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Sample records for cell mutation test

  1. Should EGFR mutations be tested in advanced lung squamous cell carcinomas to guide frontline treatment?

    Science.gov (United States)

    Chiu, Chao-Hua; Chou, Teh-Ying; Chiang, Chi-Lu; Tsai, Chun-Ming

    2014-10-01

    There is no argument over using epidermal growth factor receptor (EGFR) mutation status to guide the frontline treatment for advanced lung adenocarcinoma (LADC); however, the role of the testing in lung squamous cell carcinoma (LSQC) remains controversial. Currently, the guidelines/consensus statements regarding EGFR mutation testing in LSQC are not consistent among different oncology societies. American Society of Clinical Oncology recommends performing EGFR mutation testing in all patients; European Society for Medical Oncology, College of American Pathologists/International Association for the Study of Lung Cancer/Association for Molecular Pathology, and National Comprehensive Cancer Network suggest for some selected group. EGFR mutation is rarely found in LSQC; however, more importantly, it is not a valid predictive biomarker for EGFR tyrosine kinase inhibitors (EGFR-TKI) in LSQC as it has been shown in LADC. Available data showed that the response rate and progression-free survival in EGFR mutant LSQC patients treated with EGFR-TKI are not better than that observed in patients treated with platinum-doublet chemotherapy in the first-line setting. Therefore, in contrast to advanced LADC, EGFR mutation testing may not be necessarily performed upfront in advanced LSQC because not only the mutation rate is low, but also the predictive value is insufficient. For LSQC patients with known sensitizing-EGFR mutations, both conventional chemotherapy and EGFR-TKI are acceptable frontline treatment options.

  2. Diphtheria toxin resistance in human lymphocytes and lymphoblasts in the in vivo somatic cell mutation test

    International Nuclear Information System (INIS)

    Tomkins, D.J.; Wei, L.; Laurie, K.E.

    1985-01-01

    It has been shown that circulating peripheral blood lymphocytes can be used for the enumeration of 6-thioguanine-resistant cells that presumably arise by mutation in vivo. This somatic cell mutation test has been studied in lymphocytes from human populations exposed to known mutagens and/or carcinogens. The sensitivity of the test could be further enhanced by including other gene markers, since there is evidence for locus-specific differences in response to mutagens. Resistance to diphtheria toxin (Dip/sup r/) seemed like a potential marker to incorporate into the test because the mutation acts codominantly, can readily be selected in human diploid fibroblasts and Chinese hamster cells with no evidence for cell density or cross-feeding effects, and can be assayed for in nondividing cells by measuring protein synthesis inhibition. Blood samples were collected from seven individuals, and fresh, cryopreserved, or Epstein-Barr virus (EBV)-transformed lymphocytes were tested for continued DNA synthesis ( 3 H-thymidine, autoradiography) or protein synthesis ( 35 S-methionine, scintillation counting). Both fresh and cryopreserved lymphocytes, stimulated to divide with phytohemagglutinin (PHA), continued to synthesize DNA in the presence of high doses of diphtheria toxin (DT). Similarly, both dividing (PHA-stimulated) and nondividing fresh lymphocytes carried on significant levels of protein synthesis even 68 hr after exposure to 100 flocculating units (LF)/ml DT. The results suggest that human T and B lymphocytes may not be as sensitive to DT protein synthesis inhibition as human fibroblast and Chinese hamster cells. For this reason, Dip/sup r/ may not be a suitable marker for the somatic cell mutation test

  3. Consensus for EGFR mutation testing in non-small cell lung cancer: results from a European workshop

    DEFF Research Database (Denmark)

    Pirker, Robert; Herth, Felix J F; Kerr, Keith M

    2010-01-01

    Activating somatic mutations of the tyrosine kinase domain of epidermal growth factor receptor (EGFR) have recently been characterized in a subset of patients with advanced non-small cell lung cancer (NSCLC). Patients harboring these mutations in their tumors show excellent response to EGFR tyros...

  4. EGFR mutation testing in patients with advanced non-small cell lung cancer: a comprehensive evaluation of real-world practice in an East Asian tertiary hospital.

    Science.gov (United States)

    Choi, Yoon-La; Sun, Jong-Mu; Cho, Juhee; Rampal, Sanjay; Han, Joungho; Parasuraman, Bhash; Guallar, Eliseo; Lee, Genehee; Lee, Jeeyun; Shim, Young Mog

    2013-01-01

    Guidelines for management of non-small cell lung cancer (NSCLC) strongly recommend EGFR mutation testing. These recommendations are particularly relevant in Asians that have higher EGFR mutation prevalence. This study aims to explore current testing practices, logistics of testing, types of EGFR mutation, and prevalence of EGFR mutations in patients with advanced NSCLC in a large comprehensive cancer center in Korea. Our retrospective cohort included 1,503 NSCLC patients aged ≥18 years, with stage IIIB/IV disease, who attended the Samsung Medical Center in Seoul, Korea, from January 2007 through July 2010. Trained oncology nurses reviewed and abstracted data from electronic medical records. This cohort had a mean age (SD) of 59.6 (11.1) years, 62.7% were males, and 52.9% never-smokers. The most common NSCLC histological types were adenocarcinoma (70.5%) and squamous cell carcinoma (18.0%). Overall, 39.5% of patients were tested for EGFR mutations. The proportion of patients undergoing EGFR testing during January 2007 through July 2008, August 2008 through September 2009, and October 2009 through July 2010 were 23.3%, 38.3%, and 63.5%, respectively (Pwomen, younger patients, stage IV disease, non-smokers, and adenocarcinoma histology. Of 586 cases successfully tested for EGFR mutations, 209 (35.7%) were positive, including 118 cases with exon 19 deletions and 62 with L858R mutations. EGFR mutation positive patients were more likely to be female, never-smokers, never-drinkers and to have adenocarcinoma. In a large cancer center in Korea, the proportion of EGFR testing increased from 2007 through 2010. The high frequency of EGFR mutation positive cases warrants the need for generalized testing in Asian NSCLC patients.

  5. 40 CFR 799.9530 - TSCA in vitro mammalian cell gene mutation test.

    Science.gov (United States)

    2010-07-01

    ... sensitive to TFT, which causes the inhibition of cellular metabolism and halts further cell division. Thus... used, their inclusion should be supported by data indicating their compatibility. It is recommended... mutant frequency. Biological relevance of the results should be considered first. Statistical methods may...

  6. EGFR Mutation Testing in Patients with Advanced Non-Small Cell Lung Cancer: A Comprehensive Evaluation of Real-World Practice in an East Asian Tertiary Hospital

    Science.gov (United States)

    Cho, Juhee; Rampal, Sanjay; Han, Joungho; Parasuraman, Bhash; Guallar, Eliseo; Lee, Genehee; Lee, Jeeyun; Shim, Young Mog

    2013-01-01

    Introduction Guidelines for management of non-small cell lung cancer (NSCLC) strongly recommend EGFR mutation testing. These recommendations are particularly relevant in Asians that have higher EGFR mutation prevalence. This study aims to explore current testing practices, logistics of testing, types of EGFR mutation, and prevalence of EGFR mutations in patients with advanced NSCLC in a large comprehensive cancer center in Korea. Methods Our retrospective cohort included 1,503 NSCLC patients aged ≥18 years, with stage IIIB/IV disease, who attended the Samsung Medical Center in Seoul, Korea, from January 2007 through July 2010. Trained oncology nurses reviewed and abstracted data from electronic medical records. Results This cohort had a mean age (SD) of 59.6 (11.1) years, 62.7% were males, and 52.9% never-smokers. The most common NSCLC histological types were adenocarcinoma (70.5%) and squamous cell carcinoma (18.0%). Overall, 39.5% of patients were tested for EGFR mutations. The proportion of patients undergoing EGFR testing during January 2007 through July 2008, August 2008 through September 2009, and October 2009 through July 2010 were 23.3%, 38.3%, and 63.5%, respectively (P<0.001). The median time elapsed between cancer diagnoses and receiving EGFR testing results was 21 days. EGFR testing was most frequently ordered by oncologists (57.7%), pulmonologists (31.9%), and thoracic surgeons (6.6%). EGFR testing was more commonly requested for women, younger patients, stage IV disease, non-smokers, and adenocarcinoma histology. Of 586 cases successfully tested for EGFR mutations, 209 (35.7%) were positive, including 118 cases with exon 19 deletions and 62 with L858R mutations. EGFR mutation positive patients were more likely to be female, never-smokers, never-drinkers and to have adenocarcinoma. Conclusions In a large cancer center in Korea, the proportion of EGFR testing increased from 2007 through 2010. The high frequency of EGFR mutation positive cases warrants

  7. EGFR mutation testing in patients with advanced non-small cell lung cancer: a comprehensive evaluation of real-world practice in an East Asian tertiary hospital.

    Directory of Open Access Journals (Sweden)

    Yoon-La Choi

    Full Text Available INTRODUCTION: Guidelines for management of non-small cell lung cancer (NSCLC strongly recommend EGFR mutation testing. These recommendations are particularly relevant in Asians that have higher EGFR mutation prevalence. This study aims to explore current testing practices, logistics of testing, types of EGFR mutation, and prevalence of EGFR mutations in patients with advanced NSCLC in a large comprehensive cancer center in Korea. METHODS: Our retrospective cohort included 1,503 NSCLC patients aged ≥18 years, with stage IIIB/IV disease, who attended the Samsung Medical Center in Seoul, Korea, from January 2007 through July 2010. Trained oncology nurses reviewed and abstracted data from electronic medical records. RESULTS: This cohort had a mean age (SD of 59.6 (11.1 years, 62.7% were males, and 52.9% never-smokers. The most common NSCLC histological types were adenocarcinoma (70.5% and squamous cell carcinoma (18.0%. Overall, 39.5% of patients were tested for EGFR mutations. The proportion of patients undergoing EGFR testing during January 2007 through July 2008, August 2008 through September 2009, and October 2009 through July 2010 were 23.3%, 38.3%, and 63.5%, respectively (P<0.001. The median time elapsed between cancer diagnoses and receiving EGFR testing results was 21 days. EGFR testing was most frequently ordered by oncologists (57.7%, pulmonologists (31.9%, and thoracic surgeons (6.6%. EGFR testing was more commonly requested for women, younger patients, stage IV disease, non-smokers, and adenocarcinoma histology. Of 586 cases successfully tested for EGFR mutations, 209 (35.7% were positive, including 118 cases with exon 19 deletions and 62 with L858R mutations. EGFR mutation positive patients were more likely to be female, never-smokers, never-drinkers and to have adenocarcinoma. CONCLUSIONS: In a large cancer center in Korea, the proportion of EGFR testing increased from 2007 through 2010. The high frequency of EGFR mutation positive

  8. Mutation in cultured mammalian cells

    International Nuclear Information System (INIS)

    Nakamura, N.; Okada, S.

    1982-01-01

    Mammalian cell cultures were exposed to gamma-rays at various dose rates. Dose-rate effects were observed in cultured somatic cells of the mouse for cell killing and mutations resistant to 6-thioguanine (TGsup(r)) and to methotrexate (MTXsup(r)). Linear quadratic model may be applied to cell killing and TGsup(r) mutations in some cases but can not explain the whole data. Results at low doses with far low dose-rate were not predictable from data at high doses with acute or chronic irradiation. Radioprotective effects of dimethyl sulfoxide were seen only after acute exposure but not after chronic one, suggesting that damages by indirect action of radiations may be potentially reparable by cells. TGsup(r) mutations seem to contain gross structural changes whereas MTXsup(r) ones may have smaller alterations. (Namekawa, K.)

  9. Factor V Leiden Mutation and PT 20210 Mutation Test

    Science.gov (United States)

    ... Disorders Fibromyalgia Food and Waterborne Illness Fungal Infections Gout Graves Disease Guillain-Barré Syndrome Hashimoto Thyroiditis Heart ... Tested? To determine whether you have an inherited gene mutation that increases your risk of developing a ...

  10. Epidermal growth factor receptor tyrosine kinase (EGFR-TK) mutation testing in adults with locally advanced or metastatic non-small cell lung cancer: A systematic review and cost-effectiveness analysis

    OpenAIRE

    Westwood, Marie; Joore, Manuela; Whiting, Penny; Asselt, Thea; Ramaekers, Bram; Armstrong, Nigel; Misso, Kate; Severens, Hans; Kleijnen, Jos

    2014-01-01

    markdownabstract__Abstract__ Background: Non-small cell lung cancer (NSCLC) is the most common form of lung cancer. Some epidermal growth factor receptor tyrosine kinase (EGFR-TK) mutations make tumours responsive to treatment with EGFR-TK inhibitors (EGFR-TKIs) but less responsive to treatment with standard chemotherapy. Patients with NSCLC are therefore tested for EGFR-TK tumour gene mutations to inform treatment decisions. There are a variety of tests available to detect these mutations. T...

  11. Cost-effectiveness analysis of EGFR mutation testing and gefitinib as first-line therapy for non-small cell lung cancer.

    Science.gov (United States)

    Narita, Yusuke; Matsushima, Yukiko; Shiroiwa, Takeru; Chiba, Koji; Nakanishi, Yoichi; Kurokawa, Tatsuo; Urushihara, Hisashi

    2015-10-01

    The combination use of gefitinib and epidermal growth factor receptor (EGFR) testing is a standard first-line therapy for patients with non-small cell lung cancer (NSCLC). Here, we examined the cost-effectiveness of this approach in Japan. Our analysis compared the 'EGFR testing strategy', in which EGFR mutation testing was performed before treatment and patients with EGFR mutations received gefitinib while those without mutations received standard chemotherapy, to the 'no-testing strategy,' in which genetic testing was not conducted and all patients were treated with standard chemotherapy. A three-state Markov model was constructed to predict expected costs and outcomes for each strategy. We included only direct medical costs from the healthcare payer's perspective. Outcomes in the model were based on those reported in the Iressa Pan-Asia Study (IPASS). The incremental cost-effectiveness ratio (ICER) was calculated using quality-adjusted life-years (QALYs) gained. Sensitivity and scenario analyses were conducted. The incremental cost and effectiveness per patient of the 'EGFR testing strategy' compared to the 'no-testing strategy' was estimated to be approximately JP¥122,000 (US$1180; US$1=JP¥104 as of February 2014) and 0.036 QALYs. The ICER was then calculated to be around JP¥3.38 million (US$32,500) per QALY gained. These results suggest that the 'EGFR testing strategy' is cost-effective compared with the 'no-testing strategy' when JP¥5.0 million to 6.0 million per QALY gained is considered an acceptable threshold. These results were supported by the sensitivity and scenario analyses. The combination use of gefitinib and EGFR testing can be considered a cost-effective first-line therapy compared to chemotherapy such as carboplatin-paclitaxel for the treatment for NSCLC in Japan. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  12. EGFR T790M mutation testing of non-small cell lung cancer tissue and blood samples artificially spiked with circulating cell-free tumor DNA: results of a round robin trial.

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    Fassunke, Jana; Ihle, Michaela Angelika; Lenze, Dido; Lehmann, Annika; Hummel, Michael; Vollbrecht, Claudia; Penzel, Roland; Volckmar, Anna-Lena; Stenzinger, Albrecht; Endris, Volker; Jung, Andreas; Lehmann, Ulrich; Zeugner, Silke; Baretton, Gustavo; Kreipe, Hans; Schirmacher, Peter; Kirchner, Thomas; Dietel, Manfred; Büttner, Reinhard; Merkelbach-Bruse, Sabine

    2017-10-01

    The European Commision (EC) recently approved osimertinib for the treatment of adult patients with locally advanced or metastatic non-small-cell lung cancer (NSCLC) harboring EGFR T790M mutations. Besides tissue-based testing, blood samples containing cell-free circulating tumor DNA (ctDNA) can be used to interrogate T790M status. Herein, we describe the conditions and results of a round robin trial (RRT) for T790M mutation testing in NSCLC tissue specimens and peripheral blood samples spiked with cell line DNA mimicking tumor-derived ctDNA. The underlying objectives of this two-staged external quality assessment (EQA) approach were (a) to evaluate the accuracy of T790M mutations testing across multiple centers and (b) to investigate if a liquid biopsy-based testing for T790M mutations in spiked blood samples is feasible in routine diagnostic. Based on a successfully completed internal phase I RRT, an open RRT for EGFR T790M mutation testing in tumor tissue and blood samples was initiated. In total, 48 pathology centers participated in the EQA. Of these, 47 (97.9%) centers submitted their analyses within the pre-defined time frame and 44 (tissue), respectively, 40 (plasma) successfully passed the test. The overall success rates in the RRT phase II were 91.7% (tissue) and 83.3% (blood), respectively. Thirty-eight out of 48 participants (79.2%) successfully passed both parts of the RRT. The RRT for blood-based EGFR testing initiated in Germany is, to the best of our knowledge, the first of his kind in Europe. In summary, our results demonstrate that blood-based genotyping for EGFR resistance mutations can be successfully integrated in routine molecular diagnostics complementing the array of molecular methods already available at pathology centers in Germany.

  13. The molecular anatomy of spontaneous germline mutations in human testes.

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    Jian Qin

    2007-09-01

    Full Text Available The frequency of the most common sporadic Apert syndrome mutation (C755G in the human fibroblast growth factor receptor 2 gene (FGFR2 is 100-1,000 times higher than expected from average nucleotide substitution rates based on evolutionary studies and the incidence of human genetic diseases. To determine if this increased frequency was due to the nucleotide site having the properties of a mutation hot spot, or some other explanation, we developed a new experimental approach. We examined the spatial distribution of the frequency of the C755G mutation in the germline by dividing four testes from two normal individuals each into several hundred pieces, and, using a highly sensitive PCR assay, we measured the mutation frequency of each piece. We discovered that each testis was characterized by rare foci with mutation frequencies 10(3 to >10(4 times higher than the rest of the testis regions. Using a model based on what is known about human germline development forced us to reject (p < 10(-6 the idea that the C755G mutation arises more frequently because this nucleotide simply has a higher than average mutation rate (hot spot model. This is true regardless of whether mutation is dependent or independent of cell division. An alternate model was examined where positive selection acts on adult self-renewing Ap spermatogonial cells (SrAp carrying this mutation such that, instead of only replacing themselves, they occasionally produce two SrAp cells. This model could not be rejected given our observed data. Unlike the disease site, similar analysis of C-to-G mutations at a control nucleotide site in one testis pair failed to find any foci with high mutation frequencies. The rejection of the hot spot model and lack of rejection of a selection model for the C755G mutation, along with other data, provides strong support for the proposal that positive selection in the testis can act to increase the frequency of premeiotic germ cells carrying a mutation

  14. Cost-effectiveness analysis of EGFR mutation testing in patients with non-small cell lung cancer (NSCLC) with gefitinib or carboplatin-paclitaxel.

