, age, or sex was identified. In conclusion, APC promoter methylation was found to be significantly associated with a higher risk of developing CRC. The findings indicate that APC promoter methylation may be a potential biomarker for the carcinogenesis of CRC.

  19. [Sub-health status of middle school teachers and its correlation analysis with occupational stress].

    Science.gov (United States)

    Chang, W J; Shao, H M; Zhi, X Y; Xu, J; Xie, J

    2017-08-20

    Objective: To study the distribution of sub-health and occupational stress as well as their correlation among middle school teachers in Tianjin, then provide evidences for prevention and control of the status of sub-health. Methods: A total of 3 522 middle school teachers from six districts of Tianjin were recruited with stratified cluster sampling strategy for the investigation of Sub-Health Measurement Scale version 1.0 (SHMS V1.0) and Occupational Stress Inventory-Revised Edition (OSI-R) . Results: Detection rate of sub-health status among Tianjin middle school teachers was 58.55%. Men had significantly lower sub-health detection rate (55.19%) than women (59.71%) . Sub-health detection rate increased with age ( P occupational stress norm of China ( P occupational stress of middle school teachers showed that the scores of occupational role and personal strain were negatively correlated with the scores of sub-health state ( P occupational stress level of middle school teachers, increase personal resources, and scientific and effective health guidance and education should be strengthened.

  20. Methylation of food commodities during fumigation with methyl bromide

    International Nuclear Information System (INIS)

    Starratt, A.N.; Bond, E.J.

    1990-01-01

    Sites of methylation in several commodities (wheat, oatmeal, peanuts, almonds, apples, oranges, maize, alfalfa and potatoes) during fumigation with 14 C-methyl bromide were studied. Differences were observed in levels of the major volatiles: methanol, dimethyl sulphide and methyl mercaptan, products of O- and S-methylation, resulting from treatment of the fumigated materials with 1N sodium hydroxide. In studies of maize and wheat, histidine was the amino acid which underwent the highest level of N-methylation. (author). 24 refs, 3 tabs

  1. AN UPWARD TREND IN DNA P16INK4A METHYLATION PATTERN AND HIGH RISK HPV INFECTION ACCORDING TO THE SEVERITY OF THE CERVICAL LESION

    Directory of Open Access Journals (Sweden)

    Fernanda Nahoum Carestiato

    2013-09-01

    Full Text Available SUMMARY High-risk human papillomavirus (hr-HPV infection is necessary but not sufficient for cervical cancer development. Recently, P16INK4A gene silencing through hypermethylation has been proposed as an important cofactor in cervical carcinogenesis due to its tumor suppressor function. We aimed to investigate P16INK4A methylation status in normal and neoplastic epithelia and evaluate an association with HPV infection and genotype. This cross-sectional study was performed with 141 cervical samples from patients attending Hospital Moncorvo Filho, Rio de Janeiro. HPV detection and genotyping were performed through PCR and P16INK4A methylation by nested-methylation specific PCR (MSP. HPV frequency was 62.4% (88/141. The most common HPV were HPV16 (37%, HPV18 (16.3% and HPV33/45(15.2%. An upward trend was observed concerning P16INK4A methylation and lesion degree: normal epithelia (10.7%, low grade lesions (22.9%, high grade (57.1% and carcinoma (93.1% (p < 0.0001. A multivariate analysis was performed to evaluate an association between methylation, age, tobacco exposure, HPV infection and genotyping. A correlation was found concerning methylation with HPV infection (p < 0.0001, hr-HPV (p = 0.01, HSIL (p < 0.0007 and malignant lesions (p < 0.0001. Since viral infection and epigenetic alterations are related to cervical carcinoma, we suggest that P16INK4A methylation profile maybe thoroughly investigated as a biomarker to identify patients at risk of cancer.

  2. DNA methylation of amino acid transporter genes in the human placenta.

    Science.gov (United States)

    Simner, C; Novakovic, B; Lillycrop, K A; Bell, C G; Harvey, N C; Cooper, C; Saffery, R; Lewis, R M; Cleal, J K

    2017-12-01

    Placental transfer of amino acids via amino acid transporters is essential for fetal growth. Little is known about the epigenetic regulation of amino acid transporters in placenta. This study investigates the DNA methylation status of amino acid transporters and their expression across gestation in human placenta. BeWo cells were treated with 5-aza-2'-deoxycytidine to inhibit methylation and assess the effects on amino acid transporter gene expression. The DNA methylation levels of amino acid transporter genes in human placenta were determined across gestation using DNA methylation array data. Placental amino acid transporter gene expression across gestation was also analysed using data from publically available Gene Expression Omnibus data sets. The expression levels of these transporters at term were established using RNA sequencing data. Inhibition of DNA methylation in BeWo cells demonstrated that expression of specific amino acid transporters can be inversely associated with DNA methylation. Amino acid transporters expressed in term placenta generally showed low levels of promoter DNA methylation. Transporters with little or no expression in term placenta tended to be more highly methylated at gene promoter regions. The transporter genes SLC1A2, SLC1A3, SLC1A4, SLC7A5, SLC7A11 and SLC7A10 had significant changes in enhancer DNA methylation across gestation, as well as gene expression changes across gestation. This study implicates DNA methylation in the regulation of amino acid transporter gene expression. However, in human placenta, DNA methylation of these genes remains low across gestation and does not always play an obvious role in regulating gene expression, despite clear evidence for differential expression as gestation proceeds. Copyright © 2017. Published by Elsevier Ltd.

  3. Association of Tissue-Specific DNA Methylation Alterations with α-Thalassemia Southeast Asian Deletion

    Directory of Open Access Journals (Sweden)

    Tanapat Pangeson

    2017-11-01

    Full Text Available In the wild-type allele, DNA methylation levels of 10 consecutive CpG sites adjacent to the upstream 5′-breakpoint of α-thalassemia Southeast Asian (SEA deletion are not different between placenta and leukocytes. However, no previous study has reported the map of DNA methylation in the SEA allele. This report aims to show that the SEA mutation is associated with DNA methylation changes, resulting in differential methylation between placenta and leukocytes. Methylation-sensitive high-resolution analysis was used to compare DNA methylation among placenta, leukocytes, and unmethylated control DNA. The result indicates that the DNA methylation between placenta and leukocyte DNA is different and shows that the CpG status of both is not fully unmethylated. Mapping of individual CpG sites was performed by targeted bisulfite sequencing. The DNA methylation level of the 10 consecutive CpG sites was different between placenta and leukocyte DNA. When the 10th CpG of the mutation allele was considered as a hallmark for comparing DNA methylation level, it was totally different from the unmethylated 10th CpG of the wild-type allele. Finally, the distinct DNA methylation patterns between both DNA were extracted. In total, 24 patterns were found in leukocyte samples and 9 patterns were found in placenta samples. This report shows that the large deletion is associated with DNA methylation change. In further studies for clinical application, the distinct DNA methylation pattern might be a potential marker for detecting cell-free fetal DNA.

  4. Effects of cytosine methylation on transcription factor binding sites

    KAUST Repository

    Medvedeva, Yulia A

    2014-03-26

    Background: DNA methylation in promoters is closely linked to downstream gene repression. However, whether DNA methylation is a cause or a consequence of gene repression remains an open question. If it is a cause, then DNA methylation may affect the affinity of transcription factors (TFs) for their binding sites (TFBSs). If it is a consequence, then gene repression caused by chromatin modification may be stabilized by DNA methylation. Until now, these two possibilities have been supported only by non-systematic evidence and they have not been tested on a wide range of TFs. An average promoter methylation is usually used in studies, whereas recent results suggested that methylation of individual cytosines can also be important.Results: We found that the methylation profiles of 16.6% of cytosines and the expression profiles of neighboring transcriptional start sites (TSSs) were significantly negatively correlated. We called the CpGs corresponding to such cytosines " traffic lights" We observed a strong selection against CpG " traffic lights" within TFBSs. The negative selection was stronger for transcriptional repressors as compared with transcriptional activators or multifunctional TFs as well as for core TFBS positions as compared with flanking TFBS positions.Conclusions: Our results indicate that direct and selective methylation of certain TFBS that prevents TF binding is restricted to special cases and cannot be considered as a general regulatory mechanism of transcription. 2013 Medvedeva et al.; licensee BioMed Central Ltd.

  5. Factors affecting the retention of methyl iodide by iodide-impregnated carbon

    International Nuclear Information System (INIS)

    Hyder, M.L.; Malstrom, R.A.

    1991-01-01

    This paper comprises two sets of studies of methyl iodide retention by iodide-impregnated carbon. In the first of these, the retention of the methyl iodide on the carbon surface and its subsequent evolution were observed directly by a technique of combustion and phosphorescence. In the second, the methyl iodide retention in a standard test was compared with surface area measurements and the concentration of unreacted iodine. A correlation among these parameters was identified and characterized. Carbon quality was varied through the selection of used material with differing service histories. Air from the Savannah River Site reactor buildings is vented through carbon beds for control of radioiodine before release to the atmosphere. The carbon used is North American Carbon Co. type GX-176 coconut shell carbon impregnated with 1% triethylenedimaine (TEDA) and 2% potassium iodide by weight. Replacement intervals for the carbon have been as long as thirty months. Analysis of samples withdrawn at much shorter times has shown that the TEDA is lost after a few months, and the performance of the carbon for methyl iodide retention is dependent on the iodide impregnant. Efficient methyl iodide retention is not a requirement for carbon in this service; however, methyl iodide retention as measured by the ASTM Test D3803 (method B) has been found to correlate well with other desirable properties of the carbon such as radiation stability. The studies undertaken here were intended to shed light on the changes taking place in this carbon during long-term service and to provide a basis for simpler measurements of carbon quality

  6. Epigenetic Loss of MLH1 Expression in Normal Human Hematopoietic Stem Cell Clones is Defined by the Promoter CpG Methylation Pattern Observed by High-Throughput Methylation Specific Sequencing.

    Science.gov (United States)

    Kenyon, Jonathan; Nickel-Meester, Gabrielle; Qing, Yulan; Santos-Guasch, Gabriela; Drake, Ellen; PingfuFu; Sun, Shuying; Bai, Xiaodong; Wald, David; Arts, Eric; Gerson, Stanton L

    Normal human hematopoietic stem and progenitor cells (HPC) lose expression of MLH1 , an important mismatch repair (MMR) pathway gene, with age. Loss of MMR leads to replication dependent mutational events and microsatellite instability observed in secondary acute myelogenous leukemia and other hematologic malignancies. Epigenetic CpG methylation upstream of the MLH1 promoter is a contributing factor to acquired loss of MLH1 expression in tumors of the epithelia and proximal mucosa. Using single molecule high-throughput bisulfite sequencing we have characterized the CpG methylation landscape from -938 to -337 bp upstream of the MLH1 transcriptional start site (position +0), from 30 hematopoietic colony forming cell clones (CFC) either expressing or not expressing MLH1 . We identify a correlation between MLH1 promoter methylation and loss of MLH1 expression. Additionally, using the CpG site methylation frequencies obtained in this study we were able to generate a classification algorithm capable of sorting the expressing and non-expressing CFC. Thus, as has been previously described for many tumor cell types, we report for the first time a correlation between the loss of MLH1 expression and increased MLH1 promoter methylation in CFC derived from CD34 + selected hematopoietic stem and progenitor cells.

  7. Thermophysical study of methyl levulinate

    International Nuclear Information System (INIS)

    Lomba, Laura; Lafuente, Carlos; García-Mardones, Mónica; Gascón, Ignacio; Giner, Beatriz

    2013-01-01

    Highlights: • We have carried out a thermophysical characterization of methyl levulinate. • The study has been performed over a temperature range from (278.15 to 328.15) K. • pρT behavior has been studied over a temperature range from (333.15 to 453.15) K. • TRIDEN equation has been used to correlate pρT data. • Results have been compared with of ethyl and butyl levulinate and levulinic acid. -- Abstract: Several thermophysical properties (density, speed of sound, refractive index, surface tension, static permittivity and dynamic viscosity) of methyl levulinate have been measured under atmospheric pressure at temperatures from (278.15 to 338.15) K, while the vapor pressure was determined over a temperature range from (333.15 to 453.15) K. Furthermore, pρT behavior has been also investigated using a high-pressure, high-temperature vibrating tube densimeter over a temperature range from (283.15 to 338.15) K and a pressure range from (0.1 to 60.0) MPa. All these values obtained for methyl levulinate have been compared with other members of the levulinate family and also with levulinic acid

  8. LINE-1 methylation status in prostate cancer and non-neoplastic tissue adjacent to tumor in association with mortality.

    Science.gov (United States)

    Fiano, Valentina; Zugna, Daniela; Grasso, Chiara; Trevisan, Morena; Delsedime, Luisa; Molinaro, Luca; Gillio-Tos, Anna; Merletti, Franco; Richiardi, Lorenzo

    2017-01-02

    Aberrant DNA methylation seems to be associated with prostate cancer behavior. We investigated LINE-1 methylation in prostate cancer and non-neoplastic tissue adjacent to tumor (NTAT) in association with mortality from prostate cancer. We selected 157 prostate cancer patients with available NTAT from 2 cohorts of patients diagnosed between 1982-1988 and 1993-1996, followed up until 2010. An association between LINE-1 hypomethylation and prostate cancer mortality in tumor was suggested [hazard ratio per 5% decrease in LINE-1 methylation levels: 1.40, 95% confidence interval (CI): 0.95-2.01]. After stratification of the patients for Gleason score, the association was present only for those with a Gleason score of at least 8. Among these, low (80%) LINE-1 methylation was associated with a hazard ratio of 4.68 (95% CI: 1.03-21.34). LINE-1 methylation in the NTAT was not associated with prostate cancer mortality. Our results are consistent with the hypothesis that tumor tissue global hypomethylation may be a late event in prostate cancerogenesis and is associated with tumor progression.

  9. Potentiation of insulin release in response to amino acid methyl esters correlates to activation of islet glutamate dehydrogenase activity

    DEFF Research Database (Denmark)

    Kofod, Hans; Lernmark, A; Hedeskov, C J

    1986-01-01

    Column perifusion of mouse pancreatic islets was used to study the ability of amino acids and their methyl esters to influence insulin release and activate islet glutamate dehydrogenase activity. In the absence of L-glutamine, L-serine and the methyl ester of L-phenylalanine, but neither L...... glutamate dehydrogenase activity showed that only the two methyl esters of L-phenylalanine and L-serine activated the enzyme. It is concluded that the mechanism by which methyl esters of amino acids potentiate insulin release is most likely to be mediated by the activation of pancreatic beta-cell glutamate...

  10. No effect of folic acid supplementation on global DNA methylation in men and women with moderately elevated homocysteine.

    Directory of Open Access Journals (Sweden)

    Audrey Y Jung

    Full Text Available A global loss of cytosine methylation in DNA has been implicated in a wide range of diseases. There is growing evidence that modifications in DNA methylation can be brought about by altering the intake of methyl donors such as folate. We examined whether long-term daily supplementation with 0.8 mg of folic acid would increase global DNA methylation compared with placebo in individuals with elevated plasma homocysteine. We also investigated if these effects were modified by MTHFR C677T genotype. Two hundred sixteen participants out of 818 subjects who had participated in a randomized double-blind placebo-controlled trial were selected, pre-stratified on MTHFR C677T genotype and matched on age and smoking status. They were allocated to receive either folic acid (0.8 mg/d; n = 105 or placebo treatment (n = 111 for three years. Peripheral blood leukocyte DNA methylation and serum and erythrocyte folate were assessed. Global DNA methylation was measured using liquid chromatography-tandem mass spectrometry and expressed as a percentage of 5-methylcytosines versus the total number of cytosine. There was no difference in global DNA methylation between those randomized to folic acid and those in the placebo group (difference = 0.008, 95%CI = -0.05,0.07, P = 0.79. There was also no difference between treatment groups when we stratified for MTHFR C677T genotype (CC, n = 76; CT, n = 70; TT, n = 70, baseline erythrocyte folate status or baseline DNA methylation levels. In moderately hyperhomocysteinemic men and women, long-term folic acid supplementation does not increase global DNA methylation in peripheral blood leukocytes.ClinicalTrials.gov NCT00110604.

  11. No effect of folic acid supplementation on global DNA methylation in men and women with moderately elevated homocysteine.

    Science.gov (United States)

    Jung, Audrey Y; Smulders, Yvo; Verhoef, Petra; Kok, Frans J; Blom, Henk; Kok, Robert M; Kampman, Ellen; Durga, Jane

    2011-01-01

    A global loss of cytosine methylation in DNA has been implicated in a wide range of diseases. There is growing evidence that modifications in DNA methylation can be brought about by altering the intake of methyl donors such as folate. We examined whether long-term daily supplementation with 0.8 mg of folic acid would increase global DNA methylation compared with placebo in individuals with elevated plasma homocysteine. We also investigated if these effects were modified by MTHFR C677T genotype. Two hundred sixteen participants out of 818 subjects who had participated in a randomized double-blind placebo-controlled trial were selected, pre-stratified on MTHFR C677T genotype and matched on age and smoking status. They were allocated to receive either folic acid (0.8 mg/d; n = 105) or placebo treatment (n = 111) for three years. Peripheral blood leukocyte DNA methylation and serum and erythrocyte folate were assessed. Global DNA methylation was measured using liquid chromatography-tandem mass spectrometry and expressed as a percentage of 5-methylcytosines versus the total number of cytosine. There was no difference in global DNA methylation between those randomized to folic acid and those in the placebo group (difference = 0.008, 95%CI = -0.05,0.07, P = 0.79). There was also no difference between treatment groups when we stratified for MTHFR C677T genotype (CC, n = 76; CT, n = 70; TT, n = 70), baseline erythrocyte folate status or baseline DNA methylation levels. In moderately hyperhomocysteinemic men and women, long-term folic acid supplementation does not increase global DNA methylation in peripheral blood leukocytes.ClinicalTrials.gov NCT00110604.

  12. Alterations of global histone H4K20 methylation during prostate carcinogenesis

    Directory of Open Access Journals (Sweden)

    Behbahani Turang E

    2012-03-01

    Full Text Available Abstract Background Global histone modifications have been implicated in the progression of various tumour entities. Our study was designed to assess global methylation levels of histone 4 lysine 20 (H4K20me1-3 at different stages of prostate cancer (PCA carcinogenesis. Methods Global H4K20 methylation levels were evaluated using a tissue microarray in patients with clinically localized PCA (n = 113, non-malignant prostate disease (n = 27, metastatic hormone-naive PCA (mPCA, n = 30 and castration-resistant PCA (CRPC, n = 34. Immunohistochemistry was performed to assess global levels of H4K20 methylation levels. Results Similar proportions of the normal, PCA, and mPCA prostate tissues showed strong H4K20me3 staining. CRPC tissue analysis showed the weakest immunostaining levels of H4K20me1 and H4K20me2, compared to other prostate tissues. H4K20me2 methylation levels indicated significant differences in examined tissues except for normal prostate versus PCA tissue. H4K20me1 differentiates CRPC from other prostate tissues. H4K20me1 was significantly correlated with lymph node metastases, and H4K20me2 showed a significant correlation with the Gleason score. However, H4K20 methylation levels failed to predict PSA recurrence after radical prostatectomy. Conclusions H4K20 methylation levels constitute valuable markers for the dynamic process of prostate cancer carcinogenesis.

  13. Tumors with unmethylated MLH1 and the CpG island methylator phenotype are associated with a poor prognosis in stage II colorectal cancer patients.

    Science.gov (United States)

    Fu, Tao; Liu, Yanliang; Li, Kai; Wan, Weiwei; Pappou, Emmanouil P; Iacobuzio-Donahue, Christine A; Kerner, Zachary; Baylin, Stephen B; Wolfgang, Christopher L; Ahuja, Nita

    2016-12-27

    We previously developed a novel tumor subtype classification model for duodenal adenocarcinomas based on a combination of the CpG island methylator phenotype (CIMP) and MLH1 methylation status. Here, we tested the prognostic value of this model in stage II colorectal cancer (CRC) patients. Tumors were assigned to CIMP+/MLH1-unmethylated (MLH1-U), CIMP+/MLH1-methylated (MLH1-M), CIMP-/MLH1-U, or CIMP-/MLH1-M groups. Age, tumor location, lymphovascular invasion, and mucin production differed among the four patient subgroups, and CIMP+/MLH1-U tumors were more likely to have lymphovascular invasion and mucin production. Kaplan-Meier analyses revealed differences in both disease-free survival (DFS) and overall survival (OS) among the four groups. In a multivariate analysis, CIMP/MLH1 methylation status was predictive of both DFS and OS, and DFS and OS were shortest in CIMP+/MLH1-U stage II CRC patients. These results suggest that tumor subtype classification based on the combination of CIMP and MLH1 methylation status is informative in stage II CRC patients, and that CIMP+/MLH1-U tumors exhibit aggressive features and are associated with poor clinical outcomes.

  14. Patterns of DNA methylation in the normal colon vary by anatomical location, gender, and age

    Science.gov (United States)

    Kaz, Andrew M; Wong, Chao-Jen; Dzieciatkowski, Slavomir; Luo, Yanxin; Schoen, Robert E; Grady, William M

    2014-01-01

    Alterations in DNA methylation have been proposed to create a field cancerization state in the colon, where molecular alterations that predispose cells to transformation occur in histologically normal tissue. However, our understanding of the role of DNA methylation in field cancerization is limited by an incomplete characterization of the methylation state of the normal colon. In order to determine the colon’s normal methylation state, we extracted DNA from normal colon biopsies from the rectum, sigmoid, transverse, and ascending colon and assessed the methylation status of the DNA by pyrosequencing candidate loci as well as with HumanMethylation450 arrays. We found that methylation levels of repetitive elements LINE-1 and SAT-α showed minimal variability throughout the colon in contrast to other loci. Promoter methylation of EVL was highest in the rectum and progressively lower in the proximal segments, whereas ESR1 methylation was higher in older individuals. Genome-wide methylation analysis of normal DNA revealed 8388, 82, and 93 differentially methylated loci that distinguished right from left colon, males from females, and older vs. younger individuals, respectively. Although variability in methylation between biopsies and among different colon segments was minimal for repetitive elements, analyses of specific cancer-related genes as well as a genome-wide methylation analysis demonstrated differential methylation based on colon location, individual age, and gender. These studies advance our knowledge regarding the variation of DNA methylation in the normal colon, a prerequisite for future studies aimed at understanding methylation differences indicative of a colon field effect. PMID:24413027

  15. A relative quantitative Methylation-Sensitive Amplified Polymorphism (MSAP) method for the analysis of abiotic stress.

    Science.gov (United States)

    Bednarek, Piotr T; Orłowska, Renata; Niedziela, Agnieszka

    2017-04-21

    We present a new methylation-sensitive amplified polymorphism (MSAP) approach for the evaluation of relative quantitative characteristics such as demethylation, de novo methylation, and preservation of methylation status of CCGG sequences, which are recognized by the isoschizomers HpaII and MspI. We applied the technique to analyze aluminum (Al)-tolerant and non-tolerant control and Al-stressed inbred triticale lines. The approach is based on detailed analysis of events affecting HpaII and MspI restriction sites in control and stressed samples, and takes advantage of molecular marker profiles generated by EcoRI/HpaII and EcoRI/MspI MSAP platforms. Five Al-tolerant and five non-tolerant triticale lines were exposed to aluminum stress using the physiologicaltest. Total genomic DNA was isolated from root tips of all tolerant and non-tolerant lines before and after Al stress following metAFLP and MSAP approaches. Based on codes reflecting events affecting cytosines within a given restriction site recognized by HpaII and MspI in control and stressed samples demethylation (DM), de novo methylation (DNM), preservation of methylated sites (MSP), and preservation of nonmethylatedsites (NMSP) were evaluated. MSAP profiles were used for Agglomerative hierarchicalclustering (AHC) based on Squared Euclidean distance and Ward's Agglomeration method whereas MSAP characteristics for ANOVA. Relative quantitative MSAP analysis revealed that both Al-tolerant and non-tolerant triticale lines subjected to Al stress underwent demethylation, with demethylation of CG predominating over CHG. The rate of de novo methylation in the CG context was ~3-fold lower than demethylation, whereas de novo methylation of CHG was observed only in Al-tolerant lines. Our relative quantitative MSAP approach, based on methylation events affecting cytosines within HpaII-MspI recognition sequences, was capable of quantifying de novo methylation, demethylation, methylation, and non-methylated status in control

  16. Association of the colorectal CpG island methylator phenotype with molecular features, risk factors, and family history.

    Science.gov (United States)

    Weisenberger, Daniel J; Levine, A Joan; Long, Tiffany I; Buchanan, Daniel D; Walters, Rhiannon; Clendenning, Mark; Rosty, Christophe; Joshi, Amit D; Stern, Mariana C; LeMarchand, Loic; Lindor, Noralane M; Daftary, Darshana; Gallinger, Steven; Selander, Teresa; Bapat, Bharati; Newcomb, Polly A; Campbell, Peter T; Casey, Graham; Ahnen, Dennis J; Baron, John A; Haile, Robert W; Hopper, John L; Young, Joanne P; Laird, Peter W; Siegmund, Kimberly D

    2015-03-01

    The CpG island methylator phenotype (CIMP) represents a subset of colorectal cancers characterized by widespread aberrant DNA hypermethylation at select CpG islands. The risk factors and environmental exposures contributing to etiologic heterogeneity between CIMP and non-CIMP tumors are not known. We measured the CIMP status of 3,119 primary population-based colorectal cancer tumors from the multinational Colon Cancer Family Registry. Etiologic heterogeneity was assessed by a case-case study comparing risk factor frequency of colorectal cancer cases with CIMP and non-CIMP tumors using logistic regression to estimate the case-case odds ratio (ccOR). We found associations between tumor CIMP status and MSI-H (ccOR = 7.6), BRAF V600E mutation (ccOR = 59.8), proximal tumor site (ccOR = 9; all P CIMP status for both males and females (P = 0.0001 and P = 0.02, respectively), use of multivitamin or calcium supplements did not. Only for female colorectal cancer was CIMP status associated with increased pack-years of smoking (Ptrend CIMP status, and the associations of smoking and obesity with tumor subtype were evident only for females. Differences in the associations of a unique DNA methylation-based subgroup of colorectal cancer with important lifestyle and environmental exposures increase understanding of the molecular pathologic epidemiology of this heavily methylated subset of colorectal cancer. Cancer Epidemiol Biomarkers Prev; 24(3); 512-9. ©2015 AACR. ©2015 American Association for Cancer Research.

  17. Effect of different light quality on DNA methylation variation for brown ...

    African Journals Online (AJOL)

    Jane

    2011-07-06

    Jul 6, 2011 ... violet A (UV-A), blue, green, red and far-red (Chen et al.,. 2004; Jiao et al., 2007; Hu and Desai, ... (Tatra et al., 2000). ... methylation status (Reyna et al., 1997; Jaligot et al.,. 2004). ..... Hu JP, Desai M (2008). Light control of ...

  18. DNA methylation abnormalities in congenital heart disease.

    Science.gov (United States)

    Serra-Juhé, Clara; Cuscó, Ivon; Homs, Aïda; Flores, Raquel; Torán, Núria; Pérez-Jurado, Luis A

    2015-01-01

    Congenital heart defects represent the most common malformation at birth, occurring also in ∼50% of individuals with Down syndrome. Congenital heart defects are thought to have multifactorial etiology, but the main causes are largely unknown. We have explored the global methylation profile of fetal heart DNA in comparison to blood DNA from control subjects: an absolute correlation with the type of tissue was detected. Pathway analysis revealed a significant enrichment of differential methylation at genes related to muscle contraction and cardiomyopathies in the developing heart DNA. We have also searched for abnormal methylation profiles on developing heart-tissue DNA of syndromic and non-syndromic congenital heart defects. On average, 3 regions with aberrant methylation were detected per sample and 18 regions were found differentially methylated between groups. Several epimutations were detected in candidate genes involved in growth regulation, apoptosis and folate pathway. A likely pathogenic hypermethylation of several intragenic sites at the MSX1 gene, involved in outflow tract morphogenesis, was found in a fetus with isolated heart malformation. In addition, hypermethylation of the GATA4 gene was present in fetuses with Down syndrome with or without congenital heart defects, as well as in fetuses with isolated heart malformations. Expression deregulation of the abnormally methylated genes was detected. Our data indicate that epigenetic alterations of relevant genes are present in developing heart DNA in fetuses with both isolated and syndromic heart malformations. These epimutations likely contribute to the pathogenesis of the malformation by cis-acting effects on gene expression.

  19. Ionogels Based on Poly(methyl methacrylate) and Metal-Containing Ionic Liquids: Correlation between Structure and Mechanical and Electrical Properties.

    Science.gov (United States)

    Zehbe, Kerstin; Kollosche, Matthias; Lardong, Sebastian; Kelling, Alexandra; Schilde, Uwe; Taubert, Andreas

    2016-03-16

    Ionogels (IGs) based on poly(methyl methacrylate) (PMMA) and the metal-containing ionic liquids (ILs) bis-1-butyl-3-methlimidazolium tetrachloridocuprate(II), tetrachloride cobaltate(II), and tetrachlorido manganate(II) have been synthesized and their mechanical and electrical properties have been correlated with their microstructure. Unlike many previous examples, the current IGs show a decreasing stability in stress-strain experiments on increasing IL fractions. The conductivities of the current IGs are lower than those observed in similar examples in the literature. Both effects are caused by a two-phase structure with micrometer-sized IL-rich domains homogeneously dispersed an IL-deficient continuous PMMA phase. This study demonstrates that the IL-polymer miscibility and the morphology of the IGs are key parameters to control the (macroscopic) properties of IGs.

  20. Higher Alu methylation levels in catch-up growth in twenty-year-old offsprings.

    Directory of Open Access Journals (Sweden)

    Kittipan Rerkasem

    Full Text Available Alu elements and long interspersed element-1 (LINE-1 or L1 are two major human intersperse repetitive sequences. Lower Alu methylation, but not LINE-1, has been observed in blood cells of people in old age, and in menopausal women having lower bone mass and osteoporosis. Nevertheless, Alu methylation levels also vary among young individuals. Here, we explored phenotypes at birth that are associated with Alu methylation levels in young people. In 2010, 249 twenty-years-old volunteers whose mothers had participated in a study association between birth weight (BW and nutrition during pregnancy in 1990, were invited to take part in our present study. In this study, the LINE-1 and Alu methylation levels and patterns were measured in peripheral mononuclear cells and correlated with various nutritional parameters during intrauterine and postnatal period of offspring. This included the amount of maternal intake during pregnancy, the mother's weight gain during pregnancy, birth weight, birth length, and the rate of weight gain in the first year of life. Catch-up growth (CUG was defined when weight during the first year was >0.67 of the standard score, according to WHO data. No association with LINE-1 methylation was identified. The mean level of Alu methylation in the CUG group was significantly higher than those non-CUG (39.61% and 33.66 % respectively, P < 0.0001. The positive correlation between the history of CUG in the first year and higher Alu methylation indicates the role of Alu methylation, not only in aging cells, but also in the human growth process. Moreover, here is the first study that demonstrated the association between a phenotype during the newborn period and intersperse repetitive sequences methylation during young adulthood.

  1. MethylMix 2.0: an R package for identifying DNA methylation genes.

    Science.gov (United States)

    Cedoz, Pierre-Louis; Prunello, Marcos; Brennan, Kevin; Gevaert, Olivier

    2018-04-14

    DNA methylation is an important mechanism regulating gene transcription, and its role in carcinogenesis has been extensively studied. Hyper and hypomethylation of genes is a major mechanism of gene expression deregulation in a wide range of diseases. At the same time, high-throughput DNA methylation assays have been developed generating vast amounts of genome wide DNA methylation measurements. We developed MethylMix, an algorithm implemented in R to identify disease specific hyper and hypomethylated genes. Here we present a new version of MethylMix that automates the construction of DNA-methylation and gene expression datasets from The Cancer Genome Atlas (TCGA). More precisely, MethylMix 2.0 incorporates two major updates: the automated downloading of DNA methylation and gene expression datasets from TCGA and the automated preprocessing of such datasets: value imputation, batch correction and CpG sites clustering within each gene. The resulting datasets can subsequently be analyzed with MethylMix to identify transcriptionally predictive methylation states. We show that the Differential Methylation Values created by MethylMix can be used for cancer subtyping. olivier.gevaert@stanford.edu. https://bioconductor.org/packages/release/bioc/manuals/MethylMix/man/MethylMix.pdf. MethylMix 2.0 was implemented as an R package and is available in bioconductor.

  2. [Novel Approaches in DNA Methylation Studies - MS-HRM Analysis and Electrochemistry].

    Science.gov (United States)

    Bartošík, M; Ondroušková, E

    Cytosine methylation in DNA is an epigenetic mechanism regulating gene expression and plays a vital role in cell differentiation or proliferation. Tumor cells often exhibit aberrant DNA methylation, e.g. hypermethylation of tumor suppressor gene promoters. New methods, capable of determining methylation status of specific DNA sequences, are thus being developed. Among them, MS-HRM (methylation-specific high resolution melting) and electrochemistry offer relatively inexpensive instrumentation, fast assay times and possibility of screening multiple samples/DNA regions simultaneously. MS-HRM is due to its sensitivity and simplicity an interesting alternative to already established techniques, including methylation-specific PCR or bisulfite sequencing. Electrochemistry, when combined with suitable electroactive labels and electrode surfaces, has been applied in several unique strategies for discrimination of cytosines and methylcytosines. Both techniques were successfully tested in analysis of DNA methylation within promoters of important tumor suppressor genes and could thus help in achieving more precise diagnostics and prognostics of cancer. Aberrant methylation of promoters has already been described in hundreds of genes associated with tumorigenesis and could serve as important biomarker if new methods applicable into clinical practice are sufficiently advanced.Key words: DNA methylation - 5-methylcytosine - HRM analysis - melting temperature - DNA duplex - electrochemistry - nucleic acid hybridizationThis work was supported by MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 6. 5. 2016Accepted: 16. 5. 2016.

  3. DNA methylation and gene expression of HIF3A

    DEFF Research Database (Denmark)

    Main, Ailsa Maria; Gillberg, Linn; Jacobsen, Anna Louisa

    2016-01-01

    from 48 families, from whom we had SAT and muscle biopsies. DNA methylation of four CpG sites in the HIF3A promoter was analyzed in the blood and SAT by pyrosequencing, and HIF3A gene expression was analyzed in SAT and muscle by qPCR. An index of whole-body insulin sensitivity was estimated from oral...... individuals, and whether HIF3A gene expression in SAT and skeletal muscle biopsies showed associations with BMI and insulin resistance. Furthermore, we aimed to investigate gender specificity and heritability of these traits. METHODS: We studied 137 first-degree relatives of type 2 diabetes (T2D) patients...... glucose tolerance tests. RESULTS: BMI was associated with HIF3A methylation at one CpG site in the blood, and there was a positive association between the blood and SAT methylation levels at a different CpG site within the individuals. The SAT methylation level did not correlate with HIF3A gene expression...

  4. Increased activity of osteocyte autophagy in ovariectomized rats and its correlation with oxidative stress status and bone loss

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Yuehua, E-mail: yuesjtu@126.com; Zheng, Xinfeng, E-mail: zxf272@126.com; Li, Bo, E-mail: libo@126.com; Jiang, Shengdan, E-mail: jiangsd@126.com; Jiang, Leisheng, E-mail: leisheng_jiang@126.com

    2014-08-15

    Highlights: • Examine autophagy level in the proximal tibia of ovariectomized rats. • Investigate whether autophagy level is associated with bone loss. • Investigate whether autophagy level is associated with oxidative stress status. - Abstract: Objectives: The objectives of the present study were to investigate ovariectomy on autophagy level in the bone and to examine whether autophagy level is associated with bone loss and oxidative stress status. Methods: 36 female Sprague–Dawley rats were randomly divided into sham-operated (Sham), and ovariectomized (OVX) rats treated either with vehicle or 17-β-estradiol. At the end of the six-week treatment, bone mineral density (BMD) and bone micro-architecture in proximal tibias were assessed by micro-CT. Serum 17β-estradiol (E2) level were measured. Total antioxidant capacity (T-AOC), superoxide dismutase (SOD) activity, catalase (CAT) activity in proximal tibia was also determined. The osteocyte autophagy in proximal tibias was detected respectively by Transmission Electron Microscopy (TEM), immunofluorescent histochemistry (IH), realtime-PCR and Western blot. In addition, the spearman correlation between bone mass, oxidative stress status, serum E2 and autophagy were analyzed. Results: Ovariectomy increased Atg5, LC3, and Beclin1 mRNA and proteins expressions while decreased p62 expression. Ovariectomy also declined the activities of T-AOC, CAT, and SOD. Treatment with E2 prevented the reduction in bone mass as well as restored the autophagy level. Furthermore, LC3-II expression was inversely correlated with T-AOC, CAT, and SOD activities. A significant inverse correlation between LC3-II expression and BV/TV, Tb.N, BMD in proximal tibias was found. Conclusions: Ovariectomy induced oxidative stress, autophagy and bone loss. Autophagy of osteocyte was inversely correlated with oxidative stress status and bone loss.

  5. De novo CpG methylation on an artificial chromosome-like vector maintained for a long-term in mammalian cells.

    Science.gov (United States)

    Nishioka, Keisuke; Kishida, Tsunao; Masui, Shinji; Mazda, Osam

    2016-04-01

    To examine whether an autonomously replicating, artificial chromosome-like vector containing a long genomic DNA sequence (namely, Epigenosome-Nanog) undergoes de novo CpG methylation after maintenance in cultured cells for more than a half year. Epigenosome-Nanog efficiently replicated in iPS cells after transfection. In HeLa and C2C12 cells Epigenosome-Nanog was stably maintained for more than eight months. The CpG methylation occurred de novo at the Nanog gene promoter region on the epigenosome in C2C12 cells but the degrees of methylation were much lower than those at the same CpG sites on the chromosomes. Among the four CpG sites at the region, the upstream two CpGs underwent methylation in a correlated manner while methylation at the downstream two CpGs was also correlated to each other, and these correlations were commonly shared between the epigenosome and the chromosome. CpG methylation thus was not solely dependent on the nucleotide sequence at the DNA locus. The epigenosome may become a useful tool to study the mechanisms of epigenetic regulation of a genetic region of interest in mammalian cells.

  6. Correlates of the health statuses of the faculty at midlife

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    Galvin Alaan Galeon

    2016-01-01

    Full Text Available Background: Between the school years of 2009-2012, the turnover record of the University of San Jose-Recoletos (USJ-R, Cebu City, Philippines showed that permanent faculty members who left the institution were all midlifers. Their reasons varied from health issues to greener pasture elsewhere. Materials and Methods: This study then sought to explore the health statuses of the faculty midlifers of the USJ-R. The data were collected through survey conducted among the 106 faculty midlifers of the university. This study applied multivariate analyses to the survey data using Pearson-moment of correlation to determine the relationship between the sociodemographic profile of the research participants and their health statuses. Results: This research revealed that faculty midlifers are generally well physically. They showed emotional maturity and have positive outlook toward midlife. More so, their health conditions are significantly related with their sex, age, years of teaching, educational attainment, and income. Conclusion: At midlife, the faculty members of USJ-R can still generally be considered physically well. Thus, if they are well-managed, they can become relevant and better contributors to the attainment of the basic goals and objectives of the educational institution and the educational system in general.

  7. Quantitative promoter methylation analysis of multiple cancer-related genes in renal cell tumors

    International Nuclear Information System (INIS)

    Costa, Vera L; Henrique, Rui; Ribeiro, Franclim R; Pinto, Mafalda; Oliveira, Jorge; Lobo, Francisco; Teixeira, Manuel R; Jerónimo, Carmen

    2007-01-01

    Aberrant promoter hypermethylation of cancer-associated genes occurs frequently during carcinogenesis and may serve as a cancer biomarker. In this study we aimed at defining a quantitative gene promoter methylation panel that might identify the most prevalent types of renal cell tumors. A panel of 18 gene promoters was assessed by quantitative methylation-specific PCR (QMSP) in 85 primarily resected renal tumors representing the four major histologic subtypes (52 clear cell (ccRCC), 13 papillary (pRCC), 10 chromophobe (chRCC), and 10 oncocytomas) and 62 paired normal tissue samples. After genomic DNA isolation and sodium bisulfite modification, methylation levels were determined and correlated with standard clinicopathological parameters. Significant differences in methylation levels among the four subtypes of renal tumors were found for CDH1 (p = 0.0007), PTGS2 (p = 0.002), and RASSF1A (p = 0.0001). CDH1 hypermethylation levels were significantly higher in ccRCC compared to chRCC and oncocytoma (p = 0.00016 and p = 0.0034, respectively), whereas PTGS2 methylation levels were significantly higher in ccRCC compared to pRCC (p = 0.004). RASSF1A methylation levels were significantly higher in pRCC than in normal tissue (p = 0.035). In pRCC, CDH1 and RASSF1A methylation levels were inversely correlated with tumor stage (p = 0.031) and nuclear grade (p = 0.022), respectively. The major subtypes of renal epithelial neoplasms display differential aberrant CDH1, PTGS2, and RASSF1A promoter methylation levels. This gene panel might contribute to a more accurate discrimination among common renal tumors, improving preoperative assessment and therapeutic decision-making in patients harboring suspicious renal masses

  8. Predictive value of CpG island methylator phenotype for tumor recurrence in hepatitis B virus-associated hepatocellular carcinoma following liver transplantation

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    Zheng Shu-Sen

    2010-08-01

    Full Text Available Abstract Background CpG island methylator phenotype (CIMP, in which multiple genes concordantly methylated, has been demonstrated to be associated with progression, recurrence, as well as overall survival in some types of cancer. Methods We examined the promoter methylation status of seven genes including P16, CDH1, GSTP1, DAPK, XAF1, SOCS1 and SYK in 65 cases of HCC treated with LT by methylation-specific PCR. CIMP+ was defined as having three or more genes that are concordantly methylated. The relationship between CIMP status and clinicopathological parameters, as well as tumor recurrence was further analyzed. Results CIMP+ was more frequent in HCC with AFP > 400 ng/ml than those with AFP ≤ 400 ng/ml (P = 0.017. In addition, patients with CIMP+ were prone to have multiple tumor numbers than those with CIMP- (P = 0.007. Patients with CIMP+ tumors had significantly worse recurrence-free survival (RFS than patients with CIMP-tumors by Kaplan-Meier estimates (P = 0.004. Multivariate analysis also revealed that CIMP status might be a novel independent prognostic factor of RFS for HCC patients treated with LT (HR: 3.581; 95% CI: 1.473-8.710, P = 0.005. Conclusion Our results suggested that CIMP could serve as a new prognostic biomarker to predict the risk of tumor recurrence in HCC after transplantation.

  9. Predictive value of CpG island methylator phenotype for tumor recurrence in hepatitis B virus-associated hepatocellular carcinoma following liver transplantation

    International Nuclear Information System (INIS)

    Wu, Li-Ming; Zhang, Feng; Zhou, Lin; Yang, Zhe; Xie, Hai-Yang; Zheng, Shu-Sen

    2010-01-01

    CpG island methylator phenotype (CIMP), in which multiple genes concordantly methylated, has been demonstrated to be associated with progression, recurrence, as well as overall survival in some types of cancer. We examined the promoter methylation status of seven genes including P16, CDH1, GSTP1, DAPK, XAF1, SOCS1 and SYK in 65 cases of HCC treated with LT by methylation-specific PCR. CIMP+ was defined as having three or more genes that are concordantly methylated. The relationship between CIMP status and clinicopathological parameters, as well as tumor recurrence was further analyzed. CIMP+ was more frequent in HCC with AFP > 400 ng/ml than those with AFP ≤ 400 ng/ml (P = 0.017). In addition, patients with CIMP+ were prone to have multiple tumor numbers than those with CIMP- (P = 0.007). Patients with CIMP+ tumors had significantly worse recurrence-free survival (RFS) than patients with CIMP-tumors by Kaplan-Meier estimates (P = 0.004). Multivariate analysis also revealed that CIMP status might be a novel independent prognostic factor of RFS for HCC patients treated with LT (HR: 3.581; 95% CI: 1.473-8.710, P = 0.005). Our results suggested that CIMP could serve as a new prognostic biomarker to predict the risk of tumor recurrence in HCC after transplantation

  10. Using Sulfate-Amended Sediment Slurry Batch Reactors to Evaluate Mercury Methylation

    International Nuclear Information System (INIS)

    Harmon, S.M.