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    Arrieta, Oscar; Anaya, Pablo; Morales-Oyarvide, Vicente; Ramírez-Tirado, Laura Alejandra; Polanco, Ana C

    2016-09-01

    Assess the cost-effectiveness of an EGFR-mutation testing strategy for advanced NSCLC in first-line therapy with either gefitinib or carboplatin-paclitaxel in Mexican institutions. Cost-effectiveness analysis using a discrete event simulation (DES) model to simulate two therapeutic strategies in patients with advanced NSCLC. Strategy one included patients tested for EGFR-mutation and therapy given accordingly. Strategy two included chemotherapy for all patients without testing. All results are presented in 2014 US dollars. The analysis was made with data from the Mexican frequency of EGFR-mutation. A univariate sensitivity analysis was conducted on EGFR prevalence. Progression-free survival (PFS) transition probabilities were estimated on data from the IPASS and simulated with a Weibull distribution, run with parallel trials to calculate a probabilistic sensitivity analysis. PFS of patients in the testing strategy was 6.76 months (95 % CI 6.10-7.44) vs 5.85 months (95 % CI 5.43-6.29) in the non-testing group. The one-way sensitivity analysis showed that PFS has a direct relationship with EGFR-mutation prevalence, while the ICER and testing cost have an inverse relationship with EGFR-mutation prevalence. The probabilistic sensitivity analysis showed that all iterations had incremental costs and incremental PFS for strategy 1 in comparison with strategy 2. There is a direct relationship between the ICER and the cost of EGFR testing, with an inverse relationship with the prevalence of EGFR-mutation. When prevalence is >10 % ICER remains constant. This study could impact Mexican and Latin American health policies regarding mutation detection testing and treatment for advanced NSCLC.

  15. Variation in RNA virus mutation rates across host cells.

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    Marine Combe

    2014-01-01

    Full Text Available It is well established that RNA viruses exhibit higher rates of spontaneous mutation than DNA viruses and microorganisms. However, their mutation rates vary amply, from 10(-6 to 10(-4 substitutions per nucleotide per round of copying (s/n/r and the causes of this variability remain poorly understood. In addition to differences in intrinsic fidelity or error correction capability, viral mutation rates may be dependent on host factors. Here, we assessed the effect of the cellular environment on the rate of spontaneous mutation of the vesicular stomatitis virus (VSV, which has a broad host range and cell tropism. Luria-Delbrück fluctuation tests and sequencing showed that VSV mutated similarly in baby hamster kidney, murine embryonic fibroblasts, colon cancer, and neuroblastoma cells (approx. 10(-5 s/n/r. Cell immortalization through p53 inactivation and oxygen levels (1-21% did not have a significant impact on viral replication fidelity. This shows that previously published mutation rates can be considered reliable despite being based on a narrow and artificial set of laboratory conditions. Interestingly, we also found that VSV mutated approximately four times more slowly in various insect cells compared with mammalian cells. This may contribute to explaining the relatively slow evolution of VSV and other arthropod-borne viruses in nature.

  16. Rapid point-of-care testing for epidermal growth factor receptor gene mutations in patients with lung cancer using cell-free DNA from cytology specimen supernatants.

    Science.gov (United States)

    Asaka, Shiho; Yoshizawa, Akihiko; Saito, Kazusa; Kobayashi, Yukihiro; Yamamoto, Hiroshi; Negishi, Tatsuya; Nakata, Rie; Matsuda, Kazuyuki; Yamaguchi, Akemi; Honda, Takayuki

    2018-06-01

    Epidermal growth factor receptor (EGFR) mutations are associated with responses to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in non-small-cell lung cancer (NSCLC). Our previous study revealed a rapid point-of-care system for detecting EGFR mutations. This system analyzes cell pellets from cytology specimens using droplet-polymerase chain reaction (d-PCR), and has a reaction time of 10 min. The present study aimed to validate the performance of the EGFR d-PCR assay using cell-free DNA (cfDNA) from supernatants obtained from cytology specimens. Assay results from cfDNA supernatant analyses were compared with those from cell pellets for 90 patients who were clinically diagnosed with, or suspected of having, lung cancer (80 bronchial lavage fluid samples, nine pleural effusion samples and one spinal fluid sample). EGFR mutations were identified in 12 and 15 cases using cfDNA supernatants and cell pellets, respectively. The concordance rates between cfDNA-supernatant and cell‑pellet assay results were 96.7% [kappa coefficient (K)=0.87], 98.9% (K=0.94), 98.9% (K=0.79) and 98.9% (K=0.79) for total EGFR mutations, L858R, E746_A750del and T790M, respectively. All 15 patients with EGFR mutation-positive results, as determined by EGFR d-PCR assay using cfDNA supernatants or cell pellets, also displayed positive results by conventional EGFR assays using tumor tissue or cytology specimens. Notably, EGFR mutations were even detected in five cfDNA supernatants for which the cytological diagnoses of the corresponding cell pellets were 'suspicious for malignancy', 'atypical' or 'negative for malignancy.' In conclusion, this rapid point-of-care system may be considered a promising novel screening method that may enable patients with NSCLC to receive EGFR-TKI therapy more rapidly, whilst also reserving cell pellets for additional morphological and molecular analyses.

  17. Mutation studies upon spermatogonial stem cells of mammals and genetic tests for non-disjunction in the mouse

    International Nuclear Information System (INIS)

    Cattanach, B.M.

    1993-01-01

    Studies upon strain differences in genetic response to radiation may facilitate extrapolation of mouse data to man. The objective of the project is to investigate the basis of the genetic responses obtained with different treatment regimes. Two systems of genetic (complementation) tests were developed using Robertsonian translocations in tester animals to detect non-disjunction and chromosome loss events in normal mice. The aim is to evaluate the two methods for detecting chromosome 11 loss, and compare the frequency of chromosomes 11 and 13 loss following X-irradiation of males and females. (R.P.) 6 refs., 3 tabs

  18. BRAF mutation in hairy cell leukemia

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    Ahmad Ahmadzadeh

    2014-09-01

    Full Text Available BRAF is a serine/threonine kinase with a regulatory role in the mitogen-activated protein kinase (MAPK signaling pathway. A mutation in the RAF gene, especially in BRAF protein, leads to an increased stimulation of this cascade, causing uncontrolled cell division and development of malignancy. Several mutations have been observed in the gene coding for this protein in a variety of human malignancies, including hairy cell leukemia (HCL. BRAF V600E is the most common mutation reported in exon15 of BRAF, which is observed in almost all cases of classic HCL, but it is negative in other B-cell malignancies, including the HCL variant. Therefore it can be used as a marker to differentiate between these B-cell disorders. We also discuss the interaction between miRNAs and signaling pathways, including MAPK, in HCL. When this mutation is present, the use of BRAF protein inhibitors may represent an effective treatment. In this review we have evaluated the role of the mutation of the BRAF gene in the pathogenesis and progression of HCL.

  19. Diploid yeast cells yield homozygous spontaneous mutations

    Science.gov (United States)

    Esposito, M. S.; Bruschi, C. V.; Brushi, C. V. (Principal Investigator)

    1993-01-01

    A leucine-requiring hybrid of Saccharomyces cerevisiae, homoallelic at the LEU1 locus (leu1-12/leu1-12) and heterozygous for three chromosome-VII genetic markers distal to the LEU1 locus, was employed to inquire: (1) whether spontaneous gene mutation and mitotic segregation of heterozygous markers occur in positive nonrandom association and (2) whether homozygous LEU1/LEU1 mutant diploids are generated. The results demonstrate that gene mutation of leu1-12 to LEU1 and mitotic segregation of heterozygous chromosome-VII markers occur in strong positive nonrandom association, suggesting that the stimulatory DNA lesion is both mutagenic and recombinogenic. In addition, genetic analysis of diploid Leu+ revertants revealed that approximately 3% of mutations of leu1-12 to LEU1 result in LEU1/LEU1 homozygotes. Red-white sectored Leu+ colonies exhibit genotypes that implicate post-replicational chromatid breakage and exchange near the site of leu1-12 reversion, chromosome loss, and subsequent restitution of diploidy, in the sequence of events leading to mutational homozygosis. By analogy, diploid cell populations can yield variants homozygous for novel recessive gene mutations at biologically significant rates. Mutational homozygosis may be relevant to both carcinogenesis and the evolution of asexual diploid organisms.

  20. Roles of Mitochondrial DNA Mutations in Stem Cell Ageing

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    Tianhong Su

    2018-03-01

    Full Text Available Mitochondrial DNA (mtDNA mutations accumulate in somatic stem cells during ageing and cause mitochondrial dysfunction. In this review, we summarize the studies that link mtDNA mutations to stem cell ageing. We discuss the age-related behaviours of the somatic mtDNA mutations in stem cell populations and how they potentially contribute to stem cell ageing by altering mitochondrial properties in humans and in mtDNA-mutator mice. We also draw attention to the diverse fates of the mtDNA mutations with different origins during ageing, with potential selective pressures on the germline inherited but not the somatic mtDNA mutations.

  1. Mutational Analysis of Merkel Cell Carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Erstad, Derek J. [Department of Surgery, Massachusetts General Hospital, 55 Fruit Street, Boston, MA 02114 (United States); Cusack, James C. Jr., E-mail: jcusack@mgh.harvard.edu [Division of Surgical Oncology, Harvard Medical School, Massachusetts General Hospital, 55 Fruit Street, Boston, MA 02114 (United States)

    2014-10-17

    Merkel cell carcinoma (MCC) is an aggressive cutaneous neuroendocrine malignancy that is associated with a poor prognosis. The pathogenesis of MCC is not well understood, and despite a recent plethora of mutational analyses, we have yet to find a set of signature mutations implicated in the majority of cases. Mutations, including TP53, Retinoblastoma and PIK3CA, have been documented in subsets of patients. Other mechanisms are also likely at play, including infection with the Merkel cell polyomavirus in a subset of patients, dysregulated immune surveillance, epigenetic alterations, aberrant protein expression, posttranslational modifications and microRNAs. In this review, we summarize what is known about MCC genetic mutations and chromosomal abnormalities, and their clinical significance. We also examine aberrant protein function and microRNA expression, and discuss the therapeutic and prognostic implications of these findings. Multiple clinical trials designed to selectively target overexpressed oncogenes in MCC are currently underway, though most are still in early phases. As we accumulate more molecular data on MCC, we will be better able to understand its pathogenic mechanisms, develop libraries of targeted therapies, and define molecular prognostic signatures to enhance our clinicopathologic knowledge.

  2. Mutational Analysis of Merkel Cell Carcinoma

    International Nuclear Information System (INIS)

    Erstad, Derek J.; Cusack, James C. Jr.

    2014-01-01

    Merkel cell carcinoma (MCC) is an aggressive cutaneous neuroendocrine malignancy that is associated with a poor prognosis. The pathogenesis of MCC is not well understood, and despite a recent plethora of mutational analyses, we have yet to find a set of signature mutations implicated in the majority of cases. Mutations, including TP53, Retinoblastoma and PIK3CA, have been documented in subsets of patients. Other mechanisms are also likely at play, including infection with the Merkel cell polyomavirus in a subset of patients, dysregulated immune surveillance, epigenetic alterations, aberrant protein expression, posttranslational modifications and microRNAs. In this review, we summarize what is known about MCC genetic mutations and chromosomal abnormalities, and their clinical significance. We also examine aberrant protein function and microRNA expression, and discuss the therapeutic and prognostic implications of these findings. Multiple clinical trials designed to selectively target overexpressed oncogenes in MCC are currently underway, though most are still in early phases. As we accumulate more molecular data on MCC, we will be better able to understand its pathogenic mechanisms, develop libraries of targeted therapies, and define molecular prognostic signatures to enhance our clinicopathologic knowledge

  3. Volatility of Mutator Phenotypes at Single Cell Resolution.

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    Scott R Kennedy

    2015-04-01

    Full Text Available Mutator phenotypes accelerate the evolutionary process of neoplastic transformation. Historically, the measurement of mutation rates has relied on scoring the occurrence of rare mutations in target genes in large populations of cells. Averaging mutation rates over large cell populations assumes that new mutations arise at a constant rate during each cell division. If the mutation rate is not constant, an expanding mutator population may contain subclones with widely divergent rates of evolution. Here, we report mutation rate measurements of individual cell divisions of mutator yeast deficient in DNA polymerase ε proofreading and base-base mismatch repair. Our data are best fit by a model in which cells can assume one of two distinct mutator states, with mutation rates that differ by an order of magnitude. In error-prone cell divisions, mutations occurred on the same chromosome more frequently than expected by chance, often in DNA with similar predicted replication timing, consistent with a spatiotemporal dimension to the hypermutator state. Mapping of mutations onto predicted replicons revealed that mutations were enriched in the first half of the replicon as well as near termination zones. Taken together, our findings show that individual genome replication events exhibit an unexpected volatility that may deepen our understanding of the evolution of mutator-driven malignancies.

  4. Identifying EGFR-Expressed Cells and Detecting EGFR Multi-Mutations at Single-Cell Level by Microfluidic Chip

    Science.gov (United States)

    Li, Ren; Zhou, Mingxing; Li, Jine; Wang, Zihua; Zhang, Weikai; Yue, Chunyan; Ma, Yan; Peng, Hailin; Wei, Zewen; Hu, Zhiyuan

    2018-03-01

    EGFR mutations companion diagnostics have been proved to be crucial for the efficacy of tyrosine kinase inhibitor targeted cancer therapies. To uncover multiple mutations occurred in minority of EGFR-mutated cells, which may be covered by the noises from majority of un-mutated cells, is currently becoming an urgent clinical requirement. Here we present the validation of a microfluidic-chip-based method for detecting EGFR multi-mutations at single-cell level. By trapping and immunofluorescently imaging single cells in specifically designed silicon microwells, the EGFR-expressed cells were easily identified. By in situ lysing single cells, the cell lysates of EGFR-expressed cells were retrieved without cross-contamination. Benefited from excluding the noise from cells without EGFR expression, the simple and cost-effective Sanger's sequencing, but not the expensive deep sequencing of the whole cell population, was used to discover multi-mutations. We verified the new method with precisely discovering three most important EGFR drug-related mutations from a sample in which EGFR-mutated cells only account for a small percentage of whole cell population. The microfluidic chip is capable of discovering not only the existence of specific EGFR multi-mutations, but also other valuable single-cell-level information: on which specific cells the mutations occurred, or whether different mutations coexist on the same cells. This microfluidic chip constitutes a promising method to promote simple and cost-effective Sanger's sequencing to be a routine test before performing targeted cancer therapy.[Figure not available: see fulltext.

  5. Epidermal Growth Factor Receptor Mutation (EGFR) Testing for Prediction of Response to EGFR-Targeting Tyrosine Kinase Inhibitor (TKI) Drugs in Patients with Advanced Non-Small-Cell Lung Cancer: An Evidence-Based Analysis.

    Science.gov (United States)

    2010-01-01

    In February 2010, the Medical Advisory Secretariat (MAS) began work on evidence-based reviews of the literature surrounding three pharmacogenomic tests. This project came about when Cancer Care Ontario (CCO) asked MAS to provide evidence-based analyses on the effectiveness and cost-effectiveness of three oncology pharmacogenomic tests currently in use in Ontario.Evidence-based analyses have been prepared for each of these technologies. These have been completed in conjunction with internal and external stakeholders, including a Provincial Expert Panel on Pharmacogenetics (PEPP). Within the PEPP, subgroup committees were developed for each disease area. For each technology, an economic analysis was also completed by the Toronto Health Economics and Technology Assessment Collaborative (THETA) and is summarized within the reports.THE FOLLOWING REPORTS CAN BE PUBLICLY ACCESSED AT THE MAS WEBSITE AT: http://www.health.gov.on.ca/mas or at www.health.gov.on.ca/english/providers/program/mas/mas_about.htmlGENE EXPRESSION PROFILING FOR GUIDING ADJUVANT CHEMOTHERAPY DECISIONS IN WOMEN WITH EARLY BREAST CANCER: An Evidence-Based AnalysisEpidermal Growth Factor Receptor Mutation (EGFR) Testing for Prediction of Response to EGFR-Targeting Tyrosine Kinase Inhibitor (TKI) Drugs in Patients with Advanced Non-Small-Cell Lung Cancer: an Evidence-Based AnalysisK-RAS testing in Treatment Decisions for Advanced Colorectal Cancer: an Evidence-Based Analysis The Medical Advisory Secretariat undertook a systematic review of the evidence on the clinical effectiveness and cost-effectiveness of epidermal growth factor receptor (EGFR) mutation testing compared with no EGFR mutation testing to predict response to tyrosine kinase inhibitors (TKIs), gefitinib (Iressa(®)) or erlotinib (Tarceva(®)) in patients with advanced non-small cell lung cancer (NSCLC). TARGET POPULATION AND CONDITION With an estimated 7,800 new cases and 7,000 deaths last year, lung cancer is the leading cause of cancer

  6. KRAS mutation: should we test for it, and does it matter?

    Science.gov (United States)

    Roberts, Patrick J; Stinchcombe, Thomas E

    2013-03-10

    Lung cancer is the leading cause of cancer mortality in the United States and worldwide. Previously, lung cancer was simplistically divided into non-small-cell lung cancer (NSCLC) and small-cell lung cancer. However, in the last decade, we have gone from a simplistic binary system of classifying lung cancer to defining NSCLC on the basis of molecular subsets. KRAS mutations represent the most common molecular change in NSCLC. The presence of KRAS mutation has been shown to be associated with a poor prognosis in NSCLC, but this is of little clinical utility. More important is determining the clinical utility of KRAS mutational analysis for predicting benefit of chemotherapy, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), anti-EGFR monoclonal antibodies, or other novel therapeutics. Current data does not support the routine use of KRAS mutational analysis for predicting chemotherapy benefit. Additionally, there was significant interest in using KRAS status to select patients for EGFR TKI and anti-EGFR monoclonal antibodies. However, the EGFR mutational status has demonstrated significant predictive value in the selection of patients for EGFR TKI therapy and is now the preferred test. An association between KRAS mutational status and benefit of anti-EGFR monoclonal antibodies has not been demonstrated in NSCLC. Here we review, in the context of NSCLC, the underlying biology of KRAS mutations, the predictive value of KRAS mutations for therapeutic intervention, and the integration of KRAS mutational testing into our current clinical paradigms.

  7. Frequency and distribution of Notch mutations in tumor cell lines

    International Nuclear Information System (INIS)

    Mutvei, Anders Peter; Fredlund, Erik; Lendahl, Urban

    2015-01-01

    Deregulated Notch signaling is linked to a variety of tumors and it is therefore important to learn more about the frequency and distribution of Notch mutations in a tumor context. In this report, we use data from the recently developed Cancer Cell Line Encyclopedia to assess the frequency and distribution of Notch mutations in a large panel of cancer cell lines in silico. Our results show that the mutation frequency of Notch receptor and ligand genes is at par with that for established oncogenes and higher than for a set of house-keeping genes. Mutations were found across all four Notch receptor genes, but with notable differences between protein domains, mutations were for example more prevalent in the regions encoding the LNR and PEST domains in the Notch intracellular domain. Furthermore, an in silico estimation of functional impact showed that deleterious mutations cluster to the ligand-binding and the intracellular domains of NOTCH1. For most cell line groups, the mutation frequency of Notch genes is higher than in associated primary tumors. Our results shed new light on the spectrum of Notch mutations after in vitro culturing of tumor cells. The higher mutation frequency in tumor cell lines indicates that Notch mutations are associated with a growth advantage in vitro, and thus may be considered to be driver mutations in a tumor cell line context. The online version of this article (doi:10.1186/s12885-015-1278-x) contains supplementary material, which is available to authorized users

  8. Skin prick test reactivity to aeroallergens by filaggrin mutation status

    DEFF Research Database (Denmark)

    Hougaard, M G; Johansen, J D; Linneberg, A

    2014-01-01

    BACKGROUND: Studies have shown that filaggrin gene (FLG) mutations are positively associated with sensitization to aero allergens. We hypothesized that FLG mutations would also have an effect on the mean size of positive skin prick test (SPT) reactions as well as the number of positive reactions....... OBJECTIVE: To investigate the effect of FLG mutations on the mean size and the number of positive SPT reactions, as well as the association with positive specific IgE. METHODS: A random sample of 3335 adults from the general population in Denmark was genotyped for the R501X and 2282del4 mutations in the FLG...... mutations alone are insufficient to cause secondary sensitization to allergens. The positive association seen in patients must be explained by a combination of further barrier abnormality caused by dermatitis as well as increased allergen exposure....