    2003-01-01

    In the methylated form, mercury represents a concern to public health primarily through the consumption of contaminated fish tissue. Research conducted on the methylation of mercury strongly suggests the process is microbial in nature and facilitated principally by sulfate-reducing bacteria. This study addressed the potential for mercury methylation by varying sulfate treatments and wetland-based soil in microbial slurry reactors with available inorganic mercury. Under anoxic laboratory conditions conducive to growth of naturally occurring sulfate-reducing bacteria in the soil, it was possible to evaluate how various sulfate additions influenced the methylation of inorganic mercury added to overlying water. Treatments included sulfate amendments ranging FR-om 25 to 500 mg/L (0.26 to 5.2 mM) above the soil's natural sulfate level. This study also provided an assessment of mercury methylation relative to sulfate-reducing bacterial population growth and subsequent sulfide production. Mercury methylation in sulfate treatments did not exceed that of the non-amended control during a 35-day incubation. However, increases in methylmercury concentration were linked to bacterial growth and sulfate reduction. A time lag in methylation in the highest treatment correlated with an equivalent lag in bacterial growth

  11. DNA methylation alteration is a major consequence of genome doubling in autotetraploid Brassica rapa

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    Xu Yanhao

    2017-01-01

    Full Text Available Polyploids are typically classified as autopolyploids or allopolyploids based on the origin of their chromosome sets. Autopolyploidy is much more common than traditionally believed. Allopolyploidization, accompanied by genomic and transcriptomic changes, has been well investigated. In this study, genetic, DNA methylation and gene expression changes in autotetraploid Brassica rapa were investigated. No genetic alteration was detected using an amplified fragment length polymorphism (AFLP approach. Using a cDNA-AFLP approach, approximately 0.58% of fragments showed changes in gene expression in autotetraploid B. rapa. The methylation-sensitive amplification polymorphism (MSAP analysis showed that approximately 1.7% of the fragments underwent DNA methylation changes upon genome doubling, with hypermethylation and demethylation changes equally affected. Fragments displaying changes in gene expression and methylation status were isolated and then sequenced and characterized, respectively. This study showed that variation in cytosine methylation is a major consequence of genome doubling in autotetraploid Brassica rapa.

  12. Association between promoter methylation of MLH1 and MSH2 and reactive oxygen species in oligozoospermic men-A pilot study.

    Science.gov (United States)

    Gunes, S; Agarwal, A; Henkel, R; Mahmutoglu, A M; Sharma, R; Esteves, S C; Aljowair, A; Emirzeoglu, D; Alkhani, A; Pelegrini, L; Joumah, A; Sabanegh, E

    2018-04-01

    MLH1 and MSH2 are important genes for DNA mismatch repair and crossing over during meiosis and are implicated in male infertility. Therefore, the methylation patterns of the DNA mismatch repair genes MLH1 and MSH2 in oligozoospermic males were investigated. Ten oligozoospermic patients and 29 normozoospermic donors were analysed. Methylation profiles of the MLH1 and MSH2 promotors were analysed. In addition, sperm motility and seminal reactive oxygen species (ROS) were recorded. Receiver operating characteristic (ROC) analysis was conducted to determine the accuracy of the DNA methylation status of MLH1 and MSH2 to distinguish between oligozoospermic and normozoospermic men. In oligozoospermic men, MLH1 was significantly (p = .0013) more methylated compared to normozoospermic men. Additionally, there was a significant positive association (r = .384; p = .0159) between seminal ROS levels and MLH1 methylation. Contrary, no association between MSH2 methylation and oligozoospermia was found. ROC curve analysis for methylation status of MLH1 was significant (p = .0275) with an area under the curve of 61.1%, a sensitivity of 22.2% and a specificity of 100.0%. This pilot study indicates oligozoospermic patients have more methylation of MLH1 than normozoospermic patients. Whether hypermethylation of the MLH1 promoter plays a role in repairing relevant mismatches of sperm DNA strands in idiopathic oligozoospermia warrants further investigation. © 2017 Blackwell Verlag GmbH.

  13. Global DNA methylation in earthworms: A candidate biomarker of epigenetic risks related to the presence of metals/metalloids in terrestrial environments

    Energy Technology Data Exchange (ETDEWEB)

    Maldonado Santoyo, Maria; Rodriguez Flores, Crescencio; Lopez Torres, Adolfo; Wrobel, Kazimierz [Department of Chemistry, University of Guanajuato, L de Retana No 5, 36000 Guanajuato (Mexico); Wrobel, Katarzyna, E-mail: katarzyn@quijote.ugto.mx [Department of Chemistry, University of Guanajuato, L de Retana No 5, 36000 Guanajuato (Mexico)

    2011-10-15

    In this work, possible relationships between global DNA methylation and metal/metalloid concentrations in earthworms have been explored. Direct correlation was observed between soil and tissue As, Se, Sb, Zn, Cu, Mn, Ag, Co, Hg, Pb (p < 0.05). Speciation results obtained for As and Hg hint at the capability of earthworms for conversion of inorganic element forms present in soil to methylated species. Inverse correlation was observed between the percentage of methylated DNA cytosines and total tissue As, As + Hg, As + Hg + Se + Sb ({beta} = -0.8456, p = 0.071; {beta} = -0.9406, p = 0.017; {beta} = -0.9526, p = 0.012 respectively), as well as inorganic As + Hg ({beta} = -0.8807, p = 0.049). It was concluded that earthworms would be particularly helpful as bioindicators of elements undergoing in vivo methylation and might also be used to assess the related risk of epigenetic changes in DNA methylation. - Graphical abstract: Display Omitted Highlights: > Several metals and metalloids contribute to epigenetic gene regulation. > As, Hg, Se, Sb inversely correlated with global DNA methylation in earthworms. > Biomethylation of the above elements in worms suggested. > Elements biomethylation apparently competes with DNA methylation. > DNA methylation a biomarker of epigenetic risks related to soil metals/metalloids. - Biomethylation of As, Hg in earthworms versus DNA methylation - a candidate biomarker of epigenetic risks related to the presence of metals/metalloids in soil.

  14. The rules of gene expression in plants: Organ identity and gene body methylation are key factors for regulation of gene expression in Arabidopsis thaliana

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    Gutiérrez Rodrigo A

    2008-09-01

    Full Text Available Abstract Background Microarray technology is a widely used approach for monitoring genome-wide gene expression. For Arabidopsis, there are over 1,800 microarray hybridizations representing many different experimental conditions on Affymetrix™ ATH1 gene chips alone. This huge amount of data offers a unique opportunity to infer the principles that govern the regulation of gene expression in plants. Results We used bioinformatics methods to analyze publicly available data obtained using the ATH1 chip from Affymetrix. A total of 1887 ATH1 hybridizations were normalized and filtered to eliminate low-quality hybridizations. We classified and compared control and treatment hybridizations and determined differential gene expression. The largest differences in gene expression were observed when comparing samples obtained from different organs. On average, ten-fold more genes were differentially expressed between organs as compared to any other experimental variable. We defined "gene responsiveness" as the number of comparisons in which a gene changed its expression significantly. We defined genes with the highest and lowest responsiveness levels as hypervariable and housekeeping genes, respectively. Remarkably, housekeeping genes were best distinguished from hypervariable genes by differences in methylation status in their transcribed regions. Moreover, methylation in the transcribed region was inversely correlated (R2 = 0.8 with gene responsiveness on a genome-wide scale. We provide an example of this negative relationship using genes encoding TCA cycle enzymes, by contrasting their regulatory responsiveness to nitrate and methylation status in their transcribed regions. Conclusion Our results indicate that the Arabidopsis transcriptome is largely established during development and is comparatively stable when faced with external perturbations. We suggest a novel functional role for DNA methylation in the transcribed region as a key determinant

  15. [Association between obesity and DNA methylation among the 7-16 year-old twins].

    Science.gov (United States)

    Li, C X; Gao, Y; Gao, W J; Yu, C Q; Lyu, J; Lyu, R R; Duan, J L; Sun, Y; Guo, X H; Wang, S F; Zhou, B; Wang, G; Cao, W H; Li, L M

    2018-04-10

    Objective: On whole-genome scale, we tried to explore the correlation between obesity-related traits and DNA methylation sites, based on discordant monozygotic twin pairs. Methods: A total of 90 pairs of 6-17 year-old twins were recruited in Chaoyang district, Yanqing district and Fangshan district in Beijing in 2016. Information on twins was gathered through a self-designed questionnaire and results: from physical examination, including height, weight and waist circumference of the subjects under study. DNA methylation detection was chosen on the Illumina Human Methylation EPIC BeadChip. R 3.3.1 language was used to read the DNA methylation signal under quality control on samples and probes. Ebayes function of empirical Bayes paired moderated t -test was used to identify the differential methylated CpG sites (DMCs). VarFit function of empirical Bayes paired moderated Levene test was used to identify the differentially variables CpG sits (DVCs) in obese and normal groups. Results According to the obesity discordance criteria, we collected 23 pairs of twins (age range 7 to 16 years), including 12 male pairs. A total of 817 471 qualified CpG loci were included in the genome-wide correlation analysis. According to the significance level of FDR set as obesity traits. After multiple testing corrections, no positive sites were found to have associated with obesity. However, results from the correlation analysis demonstrated sites cg05684382 (chr: 12) and cg26188191 (chr: 16) might have played a role in the development of obesity. This study provides a methodologic reference for the studies on discordance twins related problems.

  16. Constitutional and somatic methylation status of DMRH19 and KvDMR in Wilms tumor patients

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    Leila C.A. Cardoso

    2012-01-01

    Full Text Available The most frequent epigenetic alterations in Wilms tumor (WT occur at WT2, assigned to 11p15. WT2 consists of two domains: telomeric domain 1 (DMRH19 that contains the IGF2 gene and an imprinted maternally expressed transcript (H19 and centromeric domain 2 (KvDMR that contains the genes KCNQ1, KCNQ1OT1 and CDKN1C. In this work, we used pyrosequencing and MS-MLPA to compare the methylation patterns of DMRH19/KvDMR in blood and tumor samples from 40 WT patients. Normal constitutional KvDMR methylation indicated that most of the epigenetic alterations in WT occur at DMRH19. Constitutional DMRH19 hypermethylation (HM DMRH19 was observed in two patients with Beckwith-Wiedemann syndrome. Pyrosequencing and MS-MLPA showed HM DMRH19 in 28/34 tumor samples: 16/34 with isolated HM DMRH19 and 12/34 with concomitant HM DMRH19 and KvDMR hypomethylation, indicating paternal uniparental disomy. With the exception of one blood sample, the MS-MLPA and pyrosequencing findings were concordant. Diffuse or focal anaplasia was present in five tumor samples and was associated with isolated somatic HM DMRH19 in four of them. Constitutional 11p15 methylation abnormalities were present in 5% of the samples and somatic abnormalities in the majority of tumors. Combined analysis of DMRH19/KvDMR by pyrosequencing and MS-MLPA is beneficial for characterizing epigenetic anomalies in WT, and MS-MLPA is useful and reliable for estimation of DNA methylation in a clinical setting.

  17. A novel method for identification and quantification of consistently differentially methylated regions.

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    Ching-Lin Hsiao

    Full Text Available Advances in biotechnology have resulted in large-scale studies of DNA methylation. A differentially methylated region (DMR is a genomic region with multiple adjacent CpG sites that exhibit different methylation statuses among multiple samples. Many so-called "supervised" methods have been established to identify DMRs between two or more comparison groups. Methods for the identification of DMRs without reference to phenotypic information are, however, less well studied. An alternative "unsupervised" approach was proposed, in which DMRs in studied samples were identified with consideration of nature dependence structure of methylation measurements between neighboring probes from tiling arrays. Through simulation study, we investigated effects of dependencies between neighboring probes on determining DMRs where a lot of spurious signals would be produced if the methylation data were analyzed independently of the probe. In contrast, our newly proposed method could successfully correct for this effect with a well-controlled false positive rate and a comparable sensitivity. By applying to two real datasets, we demonstrated that our method could provide a global picture of methylation variation in studied samples. R source codes to implement the proposed method were freely available at http://www.csjfann.ibms.sinica.edu.tw/eag/programlist/ICDMR/ICDMR.html.

  18. Social Crowding during Development Causes Changes in GnRH1 DNA Methylation.

    Science.gov (United States)

    Alvarado, Sebastian G; Lenkov, Kapa; Williams, Blake; Fernald, Russell D

    2015-01-01

    Gestational and developmental cues have important consequences for long-term health, behavior and adaptation to the environment. In addition, social stressors cause plastic molecular changes in the brain that underlie unique behavioral phenotypes that also modulate fitness. In the adult African cichlid, Astatotilapia burtoni, growth and social status of males are both directly regulated by social interactions in a dynamic social environment, which causes a suite of plastic changes in circuits, cells and gene transcription in the brain. We hypothesized that a possible mechanism underlying some molecular changes might be DNA methylation, a reversible modification made to cytosine nucleotides that is known to regulate gene function. Here we asked whether changes in DNA methylation of the GnRH1 gene, the central regulator of the reproductive axis, were altered during development of A. burtoni. We measured changes in methylation state of the GnRH1 gene during normal development and following the gestational and developmental stress of social crowding. We found differential DNA methylation within developing juveniles between 14-, 28- and 42-day-old. Following gestational crowding of mouth brooding mothers, we saw differential methylation and transcription of GnRH1 in their offspring. Taken together, our data provides evidence for social control of GnRH1 developmental responses to gestational cues through DNA methylation.

  19. Methylation-Sensitive Amplification Length Polymorphism (MS-AFLP) Microarrays for Epigenetic Analysis of Human Genomes.

    Science.gov (United States)

    Alonso, Sergio; Suzuki, Koichi; Yamamoto, Fumiichiro; Perucho, Manuel

    2018-01-01

    Somatic, and in a minor scale also germ line, epigenetic aberrations are fundamental to carcinogenesis, cancer progression, and tumor phenotype. DNA methylation is the most extensively studied and arguably the best understood epigenetic mechanisms that become altered in cancer. Both somatic loss of methylation (hypomethylation) and gain of methylation (hypermethylation) are found in the genome of malignant cells. In general, the cancer cell epigenome is globally hypomethylated, while some regions-typically gene-associated CpG islands-become hypermethylated. Given the profound impact that DNA methylation exerts on the transcriptional profile and genomic stability of cancer cells, its characterization is essential to fully understand the complexity of cancer biology, improve tumor classification, and ultimately advance cancer patient management and treatment. A plethora of methods have been devised to analyze and quantify DNA methylation alterations. Several of the early-developed methods relied on the use of methylation-sensitive restriction enzymes, whose activity depends on the methylation status of their recognition sequences. Among these techniques, methylation-sensitive amplification length polymorphism (MS-AFLP) was developed in the early 2000s, and successfully adapted from its original gel electrophoresis fingerprinting format to a microarray format that notably increased its throughput and allowed the quantification of the methylation changes. This array-based platform interrogates over 9500 independent loci putatively amplified by the MS-AFLP technique, corresponding to the NotI sites mapped throughout the human genome.

  20. Systemic effects of chronically administered methyl prednisolonate and methyl 17-deoxyprednisolonate.

    Science.gov (United States)

    Olejniczak, E; Lee, H J

    1984-06-01

    The systemic activities of methyl prednisolonate and methyl 17-deoxyprednisolonate (1) were studied in rats. Methyl 17-deoxyprednisolonate produced significant changes in the amount of sodium ion (decreased) and potassium ion (increased) in urine; however, methyl prednisolonate had no effect on electrolyte balance. Both methyl prednisolonate and methyl 17-deoxyprednisolonate had no effect on liver glycogen content, plasma corticosterone level and relative adrenal weight. In contrast, the parent compound prednisolone caused a significant decrease in liver glycogen content, plasma corticosterone level and relative adrenal weight.

  1. Aberrantly methylated genes in human papillary thyroid cancer and their association with BRAF/RAS mutation.

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    Yasuko eKikuchi

    2013-12-01

    Full Text Available Cancer arises through accumulation of epigenetic and genetic alteration. Aberrant promoter methylation is a common epigenetic mechanism of gene silencing in cancer cells. We here performed genome-wide analysis of DNA methylation of promoter regions by Infinium HumanMethylation27 BeadChip, using 14 clinical papillary thyroid cancer samples and 10 normal thyroid samples. Among the 14 papillary cancer cases, 11 showed frequent aberrant methylation, but the other three cases showed no aberrant methylation at all. Distribution of the hypermethylation among cancer samples was non-random, which implied existence of a subset of preferentially methylated papillary thyroid cancer. Among 25 frequently methylated genes, methylation status of six genes (HIST1H3J, POU4F2, SHOX2, PHKG2, TLX3, HOXA7 was validated quantitatively by pyrosequencing. Epigenetic silencing of these genes in methylated papillary thyroid cancer cell lines was confirmed by gene re-expression following treatment with 5-aza-2'-deoxycytidine and trichostatin A, and detected by real-time RT-PCR. Methylation of these six genes was validated by analysis of additional 20 papillary thyroid cancer and 10 normal samples. Among the 34 cancer samples in total, 26 cancer samples with preferential methylation were significantly associated with mutation of BRAF/RAS oncogene (P=0.04, Fisher’s exact test. Thus we identified new genes with frequent epigenetic hypermethylation in papillary thyroid cancer, two subsets of either preferentially methylated or hardly methylated papillary thyroid cancer, with a concomitant occurrence of oncogene mutation and gene methylation. These hypermethylated genes may constitute potential biomarkers for papillary thyroid cancer.

  2. Social Status Correlates of Reporting Racial Discrimination and Gender Discrimination among Racially Diverse Women

    OpenAIRE

    Ro, Annie E.; Choi, Kyung-Hee

    2009-01-01

    The growing body of research on discrimination and health indicates a deleterious effect of discrimination on various health outcomes. However, less is known about the sociodemographic correlates of reporting racial discrimination and gender discrimination among racially diverse women. We examined the associations of social status characteristics with lifetime experiences of racial discrimination and gender discrimination using a racially-diverse sample of 754 women attending family planning ...

  3. Promoter methylation inhibits BRD7 expression in human nasopharyngeal carcinoma cells

    International Nuclear Information System (INIS)

    Liu, Huaying; Li, Guiyuan; Zhang, Liming; Niu, Zhaoxia; Zhou, Ming; Peng, Cong; Li, Xiayu; Deng, Tan; Shi, Lei; Tan, Yixin

    2008-01-01

    Nasopharyngeal carcinoma (NPC) is a head and neck malignancy with high occurrence in South-East Asia and Southern China. Recent findings suggest that epigenetic inactivation of multiple tumor suppressor genes plays an important role in the tumourigenesis of NPC. BRD7 is a NPC-associated bromodomain gene that exhibits a much higher-level of mRNA expression in normal than in NPC biopsies and cell lines. In this study, we explored the role of DNA methylation in regulation of BRD7 transcription. The presence of CpG islands within BRD7 promoter was predicted by EMBOSS CpGplot and Softberry CpGFinder, respectively. Nested methylation-specific PCR and RT-PCR were employed to detect the methylation status of BRD7 promoter and the mRNA expression of BRD7 gene in tumor cell lines as well as clinical samples. Electrophoretic mobility shift assays (EMSA) and luciferase assay were used to detect the effects of cytosine methylation on the nuclear protein binding to BRD7 promoter. We found that DNA methylation suppresses BRD7 expression in NPC cells. In vitro DNA methylation in NPC cells silenced BRD7 promoter activity and inhibited the binding of the nuclear protein (possibly Sp1) to Sp1 binding sites in the BRD7 promoter. In contrast, inhibition of DNA methylation augments induction of endogenous BRD7 mRNA in NPC cells. We also found that methylation frequency of BRD7 promoter is much higher in the tumor and matched blood samples from NPC patients than in the blood samples from normal individuals. BRD7 promoter demethylation is a prerequisite for high level induction of BRD7 gene expression. DNA methylation of BRD7 promoter might serve as a diagnostic marker in NPC

  4. DNA Methylation at the Neonatal State and at the Time of Diagnosis: Preliminary Support for an Association with the Estrogen Receptor 1, Gamma-Aminobutyric Acid B Receptor 1, and Myelin Oligodendrocyte Glycoprotein in Female Adolescent Patients with OCD.

    Science.gov (United States)

    Nissen, Judith Becker; Hansen, Christine Søholm; Starnawska, Anna; Mattheisen, Manuel; Børglum, Anders Dupont; Buttenschøn, Henriette Nørmølle; Hollegaard, Mads

    2016-01-01

    Obsessive-compulsive disorder (OCD) is a neuropsychiatric disorder. Non-genetic factors and their interaction with genes have attracted increasing attention. Epigenetics is regarded an important interface between environmental signals and activation/repression of genomic responses. Epigenetic mechanisms have not previously been examined in OCD in children and adolescents. The aim of the present study was to examine the DNA methylation profile of selected genes in blood spots from neonates later diagnosed with OCD and in the same children/adolescents at the time of diagnosis compared with age- and sex-matched controls. Furthermore, we wanted to characterize the association of the differential methylation profiles with the severity of OCD and treatment outcome. Dried and new blood spot samples were obtained from 21 female children/adolescents with verified OCD and 12 female controls. The differential methylation was analyzed using a linear model and the correlation with the severity of OCD and treatment outcome was analyzed using the Pearson correlation. We evaluated selected Illumina Infinium HumanMethylation450 BeadChip probes within and up to 100,000 bp up- and downstream of 14 genes previously associated with OCD (SLC1A1, SLC25A12, GABBR1, GAD1, DLGAP1, MOG, BDNF, OLIG2, NTRK2 and 3, ESR1, SL6A4, TPH2, and COMT). The study found no significantly differential methylation. However, preliminary support for a difference was found for the gamma-aminobutyric acid (GABA) B receptor 1 (cg10234998, cg17099072) in blood samples at birth and for the estrogen receptor 1 (ESR1) (cg10939667), the myelin oligodendrocyte glycoprotein (MOG) (cg16650906), and the brain-derived neurotrophic factor (BDNF) (cg14080521) in blood samples at the time of diagnosis. Preliminary support for an association was observed between the methylation profiles of GABBR1 and MOG and baseline severity, treatment effect, and responder status; and between the methylation profile of ESR1 and baseline

  5. Effects of nickel, chromate, and arsenite on histone 3 lysine methylation

    International Nuclear Information System (INIS)

    Zhou Xue; Li Qin; Arita, Adriana; Sun Hong; Costa, Max

    2009-01-01

    Occupational exposure to nickel (Ni), chromium (Cr), and arsenic (As) containing compounds has been associated with lung cancer and other adverse health effects. Their carcinogenic properties may be attributable in part, to activation and/or repression of gene expression induced by changes in the DNA methylation status and histone tail post-translational modifications. Here we show that individual treatment with nickel, chromate, and arsenite all affect the gene activating mark H3K4 methylation. We found that nickel (1 mM), chromate (10 μM), and arsenite (1 μM) significantly increase tri-methyl H3K4 after 24 h exposure in human lung carcinoma A549 cells. Seven days of exposure to lower levels of nickel (50 and 100 μM), chromate (0.5 and 1 μM) or arsenite (0.1, 0.5 and 1 μM) also increased tri-methylated H3K4 in A549 cells. This mark still remained elevated and inherited through cell division 7 days following removal of 1 μM arsenite. We also demonstrate by dual staining immunofluorescence microscopy that both H3K4 tri-methyl and H3K9 di-methyl marks increase globally after 24 h exposure to each metal treatment in A549 cells. However, the tri-methyl H3K4 and di-methyl H3K9 marks localize in different regions in the nucleus of the cell. Thus, our study provides further evidence that a mechanism(s) of carcinogenicity of nickel, chromate, and arsenite metal compounds may involve alterations of various histone tail modifications that may in turn affect the expression of genes that may cause transformation

  6. DNA methylation and temperature stress in an Antarctic polychaete, Spiophanes tcherniai.

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    Marsh, Adam G; Pasqualone, Annamarie A

    2014-01-01

    Epigenetic modifications of DNA and histones are a primary mechanism by which gene expression activities may be modified in response to environmental stimuli. Here we characterize patterns of methyl-cytosine composition in the marine polychaete Spiophanes tcherniai from McMurdo Sound, Antarctica. We cultured adult worms at two temperatures, -1.5°C (ambient control) and +4°C (warm treatment), for 4 weeks. We observed a rapid capacity for S. tcherniai organismal respiration rates and underlying catalytic rates of citrate synthase at +4°C to return to control levels in less than 4 weeks. We profiled changes in the methylation states of CpG sites in these treatments using an NGS strategy to computationally reconstruct and quantify methylation status across the genome. In our analysis we recovered 120,000 CpG sites in assembled contigs from both treatments. Of those, we were able to align 28,000 CpG sites in common between the two sample groups. In comparing these aligned sites between treatments, only 3000 (11%) evidenced a change in methylation state, but over 85% of changes involved a gain of a 5-methyl group on a CpG site (net increase in methyation). The ability to score CpG sites as partially methylated among gDNA copies in a sample opens up a new avenue for assessing DNA methylation responses to changing environments. By quantitatively distinguishing a "mixed" population of copies of one CpG site, we can begin to identify dynamic, non-binary, continuous-response reactions in DNA methylation intensity or density that previously may have been overlooked as noise.

  7. DNA Methylation and Temperature Stress in an Antarctic Polychaete, Spiophanes tcherniai

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    Adam G. Marsh

    2014-05-01

    Full Text Available Epigenetic modifications of DNA and histones are a primary mechanism by which gene expression activities may be modified in response to environmental stimuli. Here we characterize patterns of methyl-cytosine composition in the marine polychaete emph{Spiophanes tcherniai} from McMurdo Sound, Antarctica. We cultured adult worms at two temperatures, -1.5 C (ambient control and +4 C (warm treatment, for four weeks. We observed a rapid capacity for emph{S. tcherniai} organismal respiration rates and underlying catalytic rates of citrate synthase to acclimate at +4 C and return to control levels. We profiled changes in the methylation states of CpG sites in these treatments using an NGS strategy to computationally reconstruct and quantify methylation status across the genome. In our analysis we recovered 120,000 CpG sites in assembled contigs from both treatments. Of those, we were able to align 28,000 CpG sites in common between the two sample groups. In comparing these aligned sites between treatments, only 3,000 (11% evidenced a change in methylation state, but over 85% of changes involved a gain of a 5-methyl group on a CpG site (net increase in methyation. The ability to score CpG sites as partially methylated among gDNA copies in a sample opens up a new avenue for assessing DNA methylation responses to changing environments. By quantitatively distinguishing a ``mixed'' population of copies of one CpG site, we can begin to identify dynamic, non-binary, continuous-response reactions in DNA methylation intensity or density that previously may have been overlooked as noise.

  8. Chlamydia trachomatis Infection Is Associated with E-Cadherin Promoter Methylation, Downregulation of E-Cadherin Expression, and Increased Expression of Fibronectin and α-SMA—Implications for Epithelial-Mesenchymal Transition

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    Jovana Rajić

    2017-06-01

    Full Text Available Chlamydia trachomatis (Ct can induce scarring disease of the ocular mucosa, known as trachoma, the most common infectious cause of blindness worldwide. We hypothesized that epithelial-mesenchymal transition (EMT contributes to the fibrotic process in trachomatous scarring. Infection of human conjunctival epithelial cells (HCjE with Ct activated signaling pathways involved in EMT induction, which was correlated with decreased expression of E-cadherin, guardian of the epithelial phenotype. In addition, Ct infection was associated with increased expression of two mesenchymal cell markers: fibronectin and α-SMA. The DNA methylation statuses of selected regions of E-cadherin, fibronectin, and α-SMA genes revealed that Ct infection was accompanied with changes in DNA methylation of the E-cadherin promoter, while the expression of the two mesenchymal markers was not related with this epigenetic event. Our data suggest that Ct infection of conjunctival epithelial cells induces EMT-like changes that go along with modification of the methylation profile of the E-cadherin promoter and could, as one of the earliest events, contribute to processes triggering conjunctival scarring.

  9. Nuclear magnetic relaxation of methyl group in liquids

    International Nuclear Information System (INIS)

    Blicharska, B.

    1986-01-01

    The theoretical description of the relaxation process of methyl group in liquids and some results of the measurements of relaxation function and relaxation times for cryoprotective solutions are presented. Starting from the application of the operator formalism the general equation for spin operators e.g. components of the nuclear spin and magnetization is founded. Next, the spin Hamiltonian is presented as contraction of the symmetry adapted spherical tensors as well as the correlation functions and spectral densities. On the basis of extended and modified Woessner model of motion the correlation functions and spectral densities are calculated for methyl group in liquids. Using these functions the relaxation matrix elements, the spin-spin and spin-lattice relaxation times can be expressed. The prediction of the theory agrees with author's previous experiments on cryoprotective solutions. The observed dependence on temperature, frequency and isotopic dilution in methanol-water, methanol-dimethyl sulfoxide (DMSO) and DMSO-water solutions is in a satisfactory agreement with theoretical equations. 34 refs. (author)

  10. Quantitative promoter methylation analysis of multiple cancer-related genes in renal cell tumors

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    Oliveira Jorge

    2007-07-01

    Full Text Available Abstract Background Aberrant promoter hypermethylation of cancer-associated genes occurs frequently during carcinogenesis and may serve as a cancer biomarker. In this study we aimed at defining a quantitative gene promoter methylation panel that might identify the most prevalent types of renal cell tumors. Methods A panel of 18 gene promoters was assessed by quantitative methylation-specific PCR (QMSP in 85 primarily resected renal tumors representing the four major histologic subtypes (52 clear cell (ccRCC, 13 papillary (pRCC, 10 chromophobe (chRCC, and 10 oncocytomas and 62 paired normal tissue samples. After genomic DNA isolation and sodium bisulfite modification, methylation levels were determined and correlated with standard clinicopathological parameters. Results Significant differences in methylation levels among the four subtypes of renal tumors were found for CDH1 (p = 0.0007, PTGS2 (p = 0.002, and RASSF1A (p = 0.0001. CDH1 hypermethylation levels were significantly higher in ccRCC compared to chRCC and oncocytoma (p = 0.00016 and p = 0.0034, respectively, whereas PTGS2 methylation levels were significantly higher in ccRCC compared to pRCC (p = 0.004. RASSF1A methylation levels were significantly higher in pRCC than in normal tissue (p = 0.035. In pRCC, CDH1 and RASSF1A methylation levels were inversely correlated with tumor stage (p = 0.031 and nuclear grade (p = 0.022, respectively. Conclusion The major subtypes of renal epithelial neoplasms display differential aberrant CDH1, PTGS2, and RASSF1A promoter methylation levels. This gene panel might contribute to a more accurate discrimination among common renal tumors, improving preoperative assessment and therapeutic decision-making in patients harboring suspicious renal masses.

  11. Multiple sporadic colorectal cancers display a unique methylation phenotype.

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    Victoria Gonzalo

    Full Text Available Epigenetics are thought to play a major role in the carcinogenesis of multiple sporadic colorectal cancers (CRC. Previous studies have suggested concordant DNA hypermethylation between tumor pairs. However, only a few methylation markers have been analyzed. This study was aimed at describing the epigenetic signature of multiple CRC using a genome-scale DNA methylation profiling. We analyzed 12 patients with synchronous CRC and 29 age-, sex-, and tumor location-paired patients with solitary tumors from the EPICOLON II cohort. DNA methylation profiling was performed using the Illumina Infinium HM27 DNA methylation assay. The most significant results were validated by Methylight. Tumors samples were also analyzed for the CpG Island Methylator Phenotype (CIMP; KRAS and BRAF mutations and mismatch repair deficiency status. Functional annotation clustering was performed. We identified 102 CpG sites that showed significant DNA hypermethylation in multiple tumors with respect to the solitary counterparts (difference in β value ≥0.1. Methylight assays validated the results for 4 selected genes (p = 0.0002. Eight out of 12(66.6% multiple tumors were classified as CIMP-high, as compared to 5 out of 29(17.2% solitary tumors (p = 0.004. Interestingly, 76 out of the 102 (74.5% hypermethylated CpG sites found in multiple tumors were also seen in CIMP-high tumors. Functional analysis of hypermethylated genes found in multiple tumors showed enrichment of genes involved in different tumorigenic functions. In conclusion, multiple CRC are associated with a distinct methylation phenotype, with a close association between tumor multiplicity and CIMP-high. Our results may be important to unravel the underlying mechanism of tumor multiplicity.

  12. The CpG island methylator phenotype is concordant between primary colorectal carcinoma and matched distant metastases.

    Science.gov (United States)

    Cohen, Stacey A; Yu, Ming; Baker, Kelsey; Redman, Mary; Wu, Chen; Heinzerling, Tai J; Wirtz, Ralph M; Charalambous, Elpida; Pentheroudakis, George; Kotoula, Vassiliki; Kalogeras, Konstantine T; Fountzilas, George; Grady, William M

    2017-01-01

    The CpG island methylator phenotype (CIMP) in stage III colon cancer (CRC) has been associated with improved survival after treatment with adjuvant irinotecan-based chemotherapy. In this analysis, we determine whether CIMP status in the primary CRC is concordant with the CIMP status of matched metastases in order to determine if assessment of CIMP status in the primary tumor can be used to predict CIMP status of metastatic disease, which is relevant for patient management as well as for understanding the biology of CIMP CRCs. We assessed the CIMP status of 70 pairs of primary CRC and matched metastases using a CRC-specific panel of five markers ( CACNA1G , IGF2 , NEUROG1 , RUNX3 , and SOCS1 ) where CIMP positive was defined as 3/5 positive markers at a percent methylated reference threshold of ≥10%. Concordance was compared using the Fisher's exact test and P  CIMP status in the primary tumor and matched metastasis; five (7.0%) of the pairs were concordantly CIMP positive. Only one pair (1.4%) had divergent CIMP status, demonstrating CIMP positivity (4/5 markers positive) in the primary tumor, while the matched metastasis was CIMP negative (0 markers positive). CIMP status is generally concordant between primary CRCs and matched metastases. Thus, CIMP status in the primary tumor is maintained in matched metastases and can be used to inform CIMP-based therapy options for the metastases.

  13. Ionogels Based on Poly(methyl methacrylate and Metal-Containing Ionic Liquids: Correlation between Structure and Mechanical and Electrical Properties

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    Kerstin Zehbe

    2016-03-01

    Full Text Available Ionogels (IGs based on poly(methyl methacrylate (PMMA and the metal-containing ionic liquids (ILs bis-1-butyl-3-methlimidazolium tetrachloridocuprate(II, tetrachloride cobaltate(II, and tetrachlorido manganate(II have been synthesized and their mechanical and electrical properties have been correlated with their microstructure. Unlike many previous examples, the current IGs show a decreasing stability in stress-strain experiments on increasing IL fractions. The conductivities of the current IGs are lower than those observed in similar examples in the literature. Both effects are caused by a two-phase structure with micrometer-sized IL-rich domains homogeneously dispersed an IL-deficient continuous PMMA phase. This study demonstrates that the IL-polymer miscibility and the morphology of the IGs are key parameters to control the (macroscopic properties of IGs.

  14. Circulating tumour DNA methylation markers for diagnosis and prognosis of hepatocellular carcinoma

    Science.gov (United States)

    Xu, Rui-Hua; Wei, Wei; Krawczyk, Michal; Wang, Wenqiu; Luo, Huiyan; Flagg, Ken; Yi, Shaohua; Shi, William; Quan, Qingli; Li, Kang; Zheng, Lianghong; Zhang, Heng; Caughey, Bennett A.; Zhao, Qi; Hou, Jiayi; Zhang, Runze; Xu, Yanxin; Cai, Huimin; Li, Gen; Hou, Rui; Zhong, Zheng; Lin, Danni; Fu, Xin; Zhu, Jie; Duan, Yaou; Yu, Meixing; Ying, Binwu; Zhang, Wengeng; Wang, Juan; Zhang, Edward; Zhang, Charlotte; Li, Oulan; Guo, Rongping; Carter, Hannah; Zhu, Jian-Kang; Hao, Xiaoke; Zhang, Kang

    2017-11-01

    An effective blood-based method for the diagnosis and prognosis of hepatocellular carcinoma (HCC) has not yet been developed. Circulating tumour DNA (ctDNA) carrying cancer-specific genetic and epigenetic aberrations may enable a noninvasive `liquid biopsy' for diagnosis and monitoring of cancer. Here, we identified an HCC-specific methylation marker panel by comparing HCC tissue and normal blood leukocytes and showed that methylation profiles of HCC tumour DNA and matched plasma ctDNA are highly correlated. Using cfDNA samples from a large cohort of 1,098 HCC patients and 835 normal controls, we constructed a diagnostic prediction model that showed high diagnostic specificity and sensitivity (P < 0.001) and was highly correlated with tumour burden, treatment response, and stage. Additionally, we constructed a prognostic prediction model that effectively predicted prognosis and survival (P < 0.001). Together, these findings demonstrate in a large clinical cohort the utility of ctDNA methylation markers in the diagnosis, surveillance, and prognosis of HCC.

  15. Brain region-specific expression of MeCP2 isoforms correlates with DNA methylation within Mecp2 regulatory elements.

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    Carl O Olson

    Full Text Available MeCP2 is a critical epigenetic regulator in brain and its abnormal expression or compromised function leads to a spectrum of neurological disorders including Rett Syndrome and autism. Altered expression of the two MeCP2 isoforms, MeCP2E1 and MeCP2E2 has been implicated in neurological complications. However, expression, regulation and functions of the two isoforms are largely uncharacterized. Previously, we showed the role of MeCP2E1 in neuronal maturation and reported MeCP2E1 as the major protein isoform in the adult mouse brain, embryonic neurons and astrocytes. Recently, we showed that DNA methylation at the regulatory elements (REs within the Mecp2 promoter and intron 1 impact the expression of Mecp2 isoforms in differentiating neural stem cells. This current study is aimed for a comparative analysis of temporal, regional and cell type-specific expression of MeCP2 isoforms in the developing and adult mouse brain. MeCP2E2 displayed a later expression onset than MeCP2E1 during mouse brain development. In the adult female and male brain hippocampus, both MeCP2 isoforms were detected in neurons, astrocytes and oligodendrocytes. Furthermore, MeCP2E1 expression was relatively uniform in different brain regions (olfactory bulb, striatum, cortex, hippocampus, thalamus, brainstem and cerebellum, whereas MeCP2E2 showed differential enrichment in these brain regions. Both MeCP2 isoforms showed relatively similar distribution in these brain regions, except for cerebellum. Lastly, a preferential correlation was observed between DNA methylation at specific CpG dinucleotides within the REs and Mecp2 isoform-specific expression in these brain regions. Taken together, we show that MeCP2 isoforms display differential expression patterns during brain development and in adult mouse brain regions. DNA methylation patterns at the Mecp2 REs may impact this differential expression of Mecp2/MeCP2 isoforms in brain regions. Our results significantly contribute

  16. DNA methylation profiling of embryonic stem cell differentiation into the three germ layers.

    Science.gov (United States)

    Isagawa, Takayuki; Nagae, Genta; Shiraki, Nobuaki; Fujita, Takanori; Sato, Noriko; Ishikawa, Shumpei; Kume, Shoen; Aburatani, Hiroyuki

    2011-01-01

    Embryogenesis is tightly regulated by multiple levels of epigenetic regulation such as DNA methylation, histone modification, and chromatin remodeling. DNA methylation patterns are erased in primordial germ cells and in the interval immediately following fertilization. Subsequent developmental reprogramming occurs by de novo methylation and demethylation. Variance in DNA methylation patterns between different cell types is not well understood. Here, using methylated DNA immunoprecipitation and tiling array technology, we have comprehensively analyzed DNA methylation patterns at proximal promoter regions in mouse embryonic stem (ES) cells, ES cell-derived early germ layers (ectoderm, endoderm and mesoderm) and four adult tissues (brain, liver, skeletal muscle and sperm). Most of the methylated regions are methylated across all three germ layers and in the three adult somatic tissues. This commonly methylated gene set is enriched in germ cell-associated genes that are generally transcriptionally inactive in somatic cells. We also compared DNA methylation patterns by global mapping of histone H3 lysine 4/27 trimethylation, and found that gain of DNA methylation correlates with loss of histone H3 lysine 4 trimethylation. Our combined findings indicate that differentiation of ES cells into the three germ layers is accompanied by an increased number of commonly methylated DNA regions and that these tissue-specific alterations in methylation occur for only a small number of genes. DNA methylation at the proximal promoter regions of commonly methylated genes thus appears to be an irreversible mark which functions to fix somatic lineage by repressing the transcription of germ cell-specific genes.

  17. Correlation of Alzheimer Disease Neuropathologic Changes With Cognitive Status: A Review of the Literature

    Science.gov (United States)

    Nelson, Peter T.; Alafuzoff, Irina; Bigio, Eileen H.; Bouras, Constantin; Braak, Heiko; Cairns, Nigel J.; Castellani, Rudolph J.; Crain, Barbara J.; Davies, Peter; Del Tredici, Kelly; Duyckaerts, Charles; Frosch, Matthew P.; Haroutunian, Vahram; Hof, Patrick R.; Hulette, Christine M.; Hyman, Bradley T.; Iwatsubo, Takeshi; Jellinger, Kurt A.; Jicha, Gregory A.; Kövari, Enikö; Kukull, Walter A.; Leverenz, James B.; Love, Seth; Mackenzie, Ian R.; Mann, David M.; Masliah, Eliezer; McKee, Ann C.; Montine, Thomas J.; Morris, John C.; Schneider, Julie A.; Sonnen, Joshua A.; Thal, Dietmar R.; Trojanowski, John Q.; Troncoso, Juan C.; Wisniewski, Thomas; Woltjer, Randall L.; Beach, Thomas G.

    2013-01-01

    Clinicopathologic correlation studies are critically important for the field of Alzheimer disease (AD) research. Studies on human subjects with autopsy confirmation entail numerous potential biases that affect both their general applicability and the validity of the correlations. Many sources of data variability can weaken the apparent correlation between cognitive status and AD neuropathologic changes. Indeed, most persons in advanced old age have significant non-AD brain lesions that may alter cognition independently of AD. Worldwide research efforts have evaluated thousands of human subjects to assess the causes of cognitive impairment in the elderly, and these studies have been interpreted in different ways. We review the literature focusing on the correlation of AD neuropathologic changes (i.e. β-amyloid plaques and neurofibrillary tangles) with cognitive impairment. We discuss the various patterns of brain changes that have been observed in elderly individuals to provide a perspective for understanding AD clinicopathologic correlation and conclude that evidence from many independent research centers strongly supports the existence of a specific disease, as defined by the presence of Aβ plaques and neurofibrillary tangles. Although Aβ plaques may play a key role in AD pathogenesis, the severity of cognitive impairment correlates best with the burden of neocortical neurofibrillary tangles. PMID:22487856

  18. Methyl chavicol: characterization of its biogenic emission rate, abundance, and oxidation products in the atmosphere

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    N. C. Bouvier-Brown

    2009-03-01

    Full Text Available We report measurements of ambient atmospheric mixing ratios for methyl chavicol and determine its biogenic emission rate. Methyl chavicol, a biogenic oxygenated aromatic compound, is abundant within and above Blodgett Forest, a ponderosa pine forest in the Sierra Nevada Mountains of California. Methyl chavicol was detected simultaneously by three in-situ instruments – a gas chromatograph with mass spectrometer detector (GC-MS, a proton transfer reaction mass spectrometer (PTR-MS, and a thermal desorption aerosol GC-MS (TAG – and found to be abundant within and above Blodgett Forest. Methyl chavicol atmospheric mixing ratios are strongly correlated with 2-methyl-3-buten-2-ol (MBO, a light- and temperature-dependent biogenic emission from the ponderosa pine trees at Blodgett Forest. Scaling from this correlation, methyl chavicol emissions account for 4–68% of the carbon mass emitted as MBO in the daytime, depending on the season. From this relationship, we estimate a daytime basal emission rate of 0.72–10.2 μgCg−1 h−1, depending on needle age and seasonality. We also present the first observations of its oxidation products (4-methoxybenzaldehyde and 4-methyoxy benzene acetaldehyde in the ambient atmosphere. Methyl chavicol is a major essential oil component of many plant species. This work suggests that methyl chavicol plays a significant role in the atmospheric chemistry of Blodgett Forest, and potentially other sites, and should be included explicitly in both biogenic volatile organic carbon emission and atmospheric chemistry models.

  19. Vitamin D status in Egyptian euthyroid multinodular non-toxic goiter patients and its correlation with TSH levels.

    Science.gov (United States)

    Aboelnaga, Mohamed M; Elshafei, Maha M; Elsayed, Eman

    2016-10-01

    Although the prevalence of MNG is widespread throughout the world, its pathogenesis is poorly understood, and the complex interactions of both genetic predisposition and the individuals' environment are likely. However, to the best of our knowledge, it remains unknown whether there is a relationship between vitamin D status and prevalence or pathogenesis of euthyroid MNG. Therefore, the goal of the present study was determination of vitamin D status in euthyroid MNG as well as exploration of the correlation between vitamin D status & TSH levels. A total of 77 patients diagnosed with euthyroid MNG and 50 subjects without goiter were matched according to age, weight and BMI as control group in this case control study. We found that patients with euthyroid MNG had statistically significant lower mean of [25(OH)D] (24.21±8.68ng/mL) in comparison with its mean in control subjects (28.37±10.91ng/mL, P value=0.019). The 28 sufficient vitamin D MNG patients had statistically significant lower level of TSH than 49 insufficient vitamin D MNG patients. Vitamin D and TSH levels correlate with vitamin D levels in MNG patients in Pearson correlation. Also 25 OH vitamin D was a significant independent predictor for TSH levels among euthyroid MNG patients in regression analysis. Patients with euthyroid MNG have lower levels of vitamin D and TSH levels correlate with vitamin D levels in euthyroid MNG patients. In addition, 25 OH vitamin D was a significant independent predictor for TSH levels among euthyroid MNG patients. We recommend hypovitaminosis D evaluation and correction in patients with MNG. Copyright © 2016 SEEN. Publicado por Elsevier España, S.L.U. All rights reserved.