  9. Sickle cell test

    Science.gov (United States)

    ... cell anemia Sickle cell trait Iron deficiency or blood transfusions within the past 3 months can cause a " ... slight risk any time the skin is broken) Alternative Names Sickledex; Hgb S test Images Red blood cells, sickle cell Red blood cells, multiple sickle ...

  10. Mutation dynamics and fitness effects followed in single cells.

    Science.gov (United States)

    Robert, Lydia; Ollion, Jean; Robert, Jerome; Song, Xiaohu; Matic, Ivan; Elez, Marina

    2018-03-16

    Mutations have been investigated for more than a century but remain difficult to observe directly in single cells, which limits the characterization of their dynamics and fitness effects. By combining microfluidics, time-lapse imaging, and a fluorescent tag of the mismatch repair system in Escherichia coli , we visualized the emergence of mutations in single cells, revealing Poissonian dynamics. Concomitantly, we tracked the growth and life span of single cells, accumulating ~20,000 mutations genome-wide over hundreds of generations. This analysis revealed that 1% of mutations were lethal; nonlethal mutations displayed a heavy-tailed distribution of fitness effects and were dominated by quasi-neutral mutations with an average cost of 0.3%. Our approach has enabled the investigation of single-cell individuality in mutation rate, mutation fitness costs, and mutation interactions. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  11. Population genetic testing for cancer susceptibility: founder mutations to genomes.

    Science.gov (United States)

    Foulkes, William D; Knoppers, Bartha Maria; Turnbull, Clare

    2016-01-01

    The current standard model for identifying carriers of high-risk mutations in cancer-susceptibility genes (CSGs) generally involves a process that is not amenable to population-based testing: access to genetic tests is typically regulated by health-care providers on the basis of a labour-intensive assessment of an individual's personal and family history of cancer, with face-to-face genetic counselling performed before mutation testing. Several studies have shown that application of these selection criteria results in a substantial proportion of mutation carriers being missed. Population-based genetic testing has been proposed as an alternative approach to determining cancer susceptibility, and aims for a more-comprehensive detection of mutation carriers. Herein, we review the existing data on population-based genetic testing, and consider some of the barriers, pitfalls, and challenges related to the possible expansion of this approach. We consider mechanisms by which population-based genetic testing for cancer susceptibility could be delivered, and suggest how such genetic testing might be integrated into existing and emerging health-care structures. The existing models of genetic testing (including issues relating to informed consent) will very likely require considerable alteration if the potential benefits of population-based genetic testing are to be fully realized.

  12. Automation of diagnostic genetic testing: mutation detection by cyclic minisequencing.

    Science.gov (United States)

    Alagrund, Katariina; Orpana, Arto K

    2014-01-01

    The rising role of nucleic acid testing in clinical decision making is creating a need for efficient and automated diagnostic nucleic acid test platforms. Clinical use of nucleic acid testing sets demands for shorter turnaround times (TATs), lower production costs and robust, reliable methods that can easily adopt new test panels and is able to run rare tests in random access principle. Here we present a novel home-brew laboratory automation platform for diagnostic mutation testing. This platform is based on the cyclic minisequecing (cMS) and two color near-infrared (NIR) detection. Pipetting is automated using Tecan Freedom EVO pipetting robots and all assays are performed in 384-well micro plate format. The automation platform includes a data processing system, controlling all procedures, and automated patient result reporting to the hospital information system. We have found automated cMS a reliable, inexpensive and robust method for nucleic acid testing for a wide variety of diagnostic tests. The platform is currently in clinical use for over 80 mutations or polymorphisms. Additionally to tests performed from blood samples, the system performs also epigenetic test for the methylation of the MGMT gene promoter, and companion diagnostic tests for analysis of KRAS and BRAF gene mutations from formalin fixed and paraffin embedded tumor samples. Automation of genetic test reporting is found reliable and efficient decreasing the work load of academic personnel.

  13. Biochip-Based Detection of KRAS Mutation in Non-Small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Barbara Ziegler

    2011-11-01

    Full Text Available This study is aimed at evaluating the potential of a biochip assay to sensitively detect KRAS mutation in DNA from non-small cell lung cancer (NSCLC tissue samples. The assay covers 10 mutations in codons 12 and 13 of the KRAS gene, and is based on mutant-enriched PCR followed by reverse-hybridization of biotinylated amplification products to an array of sequence-specific probes immobilized on the tip of a rectangular plastic stick (biochip. Biochip hybridization identified 17 (21% samples to carry a KRAS mutation of which 16 (33% were adenocarcinomas and 1 (3% was a squamous cell carcinoma. All mutations were confirmed by DNA sequencing. Using 10 ng of starting DNA, the biochip assay demonstrated a detection limit of 1% mutant sequence in a background of wild-type DNA. Our results suggest that the biochip assay is a sensitive alternative to protocols currently in use for KRAS mutation testing on limited quantity samples.

  14. Mitochondrial DNA mutations in human tumor cells

    OpenAIRE

    LI, HUI; HONG, ZE-HUI

    2012-01-01

    Mitochondria play significant roles in cellular energy metabolism, free radical generation and apoptosis. The dysfunction of mitochondria is correlated with the origin and progression of tumors; thus, mutations in the mitochondrial genome that affect mitochondrial function may be one of the causal factors of tumorigenesis. Although the role of mitochondrial DNA (mtDNA) mutations in carcinogenesis has been investigated extensively by various approaches, the conclusions remain controversial to ...

  15. The Mutational Landscape of Circulating Tumor Cells in Multiple Myeloma

    Directory of Open Access Journals (Sweden)

    Yuji Mishima

    2017-04-01

    Full Text Available The development of sensitive and non-invasive “liquid biopsies” presents new opportunities for longitudinal monitoring of tumor dissemination and clonal evolution. The number of circulating tumor cells (CTCs is prognostic in multiple myeloma (MM, but there is little information on their genetic features. Here, we have analyzed the genomic landscape of CTCs from 29 MM patients, including eight cases with matched/paired bone marrow (BM tumor cells. Our results show that 100% of clonal mutations in patient BM were detected in CTCs and that 99% of clonal mutations in CTCs were present in BM MM. These include typical driver mutations in MM such as in KRAS, NRAS, or BRAF. These data suggest that BM and CTC samples have similar clonal structures, as discordances between the two were restricted to subclonal mutations. Accordingly, our results pave the way for potentially less invasive mutation screening of MM patients through characterization of CTCs.

  16. Automated objective determination of percentage of malignant nuclei for mutation testing.

    Science.gov (United States)

    Viray, Hollis; Coulter, Madeline; Li, Kevin; Lane, Kristin; Madan, Aruna; Mitchell, Kisha; Schalper, Kurt; Hoyt, Clifford; Rimm, David L

    2014-01-01

    Detection of DNA mutations in tumor tissue can be a critical companion diagnostic test before prescription of a targeted therapy. Each method for detection of these mutations is associated with an analytic sensitivity that is a function of the percentage of tumor cells present in the specimen. Currently, tumor cell percentage is visually estimated resulting in an ordinal and highly variant result for a biologically continuous variable. We proposed that this aspect of DNA mutation testing could be standardized by developing a computer algorithm capable of accurately determining the percentage of malignant nuclei in an image of a hematoxylin and eosin-stained tissue. Using inForm software, we developed an algorithm, to calculate the percentage of malignant cells in histologic specimens of colon adenocarcinoma. A criterion standard was established by manually counting malignant and benign nuclei. Three pathologists also estimated the percentage of malignant nuclei in each image. Algorithm #9 had a median deviation from the criterion standard of 5.4% on the training set and 6.2% on the validation set. Compared with pathologist estimation, Algorithm #9 showed a similar ability to determine percentage of malignant nuclei. This method represents a potential future tool to assist in determining the percent of malignant nuclei present in a tissue section. Further validation of this algorithm or an improved algorithm may have value to more accurately assess percentage of malignant cells for companion diagnostic mutation testing.

  17. Rapid BRAF mutation tests in patients with advanced melanoma : Comparison of immunohistochemistry, Droplet Digital PCR, and the Idylla Mutation Platform

    NARCIS (Netherlands)

    Bisschop, Cornelis; Ter Elst, Arja; Bosman, Lisette J; Platteel, Inge; Jalving, Mathilde; van den Berg, Anke; Diepstra, Arjan; van Hemel, Bettien; Diercks, Gilles F H; Hospers, Geke A P; Schuuring, Ed

    BRAF mutational testing has become a common practice in the diagnostic process of patients with advanced melanoma. Although time-consuming, DNA sequencing techniques are the current gold standard for mutational testing. However, in certain clinical situations, a rapid test result is required. In

  18. Mitochondrial DNA mutations in mutator mice confer respiration defects and B-cell lymphoma development.

    Directory of Open Access Journals (Sweden)

    Takayuki Mito

    Full Text Available Mitochondrial DNA (mtDNA mutator mice are proposed to express premature aging phenotypes including kyphosis and hair loss (alopecia due to their carrying a nuclear-encoded mtDNA polymerase with a defective proofreading function, which causes accelerated accumulation of random mutations in mtDNA, resulting in expression of respiration defects. On the contrary, transmitochondrial mito-miceΔ carrying mtDNA with a large-scale deletion mutation (ΔmtDNA also express respiration defects, but not express premature aging phenotypes. Here, we resolved this discrepancy by generating mtDNA mutator mice sharing the same C57BL/6J (B6J nuclear background with that of mito-miceΔ. Expression patterns of premature aging phenotypes are very close, when we compared between homozygous mtDNA mutator mice carrying a B6J nuclear background and selected mito-miceΔ only carrying predominant amounts of ΔmtDNA, in their expression of significant respiration defects, kyphosis, and a short lifespan, but not the alopecia. Therefore, the apparent discrepancy in the presence and absence of premature aging phenotypes in mtDNA mutator mice and mito-miceΔ, respectively, is partly the result of differences in the nuclear background of mtDNA mutator mice and of the broad range of ΔmtDNA proportions of mito-miceΔ used in previous studies. We also provided direct evidence that mtDNA abnormalities in homozygous mtDNA mutator mice are responsible for respiration defects by demonstrating the co-transfer of mtDNA and respiration defects from mtDNA mutator mice into mtDNA-less (ρ(0 mouse cells. Moreover, heterozygous mtDNA mutator mice had a normal lifespan, but frequently developed B-cell lymphoma, suggesting that the mtDNA abnormalities in heterozygous mutator mice are not sufficient to induce a short lifespan and aging phenotypes, but are able to contribute to the B-cell lymphoma development during their prolonged lifespan.

  19. BRAF mutation testing in solid tumors: a methodological comparison.

    Science.gov (United States)

    Weyant, Grace W; Wisotzkey, Jeffrey D; Benko, Floyd A; Donaldson, Keri J

    2014-09-01

    Solid tumor genotyping has become standard of care for the characterization of proto-oncogene mutational status, which has traditionally been accomplished with Sanger sequencing. However, companion diagnostic assays and comparable laboratory-developed tests are becoming increasingly popular, such as the cobas 4800 BRAF V600 Mutation Test and the INFINITI KRAS-BRAF assay, respectively. This study evaluates and validates the analytical performance of the INFINITI KRAS-BRAF assay and compares concordance of BRAF status with two reference assays, the cobas test and Sanger sequencing. DNA extraction from FFPE tissue specimens was performed followed by multiplex PCR amplification and fluorescent label incorporation using allele-specific primer extension. Hybridization to a microarray, signal detection, and analysis were then performed. The limits of detection were determined by testing dilutions of mutant BRAF alleles within wild-type background DNA, and accuracy was calculated based on these results. The INFINITI KRAS-BRAF assay produced 100% concordance with the cobas test and Sanger sequencing and had sensitivity equivalent to the cobas assay. The INFINITI assay is repeatable with at least 95% accuracy in the detection of mutant and wild-type BRAF alleles. These results confirm that the INFINITI KRAS-BRAF assay is comparable to traditional sequencing and the Food and Drug Administration-approved companion diagnostic assay for the detection of BRAF mutations. Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  20. Epidermal growth factor receptor tyrosine kinase (EGFR-TK) mutation testing in adults with locally advanced or metastatic non-small cell lung cancer : a systematic review and cost-effectiveness analysis

    NARCIS (Netherlands)

    Westwood, Marie; Joore, Manuela; Whiting, Penny; van Asselt, Thea; Ramaekers, Bram; Armstrong, Nigel; Misso, Kate; Severens, Johan; Kleijnen, Jos

    BACKGROUND: Non-small cell lung cancer (NSCLC) is the most common form of lung cancer. Some epidermal growth factor receptor tyrosine kinase (EGFR-TK) mutations make tumours responsive to treatment with EGFR-TK inhibitors (EGFR-TKIs) but less responsive to treatment with standard chemotherapy.

  1. Epidermal growth factor receptor tyrosine kinase (EGFR-TK) mutation testing in adults with locally advanced or metastatic non-small cell lung cancer: A systematic review and cost-effectiveness analysis

    NARCIS (Netherlands)

    M. Westwood (Marie); M.A. Joore (Manuela); P. Whiting (Penny); T. van Asselt (Thea); B.L.T. Ramaekers (Bram); N. Armstrong (Nigel); K. Misso (Kate); J.L. Severens (Hans); J. Kleijnen (Jos)

    2014-01-01

    markdownabstract__Abstract__ Background: Non-small cell lung cancer (NSCLC) is the most common form of lung cancer. Some epidermal growth factor receptor tyrosine kinase (EGFR-TK) mutations make tumours responsive to treatment with EGFR-TK inhibitors (EGFR-TKIs) but less responsive to treatment

  2. Teaching the fluctuation test in silico by using mutate: a program to distinguish between the adaptive and spontaneous mutation hypotheses.

    Science.gov (United States)

    Carvajal-Rodríguez, Antonio

    2012-07-01

    Mutate is a program developed for teaching purposes to impart a virtual laboratory class for undergraduate students of Genetics in Biology. The program emulates the so-called fluctuation test whose aim is to distinguish between spontaneous and adaptive mutation hypotheses in bacteria. The plan is to train students in certain key multidisciplinary aspects of current genetics such as sequence databases, DNA mutations, and hypothesis testing, while introducing the fluctuation test. This seminal experiment was originally performed studying Escherichia coli resistance to the infection by bacteriophage T1. The fluctuation test initiated the modern bacterial genetics that 25 years later ushered in the era of the recombinant DNA. Nowadays we know that some deletions in fhuA, the gene responsible for E. coli membrane receptor of T1, could cause the E. coli resistance to this phage. For the sake of simplicity, we will introduce the assumption that a single mutation generates the resistance to T1. During the practical, the students use the program to download some fhuA gene sequences, manually introduce some stop codon mutations, and design a fluctuation test to obtain data for distinguishing between preadaptative (spontaneous) and induced (adaptive) mutation hypotheses. The program can be launched from a browser or, if preferred, its executable file can be downloaded from http://webs.uvigo.es/acraaj/MutateWeb/Mutate.html. It requires the Java 5.0 (or higher) Runtime Environment (freely available at http://www.java.com). Copyright © 2012 Wiley Periodicals, Inc.

  3. The rate of spontaneous mutations in human myeloid cells

    International Nuclear Information System (INIS)

    Araten, David J.; Krejci, Ondrej; DiTata, Kimberly; Wunderlich, Mark; Sanders, Katie J.; Zamechek, Leah; Mulloy, James C.

    2013-01-01

    Highlights: • We provide the first measurement of the mutation rate (μ) in human myeloid cells. • μ is measured to be 3.6–23 × 10 −7 per cell division. • The AML-ETO and MLL-AF9 fusions do not seem to increase μ. • Cooperating mutations in NRAS, FLT3 and p53 not seem to increase μ. • Hypermutability may be required to explain leukemogenesis. - Abstract: The mutation rate (μ) is likely to be a key parameter in leukemogenesis, but historically, it has been difficult to measure in humans. The PIG-A gene has some advantages for the detection of spontaneous mutations because it is X-linked, and therefore only one mutation is required to disrupt its function. Furthermore, the PIG-A-null phenotype is readily detected by flow cytometry. Using PIG-A, we have now provided the first in vitro measurement of μ in myeloid cells, using cultures of CD34+ cells that are transduced with either the AML-ETO or the MLL-AF9 fusion genes and expanded with cytokines. For the AML-ETO cultures, the median μ value was ∼9.4 × 10 −7 (range ∼3.6–23 × 10 −7 ) per cell division. In contrast, few spontaneous mutations were observed in the MLL-AF9 cultures. Knockdown of p53 or introduction of mutant NRAS or FLT3 alleles did not have much of an effect on μ. Based on these data, we provide a model to predict whether hypermutability must occur in the process of leukemogenesis

  4. KIT mutation analysis in mast cell neoplasms

    DEFF Research Database (Denmark)

    Arock, M; Sotlar, K; Akin, C

    2015-01-01

    mutations in patients with mastocytosis at diagnosis and during follow-up with sufficient precision and sensitivity in daily practice. In addition, we provide recommendations for sampling and storage of diagnostic material as well as a robust diagnostic algorithm. Using highly sensitive assays, KIT D816V...... can be detected in peripheral blood leukocytes from most patients with systemic mastocytosis (SM) that is a major step forward in screening and SM diagnosis. In addition, the KIT D816V allele burden can be followed quantitatively during the natural course or during therapy. Our recommendations should...... greatly facilitate diagnostic and follow-up investigations in SM in daily practice as well as in clinical trials. In addition, the new tools and algorithms proposed should lead to a more effective screen, early diagnosis of SM and help to avoid unnecessary referrals....

  5. TET2 mutations in B cells of patients affected by angioimmunoblastic T-cell lymphoma.

    Science.gov (United States)

    Schwartz, Friederike H; Cai, Qian; Fellmann, Eva; Hartmann, Sylvia; Mäyränpää, Mikko I; Karjalainen-Lindsberg, Marja-Liisa; Sundström, Christer; Scholtysik, René; Hansmann, Martin-Leo; Küppers, Ralf

    2017-06-01

    Angioimmunoblastic T-cell lymphomas (AITLs) frequently carry mutations in the TET2 and IDH2 genes. TET2 mutations represent early genetic lesions as they had already been detected in haematopoietic precursor cells of AITL patients. We show by analysis of whole-tissue sections and microdissected PD1 + cells that the frequency of TET2-mutated AITL is presumably even higher than reported (12/13 cases in our collection; 92%). In two-thirds of informative AITLs (6/9), a fraction of B cells was also TET2-mutated. Investigation of four AITLs by TET2 and IGHV gene sequencing of single microdissected B cells showed that between 10% and 60% of polyclonal B cells in AITL lymph nodes harboured the identical TET2 mutations of the respective T-cell lymphoma clone. Thus, TET2-mutated haematopoietic precursor cells in AITL patients not only give rise to the T-cell lymphoma but also generate a large population of mutated mature B cells. Future studies will show whether this is a reason why AITL patients frequently also develop B-cell lymphomas. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  6. Teaching the Fluctuation Test "In Silico" by Using Mutate: A Program to Distinguish between the Adaptive and Spontaneous Mutation Hypotheses

    Science.gov (United States)

    Carvajal-Rodriguez, Antonio

    2012-01-01

    Mutate is a program developed for teaching purposes to impart a virtual laboratory class for undergraduate students of Genetics in Biology. The program emulates the so-called fluctuation test whose aim is to distinguish between spontaneous and adaptive mutation hypotheses in bacteria. The plan is to train students in certain key multidisciplinary…

  7. Somatic activating ARAF mutations in Langerhans cell histiocytosis

    NARCIS (Netherlands)

    Nelson, David S.; Quispel, Willemijn; Badalian-Very, Gayane; van Halteren, Astrid G. S.; van den Bos, Cor; Bovée, Judith V. M. G.; Tian, Sara Y.; van Hummelen, Paul; Ducar, Matthew; MacConaill, Laura E.; Egeler, R. Maarten; Rollins, Barrett J.