  20. CpG island methylator phenotype in adenocarcinomas from the digestive tract: Methods, conclusions, and controversies

    Science.gov (United States)

    Sánchez-Vega, Francisco; Gotea, Valer; Chen, Yun-Ching; Elnitski, Laura

    2017-01-01

    Over the last two decades, cancer-related alterations in DNA methylation that regulate transcription have been reported for a variety of tumors of the gastrointestinal tract. Due to its relevance for translational research, great emphasis has been placed on the analysis and molecular characterization of the CpG island methylator phenotype (CIMP), defined as widespread hypermethylation of CpG islands in clinically distinct subsets of cancer patients. Here, we present an overview of previous work in this field and also explore some open questions using cross-platform data for esophageal, gastric, and colorectal adenocarcinomas from The Cancer Genome Atlas. We provide a data-driven, pan-gastrointestinal stratification of individual samples based on CIMP status and we investigate correlations with oncogenic alterations, including somatic mutations and epigenetic silencing of tumor suppressor genes. Besides known events in CIMP such as BRAF V600E mutation, CDKN2A silencing or MLH1 inactivation, we discuss the potential role of emerging actors such as Wnt pathway deregulation through truncating mutations in RNF43 and epigenetic silencing of WIF1. Our results highlight the existence of molecular similarities that are superimposed over a larger backbone of tissue-specific features and can be exploited to reduce heterogeneity of response in clinical trials. PMID:28344746

  1. Methylation of BDNF in women with bulimic eating syndromes: associations with childhood abuse and borderline personality disorder.

    Science.gov (United States)

    Thaler, Lea; Gauvin, Lise; Joober, Ridha; Groleau, Patricia; de Guzman, Rosherrie; Ambalavanan, Amirthagowri; Israel, Mimi; Wilson, Samantha; Steiger, Howard

    2014-10-03

    DNA methylation allows for the environmental regulation of gene expression and is believed to link environmental stressors to such mental-illness phenotypes as eating disorders. Numerous studies have shown an association between bulimia nervosa (BN) and variations in brain-derived neurotrophic factor (BDNF). BDNF has also been linked to borderline personality disorder (BPD) and to such traits as reward dependence. We examined the extent to which BDNF methylation corresponded to bulimic or normal-eater status, and also to the presence of comorbid borderline personality disorder (BPD) and childhood abuse. Our sample consisted of 64 women with BN and 32 normal-eater (NE) control women. Participants were assessed for eating-disorder symptoms, comorbid psychopathology, and childhood trauma, and then they were required to provide blood samples for methylation analyses. We observed a significant site×group (BN vs. NE) interaction indicating that women with BN showed increases in methylation at specific regions of the BDNF promoter. Furthermore, examining effects of childhood abuse and BPD, we observed significant site×group interactions such that groups composed of individuals with childhood abuse or BPD had particularly high levels of methylation at selected CpG sites. Our findings suggest that BN, especially when co-occurring with childhood abuse or BPD, is associated with a propensity towards elevated methylation at specific BDNF promoter region sites. These findings imply that hypermethylation of the BDNF gene may be related to eating disorder status, developmental stress exposure, and comorbid psychopathology. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Transcription and chromatin determinants of de novo DNA methylation timing in oocytes.

    Science.gov (United States)

    Gahurova, Lenka; Tomizawa, Shin-Ichi; Smallwood, Sébastien A; Stewart-Morgan, Kathleen R; Saadeh, Heba; Kim, Jeesun; Andrews, Simon R; Chen, Taiping; Kelsey, Gavin

    2017-01-01

    Gametogenesis in mammals entails profound re-patterning of the epigenome. In the female germline, DNA methylation is acquired late in oogenesis from an essentially unmethylated baseline and is established largely as a consequence of transcription events. Molecular and functional studies have shown that imprinted genes become methylated at different times during oocyte growth; however, little is known about the kinetics of methylation gain genome wide and the reasons for asynchrony in methylation at imprinted loci. Given the predominant role of transcription, we sought to investigate whether transcription timing is rate limiting for de novo methylation and determines the asynchrony of methylation events. Therefore, we generated genome-wide methylation and transcriptome maps of size-selected, growing oocytes to capture the onset and progression of methylation. We find that most sequence elements, including most classes of transposable elements, acquire methylation at similar rates overall. However, methylation of CpG islands (CGIs) is delayed compared with the genome average and there are reproducible differences amongst CGIs in onset of methylation. Although more highly transcribed genes acquire methylation earlier, the major transitions in the oocyte transcriptome occur well before the de novo methylation phase, indicating that transcription is generally not rate limiting in conferring permissiveness to DNA methylation. Instead, CGI methylation timing negatively correlates with enrichment for histone 3 lysine 4 (H3K4) methylation and dependence on the H3K4 demethylases KDM1A and KDM1B, implicating chromatin remodelling as a major determinant of methylation timing. We also identified differential enrichment of transcription factor binding motifs in CGIs acquiring methylation early or late in oocyte growth. By combining these parameters into multiple regression models, we were able to account for about a fifth of the variation in methylation timing of CGIs. Finally

  3. DNA methylation potential: dietary intake and blood concentrations of one-carbon metabolites and cofactors in rural African women123

    Science.gov (United States)

    Dominguez-Salas, Paula; Moore, Sophie E; Cole, Darren; da Costa, Kerry-Ann; Cox, Sharon E; Dyer, Roger A; Fulford, Anthony JC; Innis, Sheila M; Waterland, Robert A; Zeisel, Steven H; Prentice, Andrew M; Hennig, Branwen J

    2013-01-01

    Background: Animal models show that periconceptional supplementation with folic acid, vitamin B-12, choline, and betaine can induce differences in offspring phenotype mediated by epigenetic changes in DNA. In humans, altered DNA methylation patterns have been observed in offspring whose mothers were exposed to famine or who conceived in the Gambian rainy season. Objective: The objective was to understand the seasonality of DNA methylation patterns in rural Gambian women. We studied natural variations in dietary intake of nutrients involved in methyl-donor pathways and their effect on the respective metabolic biomarkers. Design: In 30 women of reproductive age (18–45 y), we monitored diets monthly for 1 y by using 48-h weighed records to measure intakes of choline, betaine, folate, methionine, riboflavin, and vitamins B-6 and B-12. Blood biomarkers of these nutrients, S-adenosylhomocysteine (SAH), S-adenosylmethionine (SAM), homocysteine, cysteine, and dimethylglycine were also assessed monthly. Results: Dietary intakes of riboflavin, folate, choline, and betaine varied significantly by season; the most dramatic variation was seen for betaine. All metabolic biomarkers showed significant seasonality, and vitamin B-6 and folate had the highest fluctuations. Correlations between dietary intakes and blood biomarkers were found for riboflavin, vitamin B-6, active vitamin B-12 (holotranscobalamin), and betaine. We observed a seasonal switch between the betaine and folate pathways and a probable limiting role of riboflavin in these processes and a higher SAM/SAH ratio during the rainy season. Conclusions: Naturally occurring seasonal variations in food-consumption patterns have a profound effect on methyl-donor biomarker status. The direction of these changes was consistent with previously reported differences in methylation of metastable epialleles. This trial was registered at www.clinicaltrials.gov as NCT01811641. PMID:23576045

  4. Ancestry dependent DNA methylation and influence of maternal nutrition.

    Directory of Open Access Journals (Sweden)

    Khyobeni Mozhui

    Full Text Available There is extensive variation in DNA methylation between individuals and ethnic groups. These differences arise from a combination of genetic and non-genetic influences and potential modifiers include nutritional cues, early life experience, and social and physical environments. Here we compare genome-wide DNA methylation in neonatal cord blood from African American (AA; N = 112 and European American (EA; N = 91 participants of the CANDLE Study (Conditions Affecting Neurocognitive Development and Learning in Early Childhood. Our goal is to determine if there are replicable ancestry-specific methylation patterns that may implicate risk factors for diseases that have differential prevalence between populations. To identify the most robust ancestry-specific CpG sites, we replicate our results in lymphoblastoid cell lines from Yoruba African and CEPH European panels of HapMap. We also evaluate the influence of maternal nutrition--specifically, plasma levels of vitamin D and folate during pregnancy--on methylation in newborns. We define stable ancestry-dependent methylation of genes that include tumor suppressors and cell cycle regulators (e.g., APC, BRCA1, MCC. Overall, there is lower global methylation in African ancestral groups. Plasma levels of 25-hydroxy vitamin D are also considerably lower among AA mothers and about 60% of AA and 40% of EA mothers have concentrations below 20 ng/ml. Using a weighted correlation analysis, we define a network of CpG sites that is jointly modulated by ancestry and maternal vitamin D. Our results show that differences in DNA methylation patterns are remarkably stable and maternal micronutrients can exert an influence on the child epigenome.

  5. A Hypermethylated Phenotype Is a Better Predictor of Survival than MGMT Methylation in Anaplastic Oligodendroglial Brain Tumors: A Report from EORTC Study 26951

    NARCIS (Netherlands)

    Bent, M.J. van den; Gravendeel, L.A.; Gorlia, T.; Kros, J.M.; Lapre, L.; Wesseling, P.; Teepen, J.L.; Idbaih, A.; Sanson, M.; Smitt, P.A.; French, P.J.

    2011-01-01

    PURPOSE: The MGMT promoter methylation status has been suggested to be predictive for outcome to temozolomide chemotherapy in patients with glioblastoma (GBM). Subsequent studies indicated that MGMT promoter methylation is a prognostic marker even in patients treated with radiotherapy alone, both in

  6. Biogeochemistry of arsenic in natural waters: The importance of methylated species

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, L.C.D.; Bruland, K.W. (Univ. of California, Santa Cruz (USA))

    1991-03-01

    Water samples from a number of lakes and estuaries, mostly in California, showed measurable concentrations of methylated arsenic (equivalent to 1-59% of total As) with the exception of one highly alkaline lake. Neither depleted phosphate concentrations nor high dissolved salts correlated with the appearance of methylated forms of As. A temporal study of As speciation in Davis Creek Reservoir, a seasonally anoxic lake in northern California, demonstrated that dimethylarsinic acid increased sufficiently to become the dominant form of dissolved As within the surface photic zone during late summer and fall. Methylated forms decreased while arsenate increased when the lake over-turned in early December, which suggested a degradation of dimethylarsinic acid to arsenate.

  7. Correlates of Parental Misperception of Their Child’s Weight Status: The ‘Be Active, Eat Right’ Study

    Science.gov (United States)

    Remmers, Teun; van Grieken, Amy; Renders, Carry M.; Hirasing, Remy A.; Broeren, Suzanne M. L.; Raat, Hein

    2014-01-01

    Objective This study reported on correlates of parental perception of their child’s weight status. Associations between parental misperception (i.e., underestimation of the child’s weight) and parental intention to improve their child’s overweight-related health behaviors and their child meeting guidelines regarding these behaviors were also investigated. Methods Baseline data from the population-based ‘Be active, eat right study’ were used. The population for analysis consisted of 630 overweight and 153 obese five year-old children and their parents. Questionnaires were used to measure parental perception of the child’s weight status, correlates of misperception (i.e., child age, child gender, child BMI, parental age, parental gender, parental country of birth, parental educational level and parental weight status), overweight-related health behaviors (i.e., child playing outside, having breakfast, drinking sweet beverages, and watching TV), and parental intention to improve these behaviors. Height and weight were measured using standardized protocols. Multivariable logistic regression analyses were performed. Results In total, 44.40% of the parents misperceived their child’s weight status. Parental misperception was associated with lower child BMI, the parent being the father, a foreign parental country of birth, and a lower parental education level (pParental misperception was not associated with parental intention to improve child overweight-related health behavior, nor with child meeting the guidelines of these behaviors. Discussion This study showed that almost half of the parents with an overweight or obese child misperceived their child’s weight status. A correct parental perception may be a small stepping-stone in improving the health of overweight and obese children. PMID:24551191

  8. Methylation and Expression of Immune and Inflammatory Genes in the Offspring of Bariatric Bypass Surgery Patients

    Directory of Open Access Journals (Sweden)

    Frédéric Guénard

    2013-01-01

    Full Text Available Background. Maternal obesity, excess weight gain and overnutrition during pregnancy increase risks of obesity, type 2 diabetes mellitus, and cardiovascular disease in the offspring. Maternal biliopancreatic diversion is an effective treatment for severe obesity and is beneficial for offspring born after maternal surgery (AMS. These offspring exhibit lower severe obesity prevalence and improved cardiometabolic risk factors including inflammatory marker compared to siblings born before maternal surgery (BMS. Objective. To assess relationships between maternal bariatric surgery and the methylation/expression of genes involved in the immune and inflammatory pathways. Methods. A differential gene methylation analysis was conducted in a sibling cohort of 25 BMS and 25 AMS offspring from 20 mothers. Following differential gene expression analysis (23 BMS and 23 AMS, pathway analysis was conducted. Correlations between gene methylation/expression and circulating inflammatory markers were computed. Results. Five immune and inflammatory pathways with significant overrepresentation of both differential gene methylation and expression were identified. In the IL-8 pathway, gene methylation correlated with both gene expression and plasma C-reactive protein levels. Conclusion. These results suggest that improvements in cardiometabolic risk markers in AMS compared to BMS offspring may be mediated through differential methylation of genes involved in immune and inflammatory pathways.

  9. DNA methylation analysis reveals distinct methylation signatures in pediatric germ cell tumors

    International Nuclear Information System (INIS)

    Amatruda, James F; Frazier, A Lindsay; Poynter, Jenny N; Ross, Julie A; Christensen, Brock; Fustino, Nicholas J; Chen, Kenneth S; Hooten, Anthony J; Nelson, Heather; Kuriger, Jacquelyn K; Rakheja, Dinesh

    2013-01-01

    Aberrant DNA methylation is a prominent feature of many cancers, and may be especially relevant in germ cell tumors (GCTs) due to the extensive epigenetic reprogramming that occurs in the germ line during normal development. We used the Illumina GoldenGate Cancer Methylation Panel to compare DNA methylation in the three main histologic subtypes of pediatric GCTs (germinoma, teratoma and yolk sac tumor (YST); N = 51) and used recursively partitioned mixture models (RPMM) to test associations between methylation pattern and tumor and demographic characteristics. We identified genes and pathways that were differentially methylated using generalized linear models and Ingenuity Pathway Analysis. We also measured global DNA methylation at LINE1 elements and evaluated methylation at selected imprinted loci using pyrosequencing. Methylation patterns differed by tumor histology, with 18/19 YSTs forming a distinct methylation class. Four pathways showed significant enrichment for YSTs, including a human embryonic stem cell pluripotency pathway. We identified 190 CpG loci with significant methylation differences in mature and immature teratomas (q < 0.05), including a number of CpGs in stem cell and pluripotency-related pathways. Both YST and germinoma showed significantly lower methylation at LINE1 elements compared with normal adjacent tissue while there was no difference between teratoma (mature and immature) and normal tissue. DNA methylation at imprinted loci differed significantly by tumor histology and location. Understanding methylation patterns may identify the developmental stage at which the GCT arose and the at-risk period when environmental exposures could be most harmful. Further, identification of relevant genetic pathways could lead to the development of new targets for therapy

  10. Modeling of the oxidation of methyl esters—Validation for methyl hexanoate, methyl heptanoate, and methyl decanoate in a jet-stirred reactor

    Science.gov (United States)

    Glaude, Pierre Alexandre; Herbinet, Olivier; Bax, Sarah; Biet, Joffrey; Warth, Valérie; Battin-Leclerc, Frédérique

    2013-01-01

    The modeling of the oxidation of methyl esters was investigated and the specific chemistry, which is due to the presence of the ester group in this class of molecules, is described. New reactions and rate parameters were defined and included in the software EXGAS for the automatic generation of kinetic mechanisms. Models generated with EXGAS were successfully validated against data from the literature (oxidation of methyl hexanoate and methyl heptanoate in a jet-stirred reactor) and a new set of experimental results for methyl decanoate. The oxidation of this last species was investigated in a jet-stirred reactor at temperatures from 500 to 1100 K, including the negative temperature coefficient region, under stoichiometric conditions, at a pressure of 1.06 bar and for a residence time of 1.5 s: more than 30 reaction products, including olefins, unsaturated esters, and cyclic ethers, were quantified and successfully simulated. Flow rate analysis showed that reactions pathways for the oxidation of methyl esters in the low-temperature range are similar to that of alkanes. PMID:23710076

  11. Modeling of the oxidation of methyl esters-Validation for methyl hexanoate, methyl heptanoate, and methyl decanoate in a jet-stirred reactor.

    Science.gov (United States)

    Glaude, Pierre Alexandre; Herbinet, Olivier; Bax, Sarah; Biet, Joffrey; Warth, Valérie; Battin-Leclerc, Frédérique

    2010-11-01

    The modeling of the oxidation of methyl esters was investigated and the specific chemistry, which is due to the presence of the ester group in this class of molecules, is described. New reactions and rate parameters were defined and included in the software EXGAS for the automatic generation of kinetic mechanisms. Models generated with EXGAS were successfully validated against data from the literature (oxidation of methyl hexanoate and methyl heptanoate in a jet-stirred reactor) and a new set of experimental results for methyl decanoate. The oxidation of this last species was investigated in a jet-stirred reactor at temperatures from 500 to 1100 K, including the negative temperature coefficient region, under stoichiometric conditions, at a pressure of 1.06 bar and for a residence time of 1.5 s: more than 30 reaction products, including olefins, unsaturated esters, and cyclic ethers, were quantified and successfully simulated. Flow rate analysis showed that reactions pathways for the oxidation of methyl esters in the low-temperature range are similar to that of alkanes.

  12. Region of interest methylation analysis: a comparison of MSP with MS-HRM and direct BSP.

    Science.gov (United States)

    Akika, Reem; Awada, Zainab; Mogharbil, Nahed; Zgheib, Nathalie K

    2017-07-01

    The aim of this study was to compare and contrast three DNA methylation methods of a specific region of interest (ROI): methylation-specific PCR (MSP), methylation-sensitive high resolution melting (MS-HRM) and direct bisulfite sequencing (BSP). The methylation of a CpG area in the promoter region of Estrogen receptor alpha (ESR1) was evaluated by these three methods with samples and standards of different methylation percentages. MSP data were neither reproducible nor sensitive, and the assay was not specific due to non-specific binding of primers. MS-HRM was highly reproducible and a step forward into categorizing the methylation status of the samples as percent ranges. Direct BSP was the most informative method regarding methylation percentage of each CpG site. Though not perfect, it was reproducible and sensitive. We recommend the use of either method depending on the research question and target amplicon, and provided that the designed primers and expected amplicons are within recommendations. If the research question targets a limited number of CpG sites and simple yes/no results are enough, MSP may be attempted. For short amplicons that are crowded with CpG sites and of single melting domain, MS-HRM may be the method of choice though it only indicates the overall methylation percentage of the entire amplicon. Although the assay is highly reproducible, being semi-quantitative makes it of lesser interest to study ROI methylation of samples with little methylation differences. Direct BSP is a step forward as it gives information about the methylation percentage at each CpG site.

  13. Thrombospondin-4 is a putative tumour-suppressor gene in colorectal cancer that exhibits age-related methylation

    International Nuclear Information System (INIS)

    Greco, Sonia A; Leggett, Barbara A; Whitehall, Vicki LJ; Chia, June; Inglis, Kelly J; Cozzi, Sarah-Jane; Ramsnes, Ingunn; Buttenshaw, Ronald L; Spring, Kevin J; Boyle, Glen M; Worthley, Daniel L

    2010-01-01

    Thrombospondin-4 (THBS4) is a member of the extracellular calcium-binding protein family and is involved in cell adhesion and migration. The aim of this study was to evaluate the potential role of deregulation of THBS4 expression in colorectal carcinogenesis. Of particular interest was the possible silencing of expression by methylation of the CpG island in the gene promoter. Fifty-five sporadic colorectal tumours stratified for the CpG Island Methylator Phenotype (CIMP) were studied. Immunohistochemical staining of THBS4 protein was assessed in normal and tumour specimens. Relative levels of THBS4 transcript expression in matched tumours and normal mucosa were also determined by quantitative RT-PCR. Colony forming ability was examined in 8 cell lines made to overexpress THBS4. Aberrant promoter hypermethylation was investigated as a possible mechanism of gene disruption using MethyLight. Methylation was also assessed in the normal colonic tissue of 99 patients, with samples biopsied from four regions along the length of the colon. THBS4 expression was significantly lower in tumour tissue than in matched normal tissue. Immunohistochemical examination demonstrated that THBS4 protein was generally absent from normal epithelial cells and tumours, but was occasionally expressed at low levels in the cytoplasm towards the luminal surface in vesicular structures. Forced THBS4 over-expression caused a 50-60% repression of tumour colony growth in all eight cell lines examined compared to control cell lines. Tumours exhibited significantly higher levels of methylation than matched normal mucosa, and THBS4 methylation correlated with the CpG island methylator phenotype. There was a trend towards decreased gene expression in tumours exhibiting high THBS4 methylation, but the correlation was not significant. THBS4 methylation was detectable in normal mucosal biopsies where it correlated with increasing patient age and negatively with the occurrence of adenomas elsewhere in the

  14. Thrombospondin-4 is a putative tumour-suppressor gene in colorectal cancer that exhibits age-related methylation

    Directory of Open Access Journals (Sweden)

    Greco Sonia A

    2010-09-01

    Full Text Available Abstract Background Thrombospondin-4 (THBS4 is a member of the extracellular calcium-binding protein family and is involved in cell adhesion and migration. The aim of this study was to evaluate the potential role of deregulation of THBS4 expression in colorectal carcinogenesis. Of particular interest was the possible silencing of expression by methylation of the CpG island in the gene promoter. Methods Fifty-five sporadic colorectal tumours stratified for the CpG Island Methylator Phenotype (CIMP were studied. Immunohistochemical staining of THBS4 protein was assessed in normal and tumour specimens. Relative levels of THBS4 transcript expression in matched tumours and normal mucosa were also determined by quantitative RT-PCR. Colony forming ability was examined in 8 cell lines made to overexpress THBS4. Aberrant promoter hypermethylation was investigated as a possible mechanism of gene disruption using MethyLight. Methylation was also assessed in the normal colonic tissue of 99 patients, with samples biopsied from four regions along the length of the colon. Results THBS4 expression was significantly lower in tumour tissue than in matched normal tissue. Immunohistochemical examination demonstrated that THBS4 protein was generally absent from normal epithelial cells and tumours, but was occasionally expressed at low levels in the cytoplasm towards the luminal surface in vesicular structures. Forced THBS4 over-expression caused a 50-60% repression of tumour colony growth in all eight cell lines examined compared to control cell lines. Tumours exhibited significantly higher levels of methylation than matched normal mucosa, and THBS4 methylation correlated with the CpG island methylator phenotype. There was a trend towards decreased gene expression in tumours exhibiting high THBS4 methylation, but the correlation was not significant. THBS4 methylation was detectable in normal mucosal biopsies where it correlated with increasing patient age and

  15. The Clinical Implications of Methylated p15 and p73 Genes in Adult Acute Lymphoblastic Leukemia

    International Nuclear Information System (INIS)

    ABD EL-HAMID, Th.M.; SHERISHER, M.A.; MOSSALLAM, Gh.I.