    2014-01-01

    The extracellular signal-regulated kinase (ERK) signaling pathway is activated in Langerhans cell histiocytosis (LCH) histiocytes, but only 60% of cases carry somatic activating mutations of BRAF. To identify other genetic causes of ERK pathway activation, we performed whole exome sequencing on

  8. Accumulation of neutral mutations in growing cell colonies with competition.

    Science.gov (United States)

    Sorace, Ron; Komarova, Natalia L

    2012-12-07

    Neutral mutations play an important role in many biological processes including cancer initiation and progression, the generation of drug resistance in bacterial and viral diseases as well as cancers, and the development of organs in multicellular organisms. In this paper we study how neutral mutants are accumulated in nonlinearly growing colonies of cells subject to growth constraints such as crowding or lack of resources. We investigate different types of growth control which range from "division-controlled" to "death-controlled" growth (and various mixtures of both). In division-controlled growth, the burden of handling overcrowding lies with the process of cell-divisions, the divisions slow down as the carrying capacity is approached. In death-controlled growth, it is death rate that increases to slow down expansion. We show that division-controlled growth minimizes the number of accumulated mutations, and death-controlled growth corresponds to the maximum number of mutants. We check that these results hold in both deterministic and stochastic settings. We further develop a general (deterministic) theory of neutral mutations and achieve an analytical understanding of the mutant accumulation in colonies of a given size in the absence of back-mutations. The long-term dynamics of mutants in the presence of back-mutations is also addressed. In particular, with equal forward- and back-mutation rates, if division-controlled and a death-controlled types are competing for space and nutrients, cells obeying division-controlled growth will dominate the population. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. Utility of bronchial lavage fluids for epithelial growth factor receptor mutation assay in lung cancer patients: Comparison between cell pellets, cell blocks and matching tissue specimens

    Science.gov (United States)

    Asaka, Shiho; Yoshizawa, Akihiko; Nakata, Rie; Negishi, Tatsuya; Yamamoto, Hiroshi; Shiina, Takayuki; Shigeto, Shohei; Matsuda, Kazuyuki; Kobayashi, Yukihiro; Honda, Takayuki

    2018-01-01

    The detection of epidermal growth factor receptor (EGFR) mutations is necessary for the selection of suitable patients with non-small cell lung cancer (NSCLC) for treatment with EGFR tyrosine kinase inhibitors. Cytology specimens are known to be suitable for EGFR mutation detection, although tissue specimens should be prioritized; however, there are limited studies that examine the utility of bronchial lavage fluid (BLF) in mutation detection. The purpose of the present study was to investigate the utility of BLF specimens for the detection of EGFR mutations using a conventional quantitative EGFR polymerase chain reaction (PCR) assay. Initially, quantification cycle (Cq) values of cell pellets, cell-free supernatants and cell blocks obtained from three series of 1% EGFR mutation-positive lung cancer cell line samples were compared for mutation detection. In addition, PCR analysis of BLF specimens obtained from 77 consecutive NSCLC patients, detecting EGFR mutations was validated, and these results were compared with those for the corresponding formalin-fixed paraffin-embedded (FFPE) tissue specimens obtained by surgical resection or biopsy of 49 of these patients. The Cq values for mutation detection were significantly lower in the cell pellet group (average, 29.58) compared with the other groups, followed by those in cell-free supernatants (average, 34.15) and in cell blocks (average, 37.12) for all three series (P<0.05). Mutational status was successfully analyzed in 77 BLF specimens, and the results obtained were concordant with those of the 49 matching FFPE tissue specimens. Notably, EGFR mutations were even detected in 10 cytological specimens that contained insufficient tumor cells. EGFR mutation testing with BLF specimens is therefore a useful and reliable method, particularly when sufficient cancer cells are not obtained. PMID:29399190

  10. Effect of dabrafenib on melanoma cell lines harbouring the BRAFV600D/R mutations

    Directory of Open Access Journals (Sweden)

    Gentilcore Giusy

    2013-01-01

    Full Text Available Abstract Background Conventional therapeutic agents are largely unsatisfactory into the treatment of malignant melanoma. Recently, an innovative approach based on inhibitors of the mutated BRAF gene (which represents the most prevalent alteration in melanoma patients appears very promising from the clinical point of view. On this regard, a new compound, dabrafenib (GSK2118436, has been demonstrated to be effective in patients carrying the BRAFV600E/K mutations. We here tested dabrafenib for its capability to inhibit cell growth on primary melanoma cell lines, established from patients' tumour tissues and carrying the BRAFV600D/R mutations. Methods Three melanoma cell lines were tested: M257 wild-type BRAF, LCP BRAFV600R and WM266 BRAFV600D. The MTT assays were performed using standardized approaches. To evaluate the inhibition of MAPK pathway and the consequent inhibition of cellular proliferation, the phosphorylation of ERK was examined by Western Blot analysis performed on total protein extracts from cell lines after treatment with dabrafenib. Results Our experiments demonstrated an effective action of Dabrafenib (GSK2118436 and the inhibition of MAPK pathway in melanoma cell lines carrying BRAFV600D/R mutations. Conclusion These results could be helpful to enlarge the number of melanoma patients who may benefit of a more effective targeted treatment.

  11. Detection and Analysis of EGFR and KRAS Mutations 
in the Patients with Lung Squamous Cell Carcinomas

    Directory of Open Access Journals (Sweden)

    Hui ZHANG

    2015-10-01

    Full Text Available Background and objective Activating mutations in epidermal growth factor receptor (EGFR and KRAS are important markers in non-small cell lung cancer. However, EGFR and KRAS gene mutations in lung squamous cell carcinoma are rarely reported. The aim of this study was to analyze EGFR and KRAS gene mutation rate and their relationship with clinical features in patients with lung squamous cell carcinomas. Methods A total of 139 patients undergoing treatment for naïve lung squamous cell carcinomas with tumor tissue samples available for testing were recruited. EGFR and KRAS mutation statuses of the tumor samples were detected using a mutant enriched liquid chip. Results Of the 139 cases of lung squamous cell carcinoma, EGFR mutations were detected in 25 cases (18%, KRAS mutations were detected in 7 cases (5%, and the presence of both EGFR and KRAS mutations was detected in 1 case (0.7%. EGFR mutations occurred more often in females than in males (33.3% vs 16.5% and in patients that never smoked than in those who smoke (29.6% vs 16.1%. However, the difference did not reach statistical significance (P>0.05. No significant differences were observed in age, stage, and different biopsy type. KRAS mutations occurred more often in males than in females (5.5% vs 0%, but the difference did not reach statistical significance (P>0.05. No significant differences were observed in age, stage, different biopsy type, and smoking status (P>0.05. Conclusion EGFR and KRAS mutations were low in lung squamous cell carcinomas, and had no significant correlation with clinical features. Before using tyrosine kinase inhibitor targeted therapy, EGFR and KRAS mutations should be detected in patients with lung squamous cell carcinomas.

  12. Cytotoxicity Testing: Cell Experiments

    Science.gov (United States)

    Grünert, Renate; Westendorf, Aron; Buczkowska, Magdalena; Hänsch, Mareike; Grüunert, Sybil; Bednarski, Patrick J.

    Screening for new anticancer agents has traditionally been done with in vitro cell culture methods. Even in the genomic era of target-driven drug design, screening for cytotoxic activity is still a standard tool in the search for new anticancer agents, especially if the mode of action of a substance is not yet known. A wide variety of cell culture methods with unique end-points are available for testing the anticancer potential of a substance. Each has its advantages and disadvantages, which must be weighed in the decision to use a particular method. Often several complementary methods are used to gain information on the mode of action of a substance.

  13. Quantum dots immunofluorescence histochemical detection of EGFR gene mutations in the non-small cell lung cancers using mutation-specific antibodies

    Directory of Open Access Journals (Sweden)

    Qu YG

    2014-12-01

    Full Text Available Yan-Gang Qu,1 Qian Zhang,2 Qi Pan,3 Xian-Da Zhao,4 Yan-Hua Huang,2 Fu-Chun Chen,3 Hong-Lei Chen41Department of Pathology, The Central Hospital of Enshi Autonomous Prefecture, Enshi, 2Department of Molecular Pathology, Wuhan Nano Tumor Diagnosis Engineering Research Center, Wuhan, Hubei, People’s Republic of China; 3Department of Thoracosurgery, Traditional Chinese Medical Hospital of Wenling, Wenling, Zhejiang, People’s Republic of China; 4Department of Pathology, School of Basic Medical Science, Wuhan University, Wuhan, Hubei, People’s Republic of ChinaBackground: Epidermal growth factor receptor (EGFR mutation status plays an important role in therapeutic decision making for non-small cell lung cancer (NSCLC patients. Since EGFR mutation-specific antibodies (E746-A750del and L858R have been developed, EGFR mutation detection by immunohistochemistry (IHC is a suitable screening test. On this basis, we want to establish a new screening test, quantum dots immunofluorescence histochemistry (QDs-IHC, to assess EGFR gene mutation in NSCLC tissues, and we compared it to traditional IHC and amplification refractory mutation system (ARMS.Materials and methods: EGFR gene mutations were detected by QDs-IHC, IHC, and ADx-ARMS in 65 cases of NSCLC composed of 55 formalin-fixed, paraffin-embedded specimens and ten pleural effusion cell blocks, including 13 squamous cell carcinomas, two adenosquamous carcinomas, and 50 adenocarcinomas.Results: Positive rates of EGFR gene mutations detected by QDs-IHC, IHC, and ADx-ARMS were 40.0%, 36.9%, and 46.2%, respectively, in 65 cases of NSCLC patients. The sensitivity of QDs-IHC when detecting EGFR mutations, as compared to ADx-ARMS, was 86.7% (26/30; the specificity for both antibodies was 100.0% (26/26. IHC sensitivity was 80.0% (24/30 and the specificity was 92.31% (24/26. When detecting EGFR mutations, QDs-IHC and ADx-ARMS had perfect consistency (κ=0.882; P<0.01. Excellent agreement was observed

  14. GluD1 is a common altered player in neuronal differentiation from both MECP2-mutated and CDKL5-mutated iPS cells.

    Science.gov (United States)

    Livide, Gabriella; Patriarchi, Tommaso; Amenduni, Mariangela; Amabile, Sonia; Yasui, Dag; Calcagno, Eleonora; Lo Rizzo, Caterina; De Falco, Giulia; Ulivieri, Cristina; Ariani, Francesca; Mari, Francesca; Mencarelli, Maria Antonietta; Hell, Johannes Wilhelm; Renieri, Alessandra; Meloni, Ilaria

    2015-02-01

    Rett syndrome is a monogenic disease due to de novo mutations in either MECP2 or CDKL5 genes. In spite of their involvement in the same disease, a functional interaction between the two genes has not been proven. MeCP2 is a transcriptional regulator; CDKL5 encodes for a kinase protein that might be involved in the regulation of gene expression. Therefore, we hypothesized that mutations affecting the two genes may lead to similar phenotypes by dysregulating the expression of common genes. To test this hypothesis we used induced pluripotent stem (iPS) cells derived from fibroblasts of one Rett patient with a MECP2 mutation (p.Arg306Cys) and two patients with mutations in CDKL5 (p.Gln347Ter and p.Thr288Ile). Expression profiling was performed in CDKL5-mutated cells and genes of interest were confirmed by real-time RT-PCR in both CDKL5- and MECP2-mutated cells. The only major change in gene expression common to MECP2- and CDKL5-mutated cells was for GRID1, encoding for glutamate D1 receptor (GluD1), a member of the δ-family of ionotropic glutamate receptors. GluD1 does not form AMPA or NMDA glutamate receptors. It acts like an adhesion molecule by linking the postsynaptic and presynaptic compartments, preferentially inducing the inhibitory presynaptic differentiation of cortical neurons. Our results demonstrate that GRID1 expression is downregulated in both MECP2- and CDKL5-mutated iPS cells and upregulated in neuronal precursors and mature neurons. These data provide novel insights into disease pathophysiology and identify possible new targets for therapeutic treatment of Rett syndrome.

  15. Detection of Ultra-Rare Mitochondrial Mutations in Breast Stem Cells by Duplex Sequencing.

    Directory of Open Access Journals (Sweden)

    Eun Hyun Ahn

    Full Text Available Long-lived adult stem cells could accumulate non-repaired DNA damage or mutations that increase the risk of tumor formation. To date, studies on mutations in stem cells have concentrated on clonal (homoplasmic mutations and have not focused on rarely occurring stochastic mutations that may accumulate during stem cell dormancy. A major challenge in investigating these rare mutations is that conventional next generation sequencing (NGS methods have high error rates. We have established a new method termed Duplex Sequencing (DS, which detects mutations with unprecedented accuracy. We present a comprehensive analysis of mitochondrial DNA mutations in human breast normal stem cells and non-stem cells using DS. The vast majority of mutations occur at low frequency and are not detectable by NGS. The most prevalent point mutation types are the C>T/G>A and A>G/T>C transitions. The mutations exhibit a strand bias with higher prevalence of G>A, T>C, and A>C mutations on the light strand of the mitochondrial genome. The overall rare mutation frequency is significantly lower in stem cells than in the corresponding non-stem cells. We have identified common and unique non-homoplasmic mutations between non-stem and stem cells that include new mutations which have not been reported previously. Four mutations found within the MT-ND5 gene (m.12684G>A, m.12705C>T, m.13095T>C, m.13105A>G are present in all groups of stem and non-stem cells. Two mutations (m.8567T>C, m.10547C>G are found only in non-stem cells. This first genome-wide analysis of mitochondrial DNA mutations may aid in characterizing human breast normal epithelial cells and serve as a reference for cancer stem cell mutation profiles.

  16. Sickle Cell Tests

    Science.gov (United States)

    ... Disorders Fibromyalgia Food and Waterborne Illness Fungal Infections Gout Graves Disease Guillain-Barré Syndrome Hashimoto Thyroiditis Heart ... predominant hemoglobin shortly after birth. Mutations in the genes that code for the production of hemoglobin can ...

  17. Relationship of JAK2V617F gene mutation with cell proliferation and coagulation function in myeloproliferative neoplasms

    Directory of Open Access Journals (Sweden)

    Xiao-Nan Zhang

    2017-05-01

    Full Text Available Objective: To study the relationship of JAK2V617F gene mutation with cell proliferation and coagulation function in myeloproliferative neoplasms. Methods: Patients who were diagnosed with BCR-ABL-negative myeloproliferative neoplasms in Anyang District Hospital between June 2014 and August 2016 were selected, JAK2V617F gene mutation was detected, and according to the test results, the patients were divided into mutation-positive group and mutation-negative group. The expression of JAK2/STATs signaling pathway molecules and cell proliferation genes in bone marrow fluid as well as the coagulation function indexes in peripheral blood were detected. Results: p-JAK2, p-STAT3, p-STAT5, Survivin, C-myc, CyclinD1 and ASXL1 protein expression in myeloproliferative neoplasms of mutation-positive group were significantly higher than those of mutation-negative group, and peripheral blood PT and APTT levels were significantly lower than those of mutation-negative group while TT and FIB levels were not significantly different from those of mutation-negative group. Conclusion: JAK2V617F gene mutation in myeloproliferative neoplasms can promote the cell proliferation and cause the hypercoagulable state.

  18. Detection of EGFR mutations with mutation-specific antibodies in stage IV non-small-cell lung cancer

    Directory of Open Access Journals (Sweden)

    Viteri Santiago

    2010-12-01

    Full Text Available Abstract Background Immunohistochemistry (IHC with mutation-specific antibodies may be an ancillary method of detecting EGFR mutations in lung cancer patients. Methods EGFR mutation status was analyzed by DNA assays, and compared with IHC results in five non-small-cell lung cancer (NSCLC cell lines and tumor samples from 78 stage IV NSCLC patients. Results IHC correctly identified del 19 in the H1650 and PC9 cell lines, L858R in H1975, and wild-type EGFR in H460 and A549, as well as wild-type EGFR in tumor samples from 22 patients. IHC with the mAb against EGFR with del 19 was highly positive for the protein in all 17 patients with a 15-bp (ELREA deletion in exon 19, whereas in patients with other deletions, IHC was weakly positive in 3 cases and negative in 9 cases. IHC with the mAb against the L858R mutation showed high positivity for the protein in 25/27 (93% patients with exon 21 EGFR mutations (all with L858R but did not identify the L861Q mutation in the remaining two patients. Conclusions IHC with mutation-specific mAbs against EGFR is a promising method for detecting EGFR mutations in NSCLC patients. However these mAbs should be validated with additional studies to clarify their possible role in routine clinical practice for screening EGFR mutations in NSCLC patients.

  19. BRCA1/2 mutation analysis in 41 ovarian cell lines reveals only one functionally deleterious BRCA1 mutation.

    LENUS (Irish Health Repository)

    Stordal, Britta

    2013-06-01

    Mutations in BRCA1\\/2 increase the risk of developing breast and ovarian cancer. Germline BRCA1\\/2 mutations occur in 8.6-13.7% of unselected epithelial ovarian cancers, somatic mutations are also frequent. BRCA1\\/2 mutated or dysfunctional cells may be sensitive to PARP inhibition by synthetic lethality. The aim of this study is to comprehensively characterise the BRCA1\\/2 status of a large panel of ovarian cancer cell lines available to the research community to assist in biomarker studies of novel drugs and in particular of PARP inhibitors. The BRCA1\\/2 genes were sequenced in 41 ovarian cell lines, mRNA expression of BRCA1\\/2 and gene methylation status of BRCA1 was also examined. The cytotoxicity of PARP inhibitors olaparib and veliparib was examined in 20 cell lines. The cell line SNU-251 has a deleterious BRCA1 mutation at 5564G > A, and is the only deleterious BRCA1\\/2 mutant in the panel. Two cell lines (UPN-251 and PEO1) had deleterious mutations as well as additional reversion mutations that restored the protein functionality. Heterozygous mutations in BRCA1\\/2 were relatively common, found in 14.6% of cell lines. BRCA1 was methylated in two cell lines (OVCAR8, A1847) and there was a corresponding decrease in gene expression. The BRCA1 methylated cell lines were more sensitive to PARP inhibition than wild-type cells. The SNU-251 deleterious mutant was more sensitive to PARP inhibition, but only in a long-term exposure to correct for its slow growth rate. Cell lines derived from metastatic disease are significantly more resistant to veliparib (2.0 fold p = 0.03) compared to those derived from primary tumours. Resistance to olaparib and veliparib was correlated Pearsons-R 0.5393, p = 0.0311. The incidence of BRCA1\\/2 deleterious mutations 1\\/41 cell lines derived from 33 different patients (3.0%) is much lower than the population incidence. The reversion mutations and high frequency of heterozygous mutations suggest that there is a selective

  20. Mutational analysis of the cell cycle inhibitor Kip1/p27 in childhood leukemia.

    Science.gov (United States)

    Markaki, E-A; Stiakaki, E; Zafiropoulos, A; Arvanitis, D A; Katzilakis, N; Dimitriou, H; Spandidos, D A; Kalmanti, M

    2006-07-01

    Cyclin-dependent kinases (CDKs) and cyclins, their regulatory subunits, govern cell-cycle progression in eukaryotic cells. Kip1/p27 is the main cyclin-dependent kinase inhibitor, which arrests cell division inhibiting G1-S transition. Kip1/p27 seems to play a critical role in the pathogenesis of several human malignancies and its lower expression has been shown to correlate with a poor prognosis in adult solid tumors. Bone marrow blasts from 49 children with leukemia, 37 acute lymphoblastic leukemia (ALL), and 12 acute myeloid leukemia (AML) were studied. Exon 3 of Kip1/p27 was amplified using the polymerase chain reaction technique (PCR). Single strand conformational polymorphism and heterodouplex analysis were performed to detect DNA sequence with altered conformations and were subsequently sequenced to document mutations. Mutations in Kip1/p27 gene were detected in 2 out of 3 T-ALL, 6 out of 12 AML patients, and only 1 out of 34 B lineage ALL cases. Although the patient groups are small, a highly significant relation of the mutation status with the type of leukemia (P = 0.0037) and the risk group according to treatment protocols (P = 0.00021) was estimated. A statistically significant difference in the white blood count was observed (P = 0.019) between the mutated and non-mutated patient groups although no statistically significant association of the mutation status with the hemoglobin and platelets values, karyotype, age, sex, disease progression, and outcome was determined. Based upon these results, the Kip1/p27 mutations should be considered for further prospective testing as an additional parameter for risk stratification and treatment of childhood leukemia. Copyright 2006 Wiley-Liss, Inc.

  1. Sensitive KIT D816V mutation analysis of blood as a diagnostic test in mastocytosis

    DEFF Research Database (Denmark)

    Kielsgaard Kristensen, Thomas; Vestergaard, Hanne; Bindslev-Jensen, Carsten

    2014-01-01

    The recent progress in sensitive KIT D816V mutation analysis suggests that mutation analysis of peripheral blood (PB) represents a promising diagnostic test in mastocytosis. However, there is a need for systematic assessment of the analytical sensitivity and specificity of the approach in order...... to establish its value in clinical use. We therefore evaluated sensitive KIT D816V mutation analysis of PB as a diagnostic test in an entire case-series of adults with mastocytosis. We demonstrate for the first time that by using a sufficiently sensitive KIT D816V mutation analysis, it is possible to detect...... the mutation in PB in nearly all adult mastocytosis patients. The mutation was detected in PB in 78 of 83 systemic mastocytosis (94%) and 3 of 4 cutaneous mastocytosis patients (75%). The test was 100% specific as determined by analysis of clinically relevant control patients who all tested negative. Mutation...

  2. Antiparietal cell antibody test

    Science.gov (United States)

    APCA; Anti-gastric parietal cell antibody; Atrophic gastritis - anti-gastric parietal cell antibody; Gastric ulcer - anti-gastric parietal cell antibody; Pernicious anemia - anti-gastric parietal cell antibody; ...

  3. Mitochondrial DNA deletion mutations in adult mouse cardiac side population cells

    International Nuclear Information System (INIS)

    Lushaj, Entela B.; Lozonschi, Lucian; Barnes, Maria; Anstadt, Emily; Kohmoto, Takushi

    2012-01-01

    We investigated the presence and potential role of mitochondrial DNA (mtDNA) deletion mutations in adult cardiac stem cells. Cardiac side population (SP) cells were isolated from 12-week-old mice. Standard polymerase chain reaction (PCR) was used to screen for the presence of mtDNA deletion mutations in (a) freshly isolated SP cells and (b) SP cells cultured to passage 10. When present, the abundance of mtDNA deletion mutation was analyzed in single cell colonies. The effect of different levels of deletion mutations on SP cell growth and differentiation was determined. MtDNA deletion mutations were found in both freshly isolated and cultured cells from 12-week-old mice. While there was no significant difference in the number of single cell colonies with mtDNA deletion mutations from any of the groups mentioned above, the abundance of mtDNA deletion mutations was significantly higher in the cultured cells, as determined by quantitative PCR. Within a single clonal cell population, the detectable mtDNA deletion mutations were the same in all cells and unique when compared to deletions of other colonies. We also found that cells harboring high levels of mtDNA deletion mutations (i.e. where deleted mtDNA comprised more than 60% of total mtDNA) had slower proliferation rates and decreased differentiation capacities. Screening cultured adult stem cells for mtDNA deletion mutations as a routine assessment will benefit the biomedical application of adult stem cells.

  4. Pitfalls in genetic testing: the story of missed SCN1A mutations.

    Science.gov (United States)

    Djémié, Tania; Weckhuysen, Sarah; von Spiczak, Sarah; Carvill, Gemma L; Jaehn, Johanna; Anttonen, Anna-Kaisa; Brilstra, Eva; Caglayan, Hande S; de Kovel, Carolien G; Depienne, Christel; Gaily, Eija; Gennaro, Elena; Giraldez, Beatriz G; Gormley, Padhraig; Guerrero-López, Rosa; Guerrini, Renzo; Hämäläinen, Eija; Hartmann, Corinna; Hernandez-Hernandez, Laura; Hjalgrim, Helle; Koeleman, Bobby P C; Leguern, Eric; Lehesjoki, Anna-Elina; Lemke, Johannes R; Leu, Costin; Marini, Carla; McMahon, Jacinta M; Mei, Davide; Møller, Rikke S; Muhle, Hiltrud; Myers, Candace T; Nava, Caroline; Serratosa, Jose M; Sisodiya, Sanjay M; Stephani, Ulrich; Striano, Pasquale; van Kempen, Marjan J A; Verbeek, Nienke E; Usluer, Sunay; Zara, Federico; Palotie, Aarno; Mefford, Heather C; Scheffer, Ingrid E; De Jonghe, Peter; Helbig, Ingo; Suls, Arvid

    2016-07-01

    Sanger sequencing, still the standard technique for genetic testing in most diagnostic laboratories and until recently widely used in research, is gradually being complemented by next-generation sequencing (NGS). No single mutation detection technique is however perfect in identifying all mutations. Therefore, we wondered to what extent inconsistencies between Sanger sequencing and NGS affect the molecular diagnosis of patients. Since mutations in SCN1A, the major gene implicated in epilepsy, are found in the majority of Dravet syndrome (DS) patients, we focused on missed SCN1A mutations. We sent out a survey to 16 genetic centers performing SCN1A testing. We collected data on 28 mutations initially missed using Sanger sequencing. All patients were falsely reported as SCN1A mutation-negative, both due to technical limitations and human errors. We illustrate the pitfalls of Sanger sequencing and most importantly provide evidence that SCN1A mutations are an even more frequent cause of DS than already anticipated.

  5. TALEN-mediated genetic tailoring as a tool to analyze the function of acquired mutations in multiple myeloma cells

    International Nuclear Information System (INIS)

    Wu, X; Blackburn, P R; Tschumper, R C; Ekker, S C; Jelinek, D F

    2014-01-01

    Multiple myeloma (MM) is a clonal plasma cell malignancy that is initiated by a number of mutations and the process of disease progression is characterized by further acquisition of mutations. The identification and functional characterization of these myelomagenic mutations is necessary to better understand the underlying pathogenic mechanisms in this disease. Recent advancements in next-generation sequencing have made the identification of most of these mutations a reality. However, the functional characterization of these mutations has been hampered by the lack of proper and efficient tools to dissect these mutations. Here we explored the possible utility of transcription activator-like effector nuclease (TALEN) genome engineering technology to tailoring the genome of MM cells. To test this possibility, we targeted the HPRT1 gene and found that TALENs are a very robust and efficient genome-editing tool in MM cells. Using cotransfected green fluorescent protein as an enrichment marker, single-cell subclones with desirable TALEN modifications in the HPRT1 gene were obtained in as little as 3–4 weeks of time. We believe that TALENs will greatly facilitate the functional study of somatic mutations in MM as well as other cancers

  6. Hot spot mutations in Finnish non-small cell lung cancers.

    Science.gov (United States)

    Mäki-Nevala, Satu; Sarhadi, Virinder Kaur; Rönty, Mikko; Kettunen, Eeva; Husgafvel-Pursiainen, Kirsti; Wolff, Henrik; Knuuttila, Aija; Knuutila, Sakari

    2016-09-01

    Non-small cell lung cancer (NSCLC) is a common cancer with a poor prognosis. The aim of this study was to screen Finnish NSCLC tumor samples for common cancer-related mutations by targeted next generation sequencing and to determine their concurrences and associations with clinical features. Sequencing libraries were prepared from DNA isolated from formalin-fixed, paraffin-embedded tumor material of 425 patients using the AmpliSeq Colon and Lung panel covering mutational hot spot regions of 22 cancer genes. Sequencing was performed with the Ion Torrent Personal Genome Machine (PGM). Data analysis of the hot spot mutations revealed mutations in 77% of the patients, with 7% having 3 or more mutations reported in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. Two of the most frequently mutated genes were TP53 (46%) and KRAS (25%). KRAS codon 12 mutations were the most recurrently occurring mutations. EGFR mutations were significantly associated with adenocarcinoma, female gender and never/light-smoking history; CTNNB1 mutations with light ex-smokers, PIK3CA and TP53 mutations with squamous cell carcinoma, and KRAS with adenocarcinoma. TP53 mutations were most prevalent in current smokers and ERBB2, ERBB4, PIK3CA, NRAS, NOTCH1, FBWX7, PTEN and STK11 mutations occurred exclusively in a group of ever-smokers, however the association was not statistically significant. No mutation was found that associated with asbestos exposure. Finnish NSCLC patients have a similar mutation profile as other Western patients, however with a higher frequency of BRAF mutations but a lower frequency of STK11 and ERBB2 mutations. Moreover, TP53 mutations occurred frequently with other gene mutations, most commonly with KRAS, MET, EGFR and PIK3CA mutations. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  7. Factors affecting the loss of MED12-mutated leiomyoma cells during in vitro growth

    OpenAIRE

    Bloch, Jeannine; Holzmann, Carsten; Koczan, Dirk; Helmke, Burkhard Maria; Bullerdiek, J?rn

    2017-01-01

    Uterine leiomyomas (UL) are the most prevalent symptomatic human tumors at all and somatic mutations of the gene encoding mediator subcomplex 12 (MED12) constitute the most frequent driver mutations in UL. Recently, a rapid loss of mutated cells during in vitro growth of UL-derived cell cultures was reported, resulting in doubts about the benefits of UL-derived cell cultures. To evaluate if the rapid loss of MED12-mutated cells in UL cell cultures depends on in vitro passaging, we set up cell...

  8. Preclinical test: bacterial reverse mutation test for {sup 18}F-fluorocholine produced in CDTN

    Energy Technology Data Exchange (ETDEWEB)

    Mendes, Bruno M.; Bispo, Ana Carolina A.; Campos, Danielle C.; Silva, Juliana B., E-mail: bmm@cdtn.br, E-mail: acab@cdtn.br, E-mail: dcc@cdtn.br, E-mail: silvajb@cdtn.b [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil)

    2013-07-01

    The choline labeled with fluorine-18 (18FCH) is being considered as a great importance radiopharmaceutical due to its effective detection of many type of malignant neoplasm. The research related to {sup 18}F-fluorocholine synthesis in CDTN was initiated in 2010. In order to obtain clinical research approval, as well as to register {sup 18}FCH for marketing, safety and efficacy preclinical testing are required. The present work evaluated the {sup 18}FCH genotoxic potential through the bacterial reverse mutation test (Ames test) using Salmonella typhimurium TA-98, TA-100, TA-1535 and TA-1537 strains and Escherichia coli WP2 uvrA strain. The reverse mutation test in bacteria for fluorcolina was conducted in two stages. Initially the method was applied to 'cold' fluorocholine molecule (19FCH). Subsequently, the decayed product of {sup 18}FCH synthesis was evaluated. The first step was performed in order to examine the FCH molecule mutagenicity. The second was carried out to determine the mutagenic potential of final product. All strains were tested in triplicate for each exposure concentration, in the presence and absence of metabolic activation (S-9 mix - 10%). There were no statistically significant increases in revertant colonies rate for any strains tested after their exposure to decayed {sup 18}FCH or {sup 19}FCH. The number of revertant colonies in positive controls was significantly higher than that observed in significant increases in revertant colonies rate for any strains tested after their exposure to decayed {sup 18}FCH or {sup 19}FCH. The number of revertant colonies in positive controls was significantly higher than that observed in negative controls. Based on results of this assay, {sup 18}FCH and {sup 19}FCH, at tested doses, were found to be non-mutagenic in bacterial reverse mutation test. (author)

  9. Preclinical test: bacterial reverse mutation test for 18F-fluorocholine produced in CDTN

    International Nuclear Information System (INIS)

    Mendes, Bruno M.; Bispo, Ana Carolina A.; Campos, Danielle C.; Silva, Juliana B.

    2013-01-01

    The choline labeled with fluorine-18 (18FCH) is being considered as a great importance radiopharmaceutical due to its effective detection of many type of malignant neoplasm. The research related to 18 F-fluorocholine synthesis in CDTN was initiated in 2010. In order to obtain clinical research approval, as well as to register 18 FCH for marketing, safety and efficacy preclinical testing are required. The present work evaluated the 18 FCH genotoxic potential through the bacterial reverse mutation test (Ames test) using Salmonella typhimurium TA-98, TA-100, TA-1535 and TA-1537 strains and Escherichia coli WP2 uvrA strain. The reverse mutation test in bacteria for fluorcolina was conducted in two stages. Initially the method was applied to 'cold' fluorocholine molecule (19FCH). Subsequently, the decayed product of 18 FCH synthesis was evaluated. The first step was performed in order to examine the FCH molecule mutagenicity. The second was carried out to determine the mutagenic potential of final product. All strains were tested in triplicate for each exposure concentration, in the presence and absence of metabolic activation (S-9 mix - 10%). There were no statistically significant increases in revertant colonies rate for any strains tested after their exposure to decayed 18 FCH or 19 FCH. The number of revertant colonies in positive controls was significantly higher than that observed in significant increases in revertant colonies rate for any strains tested after their exposure to decayed 18 FCH or 19 FCH. The number of revertant colonies in positive controls was significantly higher than that observed in negative controls. Based on results of this assay, 18 FCH and 19 FCH, at tested doses, were found to be non-mutagenic in bacterial reverse mutation test. (author)

  10. Matrilin-3 Chondrodysplasia Mutations Cause Attenuated Chondrogenesis, Premature Hypertrophy and Aberrant Response to TGF-β in Chondroprogenitor Cells

    OpenAIRE

    Chathuraka T. Jayasuriya; Fiona H. Zhou; Ming Pei; Zhengke Wang; Nicholas J. Lemme; Paul Haines; Qian Chen

    2014-01-01

    Studies have shown that mutations in the matrilin-3 gene (MATN3) are associated with multiple epiphyseal dysplasia (MED) and spondyloepimetaphyseal dysplasia (SEMD). We tested whether MATN3 mutations affect the differentiation of chondroprogenitor and/or mesenchymal stem cells, which are precursors to chondrocytes. ATDC5 chondroprogenitors stably expressing wild-type (WT) MATN3 underwent spontaneous chondrogenesis. Expression of chondrogenic markers collagen II and aggrecan was inhibited in c...

  11. Terrestrial photovoltaic cell process testing

    Science.gov (United States)

    Burger, D. R.

    1985-01-01

    The paper examines critical test parameters, criteria for selecting appropriate tests, and the use of statistical controls and test patterns to enhance PV-cell process test results. The coverage of critical test parameters is evaluated by examining available test methods and then screening these methods by considering the ability to measure those critical parameters which are most affected by the generic process, the cost of the test equipment and test performance, and the feasibility for process testing.

  12. TP53 Mutations in Nonsmall Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Akira Mogi

    2011-01-01

    Full Text Available The tumor suppressor gene TP53 is frequently mutated in human cancers. Abnormality of the TP53 gene is one of the most significant events in lung cancers and plays an important role in the tumorigenesis of lung epithelial cells. Human lung cancers are classified into two major types, small cell lung cancer (SCLC and nonsmall cell lung cancer (NSCLC. The latter accounts for approximately 80% of all primary lung cancers, and the incidence of NSCLC is increasing yearly. Most clinical studies suggest that NSCLC with TP53 alterations carries a worse prognosis and may be relatively more resistant to chemotherapy and radiation. A deep understanding of the role of TP53 in lung carcinogenesis may lead to a more reasonably targeted clinical approach, which should be exploited to enhance the survival rates of patients with lung cancer. This paper will focus on the role of TP53 in the molecular pathogenesis, epidemiology, and therapeutic strategies of TP53 mutation in NSCLC.

  13. Filaggrin Gene Mutations and Risk of Basal Cell Carcinoma

    DEFF Research Database (Denmark)

    Kaae, Jesper Rabølle; Thyssen, J P; Johansen, J D

    2013-01-01

    ) . Mice with knockdown of filaggrin, or lack of functional histidase, show decreased epidermal trans-UCA levels and increased UVB-induced skin damage (5) . FLG mutation carriers also have 10% increased serum vitamin D levels suggesting increased penetration of UVB (6) . We evaluated the prevalence of FLG......Basal cell carcinoma (BCC) is prevalent in lightly-pigmented Europeans. While ultraviolet (UV) radiation is an important risk factor, genetic predispositions to BCC have also been identified (1) . Atopic dermatitis (AD), a condition with a heritability that reaches 71-84%, might increase the risk...

  14. Higher quality of molecular testing, an unfulfilled priority: Results from external quality assessment for KRAS mutation testing in colorectal cancer.