    2010-01-01

    Aberrant methylation of promoter associated CpG islands is an epigenetic modification of DNA which is associated with gene silencing. It plays an important role in the leukemia pathogenesis. This phenomenon is frequently observed in acute lymphoblastic leukemia (ALL) and results in the functional inactivation of its associated genes. The aim of this study is to investigate the frequency and the prognostic impact of p15 and p73 genes methylation in adult acute lymphoblastic leukemia patients. Patients and Methods: Methylation-specific polymerase chain reaction (PCR) was used to analyze methylation of the p15 and p73 genes in 51 newly diagnosed adult ALL patients. Results: The methylation frequencies of p15 and p73 genes at diagnosis were 41.2% and 27.5% respectively, while concomitant methylation was detected in 14% of the patients. Concomitant methylation of p15 and p73 genes was associated with significant lower rate of CR compared to patients without methylation (57% versus 90%), p=0.008. Overall survival (OS) was not affected by p15 methylation, but was poorer with p73 methylation and the difference was near significant (p=0.059). For patients without meyhylation, the survival benefit was significant when compared to patients with p15, p73 or both genes methylation (p=0.047). The leukemia free survival was not affected by the methylation status of single gene p15 or p73, but tended to be worse in patients with methylated p15, p73 or both genes when compared to patients without methylation (p= 0.08). Conclusion: Aberrant p73 promoter methylation is a potential prognostic factor in adult ALL patients. P15 methylation is frequent in Egyptian adult ALL patients, its concomitant methylation with p73 is of poor prognostic significance. Identification of these molecular targets improve risk assessment and selection of appropriate therapy.

  16. Hepatitis B virus X protein suppresses caveolin-1 expression in hepatocellular carcinoma by regulating DNA methylation

    International Nuclear Information System (INIS)

    Yan, Jun; Lu, Qian; Dong, Jiahong; Li, Xiaowu; Ma, Kuansheng; Cai, Lei

    2012-01-01

    To understand the molecular mechanisms of caveolin-1 downregulation by hepatitis B virus X protein (HBx). The DNA methylation status of the caveolin-1 promoter was examined by nested methylation-specific PCR of 33 hepatitis B virus (HBV)-infected hepatocellular carcinoma (HCC) samples. The SMMC-7721 hepatoma cell line was transfected with a recombinant HBx adenoviral vector, and the effects of HBx protein on caveolin-1 expression and promoter methylation were examined and confirmed by sequencing. A reporter gene containing the caveolin-1 promoter region was constructed, and the effects of HBx on the transcriptional activity of the promoter were also studied. Methylation of the caveolin-1 promoter was detected in 84.8% (28/33) of HBV-infected HCC samples. Expression of caveolin-1 was significantly downregulated (P = 0.022), and multiple CpG sites in the promoter region of caveolin-1 were methylated in SMMC-7721 cells after HBx transfection. Transfected HBx significantly suppressed caveolin-1 promoter activity (P = 0.001). HBx protein induces methylation of the caveolin-1 promoter region and suppresses its expression

  17. Holocaust Exposure Induced Intergenerational Effects on FKBP5 Methylation.

    Science.gov (United States)

    Yehuda, Rachel; Daskalakis, Nikolaos P; Bierer, Linda M; Bader, Heather N; Klengel, Torsten; Holsboer, Florian; Binder, Elisabeth B

    2016-09-01

    The involvement of epigenetic mechanisms in intergenerational transmission of stress effects has been demonstrated in animals but not in humans. Cytosine methylation within the gene encoding for FK506 binding protein 5 (FKBP5) was measured in Holocaust survivors (n = 32), their adult offspring (n = 22), and demographically comparable parent (n = 8) and offspring (n = 9) control subjects, respectively. Cytosine-phosphate-guanine sites for analysis were chosen based on their spatial proximity to the intron 7 glucocorticoid response elements. Holocaust exposure had an effect on FKBP5 methylation that was observed in exposed parents as well in their offspring. These effects were observed at bin 3/site 6. Interestingly, in Holocaust survivors, methylation at this site was higher in comparison with control subjects, whereas in Holocaust offspring, methylation was lower. Methylation levels for exposed parents and their offspring were significantly correlated. In contrast to the findings at bin 3/site 6, offspring methylation at bin 2/sites 3 to 5 was associated with childhood physical and sexual abuse in interaction with an FKBP5 risk allele previously associated with vulnerability to psychological consequences of childhood adversity. The findings suggest the possibility of site specificity to environmental influences, as sites in bins 3 and 2 were differentially associated with parental trauma and the offspring's own childhood trauma, respectively. FKBP5 methylation averaged across the three bins examined was associated with wake-up cortisol levels, indicating functional relevance of the methylation measures. This is the first demonstration of an association of preconception parental trauma with epigenetic alterations that is evident in both exposed parent and offspring, providing potential insight into how severe psychophysiological trauma can have intergenerational effects. Published by Elsevier Inc.

  18. Research on DNA methylation of human osteosarcoma cell MGMT and its relationship with cell resistance to alkylating agents.

    Science.gov (United States)

    Guo, Jun; Cui, Qiu; Jiang, WeiHao; Liu, Cheng; Li, DingFeng; Zeng, Yanjun

    2013-08-01

    The objective of this study was to explore the O(6)-methylguanine-DNA methyltransferase (MGMT) gene methylation status and its protein expression, as well as the effects of demethylating agent 5-Aza-2'-deoxycytidine (5-Aza-CdR) on MGMT gene expression and its resistance to alkylating agents, and to elucidate MGMT expression mechanism and significance in osteosarcoma. The human osteosarcoma cell lines Saos-2 and MG-63 were collected and treated with 5-Aza-CdR for 6 days. The cells not treated with 5-Aza-CdR were set as a negative control. The genomic DNA was extracted from the Saos-2 and MG-63 cells using methylation-specific PCR to detect the promoter CpG island methylation status of the MGMT gene. Cell sensitivity to alkylating agents before and after drug administration was detected by the MTT method. The variation in MGMT gene mRNA and protein was detected by reverse transcription PCR (RT-PCR) and Western blotting. The MGMT promoter gene of normal Saos-2 cells was methylated, with reduced MGMT mRNA and protein expression; the MGMT mRNA and protein expression of Saos-2 cells treated with 5-Aza-CdR was obviously enhanced, and its sensitivity to alkylating agents was reversed. Meanwhile, with promoter CpG island unmethylation of the MGMT gene, MGMT protein was expressed in the normal MG-63 cells and the MG-63 cells treated with 5-Aza-CdR, and both showed resistance to alkylating agents. The methylation status of the MGMT gene promoter in human osteosarcoma cells reflected the cells' ability to induce MGMT protein expression and can be used as a molecular marker to project the sensitivity of cancer tissues to alkylating agent drugs.

  19. Genome-wide DNA methylation sequencing reveals miR-663a is a novel epimutation candidate in CIMP-high endometrial cancer.

    Science.gov (United States)

    Yanokura, Megumi; Banno, Kouji; Adachi, Masataka; Aoki, Daisuke; Abe, Kuniya

    2017-06-01

    Aberrant DNA methylation is widely observed in many cancers. Concurrent DNA methylation of multiple genes occurs in endometrial cancer and is referred to as the CpG island methylator phenotype (CIMP). However, the features and causes of CIMP-positive endometrial cancer are not well understood. To investigate DNA methylation features characteristic to CIMP-positive endometrial cancer, we first classified samples from 25 patients with endometrial cancer based on the methylation status of three genes, i.e. MLH1, CDH1 (E-cadherin) and APC: CIMP-high (CIMP-H, 2/25, 8.0%), CIMP-low (CIMP-L, 7/25, 28.0%) and CIMP-negative (CIMP(-), 16/25, 64.0%). We then selected two samples each from CIMP-H and CIMP(-) classes, and analyzed DNA methylation status of both normal (peripheral blood cells: PBCs) and cancer tissues by genome-wide, targeted bisulfite sequencing. Genomes of the CIMP-H cancer tissues were significantly hypermethylated compared to those of the CIMP(-). Surprisingly, in normal tissues of the CIMP-H patients, promoter region of the miR-663a locus is hypermethylated relative to CIMP(-) samples. Consistent with this finding, miR-663a expression was lower in the CIMP-H PBCs than in the CIMP(-) PBCs. The same region of the miR663a locus is found to be highly methylated in cancer tissues of both CIMP-H and CIMP(-) cases. This is the first report showing that aberrant DNA methylation of the miR-663a promoter can occur in normal tissue of the cancer patients, suggesting a possible link between this epigenetic abnormality and endometrial cancer. This raises the possibility that the hypermethylation of the miR-663a promoter represents an epimutation associated with the CIMP-H endometrial cancers. Based on these findings, relationship of the aberrant DNA methylation and CIMP-H phenotype is discussed.

  20. Prevalence and correlates of vitamin D status in African American men

    Directory of Open Access Journals (Sweden)

    Giri Veda

    2009-06-01

    Full Text Available Abstract Background Few studies have examined vitamin D insufficiency in African American men although they are at very high risk. We examined the prevalence and correlates of vitamin D insufficiency among African American men in Philadelphia. Methods Participants in this cross-sectional analysis were 194 African American men in the Philadelphia region who were enrolled in a risk assessment program for prostate cancer from 10/96–10/07. All participants completed diet and health history questionnaires and provided plasma samples, which were assessed for 25-hydroxyvitamin D (25(OHD concentrations. We used linear regression models to examine associations with 25(OHD concentrations and logistic regression to estimate odds ratios (OR for having 25(OHD ≥ 15 ng/mL. Results Mean 25(OHD was 13.7 ng/mL, and 61% of men were classified as having vitamin D insufficiency (25(OHD 400 vs. 0 IU/day, milk consumption (OR 5.9, 95% CI 2.2, 16.0 for ≥ 3.5 vs. Conclusion The problem of low vitamin D status in African American men may be more severe than previously reported. Future efforts to increase vitamin D recommendations and intake, such as through supplementation, are warranted to improve vitamin D status in this particularly vulnerable population.

  1. A simple biosynthetic method for stereospecific resonance assignment of prochiral methyl groups in proteins

    International Nuclear Information System (INIS)

    Plevin, Michael J.; Hamelin, Olivier; Boisbouvier, Jérôme; Gans, Pierre

    2011-01-01

    A new method for stereospecific assignment of prochiral methyl groups in proteins is presented in which protein samples are produced using U-[ 13 C]glucose and subsaturating amounts of 2-[ 13 C]methyl-acetolactate. The resulting non-uniform labeling pattern allows proR and proS methyl groups to be easily distinguished by their different phases in a constant-time two-dimensional 1 H- 13 C correlation spectra. Protein samples are conveniently prepared using the same media composition as the main uniformly-labeled sample and contain higher levels of isotope-enrichment than fractional labeling approaches. This new strategy thus represents an economically-attractive, robust alternative for obtaining isotopically-encoded stereospecific NMR assignments of prochiral methyl groups.

  2. PRMT1-mediated methylation of the EGF receptor regulates signaling and cetuximab response

    KAUST Repository

    Liao, Hsin-Wei; Hsu, Jung-Mao; Xia, Weiya; Wang, Hung-Ling; Wang, Ying-Nai; Chang, Wei-Chao; Arold, Stefan T.; Chou, Chao-Kai; Tsou, Pei-Hsiang; Yamaguchi, Hirohito; Fang, Yueh-Fu; Lee, Hong-Jen; Lee, Heng-Huan; Tai, Shyh-Kuan; Yang, Mhu-Hwa; Morelli, Maria P.; Sen, Malabika; Ladbury, John E.; Chen, Chung-Hsuan; Grandis, Jennifer R.; Kopetz, Scott; Hung, Mien-Chie

    2015-01-01

    Posttranslational modifications to the intracellular domain of the EGFR are known to regulate EGFR functions; however, modifications to the extracellular domain and their effects remain relatively unexplored. Here, we determined that methylation at R198 and R200 of the EGFR extracellular domain by protein arginine methyltransferase 1 (PRMT1) enhances binding to EGF and subsequent receptor dimerization and signaling activation. In a mouse orthotopic colorectal cancer xenograft model, expression of a methylation-defective EGFR reduced tumor growth. Moreover, increased EGFR methylation sustained signaling activation and cell proliferation in the presence of the therapeutic EGFR monoclonal antibody cetuximab. In colorectal cancer patients, EGFR methylation level also correlated with a higher recurrence rate after cetuximab treatment and reduced overall survival. Together, these data indicate that R198/R200 methylation of the EGFR plays an important role in regulating EGFR functionality and resistance to cetuximab treatment.

  3. PRMT1-mediated methylation of the EGF receptor regulates signaling and cetuximab response

    KAUST Repository

    Liao, Hsin-Wei

    2015-11-16

    Posttranslational modifications to the intracellular domain of the EGFR are known to regulate EGFR functions; however, modifications to the extracellular domain and their effects remain relatively unexplored. Here, we determined that methylation at R198 and R200 of the EGFR extracellular domain by protein arginine methyltransferase 1 (PRMT1) enhances binding to EGF and subsequent receptor dimerization and signaling activation. In a mouse orthotopic colorectal cancer xenograft model, expression of a methylation-defective EGFR reduced tumor growth. Moreover, increased EGFR methylation sustained signaling activation and cell proliferation in the presence of the therapeutic EGFR monoclonal antibody cetuximab. In colorectal cancer patients, EGFR methylation level also correlated with a higher recurrence rate after cetuximab treatment and reduced overall survival. Together, these data indicate that R198/R200 methylation of the EGFR plays an important role in regulating EGFR functionality and resistance to cetuximab treatment.

  4. PRMT1-mediated methylation of the EGF receptor regulates signaling and cetuximab response

    Science.gov (United States)

    Liao, Hsin-Wei; Hsu, Jung-Mao; Xia, Weiya; Wang, Hung-Ling; Wang, Ying-Nai; Chang, Wei-Chao; Arold, Stefan T.; Chou, Chao-Kai; Tsou, Pei-Hsiang; Yamaguchi, Hirohito; Fang, Yueh-Fu; Lee, Hong-Jen; Lee, Heng-Huan; Tai, Shyh-Kuan; Yang, Mhu-Hwa; Morelli, Maria P.; Sen, Malabika; Ladbury, John E.; Chen, Chung-Hsuan; Grandis, Jennifer R.; Kopetz, Scott; Hung, Mien-Chie

    2015-01-01

    Posttranslational modifications to the intracellular domain of the EGFR are known to regulate EGFR functions; however, modifications to the extracellular domain and their effects remain relatively unexplored. Here, we determined that methylation at R198 and R200 of the EGFR extracellular domain by protein arginine methyltransferase 1 (PRMT1) enhances binding to EGF and subsequent receptor dimerization and signaling activation. In a mouse orthotopic colorectal cancer xenograft model, expression of a methylation-defective EGFR reduced tumor growth. Moreover, increased EGFR methylation sustained signaling activation and cell proliferation in the presence of the therapeutic EGFR monoclonal antibody cetuximab. In colorectal cancer patients, EGFR methylation level also correlated with a higher recurrence rate after cetuximab treatment and reduced overall survival. Together, these data indicate that R198/R200 methylation of the EGFR plays an important role in regulating EGFR functionality and resistance to cetuximab treatment. PMID:26571401

  5. Genome-wide DNA Methylation Profiling of Cell-Free Serum DNA in Esophageal Adenocarcinoma and Barrett Esophagus

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    Rihong Zhai

    2012-01-01

    Full Text Available Aberrant DNA methylation (DNAm is a feature of most types of cancers. Genome-wide DNAm profiling has been performed successfully on tumor tissue DNA samples. However, the invasive procedure limits the utility of tumor tissue for epidemiological studies. While recent data indicate that cell-free circulating DNAm (cfDNAm profiles reflect DNAm status in corresponding tumor tissues, no studies have examined the association of cfDNAm with cancer or precursors on a genome-wide scale. The objective of this pilot study was to evaluate the putative significance of genome-wide cfDNAm profiles in esophageal adenocarcinoma (EA and Barrett esophagus (BE, EA precursor. We performed genome-wide DNAm profiling in EA tissue DNA (n = 8 and matched serum DNA (n = 8, in serum DNA of BE (n = 10, and in healthy controls (n = 10 using the Infinium HumanMethylation27 BeadChip that covers 27,578 CpG loci in 14,495 genes. We found that cfDNAm profiles were highly correlated to DNAm profiles in matched tumor tissue DNA (r = 0.92 in patients with EA. We selected the most differentially methylated loci to perform hierarchical clustering analysis. We found that 911 loci can discriminate perfectly between EA and control samples, 554 loci can separate EA from BE samples, and 46 loci can distinguish BE from control samples. These results suggest that genome-wide cfDNAm profiles are highly consistent with DNAm profiles detected in corresponding tumor tissues. Differential cfDNAm profiling may be a useful approach for the noninvasive screening of EA and EA premalignant lesions.

  6. Epigenetic control of viral life-cycle by a DNA-methylation dependent transcription factor.

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    Kirsty Flower

    Full Text Available Epstein-Barr virus (EBV encoded transcription factor Zta (BZLF1, ZEBRA, EB1 is the prototype of a class of transcription factor (including C/EBPalpha that interact with CpG-containing DNA response elements in a methylation-dependent manner. The EBV genome undergoes a biphasic methylation cycle; it is extensively methylated during viral latency but is reset to an unmethylated state following viral lytic replication. Zta is expressed transiently following infection and again during the switch between latency and lytic replication. The requirement for CpG-methylation at critical Zta response elements (ZREs has been proposed to regulate EBV replication, specifically it could aid the activation of viral lytic gene expression from silenced promoters on the methylated genome during latency in addition to preventing full lytic reactivation from the non-methylated EBV genome immediately following infection. We developed a computational approach to predict the location of ZREs which we experimentally assessed using in vitro and in vivo DNA association assays. A remarkably different binding motif is apparent for the CpG and non-CpG ZREs. Computational prediction of the location of these binding motifs in EBV revealed that the majority of lytic cycle genes have at least one and many have multiple copies of methylation-dependent CpG ZREs within their promoters. This suggests that the abundance of Zta protein coupled with the methylation status of the EBV genome act together to co-ordinate the expression of lytic cycle genes at the majority of EBV promoters.

  7. To What Extent Does DNA Methylation Affect Phenotypic Variation in Cattle?

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    Stephanie McKAY

    2015-07-01

    Full Text Available DNA methylation is an environmentally influenced epigenetic modification that regulates gene transcription and has the potential to influence variation in economically important phenotypes in agricultural species. We have utilized a novel approach to evaluate the relationship between genetic and epigenetic variation and downstream phenotypes. To begin with, we have integrated RNA-Seq and methyl binding domain sequencing (MBD-Seq data in order to determine the extent to which DNA methylation affects phenotypic variation in economically important traits of cattle. MBD-Seq is a technique that involves the sample enrichment of methylated genomic regions followed by their next-generation sequencing. This study utilized Illumina next generation sequencing technology to perform both RNA-Seq and MBD-Seq. NextGENe software (SoftGenetics, State College, PA was employed for quality trimming and aligning the sequence reads to the UMD3.1 bovine reference genome, generating counts of matched reads and methylated peak identification. Subsequently, we identified and quantified genome-wide methylated regions and characterized the extent of differential methylation and differential expression between two groups of animals with extreme phenotypes. The program edgeR from the R software package (version 3.0.1 was employed for identifying differentially methylated regions and regions of differential expression. Finally, Partial Correlation with Information Theory (PCIT was performed to identify transcripts and methylation events that exhibit differential hubbing. A differential hub is defined as a gene network hub that is more highly connected in one treatment group than the other. This analysis produced every possible pair-wise interaction that subsequently enabled us to look at network interactions of how methylation affects expression. (co-expression, co-methylation, methylation x expression. Genomic regions of interest derived from this analysis were then aligned

  8. Interindividual concordance of methylation profiles in human genes for tumor necrosis factors α and β

    International Nuclear Information System (INIS)

    Kochanek, S.; Toth, M.; Dehmel, A.; Renz, D.; Doerfler, W.

    1990-01-01

    The DNA in mammalian genomes is characterized by complex patterns of DNA methylation that reflect the states of all genetic activities of that genome. The modified nucleotide 5-methyldeoxycytidine ( 5 mdC) can affect the interactions of specific proteins with DNA sequence motifs. The most extensively studied effect of sequence-specific methylations is that of the long-term silencing of eukaryotic (mammalian) promoters. The authors have initiated studies on the methylation status of parts of the human genome to view patterns of DNA methylation as indicators for genetic activities. In this report, analyses using both restriction enzyme-Southern blotting and the very precise genomic sequencing technique have been done. The results are characterized by a remarkable interindividual concordance of DNA methylation in specific human cell types. The patterns are identical in the DNA from one cell type for different individuals even of different genetic origins but different in the DNA from different cell types. The TNF-β promoter is methylated in granulocytes from 9 different individuals, and TNF-β is not expressed. In human lymphocytes, the main source of TNF-β, the TNF-β promoter is free of 5 mdC residues

  9. Association of diminished expression of RASSF1A with promoter methylation in primary gastric cancer from patients of central China

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    Zhou Feng

    2007-07-01

    Full Text Available Abstract Background Although methylation-mediated inactivation of expression of RASSF1A, a candidate tumor suppressor gene, has been observed in several human cancers, the data concerning alteration of RASSF1A expression and methylation in Chinese primary gastric cancer are scarce. Moreover, direct evidence showing the association between protein expression of RASSF1A and primary human cancers is lacking. The aim of this study was to investigate RASSF1A expression in tissue of primary gastric cancer (GC at mRNA and protein levels, and to establish the possible relationship between DNA methylation status and protein expression of RASSF1A in Chinese. Methods Fifty-four patients with primary gastric cancers were included in the study of RASSF1A mRNA expression and methylation status between the cancer tissue and the corresponding adjacent normal tissue. 20 out of 54 patients were included for study of RASSF1A protein expression. The expression of RASSF1A at mRNA and protein levels was determined by RT-PCR and Western-blotting, respectively. The RASSF1A promoter methylation was detected by methylation-specific PCR. Results RASSF1A mRNA and protein expressions in GC were reduced significantly with comparison to the corresponding normal tissues (OD value: 0.2589 ± 0.2407 vs 0.5448 ± 0.2971, P P P P Conclusion Expression of RASSF1A was reduced in tissue of GC at mRNA and protein levels. Diminished expression of RASSF1A was associated with the promoter methylation.

  10. CORRELATION BETWEEN ANTHROPOMETRY, BIOCHEMICAL MARKERS AND SUBJECTIVE GLOBAL ASSESSMENT – DIALYSIS MALNUTRITION SCORE AS PREDICTORS OF NUTRITIONAL STATUS OF THE MAINTENANCE HEMODIALYSIS PATIENTS

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    Vanitha Rani N, S. Kavimani, Soundararajan P, Chamundeeswari D, Kannan Gopal

    2015-10-01

    Full Text Available Background: Protein energy malnutrition is the major cause of poor prognostic outcome in patients on maintenance hemodialysis (MHD. The assessment of nutritional status in patients on maintenance hemodialysis should be done both subjectively and objectively by integrating clinical, biochemical and anthropometric measurements. A study was conducted to assess the possible correlations between the subjective global assessment-dialysis malnutrition score (SGA-DMS, anthropometric measurements, and biochemical parameters in hemodialysis patients. Methods: The study included 90 patients (55 males and 35 females; age range of 25 to 73 years; mean age 52.62 ± 11.7 years undergoing twice/thrice weekly maintenance hemodialysis for six months and above in the dialysis unit of a tertiary care teaching hospital. The MHD patients were assessed by SGA -DMS, anthropometry and biochemical indicators (serum albumin, iron, ferritin and transferrin of nutritional status. Results: According to the SGA-DMS 54.4 % were moderate to severely malnourished, 31% were mild to moderately nourished and 14.4% were well nourished. There was a highly significant negative correlation between SGA –DMS and serum albumin, iron, transferrin; positive correlation between SGA-DMS and ferritin (P<0.0001. Body mass index, upper arm circumferences, and skin fold thickness had a highly significant negative correlation with SGA-DMS (P<0.001, where as the lean body mass, total body water and the fat free mass had a significant negative correlation (P<0.05. Conclusion: SGA-DMS correlated with anthropometric and biochemical parameters that are indicative of nutritional status. SGA –DMS used in conjunction with other objective nutritional assessment methods may be of greater impact in determining nutritional status of hemodialysis patients.

  11. The Helicase Activity of Hyperthermophilic Archaeal MCM is Enhanced at High Temperatures by Lysine Methylation.

    Science.gov (United States)

    Xia, Yisui; Niu, Yanling; Cui, Jiamin; Fu, Yang; Chen, Xiaojiang S; Lou, Huiqiang; Cao, Qinhong

    2015-01-01

    Lysine methylation and methyltransferases are widespread in the third domain of life, archaea. Nevertheless, the effects of methylation on archaeal proteins wait to be defined. Here, we report that recombinant sisMCM, an archaeal homolog of Mcm2-7 eukaryotic replicative helicase, is methylated by aKMT4 in vitro. Mono-methylation of these lysine residues occurs coincidently in the endogenous sisMCM protein purified from the hyperthermophilic Sulfolobus islandicus cells as indicated by mass spectra. The helicase activity of mini-chromosome maintenance (MCM) is stimulated by methylation, particularly at temperatures over 70°C. The methylated MCM shows optimal DNA unwinding activity after heat-treatment between 76 and 82°C, which correlates well with the typical growth temperatures of hyperthermophilic Sulfolobus. After methylation, the half life of MCM helicase is dramatically extended at 80°C. The methylated sites are located on the accessible protein surface, which might modulate the intra- and inter- molecular interactions through changing the hydrophobicity and surface charge. Furthermore, the methylation-mimic mutants of MCM show heat resistance helicase activity comparable to the methylated MCM. These data provide the biochemical evidence that posttranslational modifications such as methylation may enhance kinetic stability of proteins under the elevated growth temperatures of hyperthermophilic archaea.

  12. Divergent methylation pattern in adult stage between two forms of Tetranychus urticae (Acari: Tetranychidae).

    Science.gov (United States)

    Yang, Si-Xia; Guo, Chao; Zhao, Xiu-Ting; Sun, Jing-Tao; Hong, Xiao-Yue

    2017-02-19

    The two-spotted spider mite, Tetranychus urticae Koch has two forms: green form and red form. Understanding the molecular basis of how these two forms established without divergent genetic background is an intriguing area. As a well-known epigenetic process, DNA methylation has particularly important roles in gene regulation and developmental variation across diverse organisms that do not alter genetic background. Here, to investigate whether DNA methylation could be associated with different phenotypic consequences in the two forms of T. urticae, we surveyed the genome-wide cytosine methylation status and expression level of DNA methyltransferase 3 (Tudnmt3) throughout their entire life cycle. Methylation-sensitive amplification polymorphism (MSAP) analyses of 585 loci revealed variable methylation patterns in the different developmental stages. In particular, principal coordinates analysis (PCoA) indicates a significant epigenetic differentiation between female adults of the two forms. The gene expression of Tudnmt3 was detected in all examined developmental stages, which was significantly different in the adult stage of the two forms. Together, our results reveal the epigenetic distance between the two forms of T. urticae, suggesting that DNA methylation might be implicated in different developmental demands, and contribute to different phenotypes in the adult stage of these two forms. © 2017 Institute of Zoology, Chinese Academy of Sciences.

  13. Transmission of epi-alleles with MET1-dependent dense methylation in Arabidopsis thaliana.

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    Michael Watson

    Full Text Available DNA methylation in plants targets cytosines in three sequence contexts, CG, CHG and CHH (H representing A, C or T. Each of these patterns has traditionally been associated with distinct DNA methylation pathways with CHH methylation being controlled by the RNA dependent DNA methylation (RdDM pathway employing small RNAs as a guide for the de novo DOMAINS REARRANGED METHYLTRANSFERASE (DRM2, and maintenance DNA METHYLTRANSFERASE1 (MET1 being responsible for faithful propagation of CG methylation. Here we report an unusual 'dense methylation' pattern under the control of MET1, with methylation in all three sequence contexts. We identified epi-alleles of dense methylation at a non coding RNA locus (At4g15242 in Arabidopsis ecotypes, with distinct dense methylation and expression characteristics, which are stably maintained and transmitted in genetic crosses and which can be heritably altered by depletion of MET1. This suggests that, in addition to its classical CG maintenance function, at certain loci MET1 plays a role in creating transcriptional diversity based on the generation of independent epi-alleles. Database inspection identified several other loci with MET1-dependent dense methylation patterns. Arabidopsis ecotypes contain distinct epi-alleles of these loci with expression patterns that inversely correlate with methylation density, predominantly within the transcribed region. In Arabidopsis, dense methylation appears to be an exception as it is only found at a small number of loci. Its presence does, however, highlight the potential for MET1 as a contributor to epigenetic diversity, and it will be interesting to investigate the representation of dense methylation in other plant species.

  14. Evidence that steroid 5alpha-reductase isozyme genes are differentially methylated in human lymphocytes.

    Science.gov (United States)

    Rodríguez-Dorantes, M; Lizano-Soberón, M; Camacho-Arroyo, I; Calzada-León, R; Morimoto, S; Téllez-Ascencio, N; Cerbón, M A

    2002-03-01

    The synthesis of dihydrotestosterone (DHT) is catalyzed by steroid 5alpha-reductase isozymes 1 and 2, and this function determines the development of the male phenotype during embriogenesis and the growth of androgen sensitive tissues during puberty. The aim of this study was to determine the cytosine methylation status of 5alpha-reductase isozymes types 1 and 2 genes in normal and in 5alpha-reductase deficient men. Genomic DNA was obtained from lymphocytes of both normal subjects and patients with primary 5alpha-reductase deficiency due to point mutations in 5alpha-reductase 2 gene. Southern blot analysis of 5alpha-reductase types 1 and 2 genes from DNA samples digested with HpaII presented a different cytosine methylation pattern compared to that observed with its isoschizomer MspI, indicating that both genes are methylated in CCGG sequences. The analysis of 5alpha-reductase 1 gene from DNA samples digested with Sau3AI and its isoschizomer MboI which recognize methylation in GATC sequences showed an identical methylation pattern. In contrast, 5alpha-reductase 2 gene digested with Sau3AI presented a different methylation pattern to that of the samples digested with MboI, indicating that steroid 5alpha-reductase 2 gene possess methylated cytosines in GATC sequences. Analysis of exon 4 of 5alpha-reductase 2 gene after metabisulfite PCR showed that normal and deficient subjects present a different methylation pattern, being more methylated in patients with 5alpha-reductase 2 mutated gene. The overall results suggest that 5alpha-reductase genes 1 and 2 are differentially methylated in lymphocytes from normal and 5alpha-reductase deficient patients. Moreover, the extensive cytosine methylation pattern observed in exon 4 of 5alpha-reductase 2 gene in deficient patients, points out to an increased rate of mutations in this gene.

  15. Investigation of HOXA9 promoter methylation as a biomarker to distinguish oral cancer patients at low risk of neck metastasis

    International Nuclear Information System (INIS)

    Uchida, Kenichiro; Veeramachaneni, Ratna; Huey, Bing; Bhattacharya, Aditi; Schmidt, Brian L; Albertson, Donna G

    2014-01-01

    Metastasis to the cervical (neck) lymph nodes is one of the most significant clinical factors responsible for death from oral squamous cell carcinoma (SCC). Therefore, the lymph nodes are frequently removed when the tumor is excised (neck dissection), even though the majority of patients will not benefit from the extra surgery. Two subtypes of oral SCC distinguished by the presence of tumor genomic aberrations +3q, -8p, +8q and/or +20 differ in risk for metastasis – high for the 3q8pq20 subtype, harboring one or more of the aberrations and low for the non-3q8pq20 subtype, lacking these alterations. A prior analysis of the literature suggested genes differentially methylated in the two subtypes. Therefore, the goal of this study was to further investigate the methylation status of candidate biomarkers of the non-3q8pq20 subtype, and evaluate their utility for identifying patients at low risk for metastasis. Methylation status of genes in a cohort of 52 oral SCC patients with at least five year follow up was determined by pyrosequencing. Gene expression levels were determined by quantitative RT-PCR. Growth following re-expression of HOXA9 in cultured oral SCC cells was assessed by proliferation and colony formation assays. A pilot study evaluating methylation levels of HOXA9, MT1A and HOXA11 promoters in DNA from 12 tumors (six each of the 3q8pq20 and non-3q8pq20 subtypes) revealed that only HOXA9 was differentially methylated. Significant differences in methylation levels of HOXA9 were observed amongst the 52 oral SCCs with respect to genomic subtype and nodal status (p = 0.014, and p = 0.024, respectively, Wilcoxon rank sum test). High levels of HOXA9 methylation and low levels of expression in oral SCC cell lines were observed compared to HaCaT, a non-tumorigenic keratinocyte cell line. Re-expression of HOXA9 in the SCC4 oral cancer cell line resulted in diminished proliferation and colony formation. HOXA9 methylation is frequent in oral cancers and levels are

  16. DNA methylation and transcriptional trajectories during human development and reprogramming of isogenic pluripotent stem cells

    NARCIS (Netherlands)

    Roost, Matthias S; Slieker, Roderick C; Bialecka, Monika; van Iperen, Liesbeth; Gomes Fernandes, Maria M; He, Nannan; Suchiman, H Eka D; Szuhai, Karoly; Carlotti, Françoise; de Koning, Eelco J P; Mummery, Christine L; Heijmans, Bastiaan T; Chuva de Sousa Lopes, Susana M

    2017-01-01

    Determining cell identity and maturation status of differentiated pluripotent stem cells (PSCs) requires knowledge of the transcriptional and epigenetic trajectory of organs during development. Here, we generate a transcriptional and DNA methylation atlas covering 21 organs during human fetal

  17. The influence of DNA degradation in formalin-fixed, paraffin-embedded (FFPE) tissue on locus-specific methylation assessment by MS-HRM.

    Science.gov (United States)

    Daugaard, Iben; Kjeldsen, Tina E; Hager, Henrik; Hansen, Lise Lotte; Wojdacz, Tomasz K

    2015-12-01

    Readily accessible formalin-fixed paraffin embedded (FFPE) tissues are a highly valuable source of genetic material for molecular analyses in both research and in vitro diagnostics but frequently genetic material in those samples is highly degraded. With locus-specific methylation changes being widely investigated for use as biomarkers in various aspects of clinical disease management, we aimed to evaluate to what extent standard laboratory procedures can approximate the quality of the DNA extracted from FFPE samples prior to methylation analyses. DNA quality in 107 FFPE non-small cell lung cancer (NSCLC) samples was evaluated using spectrophotometry and gel electrophoresis. Subsequently, the quality assessment results were correlated with the results of locus specific methylation assessment with methylation sensitive high resolution melting (MS-HRM). The correlation of template quality with PCR amplification performance and HRM based methylation detection indicated a significant influence of DNA quality on PCR amplification but not on methylation assessment. In conclusion, standard laboratory procedures fairly well approximate DNA degradation of FFPE samples and DNA degradation does not seem to considerably affect locus-specific methylation assessment by MS-HRM. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Automated sequence- and stereo-specific assignment of methyl-labeled proteins by paramagnetic relaxation and methyl-methyl nuclear overhauser enhancement spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Venditti, Vincenzo; Fawzi, Nicolas L.; Clore, G. Marius, E-mail: mariusc@mail.nih.gov [National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Laboratory of Chemical Physics (United States)

    2011-11-15

    Methyl-transverse relaxation optimized spectroscopy is rapidly becoming the preferred NMR technique for probing structure and dynamics of very large proteins up to {approx}1 MDa in molecular size. Data interpretation, however, necessitates assignment of methyl groups which still presents a very challenging and time-consuming process. Here we demonstrate that, in combination with a known 3D structure, paramagnetic relaxation enhancement (PRE), induced by nitroxide spin-labels incorporated at only a few surface-exposed engineered cysteines, provides fast, straightforward and robust access to methyl group resonance assignments, including stereoassignments for the methyl groups of leucine and valine. Neither prior assignments, including backbone assignments, for the protein, nor experiments that transfer magnetization between methyl groups and the protein backbone, are required. PRE-derived assignments are refined by 4D methyl-methyl nuclear Overhauser enhancement data, eliminating ambiguities and errors that may arise due to the high sensitivity of PREs to the potential presence of sparsely-populated transient states.

  19. Methyl CpG–binding proteins induce large-scale chromatin reorganization during terminal differentiation

    Science.gov (United States)

    Brero, Alessandro; Easwaran, Hariharan P.; Nowak, Danny; Grunewald, Ingrid; Cremer, Thomas; Leonhardt, Heinrich; Cardoso, M. Cristina

    2005-01-01

    Pericentric heterochromatin plays an important role in epigenetic gene regulation. We show that pericentric heterochromatin aggregates during myogenic differentiation. This clustering leads to the formation of large chromocenters and correlates with increased levels of the methyl CpG–binding protein MeCP2 and pericentric DNA methylation. Ectopic expression of fluorescently tagged MeCP2 mimicked this effect, causing a dose-dependent clustering of chromocenters in the absence of differentiation. MeCP2-induced rearrangement of heterochromatin occurred throughout interphase, did not depend on the H3K9 histone methylation pathway, and required the methyl CpG–binding domain (MBD) only. Similar to MeCP2, another methyl CpG–binding protein, MBD2, also increased during myogenic differentiation and could induce clustering of pericentric regions, arguing for functional redundancy. This MeCP2- and MBD2-mediated chromatin reorganization may thus represent a molecular link between nuclear genome topology and the epigenetic maintenance of cellular differentiation. PMID:15939760

  20. Genome-Wide Expression of MicroRNAs Is Regulated by DNA Methylation in Hepatocarcinogenesis

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    Jing Shen

    2015-01-01

    Full Text Available Background. Previous studies, including ours, have examined the regulation of microRNAs (miRNAs by DNA methylation, but whether this regulation occurs at a genome-wide level in hepatocellular carcinoma (HCC is unclear. Subjects/Methods. Using a two-phase study design, we conducted genome-wide screening for DNA methylation and miRNA expression to explore the potential role of methylation alterations in miRNAs regulation. Results. We found that expressions of 25 miRNAs were statistically significantly different between tumor and nontumor tissues and perfectly differentiated HCC tumor from nontumor. Six miRNAs were overexpressed, and 19 were repressed in tumors. Among 133 miRNAs with inverse correlations between methylation and expression, 8 miRNAs (6% showed statistically significant differences in expression between tumor and nontumor tissues. Six miRNAs were validated in 56 additional paired HCC tissues, and significant inverse correlations were observed for miR-125b and miR-199a, which is consistent with the inactive chromatin pattern found in HepG2 cells. Conclusion. These data suggest that the expressions of miR-125b and miR-199a are dramatically regulated by DNA hypermethylation that plays a key role in hepatocarcinogenesis.

  1. [Analysis of tissue-specific differentially methylated genes with differential gene expression in non-small cell lung cancer].

    Science.gov (United States)

    Yin, L G; Zou, Z Q; Zhao, H Y; Zhang, C L; Shen, J G; Qi, L; Qi, M; Xue, Z Q

    2014-01-01

    Adenocarcinoma (ADC) and squamous cell carcinomas (SCC) are two subtypes of non-small cell lung carcinomas which are regarded as the leading cause of cancer-related malignancy worldwide. The aim of this study is to detect the differentially methylated loci (DMLs) and differentially methylated genes (DMGs) of these two tumor sets, and then to illustrate the different expression level of specific methylated genes. Using TCGA database and Illumina HumanMethylation 27 arrays, we first screened the DMGs and DMLs in tumor samples. Then, we explored the BiologicalProcess terms of hypermethylated and hypomethylated genes using Functional Gene Ontology (GO) catalogues. Hypermethylation intensively occurred in CpG-island, whereas hypomethylation was located in non-CpG-island. Most SCC and ADC hypermethylated genes involved GO function of DNA dependenit regulation of transcription, and hypomethylated genes mainly 'enriched in the term of immune responses. Additionally, the expression level of specific differentially methylated genesis distinctbetween ADC and SCC. It is concluded that ADC and SCC have different methylated status that might play an important role in carcinogenesis.

  2. Telomerase activity, telomere length and hTERT DNA methylation in peripheral blood mononuclear cells from monozygotic twins with discordant smoking habits.

    Science.gov (United States)

    Marcon, Francesca; Siniscalchi, Ester; Andreoli, Cristina; Allione, Alessandra; Fiorito, Giovanni; Medda, Emanuela; Guarrera, Simonetta; Matullo, Giuseppe; Crebelli, Riccardo

    2017-10-01

    Increased telomerase expression has been implicated in the pathogenesis of lung cancer and, since the primary cause of lung cancer is smoking, an association between telomerase reactivation and tobacco smoke has been proposed. In this work an investigation has been performed to assess the relationship between tobacco smoke exposure and telomerase activity (TA) in peripheral blood mononuclear cells of healthy smokers. The methylation status of the catalytic subunit of telomerase hTERT was concurrently investigated to assess the possible association between epigenetic modifications of hTERT and TA. Besides, the association between smoke and telomere length (TL) has been evaluated. Healthy monozygotic twins with discordant smoking habits were selected as study population to minimize inter-individual differences because of demographic characteristics and genetic heterogeneity. Statistically significant higher values of TA and TL were observed in smokers compared to nonsmoker co-twins. The multivariate analysis of data showed, besides smoking habits (P = 0.02), an influence of gender (P = 0.006) and BMI (P = 0.001) on TA and a borderline effect of gender (P = 0.05) on TL. DNA methylation analysis, focused on 100 CpG sites mapping in hTERT, highlighted nine CpG sites differentially methylated in smokers. When co-twins were contrasted, selecting as variables the intra-twin difference in TA and hTERT DNA methylation, a statistically significant inverse correlation (P = 0.003) was observed between TA and DNA methylation at the cg05521538 site. In conclusion, these results indicate an association of tobacco smoke with TA and TL and suggest a possible association between smoke-induced epigenetic effects and TA in healthy smokers. Environ. Mol. Mutagen. 58:551-559, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  3. Methylation diet and methyl group genetics in risk for adenomatous polyp occurrence

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    Mark Lucock

    2015-06-01

    Conclusion: A methylation diet influences methyl group synthesis in the regulation of blood homocysteine level, and is modulated by genetic interactions. Methylation-related nutrients also interact with key genes to modify risk of AP, a precursor of colorectal cancer. Independent of diet, two methylation-related genes (A2756G-MS and A66G-MSR were directly associated with AP occurrence.

  4. Reactions of guanine with methyl chloride and methyl bromide: O6-methylation versus charge transfer complex formation

    Science.gov (United States)

    Shukla, P. K.; Mishra, P. C.; Suhai, S.