    Science.gov (United States)

    Tembuyser, Lien; Ligtenberg, Marjolijn J L; Normanno, Nicola; Delen, Sofie; van Krieken, J Han; Dequeker, Elisabeth M C

    2014-05-01

    Precision medicine is now a key element in clinical oncology. RAS mutational status is a crucial predictor of responsiveness to anti-epidermal growth factor receptor agents in metastatic colorectal cancer. In an effort to guarantee high-quality testing services in molecular pathology, the European Society of Pathology has been organizing an annual KRAS external quality assessment program since 2009. In 2012, 10 formalin-fixed, paraffin-embedded samples, of which 8 from invasive metastatic colorectal cancer tissue and 2 artificial samples of cell line material, were sent to more than 100 laboratories from 26 countries with a request for routine KRAS testing. Both genotyping and clinical reports were assessed independently. Twenty-seven percent of the participants genotyped at least 1 of 10 samples incorrectly. In total, less than 5% of the distributed specimens were genotyped incorrectly. Genotyping errors consisted of false negatives, false positives, and incorrectly genotyped mutations. Twenty percent of the laboratories reported a technical error for one or more samples. A review of the written reports showed that several essential elements were missing, most notably a clinical interpretation of the test result, the method sensitivity, and the use of a reference sequence. External quality assessment serves as a valuable educational tool in assessing and improving molecular testing quality and is an important asset for monitoring quality assurance upon incorporation of new biomarkers in diagnostic services. Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  15. Male Hypogonadism and Germ Cell Loss Caused by a Mutation in Polo-Like Kinase 4

    Science.gov (United States)

    Harris, Rebecca M.; Weiss, Jeffrey

    2011-01-01

    The genetic etiologies of male infertility remain largely unknown. To identify genes potentially involved in spermatogenesis and male infertility, we performed genome-wide mutagenesis in mice with N-ethyl-N-nitrosourea and identified a line with dominant hypogonadism and patchy germ cell loss. Genomic mapping and DNA sequence analysis identified a novel heterozygous missense mutation in the kinase domain of Polo-like kinase 4 (Plk4), altering an isoleucine to asparagine at residue 242 (I242N). Genetic complementation studies using a gene trap line with disruption in the Plk4 locus confirmed that the putative Plk4 missense mutation was causative. Plk4 is known to be involved in centriole formation and cell cycle progression. However, a specific role in mammalian spermatogenesis has not been examined. PLK4 was highly expressed in the testes both pre- and postnatally. In the adult, PLK4 expression was first detected in stage VIII pachytene spermatocytes and was present through step 16 elongated spermatids. Because the homozygous Plk4I242N/I242N mutation was embryonic lethal, all analyses were performed using the heterozygous Plk4+/I242N mice. Testis size was reduced by 17%, and histology revealed discrete regions of germ cell loss, leaving only Sertoli cells in these defective tubules. Testis cord formation (embryonic day 13.5) was normal. Testis histology was also normal at postnatal day (P)1, but germ cell loss was detected at P10 and subsequent ages. We conclude that the I242N heterozygous mutation in PLK4 is causative for patchy germ cell loss beginning at P10, suggesting a role for PLK4 during the initiation of spermatogenesis. PMID:21791561

  16. Dimethyl fumarate is highly cytotoxic in KRAS mutated cancer cells but spares non-tumorigenic cells

    Science.gov (United States)

    Bennett Saidu, Nathaniel Edward; Bretagne, Marie; Mansuet, Audrey Lupo; Just, Pierre-Alexandre; Leroy, Karen; Cerles, Olivier; Chouzenoux, Sandrine; Nicco, Carole; Damotte, Diane; Alifano, Marco; Borghese, Bruno; Goldwasser, François; Batteux, Frédéric; Alexandre, Jérôme

    2018-01-01

    KRAS mutation, one of the most common molecular alterations observed in adult carcinomas, was reported to activate the anti-oxidant program driven by the transcription factor NRF2 (Nuclear factor-erythroid 2-related factor 2). We previously observed that the antitumoral effect of Dimethyl fumarate (DMF) is dependent of NRF2 pathway inhibition. We used in vitro methods to examine the effect of DMF on cell death and the activation of the NRF2/DJ-1 antioxidant pathway. We report here that DMF is preferentially cytotoxic against KRAS mutated cancer cells. This effect was observed in patient-derived cancer cell lines harbouring a G12V KRAS mutation, compared with cell lines without such a mutation. In addition, KRAS*G12V over-expression in the human Caco-2 colon cancer cell line significantly promoted DMF-induced cell death, as well as DMF-induced- reactive oxygen species (ROS) formation and -glutathione (GSH) depletion. Moreover, in contrast to malignant cells, our data confirms that the same concentration of DMF has no significant cytotoxic effects on non-tumorigenic human ARPE-19 retinal epithelial, murine 3T3 fibroblasts and primary mice bone marrow cells; but is rather associated with NRF2 activation, decreased ROS and increased GSH levels. Furthermore, DJ-1 down-regulation experiments showed that this protein does not play a protective role against NRF2 in non-tumorigenic cells, as it does in malignant ones. This, interestingly, could be at the root of the differential effect of DMF observed between malignant and non-tumorigenic cells. Our results suggest for the first time that the dependence on NRF2 observed in mutated KRAS malignant cells makes them more sensitive to the cytotoxic effect of DMF, which thus opens up new prospects for the therapeutic applications of DMF. PMID:29507676

  17. Dimethyl fumarate is highly cytotoxic in KRAS mutated cancer cells but spares non-tumorigenic cells.

    Science.gov (United States)

    Bennett Saidu, Nathaniel Edward; Bretagne, Marie; Mansuet, Audrey Lupo; Just, Pierre-Alexandre; Leroy, Karen; Cerles, Olivier; Chouzenoux, Sandrine; Nicco, Carole; Damotte, Diane; Alifano, Marco; Borghese, Bruno; Goldwasser, François; Batteux, Frédéric; Alexandre, Jérôme

    2018-02-06

    KRAS mutation, one of the most common molecular alterations observed in adult carcinomas, was reported to activate the anti-oxidant program driven by the transcription factor NRF2 (Nuclear factor-erythroid 2-related factor 2). We previously observed that the antitumoral effect of Dimethyl fumarate (DMF) is dependent of NRF2 pathway inhibition. We used in vitro methods to examine the effect of DMF on cell death and the activation of the NRF2/DJ-1 antioxidant pathway. We report here that DMF is preferentially cytotoxic against KRAS mutated cancer cells. This effect was observed in patient-derived cancer cell lines harbouring a G12V KRAS mutation, compared with cell lines without such a mutation. In addition, KRAS*G12V over-expression in the human Caco-2 colon cancer cell line significantly promoted DMF-induced cell death, as well as DMF-induced- reactive oxygen species (ROS) formation and -glutathione (GSH) depletion. Moreover, in contrast to malignant cells, our data confirms that the same concentration of DMF has no significant cytotoxic effects on non-tumorigenic human ARPE-19 retinal epithelial, murine 3T3 fibroblasts and primary mice bone marrow cells; but is rather associated with NRF2 activation, decreased ROS and increased GSH levels. Furthermore, DJ-1 down-regulation experiments showed that this protein does not play a protective role against NRF2 in non-tumorigenic cells, as it does in malignant ones. This, interestingly, could be at the root of the differential effect of DMF observed between malignant and non-tumorigenic cells. Our results suggest for the first time that the dependence on NRF2 observed in mutated KRAS malignant cells makes them more sensitive to the cytotoxic effect of DMF, which thus opens up new prospects for the therapeutic applications of DMF.

  18. Minisatellite mutations and retrospective biodosimetry of population living close to the Semipalatinsk nuclear test site

    International Nuclear Information System (INIS)

    Lindholm, C.; Bersimbacv, R. I.; Dubrova, Y. E.; Hulten, M.; Bigbee, W. I.; Murphy, B. P.; Koivistoinen, A.; Tankimonova, M.; Mamyrbaeva, Z.; Djansugarova, L.; Mustonen, R.; Salomaa, S.

    2004-01-01

    The objectives of the study were to determine minisatellite mutation rates in families in three generations and to perform retrospective biodosimetry of individuals in these families living close to the Semipalatinsk nuclear test site in Kazakhstan. The oldes generation (Po) lived in the area at the time of the first Soviet nuclear test in 1949 whereas the younger generations (F1,F2) were exposed to smaller doses from the residual fallout and later tests. Matched control families in three generations living in non-contamianted areas were analysed in parallel. The retrospective biodosimetry comprehended two endpoints; chromosomal translocations determined by FISH chromosome painting and the glycophorin A (GPA) somatic mutation assay. The minisatellite mutation rate in the cohort of P0 parents was 1-8-fold higher than in the control non-exposed population. Moreover, the minisatellite mutatin rate in the cohort of f1 parents from the exposed area showed a significant negative correlation with with the year of birth, fully consistent with the decay of radioisotopes after the cessation of surface and atmospheric nuclear tests. The results from the FISH painting analysis showed similar translocation frequencies in the Semipalatinsk cohort and the control group. Based on the FISH results it can be concluded that the P0 generation has received a cumulative mean dose of less than 0.5 Gy. The GPA assay did not reveal significant diffrences in the variant cell frequencies for all subjects from the Semipalatinsk area compared with the matched controls. However, a significant increase (P<0.05) of the mean allele-loss φN variant frequency was observed among the exposed P0 generation in comparison to controls. Considering the sensitivity of the GPA assay, the results suggest that the mean dose to the P0 generation of the affected villages was relatively low and in accordance to the results obtained using FISH. (Author) 17 refs

  19. Minisatellite mutations and retrospective biodosimetry of population living close to the Semipalatinsk nuclear test site

    Energy Technology Data Exchange (ETDEWEB)

    Lindholm, C.; Bersimbacv, R. I.; Dubrova, Y. E.; Hulten, M.; Bigbee, W. I.; Murphy, B. P.; Koivistoinen, A.; Tankimonova, M.; Mamyrbaeva, Z.; Djansugarova, L.; Mustonen, R.; Salomaa, S.

    2004-07-01

    The objectives of the study were to determine minisatellite mutation rates in families in three generations and to perform retrospective biodosimetry of individuals in these families living close to the Semipalatinsk nuclear test site in Kazakhstan. The oldes generation (Po) lived in the area at the time of the first Soviet nuclear test in 1949 whereas the younger generations (F1,F2) were exposed to smaller doses from the residual fallout and later tests. Matched control families in three generations living in non-contamianted areas were analysed in parallel. The retrospective biodosimetry comprehended two endpoints; chromosomal translocations determined by FISH chromosome painting and the glycophorin A (GPA) somatic mutation assay. The minisatellite mutation rate in the cohort of P0 parents was 1-8-fold higher than in the control non-exposed population. Moreover, the minisatellite mutatin rate in the cohort of f1 parents from the exposed area showed a significant negative correlation with with the year of birth, fully consistent with the decay of radioisotopes after the cessation of surface and atmospheric nuclear tests. The results from the FISH painting analysis showed similar translocation frequencies in the Semipalatinsk cohort and the control group. Based on the FISH results it can be concluded that the P0 generation has received a cumulative mean dose of less than 0.5 Gy. The GPA assay did not reveal significant diffrences in the variant cell frequencies for all subjects from the Semipalatinsk area compared with the matched controls. However, a significant increase (P<0.05) of the mean allele-loss {phi}N variant frequency was observed among the exposed P0 generation in comparison to controls. Considering the sensitivity of the GPA assay, the results suggest that the mean dose to the P0 generation of the affected villages was relatively low and in accordance to the results obtained using FISH. (Author) 17 refs.

  20. IDH mutations in liver cell plasticity and biliary cancer

    Science.gov (United States)

    Saha, Supriya K; Parachoniak, Christine A; Bardeesy, Nabeel

    2014-01-01

    Intrahepatic cholangiocarcinoma (ICC) is an aggressive cancer associated with the bile ducts within the liver. These tumors are characterized by frequent gain-of-function mutations in the isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) genes—that are also common in subsets of neural, haematopoietic and bone tumors, but rare or absent in the other types of gastrointestinal malignancy. Mutant IDH acts through a novel mechanism of oncogenesis, producing high levels of the metabolite 2-hydroxyglutarate, which interferes with the function of α-ketoglutarate-dependent enzymes that regulate diverse cellular processes including histone demethylation and DNA modification. Recently, we used in vitro stem cell systems and genetically engineered mouse models (GEMMs) to demonstrate that mutant IDH promotes ICC formation by blocking hepatocyte differentiation and increasing pools of hepatic progenitors that are susceptible to additional oncogenic hits leading to ICC. We found that silencing of HNF4A—encoding a master transcriptional regulator of hepatocyte identity and quiescence—was critical to mutant IDH-mediated inhibition of liver differentiation. In line with these findings, human ICC with IDH mutations are characterized by a hepatic progenitor cell transcriptional signature suggesting that they are a distinct ICC subtype as compared to IDH wild type tumors. The role of mutant IDH in controlling hepatic differentiation state suggests the potential of newly developed inhibitors of the mutant enzyme as a form of differentiation therapy in a solid tumor. PMID:25485496

  1. The non-small cell lung cancer EGFR extracellular domain mutation, M277E, is oncogenic and drug-sensitive

    Directory of Open Access Journals (Sweden)

    Yu S

    2017-09-01

    Full Text Available Su Yu,1,2 Yang Zhang,1 Yunjian Pan,1 Chao Cheng,1,3 Yihua Sun,1,3 Haiquan Chen1–4 1Department of Thoracic Surgery, Fudan University Shanghai Cancer Center, Shanghai, China; 2Cancer Research Center, Fudan University Shanghai Cancer Center, Shanghai, China; 3Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China; 4Institutes of Biomedical Sciences, Fudan University, Shanghai, China Purpose: To identify novel oncogenic mutations in non-small cell lung cancer patient specimens that lack mutations in known targetable genes (“pan-negative” patients.Methods: Comprehensive mutational analyses were performed on 1,356 lung adenocarcinoma specimens. In this cohort of patients, common lung cancer oncogenic driver mutations were detected in the epidermal growth factor receptor (EGFR kinase domain, the human epidermal growth factor receptor 2 kinase domain, as well as the KRAS, BRAF, ALK, ROS1 and RET genes. A sub-cohort of pan-negative patient specimens was assayed for mutations in the EGFR extracellular domain (ECD. Additionally, EGFR mutant NIH-3T3 stable cell lines were constructed and assessed for protein content, anchorage-independent growth, and tumor formation in xenograft models to identify oncogenic mutations. BaF3 lymphocytes were also used to test sensitivities of the mutations to tyrosine kinase inhibitors.Results: In pan-negative lung adenocarcinoma cases, a novel oncogenic EGFR ECD mutation was identified (M277E. EGFR M277E mutations encoded oncoproteins that transformed NIH-3T3 cells to grow in the absence of exogenous epidermal growth factor. Transformation was further evidenced by anchorage-independent growth and tumor formation in immunocompromised xenograft mouse models. Finally, as seen in the canonical EGFR L858R mutation, the M277E mutation conferred sensitivity to both erlotinib and cetuximab in BaF3 cell lines and to erlotinib in xenograft models.Conclusion: Here, a new EGFR driver mutation, M277E

  2. Role of DNA deletion length in mutation and cell survival

    International Nuclear Information System (INIS)

    Braby, L.A.; Morgan, T.L.

    1992-01-01

    A model is presented which is based on the assumption that malignant transformation, mutation, chromosome aberration, and reproductive death of cells are all manifestations of radiation induced deletions in the DNA of the cell, and that the size of the deletion in relation to the spacing of essential genes determines the consequences of that deletion. It is assumed that two independent types of potentially lethal lesions can result in DNA deletions, and that the relative numbers of these types of damage is dependent on radiation quality. The repair of the damage reduces the length of a deletion, but does not always eliminate it. The predictions of this model are in good agreement with a wide variety of experimental evidence. (author)

  3. Pitfalls in genetic testing: the story of missed SCN1A mutations

    OpenAIRE

    Djémié, T.; Weckhuysen, S.; von Spiczak, S.; Carvill, G. L.; Jaehn, J.; Anttonen, A-K; Brilstra, E.; Caglayan, H. S.; de Kovel, C. G.; Depienne, C.; Gaily, E.; Gennaro, E.; Giraldez, B. G.; Gormley, P.; Guerrero-López, R.

    2016-01-01

    BACKGROUND: Sanger sequencing, still the standard technique for genetic testing in most diagnostic laboratories and until recently widely used in research, is gradually being complemented by next-generation sequencing (NGS). No single mutation detection technique is however perfect in identifying all mutations. Therefore, we wondered to what extent inconsistencies between Sanger sequencing and NGS affect the molecular diagnosis of patients. Since mutations in SCN1A, the major gene implicated ...

  4. Pitfalls in genetic testing : the story of missed SCN1A mutations

    OpenAIRE

    Djémié, Tania; Weckhuysen, Sarah; von Spiczak, Sarah; Carvill, Gemma L; Jaehn, Johanna; Anttonen, Anna-Kaisa; Brilstra, Eva; Caglayan, Hande S; de Kovel, Carolien G; Depienne, Christel; Gaily, Eija; Gennaro, Elena; Giraldez, Beatriz G; Gormley, Padhraig; Guerrero-López, Rosa

    2016-01-01

    BACKGROUND: Sanger sequencing, still the standard technique for genetic testing in most diagnostic laboratories and until recently widely used in research, is gradually being complemented by next-generation sequencing (NGS). No single mutation detection technique is however perfect in identifying all mutations. Therefore, we wondered to what extent inconsistencies between Sanger sequencing and NGS affect the molecular diagnosis of patients. Since mutations in SCN1A, the major gene implicated ...

  5. Dealing with the unexpected: consumer responses to direct-access BRCA mutation testing

    Directory of Open Access Journals (Sweden)

    Uta Francke

    2013-02-01

    Full Text Available Background. Inherited BRCA gene mutations convey a high risk for breast and ovarian cancer, but current guidelines limit BRCA mutation testing to women with early-onset cancer and relatives of mutation-positive cases. Benefits and risks of providing this information directly to consumers are unknown.Methods. To assess and quantify emotional and behavioral reactions of consumers to their 23andMe Personal Genome Service® report of three BRCA mutations that are common in Ashkenazi Jews, we invited all 136 BRCA1 and BRCA2 mutation-positive individuals in the 23andMe customer database who had chosen to view their BRCA reports to participate in this IRB-approved study. We also invited 160 mutation-negative customers who were matched for age, sex and ancestry. Semi-structured phone interviews were completed for 32 mutation carriers, 16 women and 16 men, and 31 non-carriers. Questions addressed personal and family history of cancer, decision and timing of viewing the BRCA report, recollection of the result, emotional responses, perception of personal cancer risk, information sharing, and actions taken or planned.Results. Eleven women and 14 men had received the unexpected result that they are carriers of a BRCA1 185delAG or 5382insC, or BRCA2 6174delT mutation. None of them reported extreme anxiety and four experienced moderate anxiety that was transitory. Remarkably, five women and six men described their response as neutral. Most carrier women sought medical advice and four underwent risk-reducing procedures after confirmatory mutation testing. Male carriers realized that their test results implied genetic risk for female relatives, and several of them felt considerably burdened by this fact. Sharing mutation information with family members led to screening of at least 30 relatives and identification of 13 additional carriers. Non-carriers did not report inappropriate actions, such as foregoing cancer screening. All but one of the 32 mutation

  6. Dealing with the unexpected: consumer responses to direct-access BRCA mutation testing.

    Science.gov (United States)

    Francke, Uta; Dijamco, Cheri; Kiefer, Amy K; Eriksson, Nicholas; Moiseff, Bianca; Tung, Joyce Y; Mountain, Joanna L

    2013-01-01

    Background. Inherited BRCA gene mutations convey a high risk for breast and ovarian cancer, but current guidelines limit BRCA mutation testing to women with early-onset cancer and relatives of mutation-positive cases. Benefits and risks of providing this information directly to consumers are unknown. Methods. To assess and quantify emotional and behavioral reactions of consumers to their 23andMe Personal Genome Service(®) report of three BRCA mutations that are common in Ashkenazi Jews, we invited all 136 BRCA1 and BRCA2 mutation-positive individuals in the 23andMe customer database who had chosen to view their BRCA reports to participate in this IRB-approved study. We also invited 160 mutation-negative customers who were matched for age, sex and ancestry. Semi-structured phone interviews were completed for 32 mutation carriers, 16 women and 16 men, and 31 non-carriers. Questions addressed personal and family history of cancer, decision and timing of viewing the BRCA report, recollection of the result, emotional responses, perception of personal cancer risk, information sharing, and actions taken or planned. Results. Eleven women and 14 men had received the unexpected result that they are carriers of a BRCA1 185delAG or 5382insC, or BRCA2 6174delT mutation. None of them reported extreme anxiety and four experienced moderate anxiety that was transitory. Remarkably, five women and six men described their response as neutral. Most carrier women sought medical advice and four underwent risk-reducing procedures after confirmatory mutation testing. Male carriers realized that their test results implied genetic risk for female relatives, and several of them felt considerably burdened by this fact. Sharing mutation information with family members led to screening of at least 30 relatives and identification of 13 additional carriers. Non-carriers did not report inappropriate actions, such as foregoing cancer screening. All but one of the 32 mutation-positive participants

  7. Gender differences in the induction of chromosomal aberrations and gene mutations in rodent germ cells

    International Nuclear Information System (INIS)

    Adler, Ilse-Dore; Carere, Angelo; Eichenlaub-Ritter, Ursula; Pacchierotti, Francesca

    2007-01-01

    Germ cell mutagenicity testing provides experimental data to quantify genetic risk for exposed human populations. The majority of tests are performed with exposure of males, and female data are relatively rare. The reason for this paucity lies in the differences between male and female germ cell biology. Male germ cells are produced throughout reproductive life and all developmental stages can be ascertained by appropriate breeding schemes. In contrast, the female germ cell pool is limited, meiosis begins during embryogenesis and oocytes are arrested over long periods of time until maturation processes start for small numbers of oocytes during the oestrus cycle in mature females. The literature data are reviewed to point out possible gender differences of germ cells to exogenous agents such as chemicals or ionizing radiation. From the limited information, it can be concluded that male germ cells are more sensitive than female germ cells to the induction of chromosomal aberrations and gene mutations. However, exceptions are described which shed doubt on the extrapolation of experimental data from male rodents to the genetic risk of the human population. Furthermore, the female genome may be more sensitive to mutation induction during peri-conceptional stages compared to the male genome of the zygote. With few exceptions, germ cell experiments have been carried out under high acute exposure to optimize the effects and to compensate for the limited sample size in animal experiments. Human exposure to environmental agents, on the other hand, is usually chronic and involves low doses. Under these conditions, gender differences may become apparent that have not been studied so far. Additionally, data are reviewed that suggest a false impression of safety when responses are negative under high acute exposure of male rodents while a mutational response is induced by low chronic exposure. The classical (morphological) germ cell mutation tests are not performed anymore

  8. Matrilin-3 chondrodysplasia mutations cause attenuated chondrogenesis, premature hypertrophy and aberrant response to TGF-β in chondroprogenitor cells.

    Science.gov (United States)

    Jayasuriya, Chathuraka T; Zhou, Fiona H; Pei, Ming; Wang, Zhengke; Lemme, Nicholas J; Haines, Paul; Chen, Qian

    2014-08-21

    Studies have shown that mutations in the matrilin-3 gene (MATN3) are associated with multiple epiphyseal dysplasia (MED) and spondyloepimetaphyseal dysplasia (SEMD). We tested whether MATN3 mutations affect the differentiation of chondroprogenitor and/or mesenchymal stem cells, which are precursors to chondrocytes. ATDC5 chondroprogenitors stably expressing wild-type (WT) MATN3 underwent spontaneous chondrogenesis. Expression of chondrogenic markers collagen II and aggrecan was inhibited in chondroprogenitors carrying the MED or SEMD MATN3 mutations. Hypertrophic marker collagen X remained attenuated in WT MATN3 chondroprogenitors, whereas its expression was elevated in chondroprogenitors expressing the MED or SEMD mutant MATN3 gene suggesting that these mutations inhibit chondrogenesis but promote hypertrophy. TGF-β treatment failed to rescue chondrogenesis markers but dramatically increased collagen X mRNA expression in mutant MATN3 expressing chondroprogenitors. Synovium derived mesenchymal stem cells harboring the SEMD mutation exhibited lower glycosaminoglycan content than those of WT MATN3 in response to TGF-β. Our results suggest that the properties of progenitor cells harboring MATN3 chondrodysplasia mutations were altered, as evidenced by attenuated chondrogenesis and premature hypertrophy. TGF-β treatment failed to completely rescue chondrogenesis but instead induced hypertrophy in mutant MATN3 chondroprogenitors. Our data suggest that chondroprogenitor cells should be considered as a potential target of chondrodysplasia therapy.

  9. Matrilin-3 Chondrodysplasia Mutations Cause Attenuated Chondrogenesis, Premature Hypertrophy and Aberrant Response to TGF-β in Chondroprogenitor Cells

    Directory of Open Access Journals (Sweden)

    Chathuraka T. Jayasuriya

    2014-08-01

    Full Text Available Studies have shown that mutations in the matrilin-3 gene (MATN3 are associated with multiple epiphyseal dysplasia (MED and spondyloepimetaphyseal dysplasia (SEMD. We tested whether MATN3 mutations affect the differentiation of chondroprogenitor and/or mesenchymal stem cells, which are precursors to chondrocytes. ATDC5 chondroprogenitors stably expressing wild-type (WT MATN3 underwent spontaneous chondrogenesis. Expression of chondrogenic markers collagen II and aggrecan was inhibited in chondroprogenitors carrying the MED or SEMD MATN3 mutations. Hypertrophic marker collagen X remained attenuated in WT MATN3 chondroprogenitors, whereas its expression was elevated in chondroprogenitors expressing the MED or SEMD mutant MATN3 gene suggesting that these mutations inhibit chondrogenesis but promote hypertrophy. TGF-β treatment failed to rescue chondrogenesis markers but dramatically increased collagen X mRNA expression in mutant MATN3 expressing chondroprogenitors. Synovium derived mesenchymal stem cells harboring the SEMD mutation exhibited lower glycosaminoglycan content than those of WT MATN3 in response to TGF-β. Our results suggest that the properties of progenitor cells harboring MATN3 chondrodysplasia mutations were altered, as evidenced by attenuated chondrogenesis and premature hypertrophy. TGF-β treatment failed to completely rescue chondrogenesis but instead induced hypertrophy in mutant MATN3 chondroprogenitors. Our data suggest that chondroprogenitor cells should be considered as a potential target of chondrodysplasia therapy.

  10. Aging of hematopoietic stem cells: DNA damage and mutations?

    Science.gov (United States)

    Moehrle, Bettina M; Geiger, Hartmut

    2016-10-01

    Aging in the hematopoietic system and the stem cell niche contributes to aging-associated phenotypes of hematopoietic stem cells (HSCs), including leukemia and aging-associated immune remodeling. Among others, the DNA damage theory of aging of HSCs is well established, based on the detection of a significantly larger amount of γH2AX foci and a higher tail moment in the comet assay, both initially thought to be associated with DNA damage in aged HSCs compared with young cells, and bone marrow failure in animals devoid of DNA repair factors. Novel data on the increase in and nature of DNA mutations in the hematopoietic system with age, the quality of the DNA damage response in aged HSCs, and the nature of γH2AX foci question a direct link between DNA damage and the DNA damage response and aging of HSCs, and rather favor changes in epigenetics, splicing-factors or three-dimensional architecture of the cell as major cell intrinsic factors of HSCs aging. Aging of HSCs is also driven by a strong contribution of aging of the niche. This review discusses the DNA damage theory of HSC aging in the light of these novel mechanisms of aging of HSCs. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  11. PMS2 gene mutation results in DNA mismatch repair system failure in a case of adult granulosa cell tumor.

    Science.gov (United States)

    Wang, Wen-Chung; Lee, Ya-Ting; Lai, Yen-Chein

    2017-03-27

    Granulosa cell tumors are rare ovarian malignancies. Their characteristics include unpredictable indolent growth with malignant potential and late recurrence. Approximately 95% are of adult type. Recent molecular studies have characterized the FOXL2 402C > G mutation in adult granulosa cell tumor. Our previous case report showed that unique FOXL2 402C > G mutation and defective DNA mismatch repair system are associated with the development of adult granulosa cell tumor. In this study, the DNA sequences of four genes, MSH2, MLH1, MSH6, and PMS2, in the DNA mismatch repair system were determined via direct sequencing to elucidate the exact mechanism for the development of this granulosa cell tumor. The results showed that two missense germline mutations, T485K and N775L, inactivate the PMS2 gene. The results of this case study indicated that although FOXL2 402C > G mutation determines the development of granulosa cell tumor, PMS2 mutation may be the initial driver of carcinogenesis. Immunohistochemistry-based tumor testing for mismatch repair gene expression may be necessary for granulosa cell tumors to determine their malignant potential or if they are part of Lynch syndrome.

  12. Combining magnetic sorting of mother cells and fluctuation tests to analyze genome instability during mitotic cell aging in Saccharomyces cerevisiae.

    Science.gov (United States)

    Patterson, Melissa N; Maxwell, Patrick H

    2014-10-16

    Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on

  13. Novel somatic and germline mutations in intracranial germ cell tumours.

    Science.gov (United States)

    Wang, Linghua; Yamaguchi, Shigeru; Burstein, Matthew D; Terashima, Keita; Chang, Kyle; Ng, Ho-Keung; Nakamura, Hideo; He, Zongxiao; Doddapaneni, Harshavardhan; Lewis, Lora; Wang, Mark; Suzuki, Tomonari; Nishikawa, Ryo; Natsume, Atsushi; Terasaka, Shunsuke; Dauser, Robert; Whitehead, William; Adekunle, Adesina; Sun, Jiayi; Qiao, Yi; Marth, Gábor; Muzny, Donna M; Gibbs, Richard A; Leal, Suzanne M; Wheeler, David A; Lau, Ching C

    2014-07-10

    Intracranial germ cell tumours (IGCTs) are a group of rare heterogeneous brain tumours that are clinically and histologically similar to the more common gonadal GCTs. IGCTs show great variation in their geographical and gender distribution, histological composition and treatment outcomes. The incidence of IGCTs is historically five- to eightfold greater in Japan and other East Asian countries than in Western countries, with peak incidence near the time of puberty. About half of the tumours are located in the pineal region. The male-to-female incidence ratio is approximately 3-4:1 overall, but is even higher for tumours located in the pineal region. Owing to the scarcity of tumour specimens available for research, little is currently known about this rare disease. Here we report the analysis of 62 cases by next-generation sequencing, single nucleotide polymorphism array and expression array. We find the KIT/RAS signalling pathway frequently mutated in more than 50% of IGCTs, including novel recurrent somatic mutations in KIT, its downstream mediators KRAS and NRAS, and its negative regulator CBL. Novel somatic alterations in the AKT/mTOR pathway included copy number gains of the AKT1 locus at 14q32.33 in 19% of patients, with corresponding upregulation of AKT1 expression. We identified loss-of-function mutations in BCORL1, a transcriptional co-repressor and tumour suppressor. We report significant enrichment of novel and rare germline variants in JMJD1C, which codes for a histone demethylase and is a coactivator of the androgen receptor, among Japanese IGCT patients. This study establishes a molecular foundation for understanding the biology of IGCTs and suggests potentially promising therapeutic strategies focusing on the inhibition of KIT/RAS activation and the AKT1/mTOR pathway.

  14. Novel somatic and germline mutations in intracranial germ cell tumors

    Science.gov (United States)

    Wang, Linghua; Yamaguchi, Shigeru; Burstein, Matthew D.; Terashima, Keita; Chang, Kyle; Ng, Ho-Keung; Nakamura, Hideo; He, Zongxiao; Doddapaneni, Harshavardhan; Lewis, Lora; Wang, Mark; Suzuki, Tomonari; Nishikawa, Ryo; Natsume, Atsushi; Terasaka, Shunsuke; Dauser, Robert; Whitehead, William; Adekunle, Adesina; Sun, Jiayi; Qiao, Yi; Marth, Gábor; Muzny, Donna M.; Gibbs, Richard A.; Leal, Suzanne M.; Wheeler, David A.; Lau, Ching C.

    2015-01-01

    Intracranial germ cell tumors (IGCTs) are a group of rare heterogeneous brain tumors which are clinically and histologically similar to the more common gonadal GCTs. IGCTs show great variation in their geographic and gender distribution, histological composition and treatment outcomes. The incidence of IGCTs is historically 5–8 fold greater in Japan and other East Asian countries than in Western countries1 with peak incidence near the time of puberty2. About half of the tumors are located in the pineal region. The male-to-female incidence ratio is approximately 3–4:1 overall but even higher for tumors located in the pineal region3. Due to the scarcity of tumor specimens available for research, little is currently known about this rare disease. Here we report the analysis of 62 cases by next generation sequencing, SNP array and expression array. We find the KIT/RAS signaling pathway frequently mutated in over 50% of IGCTs including novel recurrent somatic mutations in KIT, its downstream mediators KRAS and NRAS, and its negative regulator CBL. Novel somatic alterations in the AKT/mTOR pathway included copy number gain of the AKT1 locus at 14q32.33 in 19% of patients, with corresponding upregulation of AKT1 expression. We identified loss-of-function mutations in BCORL1, a transcriptional corepressor and tumor suppressor. We report significant enrichment of novel and rare germline variants in JMJD1C, a histone demethylase and coactivator of the androgen receptor, among Japanese IGCT patients. This study establishes a molecular foundation for understanding the biology of IGCTs and suggests potentially promising therapeutic strategies focusing on the inhibition of KIT/RAS activation and the AKT1/mTOR pathway. PMID:24896186

  15. CT radiogenomic characterization of EGFR, K-RAS, and ALK mutations in non-small cell lung cancer

    Energy Technology Data Exchange (ETDEWEB)

    Rizzo, Stefania; Rampinelli, Cristiano [European Institute of Oncology, Department of Radiology, Milan (Italy); Petrella, Francesco; Spaggiari, Lorenzo [European Institute of Oncology, Department of Thoracic Surgery, Milan (Italy); Buscarino, Valentina; De Maria, Federica [University of Milan, Department of Health Sciences, Milan (Italy); Raimondi, Sara [European Institute of Oncology, Department of Epidemiology and Biostatistics, Milan (Italy); Barberis, Massimo; Fumagalli, Caterina [European Institute of Oncology, Department of Pathology, Milan (Italy); Spitaleri, Gianluca; De Marinis, Filippo [European Institute of Oncology, Department of Thoracic Oncology, Milan (Italy); Bellomi, Massimo [European Institute of Oncology, Department of Radiology, Milan (Italy); University of Milan, Department of Health Sciences, Milan (Italy)

    2016-01-15

    To assess the association between CT features and EGFR, ALK, KRAS mutations in non-small cell lung cancer. Patients undergoing chest CT and testing for the above gene mutations were included. Qualitative evaluation of CTs included: lobe; lesion diameter; shape; margins; ground-glass opacity; density; cavitation; air bronchogram; pleural thickening; intratumoral necrosis; nodules in tumour lobe; nodules in non-tumour lobes; pleural retraction; location; calcifications; emphysema; fibrosis; pleural contact; pleural effusion. Statistical analysis was performed to assess association of features with each gene mutation. ROC curves for gene mutations were drawn; the corresponding area under the curve was calculated. P-values <0.05 were considered significant. Of 285 patients, 60/280 (21.43 %) were positive for EGFR mutation; 31/270 (11.48 %) for ALK rearrangement; 64/240 (26.67 %) for KRAS mutation. EGFR mutation was associated with air bronchogram, pleural retraction, females, non-smokers, small lesion size, and absence of fibrosis. ALK rearrangements were associated with age and pleural effusion. KRAS mutation was associated with round shape, nodules in non-tumour lobes, and smoking. This study disclosed associations between CT features and alterations of EGFR (air bronchogram, pleural retraction, small lesion size, absence of fibrosis), ALK (pleural effusion) and KRAS (round lesion shape, nodules in non-tumour lobes). (orig.)

  16. High-throughput genotyping in metastatic esophageal squamous cell carcinoma identifies phosphoinositide-3-kinase and BRAF mutations.

    Directory of Open Access Journals (Sweden)

    Chi Hoon Maeng

    Full Text Available Given the high incidence of metastatic esophageal squamous cell carcinoma, especially in Asia, we screened for the presence of somatic mutations using OncoMap platform with the aim of defining subsets of patients who may be potential candidate for targeted therapy.We analyzed 87 tissue specimens obtained from 80 patients who were pathologically confirmed with esophageal squamous cell carcinoma and received 5-fluoropyrimidine/platinum-based chemotherapy. OncoMap 4.0, a mass-spectrometry based assay, was used to interrogate 471 oncogenic mutations in 41 commonly mutated genes. Tumor specimens were prepared from primary cancer sites in 70 patients and from metastatic sites in 17 patients. In order to test the concordance between primary and metastatic sites from the patient for mutations, we analyzed 7 paired (primary-metastatic specimens. All specimens were formalin-fixed paraffin embedded tissues and tumor content was >70%.In total, we have detected 20 hotspot mutations out of 80 patients screened. The most frequent mutation was PIK3CA mutation (four E545K, five H1047R and one H1047L (N = 10, 11.5% followed by MLH1 V384D (N = 7, 8.0%, TP53 (R306, R175H and R273C (N = 3, 3.5%, BRAF V600E (N = 1, 1.2%, CTNNB1 D32N (N = 1, 1.2%, and EGFR P733L (N = 1, 1.2%. Distributions of somatic mutations were not different according to anatomic sites of esophageal cancer (cervical/upper, mid, lower. In addition, there was no difference in frequency of mutations between primary-metastasis paired samples.Our study led to the detection of potentially druggable mutations in esophageal SCC which may guide novel therapies in small subsets of esophageal cancer patients.

  17. High specificity but low sensitivity of mutation-specific antibodies against EGFR mutations in non-small-cell lung cancer

    DEFF Research Database (Denmark)

    Bondgaard, Anna-Louise; Høgdall, Estrid; Mellemgaard, Anders

    2014-01-01

    of more sensitive methods including real-time PCR (RT-PCR). Immunohistochemistry with mutation-specific antibodies might be a promising detection method. We evaluated 210 samples with NSCLC from an unselected Caucasian population. Extracted DNA was analyzed for EGFR mutations by RT-PCR (Therascreen EGFR......, and staining score (multipum of intensity (graded 0-3) and percentages (0-100%) of stained tumor cells) was calculated. Positivity was defined as staining score >0. Specificity of exon19 antibody was 98.8% (95% confidence interval=95.9-99.9%) and of exon21 antibody 97.8% (95% confidence interval=94...... was demonstrated. However, sensitivity was low, especially for exon19 deletions, and thus these antibodies cannot yet be used as screening method for EGFR mutations in NSCLC. Refinement of sensitivity for the mutation-specific antibodies is warranted to improve molecular diagnosis using EGFR immunohistochemistry....

  18. KRAS mutation is a predictor of oxaliplatin sensitivity in colon cancer cells.

    Directory of Open Access Journals (Sweden)

    Yu-Lin Lin

    Full Text Available Molecular biomarkers to determine the effectiveness of targeted therapies in cancer treatment have been widely adopted in colorectal cancer (CRC, but those to predict chemotherapy sensitivity remain poorly defined. We tested our hypothesis that KRAS mutation may be a predictor of oxaliplatin sensitivity in CRC. KRAS was knocked-down in KRAS-mutant CRC cells (DLD-1(G13D and SW480(G12V by small interfering RNAs (siRNA and overexpressed in KRAS-wild-type CRC cells (COLO320DM by KRAS-mutant vectors to generate paired CRC cells. These paired CRC cells were tested by oxaliplatin, irinotecan and 5FU to determine the change in drug sensitivity by MTT assay and flow cytometry. Reasons for sensitivity alteration were further determined by western blot and real-time quantitative reverse transcriptase polymerase chain reaction (qRT -PCR. In KRAS-wild-type CRC cells (COLO320DM, KRAS overexpression by mutant vectors caused excision repair cross-complementation group 1 (ERCC1 downregulation in protein and mRNA levels, and enhanced oxaliplatin sensitivity. In contrast, in KRAS-mutant CRC cells (DLD-1(G13D and SW480(G12V, KRAS knocked-down by KRAS-siRNA led to ERCC1 upregulation and increased oxaliplatin resistance. The sensitivity of irinotecan and 5FU had not changed in the paired CRC cells. To validate ERCC1 as a predictor of sensitivity for oxaliplatin, ERCC1 was knocked-down by siRNA in KRAS-wild-type CRC cells, which restored oxaliplatin sensitivity. In contrast, ERCC1 was overexpressed by ERCC1-expressing vectors in KRAS-mutant CRC cells, and caused oxaliplatin resistance. Overall, our findings suggest that KRAS mutation is a predictor of oxaliplatin sensitivity in colon cancer cells by the mechanism of ERCC1 downregulation.