    Density functional theory (DFT) at the B3LYP/6-31+G* and B3LYP/AUG-cc-pVDZ levels was employed to study O6-methylation of guanine due to its reactions with methyl chloride and methyl bromide and to obtain explanation as to why the methyl halides cause genotoxicity and possess mutagenic and carcinogenic properties. Geometries of the various isolated species involved in the reactions, reactant complexes (RCs), and product complexes (PCs) were optimized in gas phase. Transition states connecting the reactant complexes with the product complexes were also optimized in gas phase at the same levels of theory. The reactant complexes, product complexes, and transition states were solvated in aqueous media using the polarizable continuum model (PCM) of the self-consistent reaction field theory. Zero-point energy (ZPE) correction to total energy and the corresponding thermal energy correction to enthalpy were made in each case. The reactant complexes of the keto form of guanine with methyl chloride and methyl bromide in water are appreciably more stable than the corresponding complexes involving the enol form of guanine. The nature of binding in the product complexes was found to be of the charge transfer type (O6mG+ · X-, X dbond Cl, Br). Binding of HCl, HBr, and H2O molecules to the PCs obtained with the keto form of guanine did not alter the positions of the halide anions in the PCs, and the charge transfer character of the PCs was also not modified due to this binding. Further, the complexes obtained due to the binding of HCl, HBr, and H2O molecules to the PCs had greater stability than the isolated PCs. The reaction barriers involved in the formation of PCs were found to be quite high (?50 kcal/mol). Mechanisms of genotoxicity, mutagenesis and carcinogenesis caused by the methyl halides appear to involve charge transfer-type complex formation. Thus the mechanisms of these processes involving the methyl halides appear to be quite different from those that involve the

  5. Trans-methylation reactions in plants: focus on the activated methyl cycle.

    Science.gov (United States)

    Rahikainen, Moona; Alegre, Sara; Trotta, Andrea; Pascual, Jesús; Kangasjärvi, Saijaliisa

    2018-02-01

    Trans-methylation reactions are vital in basic metabolism, epigenetic regulation, RNA metabolism, and posttranslational control of protein function and therefore fundamental in determining the physiological processes in all living organisms. The plant kingdom is additionally characterized by the production of secondary metabolites that undergo specific hydroxylation, oxidation and methylation reactions to obtain a wide array of different chemical structures. Increasing research efforts have started to reveal the enzymatic pathways underlying the biosynthesis of complex metabolites in plants. Further engineering of these enzymatic machineries offers significant possibilities in the development of bio-based technologies, but necessitates deep understanding of their potential metabolic and regulatory interactions. Trans-methylation reactions are tightly coupled with the so-called activated methyl cycle (AMC), an essential metabolic circuit that maintains the trans-methylation capacity in all living cells. Tight regulation of the AMC is crucial in ensuring accurate trans-methylation reactions in different subcellular compartments, cell types, developmental stages and environmental conditions. This review addresses the organization and posttranslational regulation of the AMC and elaborates its critical role in determining metabolic regulation through modulation of methyl utilization in stress-exposed plants. © 2017 Scandinavian Plant Physiology Society.

  6. NDN and CD1A are novel prognostic methylation markers in patients with head and neck squamous carcinomas

    International Nuclear Information System (INIS)

    Virani, Shama; Bellile, Emily; Bradford, Carol R.; Carey, Thomas E.; Chepeha, Douglas B.; Colacino, Justin A.; Helman, Joseph I.; McHugh, Jonathan B.; Peterson, Lisa A.; Sartor, Maureen A.; Taylor, Jeremy MG; Walline, Heather M.; Wolf, Greg T.; Rozek, Laura S.

    2015-01-01

    HPV-associated HNSCCs have a distinct etiologic mechanism and better prognosis than those with non-HPV associated HNSCCs. However, even within the each group, there is heterogeneity in survival time. Here, we test the hypothesis that specific candidate gene methylation markers (CCNA1, NDN, CD1A, DCC, p16, GADD45A) are associated with tumor recurrence and survival, in a well-characterized, prospective, cohort of 346 HNSCC patients. Kaplan-Meier curves were used to estimate survival time distributions. Multivariable Cox Proportional Hazards models were used to test associations between each methylation marker and OST/RPFT after adjusting for known or identified prognostic factors. Stratified Cox models included an interaction term between HPV and methylation marker to test for differences in the associations of the biomarker with OST or RPFT across HPV status. Methylation markers were differentially associated with patient characteristics. DNA hypermethylation of NDN and CD1A was found to be significantly associated with overall survival time (OST) in all HNSCC patients (NDN hazard ratio (HR): 2.35, 95 % CI: 1.40-3.94; CD1A HR: 1.31, 95 % CI: 1.01-1.71). Stratification by HPV status revealed hypermethylation of CD1A was associated with better OST and recurrence/persistence-free time (RPFT) (OST HR: 3.34, 95 % CI: 1.88-5.93; RPFT HR: 2.06, 95 % CI: 1.21-3.49), while hypomethylation of CCNA1 was associated with increased RPFT in HPV (+) patients only (HR: 0.31, 95 % CI: 0.13-0.74). This study is the first to describe novel epigenetic alterations associated with survival in an unselected, prospectively collected, consecutive cohort of patients with HNSCC. DNA hypermethylation of NDN and CD1A was found to be significantly associated with increased overall survival time in all HNSCC patients. However, stratification by the important prognostic factor of HPV status revealed the immune marker, CD1A, and the cell cycle regulator, CCNA1 to be associated with prognosis in HPV

  7. 21 CFR 177.2000 - Vinylidene chloride/methyl acrylate/methyl methacrylate polymers.

    Science.gov (United States)

    2010-04-01

    ... methacrylate polymers. 177.2000 Section 177.2000 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF...: POLYMERS Substances for Use as Basic Components of Single and Repeated Use Food Contact Surfaces § 177.2000 Vinylidene chloride/methyl acrylate/methyl methacrylate polymers. The vinylidene chloride/methyl acrylate...

  8. Integration of CpG-free DNA induces de novo methylation of CpG islands in pluripotent stem cells

    KAUST Repository

    Takahashi, Yuta

    2017-05-05

    CpG islands (CGIs) are primarily promoter-associated genomic regions and are mostly unmethylated within highly methylated mammalian genomes. The mechanisms by which CGIs are protected from de novo methylation remain elusive. Here we show that insertion of CpG-free DNA into targeted CGIs induces de novo methylation of the entire CGI in human pluripotent stem cells (PSCs). The methylation status is stably maintained even after CpG-free DNA removal, extensive passaging, and differentiation. By targeting the DNA mismatch repair gene MLH1 CGI, we could generate a PSC model of a cancer-related epimutation. Furthermore, we successfully corrected aberrant imprinting in induced PSCs derived from an Angelman syndrome patient. Our results provide insights into how CpG-free DNA induces de novo CGI methylation and broaden the application of targeted epigenome editing for a better understanding of human development and disease.

  9. Why do results conflict regarding the prognostic value of the methylation status in colon cancers? the role of the preservation method

    Directory of Open Access Journals (Sweden)

    Tournier Benjamin

    2012-01-01

    Full Text Available Abstract Background In colorectal carcinoma, extensive gene promoter hypermethylation is called the CpG island methylator phenotype (CIMP. Explaining why studies on CIMP and survival yield conflicting results is essential. Most experiments to measure DNA methylation rely on the sodium bisulfite conversion of unmethylated cytosines into uracils. No study has evaluated the performance of bisulfite conversion and methylation levels from matched cryo-preserved and Formalin-Fixed Paraffin Embedded (FFPE samples using pyrosequencing. Methods Couples of matched cryo-preserved and FFPE samples from 40 colon adenocarcinomas were analyzed. Rates of bisulfite conversion and levels of methylation of LINE-1, MLH1 and MGMT markers were measured. Results For the reproducibility of bisulfite conversion, the mean of bisulfite-to-bisulfite standard deviation (SD was 1.3%. The mean of run-to-run SD of PCR/pyrosequencing was 0.9%. Of the 40 DNA couples, only 67.5%, 55.0%, and 57.5% of FFPE DNA were interpretable for LINE-1, MLH1, and MGMT markers, respectively, after the first analysis. On frozen samples the proportion of well converted samples was 95.0%, 97.4% and 87.2% respectively. For DNA showing a total bisulfite conversion, 8 couples (27.6% for LINE-1, 4 couples (15.4% for MLH1 and 8 couples (25.8% for MGMT displayed significant differences in methylation levels. Conclusions Frozen samples gave reproducible results for bisulfite conversion and reliable methylation levels. FFPE samples gave unsatisfactory and non reproducible bisulfite conversions leading to random results for methylation levels. The use of FFPE collections to assess DNA methylation by bisulfite methods must not be recommended. This can partly explain the conflicting results on the prognosis of CIMP colon cancers.

  10. Genome-wide screening identifies Plasmodium chabaudi-induced modifications of DNA methylation status of Tlr1 and Tlr6 gene promoters in liver, but not spleen, of female C57BL/6 mice.

    Science.gov (United States)

    Al-Quraishy, Saleh; Dkhil, Mohamed A; Abdel-Baki, Abdel Azeem S; Delic, Denis; Santourlidis, Simeon; Wunderlich, Frank

    2013-11-01

    Epigenetic reprogramming of host genes via DNA methylation is increasingly recognized as critical for the outcome of diverse infectious diseases, but information for malaria is not yet available. Here, we investigate the effect of blood-stage malaria of Plasmodium chabaudi on the DNA methylation status of host gene promoters on a genome-wide scale using methylated DNA immunoprecipitation and Nimblegen microarrays containing 2,000 bp oligonucleotide features that were split into -1,500 to -500 bp Ups promoters and -500 to +500 bp Cor promoters, relative to the transcription site, for evaluation of differential DNA methylation. Gene expression was analyzed by Agilent and Affymetrix microarray technology. Challenging of female C57BL/6 mice with 10(6) P. chabaudi-infected erythrocytes resulted in a self-healing outcome of infections with peak parasitemia on day 8 p.i. These infections induced organ-specific modifications of DNA methylation of gene promoters. Among the 17,354 features on Nimblegen arrays, only seven gene promoters were identified to be hypermethylated in the spleen, whereas the liver exhibited 109 hyper- and 67 hypomethylated promoters at peak parasitemia in comparison with non-infected mice. Among the identified genes with differentially methylated Cor-promoters, only the 7 genes Pigr, Ncf1, Klkb1, Emr1, Ndufb11, and Tlr6 in the liver and Apol6 in the spleen were detected to have significantly changed their expression. Remarkably, the Cor promoter of the toll-like receptor Tlr6 became hypomethylated and Tlr6 expression increased by 3.4-fold during infection. Concomitantly, the Ups promoter of the Tlr1 was hypermethylated, but Tlr1 expression also increased by 11.3-fold. TLR6 and TLR1 are known as auxillary receptors to form heterodimers with TLR2 in plasma membranes of macrophages, which recognize different pathogen-associated molecular patterns (PAMPs), as, e.g., intact 3-acyl and sn-2-lyso-acyl glycosylphosphatidylinositols of P. falciparum

  11. Whole-genome methylation caller designed for methyl- DNA ...

    African Journals Online (AJOL)

    etchie

    2013-02-20

    Feb 20, 2013 ... Key words: Methyl-DNA immunoprecipitation, next-generation sequencing, Hidden ... its response to environmental cues. .... have a great potential to become the most cost-effective ... hg18 reference genome (set to 0 if not present in retrieved reads). ..... DNA methylation patterns and epigenetic memory.

  12. Genome-wide methylation analysis identifies genes silenced in non-seminoma cell lines.

    Science.gov (United States)

    Noor, Dzul Azri Mohamed; Jeyapalan, Jennie N; Alhazmi, Safiah; Carr, Matthew; Squibb, Benjamin; Wallace, Claire; Tan, Christopher; Cusack, Martin; Hughes, Jaime; Reader, Tom; Shipley, Janet; Sheer, Denise; Scotting, Paul J

    2016-01-01

    Silencing of genes by DNA methylation is a common phenomenon in many types of cancer. However, the genome-wide effect of DNA methylation on gene expression has been analysed in relatively few cancers. Germ cell tumours (GCTs) are a complex group of malignancies. They are unique in developing from a pluripotent progenitor cell. Previous analyses have suggested that non-seminomas exhibit much higher levels of DNA methylation than seminomas. The genomic targets that are methylated, the extent to which this results in gene silencing and the identity of the silenced genes most likely to play a role in the tumours' biology have not yet been established. In this study, genome-wide methylation and expression analysis of GCT cell lines was combined with gene expression data from primary tumours to address this question. Genome methylation was analysed using the Illumina infinium HumanMethylome450 bead chip system and gene expression was analysed using Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays. Regulation by methylation was confirmed by demethylation using 5-aza-2-deoxycytidine and reverse transcription-quantitative PCR. Large differences in the level of methylation of the CpG islands of individual genes between tumour cell lines correlated well with differential gene expression. Treatment of non-seminoma cells with 5-aza-2-deoxycytidine verified that methylation of all genes tested played a role in their silencing in yolk sac tumour cells and many of these genes were also differentially expressed in primary tumours. Genes silenced by methylation in the various GCT cell lines were identified. Several pluripotency-associated genes were identified as a major functional group of silenced genes.

  13. The role of cytosine methylation on charge transport through a DNA strand

    Energy Technology Data Exchange (ETDEWEB)

    Qi, Jianqing, E-mail: jqqi@uw.edu; Anantram, M. P., E-mail: anantmp@uw.edu [Department of Electrical Engineering, University of Washington, Seattle, Washington 98195-2500 (United States); Govind, Niranjan, E-mail: niri.govind@pnnl.gov [William R. Wiley Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352 (United States)

    2015-09-07

    Cytosine methylation has been found to play a crucial role in various biological processes, including a number of human diseases. The detection of this small modification remains challenging. In this work, we computationally explore the possibility of detecting methylated DNA strands through direct electrical conductance measurements. Using density functional theory and the Landauer-Büttiker method, we study the electronic properties and charge transport through an eight base-pair methylated DNA strand and its native counterpart. We first analyze the effect of cytosine methylation on the tight-binding parameters of two DNA strands and then model the transmission of the electrons and conductance through the strands both with and without decoherence. We find that the main difference of the tight-binding parameters between the native DNA and the methylated DNA lies in the on-site energies of (methylated) cytosine bases. The intra- and inter-strand hopping integrals between two nearest neighboring guanine base and (methylated) cytosine base also change with the addition of the methyl groups. Our calculations show that in the phase-coherent limit, the transmission of the methylated strand is close to the native strand when the energy is nearby the highest occupied molecular orbital level and larger than the native strand by 5 times in the bandgap. The trend in transmission also holds in the presence of the decoherence with the same rate. The lower conductance for the methylated strand in the experiment is suggested to be caused by the more stable structure due to the introduction of the methyl groups. We also study the role of the exchange-correlation functional and the effect of contact coupling by choosing coupling strengths ranging from weak to strong coupling limit.

  14. DNA Methylation and Methylation Polymorphism in Genetically Stable In vitro Regenerates of Jatropha curcas L. Using Methylation-Sensitive AFLP Markers.

    Science.gov (United States)

    Rathore, Mangal S; Jha, Bhavanath

    2016-03-01

    The present investigation aimed to evaluate the degree and pattern of DNA methylation using methylation-sensitive AFLP (MS-AFLP) markers in genetically stable in vitro regenerates of Jatropha curcas L.. The genetically stable in vitro regenerates were raised through direct organogenesis via enhanced axillary shoot bud proliferation (Protocol-1) and in vitro-derived leaf regeneration (Protocol-2). Ten selective combinations of MS-AFLP primers produced 462 and 477 MS-AFLP bands in Protocol-1 (P-1) and Protocol-2 (P-2) regenerates, respectively. In P-1 regenerates, 15.8-31.17 % DNA was found methylated with an average of 25.24 %. In P-2 regenerates, 15.93-32.7 % DNA was found methylated with an average of 24.11 %. Using MS-AFLP in P-1 and P-2 regenerates, 11.52-25.53 % and 13.33-25.47 % polymorphism in methylated DNA was reported, respectively. Compared to the mother plant, P-1 regenerates showed hyper-methylation while P-2 showed hypo-methylation. The results clearly indicated alternation in degree and pattern of DNA methylation; hence, epigenetic instability in the genetically stable in vitro regenerates of J. curcas, developed so far using two different regeneration systems and explants of two different origins. The homologous nucleotide fragments in genomes of P-1 and P-2 regenerates showing methylation re-patterning might be involved in immediate adaptive responses and developmental processes through differential regulation of transcriptome under in vitro conditions.

  15. Clinical Relevance of CDH1 and CDH13 DNA-Methylation in Serum of Cervical Cancer Patients

    Directory of Open Access Journals (Sweden)

    Günther K. Bonn

    2012-07-01

    Full Text Available This study was designed to investigate the DNA-methylation status of E-cadherin (CDH1 and H-cadherin (CDH13 in serum samples of cervical cancer patients and control patients with no malignant diseases and to evaluate the clinical utility of these markers. DNA-methylation status of CDH1 and CDH13 was analyzed by means of MethyLight-technology in serum samples from 49 cervical cancer patients and 40 patients with diseases other than cancer. To compare this methylation analysis with another technique, we analyzed the samples with a denaturing high performance liquid chromatography (DHPLC PCR-method. The specificity and sensitivity of CDH1 DNA-methylation measured by MethyLight was 75% and 55%, and for CDH13 DNA-methylation 95% and 10%. We identified a specificity of 92.5% and a sensitivity of only 27% for the CDH1 DHPLC-PCR analysis. Multivariate analysis showed that serum CDH1 methylation-positive patients had a 7.8-fold risk for death (95% CI: 2.2–27.7; p = 0.001 and a 92.8-fold risk for relapse (95% CI: 3.9–2207.1; p = 0.005. We concluded that the serological detection of CDH1 and CDH13 DNA-hypermethylation is not an ideal diagnostic tool due to low diagnostic specificity and sensitivity. However, it was validated that CDH1 methylation analysis in serum samples may be of potential use as a prognostic marker for cervical cancer patients.

  16. Isothermal (vapor + liquid) equilibria and excess enthalpy data of {1-hexene + methyl butyl ether (MBE)} and {1-hexene + methyl tert-butyl ether (MTBE)} binary systems at several temperatures

    International Nuclear Information System (INIS)

    Hani, Rachida; Solimando, Roland; Negadi, Latifa; Jose, Jacques; Ait Kaci, Ahmed

    2012-01-01

    Highlights: ► Vapor pressures of (1-hexene + methyl butyl ether) or (1-hexene + methyl tert-butyl ether) are reported between (263 and 363) K. ► The two mixtures exhibit positive G E . ► Additionally, molar excess enthalpies, H E , for the two binary systems have been measured at 303.15. - Abstract: The vapor pressures of {1-hexene + methyl butyl ether (MBE)} and {1-hexene + methyl tert-butyl ether (MTBE)} binary mixtures and of the three pure components were measured by means of a static device at temperatures between (263 and 333) K. The data were correlated with the Antoine equation. From these data, excess Gibbs functions were calculated for several constant temperatures and fitted to a third-order Redlich–Kister equation using the Barker’s method. Additionally, molar excess enthalpies, H E , for the two binary systems have been measured at 303.15 K using an isothermal flow calorimeter.

  17. Prenatal exposure to maternal depressed mood and the MTHFR C677T variant affect SLC6A4 methylation in infants at birth.

    Directory of Open Access Journals (Sweden)

    Angela M Devlin

    2010-08-01

    Full Text Available Prenatal and early postnatal exposure to maternal depression may "program" childhood behavior via epigenetic processes such as DNA methylation. Methylenetetrahydro-folate reductase (MTHFR is an important enzyme in the generation of methyl groups for DNA methylation. The common MTHFR C677T variant is associated with depression in men and non-pregnant women, and with global changes in DNA methylation. This study investigated the effect of maternal MTHFR C677T genotype on antenatal maternal mood, and their impact on the gene-specific methylation in pregnant women and their newborn infants. The methylation status of SLC6A4, which encodes the transmembrane serotonin transporter, and BDNF, which encodes brain derived neurotrophic factor, were assessed because of their potential role in behaviour.Depressed mood was assessed by the Edinburgh Postnatal Depression Scale (EPDS and the Hamilton Rating Scale for Depression (HAM-D in women (n = 82, all taking folate during the 2(nd and 3(rd trimesters of pregnancy. The methylation status of SLC6A4 and BDNF were assessed in 3rd trimester maternal peripheral leukocytes and in umbilical cord leukocytes collected from their infants at birth. Women with the MTHFR 677TT genotype had greater 2(nd trimester depressed mood (p<0.05. Increased 2(nd trimester maternal depressed mood (EPDS scores was associated with decreased maternal and infant SLC6A4 promoter methylation (p<0.05, but had no effect on BDNF promoter methylation.These findings show that the MTHFR C677T variant is associated with greater depressed mood during pregnancy. We further showed that prenatal exposure to maternal depressed mood affects gene-specific DNA methylation patterns. These findings support the concept that alterations in epigenetic processes may contribute to developmental programming of behaviour by maternal depression.

  18. Clinical Utility of promoter methylation of the tumor suppressor genes DKK3, and RASSF1A in breast cancer patients

    Directory of Open Access Journals (Sweden)

    Marwa H. Saied

    2018-04-01

    Full Text Available Background: DNA methylation is the commonest known epigenetic change that results in silencing of tumor suppressor genes. Promoter methylation of tumor suppressor genes has the potential for early detection of breast cancer. Aim: Aim is to examine the potential usefulness of blood based methylation specific polymerase chain reaction (MSP of methylated DKK3 and RASSF1A genes in early detection of breast cancer. Method: Methylation status of DKK3 and RASSF1 was investigated in forty breast cancer patients, twenty fibroadenoma patients and twenty healthy ladies as control group using MSP. Results: Methylation of DKK3 promoter was found in 22.5% of breast cancer patients, while DKK3 methylation was absent in both fibroadenoma patients and control group. Similarly, methylation of RASSF1 promoter was found in 17.5% of breast cancer patients and in none of fibroadenoma and control group. Conclusion: Promoter methylation of DKK3 and RASSF1 was found in breast cancer patients while absent in control group suggesting that tumorspecific methylation of the two genes (DKK3 and RASSF1A might be a valuable biomarker for the early detection of breast cancer. Keywords: DNA methylation, Breast cancer, DKK3, RASSF1

  19. Aberrant Methylation of Preproenkephalin and p16 Genes in Pancreatic Intraepithelial Neoplasia and Pancreatic Ductal Adenocarcinoma

    OpenAIRE

    Fukushima, Noriyoshi; Sato, Norihiro; Ueki, Takashi; Rosty, Christophe; Walter, Kimberly M.; Wilentz, Robb E.; Yeo, Charles J.; Hruban, Ralph H.; Goggins, Michael

    2002-01-01

    Pancreatic intraductal neoplasia (PanIN) is thought to be the precursor to infiltrating pancreatic ductal adenocarcinoma. We have previously shown that the preproenkephalin (ppENK) and p16 genes are aberrantly methylated in pancreatic adenocarcinoma. In this study we define the methylation status of the ppENK and p16 genes in various grades of PanINs. One hundred seventy-four samples (28 nonneoplastic pancreatic epithelia, 7 reactive epithelia, 29 PanIN-1A, 48 PanIN-1B, 27 PanIN-2, 14 PanIN-3...

  20. Aberrant DNA methylation associated with MTHFR C677T genetic polymorphism in cutaneous squamous cell carcinoma in renal transplant patients.

    LENUS (Irish Health Repository)

    Laing, M E

    2010-08-01

    Changes in genomic DNA methylation associated with cancer include global DNA hypomethylation and gene-specific hyper- or hypomethylation. We have previously identified a genetic variant in the MTHFR gene involved in the methylation pathway which confers risk for the development of squamous cell carcinoma (SCC) in renal transplant patients. This genetic variant has also been discovered to confer SCC risk in nontransplant patients with low folate status.