  19. Population genetics inside a cell: Mutations and mitochondrial genome maintenance

    Science.gov (United States)

    Goyal, Sidhartha; Shraiman, Boris; Gottschling, Dan

    2012-02-01

    In realistic ecological and evolutionary systems natural selection acts on multiple levels, i.e. it acts on individuals as well as on collection of individuals. An understanding of evolutionary dynamics of such systems is limited in large part due to the lack of experimental systems that can challenge theoretical models. Mitochondrial genomes (mtDNA) are subjected to selection acting on cellular as well as organelle levels. It is well accepted that mtDNA in yeast Saccharomyces cerevisiae is unstable and can degrade over time scales comparable to yeast cell division time. We utilize a recent technology designed in Gottschling lab to extract DNA from populations of aged yeast cells and deep sequencing to characterize mtDNA variation in a population of young and old cells. In tandem, we developed a stochastic model that includes the essential features of mitochondrial biology that provides a null model for expected mtDNA variation. Overall, we find approximately 2% of the polymorphic loci that show significant increase in frequency as cells age providing direct evidence for organelle level selection. Such quantitative study of mtDNA dynamics is absolutely essential to understand the propagation of mtDNA mutations linked to a spectrum of age-related diseases in humans.

  20. Correlation of Merkel cell polyomavirus positivity with PDGFRα mutations and survivin expression in Merkel cell carcinoma.

    Science.gov (United States)

    Batinica, M; Akgül, B; Silling, S; Mauch, C; Zigrino, P

    2015-07-01

    Merkel cell carcinoma (MCC) is a neuroendocrine cancer of the skin postulated to originate through Merkel cell polyomavirus (MCPyV) oncogenesis and/or by mutations in molecules implicated in the regulation of cell growth and survival. Despite the fact that MCPvV is detected more broadly within the population, only a part of the infected people also develop MCC. It is thus conceivable that together, virus and for example mutations, are necessary for disease development. However, apart from a correlation between MCPyV positivity or mutations and MCC development, less is known about the association of these factors with progressive disease. To analyze MCPyV positivity, load and integration in MCC as well as presence of mutations in PDGFRα and TP53 genes and correlate these with clinical features and disease progression to identify features with prognostic value for clinical progression. This is a study on a MCC population group of 64 patients. MCPyV positivity, load and integration in parallel to mutations in the PDGFRα and TP53 were analyzed on genomic DNA from MCC specimens. In addition, expression of PDGFRα, survivin and p53 proteins was analyzed by immunodetection in tissues specimens. All these parameters were analyzed as function of patient's disease progression status. 83% of MCCs were positive for the MCPyV and among these 36% also displayed virus-T integration. Viral load ranged from 0.006 to 943 viral DNA copies/β-globin gene and was highest in patients with progressive disease. We detected more than one mutation within the PDGFRα gene and identified two new SNPs in 36% of MCC patients, whereas no mutations were found in TP53 gene. Survivin was expressed in 78% of specimens. We could not correlate either mutations in PDGFR or expression of PDGFR, p53 and surviving either to the disease progression or to the MCPyV positivity. In conclusion, our data indicate that the viral positivity when associated with high viral load, correlates with poor disease

  1. KIT polymorphisms and mutations determine responses of neoplastic mast cells to bafetinib (INNO-406).

    Science.gov (United States)

    Peter, Barbara; Hadzijusufovic, Emir; Blatt, Katharina; Gleixner, Karoline V; Pickl, Winfried F; Thaiwong, Tuddow; Yuzbasiyan-Gurkan, Vilma; Willmann, Michael; Valent, Peter

    2010-09-01

    Advanced systemic mastocytosis (SM) is characterized by uncontrolled growth of neoplastic mast cells (MC) and drug resistance. The tyrosine kinase receptor KIT is often mutated and activated and thus contributes to malignant growth of MC. Therefore, KIT-targeting drugs are currently tested for their ability to block growth of malignant MC. We determined the effects of the multikinase inhibitor INNO-406 (bafetinib) on primary neoplastic MC, the canine mastocytoma cell line C2, the human MC leukemia cell line HMC-1.1 bearing the KIT mutant V560G, and HMC-1.2 cells harboring KIT V560G and KIT D816V. INNO-406 was found to inhibit proliferation in HMC-1.1 cells (IC(50): 30-40 nM), but not in HMC-1.2 cells or primary neoplastic cells in patients with KIT D816V-positive SM. In canines, growth-inhibitory effects of INNO-406 were seen in C2 cells (IC(50): 50-100 nM) exhibiting a KIT exon 11 internal tandem-duplication and in primary neoplastic MC harboring wild-type exon 11, whereas no effects were seen in MC exhibiting a polymorphism at amino acid 581 in exon 11. INNO-406 was found to block KIT phosphorylation and expression in HMC-1.1 cells and C2 cells, but not in HMC-1.2 cells, whereas Lyn-phosphorylation was blocked by INNO-406 in all types of MC. In neoplastic MC, the major target of INNO-406 appears to be KIT. Drug responses may depend on the presence and type of KIT mutation. In human MC, the KIT D816V mutant introduces resistance, and in canine mastocytomas, an exon 11 polymorphism may be indicative of resistance against INNO-406.

  2. SF3B1-initiating mutations in MDS-RSs target lymphomyeloid hematopoietic stem cells.

    Science.gov (United States)

    Mortera-Blanco, Teresa; Dimitriou, Marios; Woll, Petter S; Karimi, Mohsen; Elvarsdottir, Edda; Conte, Simona; Tobiasson, Magnus; Jansson, Monika; Douagi, Iyadh; Moarii, Matahi; Saft, Leonie; Papaemmanuil, Elli; Jacobsen, Sten Eirik W; Hellström-Lindberg, Eva

    2017-08-17

    Mutations in the RNA splicing gene SF3B1 are found in >80% of patients with myelodysplastic syndrome with ring sideroblasts (MDS-RS). We investigated the origin of SF3B1 mutations within the bone marrow hematopoietic stem and progenitor cell compartments in patients with MDS-RS. Screening for recurrently mutated genes in the mononuclear cell fraction revealed mutations in SF3B1 in 39 of 40 cases (97.5%), combined with TET2 and DNMT3A in 11 (28%) and 6 (15%) patients, respectively. All recurrent mutations identified in mononuclear cells could be tracked back to the phenotypically defined hematopoietic stem cell (HSC) compartment in all investigated patients and were also present in downstream myeloid and erythroid progenitor cells. While in agreement with previous studies, little or no evidence for clonal ( SF3B1 mutation) involvement could be found in mature B cells, consistent involvement at the pro-B-cell progenitor stage was established, providing definitive evidence for SF3B1 mutations targeting lymphomyeloid HSCs and compatible with mutated SF3B1 negatively affecting lymphoid development. Assessment of stem cell function in vitro as well as in vivo established that only HSCs and not investigated progenitor populations could propagate the SF3B1 mutated clone. Upon transplantation into immune-deficient mice, SF3B1 mutated MDS-RS HSCs differentiated into characteristic ring sideroblasts, the hallmark of MDS-RS. Our findings provide evidence of a multipotent lymphomyeloid HSC origin of SF3B1 mutations in MDS-RS patients and provide a novel in vivo platform for mechanistically and therapeutically exploring SF3B1 mutated MDS-RS. © 2017 by The American Society of Hematology.

  3. Problem-Solving Test: Analysis of DNA Damage Recognizing Proteins in Yeast and Human Cells

    Science.gov (United States)

    Szeberenyi, Jozsef

    2013-01-01

    The experiment described in this test was aimed at identifying DNA repair proteins in human and yeast cells. Terms to be familiar with before you start to solve the test: DNA repair, germline mutation, somatic mutation, inherited disease, cancer, restriction endonuclease, radioactive labeling, [alpha-[superscript 32]P]ATP, [gamma-[superscript…

  4. Cell cultures in uterine leiomyomas: rapid disappearance of cells carrying MED12 mutations.

    Science.gov (United States)

    Nadine Markowski, Dominique; Tadayyon, Mahboobeh; Bartnitzke, Sabine; Belge, Gazanfer; Maria Helmke, Burkhard; Bullerdiek, Jörn

    2014-04-01

    Uterine leiomyomas (UL) are the most frequent symptomatic human tumors. Nevertheless, their molecular pathogenesis is not yet fully understood. To learn more about the biology of these common neoplasms and their response to treatment, cell cultures derived from UL are a frequently used model system, but until recently appropriate genetic markers confirming their origin from the tumor cell population were lacking for most UL, i.e., those not displaying karyotypic abnormalities. The identification of MED12 mutations in the majority of UL makes it possible to trace the tumor cell population during in vitro passaging in the absence of cytogenetic abnormalities. The present study is addressing the in vitro survival of cells carrying MED12 mutations and its association with karyotypic alterations. The results challenge numerous in vitro studies into the biology and behavior of leiomyomas. Cells of one genetic subtype of UL, i.e., those with rearrangements of the high mobility AT-hook 2 protein gene (HMGA2), seem to be able to proliferate in vitro for many passages whereas tumor cells from the much more frequent MED12-mutated lesions barely survive even the first passages. Apparently, for the most frequent type of human UL no good in vitro model seems to exist because cells do not survive culturing. On the other hand, this inability may point to an Achilles' heel of this type of UL. Copyright © 2014 Wiley Periodicals, Inc.

  5. Empirical evaluation of cell critical volume dose vs. cell response function for pink mutations in tradescantia

    International Nuclear Information System (INIS)

    Varma, M.N.; Bond, V.P.

    1982-01-01

    Microdosimetric spectra for 0.43, 1.8, and 14.7 MeV neutrons, and for 215 kVp x rays and 1250 keV gammas were used in conjunction with relative biological effectiveness (RBE) values for pink mutations in Tradescantia to obtain an effectiveness function (i.e., a cell critical volume dose vs. cell response function). This effectiveness function (or hit size weighting function) provides the probability of inducing a biological effect of interest (in the present study, pink mutations in Tradescantia) as a function of lineal energy density y. In a preliminary analysis the critical value of y above which pink mutations are seen was 4.5 keV/μm, and the value of y at which the probability reaches unity was 115 keV/μm. Idealized but approximate event size distributions for mono-LET particles ranging from 10 to 5000 keV/μm were generated, and these distributions were weighted by the effectiveness function to determine the pink mutation frequencies. Results are compared with measured pink mutation frequencies for 11 keV/μm ( 12 C) and 31 keV/μm ( 20 Ne) ions

  6. Evidence of heritable lethal mutations in progeny of X-irradiated CHO cells by micronucleus count in clon-cells

    International Nuclear Information System (INIS)

    Hagemann, G.; Kreczik, A.; Treichel, M.

    1996-01-01

    Low doses of ionizing radiation reduce the growth rates of clones following irradiation of the progenitor cells. Such reductions of clone growth have been proven by means of measurements of clone size distributions. The medians of such distributions can be used to quantify the radiation damage. Prolongations of generation times and cell death as result of heritable lethal mutations have been discussed as causes for the reduction of clone growth. The cell number of a clone of hypotetraploid CHO-cells was compared to the frequency of micronucleated binucleated cells in the same clone using the cytokinesis-block-micronucleus method. The dose dependent reduction of clone sizes is measured by the difference of the medians (after log transformation) of the clone size distributions. At cytochalasin-B concentrations of 1 μg/ml and after an incubation time of 16 h a yield of binucleated cells of about 50% was obtained. Median clone size differences as a measure of clonal radiation damage increased linearly with incubation times of 76, 100, 124, and 240 h following irradiation with 3, 5, 7, and 12 Gy. The frequency of binucleated clone cells with micronuclei strongly increased with decreasing clone size by a factor up to 20 following irradiation with 3, 5, and 7 Gy. The frequency of micronucleated binucleated clone cells was found to be independent of incubation time after irradiation. Radiation induced clone size reductions result from cell losses caused by intraclonal expression of micronuclei which have its origin in heritable lethal mutations. Measurements of clone size distributions can be done automatically. They can serve as predictive test for determination of median cell loss rates of surviving cell clones. (orig./MG) [de

  7. Arsenic trioxide promotes mitochondrial DNA mutation and cell apoptosis in primary APL cells and NB4 cell line.

    Science.gov (United States)

    Meng, Ran; Zhou, Jin; Sui, Meng; Li, ZhiYong; Feng, GuoSheng; Yang, BaoFeng

    2010-01-01

    This study aimed to investigate the effects of arsenic trioxide (As(2)O(3)) on the mitochondrial DNA (mtDNA) of acute promyelocytic leukemia (APL) cells. The NB4 cell line was treated with 2.0 micromol/L As(2)O(3) in vitro, and the primary APL cells were treated with 2.0 micromol/L As(2)O(3) in vitro and 0.16 mg kg(-1) d(-1) As(2)O(3) in vivo. The mitochondrial DNA of all the cells above was amplified by PCR, directly sequenced and analyzed by Sequence Navigatore and Factura software. The apoptosis rates were assayed by flow cytometry. Mitochondrial DNA mutation in the D-loop region was found in NB4 and APL cells before As(2)O(3) use, but the mutation spots were remarkably increased after As(2)O(3) treatment, which was positively correlated to the rates of cellular apoptosis, the correlation coefficient: r (NB4-As2O3)=0.973818, and r (APL-As2O3)=0.934703. The mutation types include transition, transversion, codon insertion or deletion, and the mutation spots in all samples were not constant and regular. It is revealed that As(2)O(3) aggravates mtDNA mutation in the D-loop region of acute promyelocytic leukemia cells both in vitro and in vivo. Mitochondrial DNA might be one of the targets of As(2)O(3) in APL treatment.

  8. Epidermal growth factor receptor (EGFR) mutations and expression in squamous cell carcinoma of the esophagus in central Asia

    International Nuclear Information System (INIS)

    Abedi-Ardekani, Behnoush; Malekzadeh, Reza; Hainaut, Pierre; Dar, Nazir Ahmad; Mir, Mohammad Muzaffar; Zargar, Showkat Ahmad; Lone, M Muqbool; Martel-Planche, Ghyslaine; Villar, Stéphanie; Mounawar, Mounia; Saidi, Farrokh

    2012-01-01

    Esophageal squamous cell carcinoma (ESCC) shows geographic variations in incidence, with high incidences (>50/10 5 person-years) in central Asia, including North Eastern Iran (Golestan) and Northern India (Kashmir). In contrast to Western countries, smoking does not appear to be a significant risk factor for ESCC in central Asia. In lung adenocarcinoma, activating mutations in the gene encoding epidermal growth factor receptor (EGFR) are frequent in tumors of never smokers of Asian origin, predicting therapeutic sensitivity to Egfr-targeting drugs. In this study 152 cases of histologically confirmed ESCC from Iran (Tehran and Golestan Province) and North India (Kashmir Valley) have been analyzed for EGFR mutation by direct sequencing of exons 18–21. Egfr protein expression was evaluated by immunohistochemistry in 34 samples from Tehran and HER2 mutations were analyzed in 54 cases from Kashmir. A total of 14 (9.2%) EGFR variations were detected, including seven variations in exons. Among those, four (2.6%) were already documented in lung cancers, two were reported as polymorphisms and one was a potentially new activating mutation. All but one variation in introns were previously identified as polymorphisms. Over-expression of Egfr was detected in 22/34 (65%) of tested cases whereas no HER2 mutation was found in 54 cases from Kashmir. Overall, EGFR mutations appear to be a rare event in ESCC in high incidence areas of central Asia, although a very small proportion of cases may harbor mutations predicting sensitivity to anti-Egfr drugs

  9. Inhibition of Bcl-2 or IAP proteins does not provoke mutations in surviving cells

    International Nuclear Information System (INIS)

    Shekhar, Tanmay M.; Green, Maja M.; Rayner, David M.; Miles, Mark A.; Cutts, Suzanne M.; Hawkins, Christine J.

    2015-01-01

    Graphical abstract: - Highlights: • Mutagenicities of anti-cancer drugs were tested using HPRT, γH2AX and comet assays. • TRAIL, doxorubicin and etoposide were more mutagenic than BH3- or Smac-mimetics. • Physiologically achievable levels of the BH3-mimetic ABT-737 were not mutagenic. • High concentrations of ABT-737 provoked mutations via an off-target mechanism. • Even very high concentrations of IAP antagonists were not mutagenic. - Abstract: Chemotherapy and radiotherapy can cause permanent damage to the genomes of surviving cells, provoking severe side effects such as second malignancies in some cancer survivors. Drugs that mimic the activity of death ligands, or antagonise pro-survival proteins of the Bcl-2 or IAP families have yielded encouraging results in animal experiments and early phase clinical trials. Because these agents directly engage apoptosis pathways, rather than damaging DNA to indirectly provoke tumour cell death, we reasoned that they may offer another important advantage over conventional therapies: minimisation or elimination of side effects such as second cancers that result from mutation of surviving normal cells. Disappointingly, however, we previously found that concentrations of death receptor agonists like TRAIL that would be present in vivo in clinical settings provoked DNA damage in surviving cells. In this study, we used cell line model systems to investigate the mutagenic capacity of drugs from two other classes of direct apoptosis-inducing agents: the BH3-mimetic ABT-737 and the IAP antagonists LCL161 and AT-406. Encouragingly, our data suggest that IAP antagonists possess negligible genotoxic activity. Doses of ABT-737 that were required to damage DNA stimulated Bax/Bak-independent signalling and exceeded concentrations detected in the plasma of animals treated with this drug. These findings provide hope that cancer patients treated by BH3-mimetics or IAP antagonists may avoid mutation-related illnesses that afflict

  10. Inhibition of Bcl-2 or IAP proteins does not provoke mutations in surviving cells

    Energy Technology Data Exchange (ETDEWEB)

    Shekhar, Tanmay M. [Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora 3083 (Australia); Green, Maja M. [Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora 3083 (Australia); Department of Anatomy & Neuroscience, The University of Melbourne, Parkville 3010 (Australia); Rayner, David M.; Miles, Mark A.; Cutts, Suzanne M. [Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora 3083 (Australia); Hawkins, Christine J., E-mail: c.hawkins@latrobe.edu.au [Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora 3083 (Australia)

    2015-07-15

    Graphical abstract: - Highlights: • Mutagenicities of anti-cancer drugs were tested using HPRT, γH2AX and comet assays. • TRAIL, doxorubicin and etoposide were more mutagenic than BH3- or Smac-mimetics. • Physiologically achievable levels of the BH3-mimetic ABT-737 were not mutagenic. • High concentrations of ABT-737 provoked mutations via an off-target mechanism. • Even very high concentrations of IAP antagonists were not mutagenic. - Abstract: Chemotherapy and radiotherapy can cause permanent damage to the genomes of surviving cells, provoking severe side effects such as second malignancies in some cancer survivors. Drugs that mimic the activity of death ligands, or antagonise pro-survival proteins of the Bcl-2 or IAP families have yielded encouraging results in animal experiments and early phase clinical trials. Because these agents directly engage apoptosis pathways, rather than damaging DNA to indirectly provoke tumour cell death, we reasoned that they may offer another important advantage over conventional therapies: minimisation or elimination of side effects such as second cancers that result from mutation of surviving normal cells. Disappointingly, however, we previously found that concentrations of death receptor agonists like TRAIL that would be present in vivo in clinical settings provoked DNA damage in surviving cells. In this study, we used cell line model systems to investigate the mutagenic capacity of drugs from two other classes of direct apoptosis-inducing agents: the BH3-mimetic ABT-737 and the IAP antagonists LCL161 and AT-406. Encouragingly, our data suggest that IAP antagonists possess negligible genotoxic activity. Doses of ABT-737 that were required to damage DNA stimulated Bax/Bak-independent signalling and exceeded concentrations detected in the plasma of animals treated with this drug. These findings provide hope that cancer patients treated by BH3-mimetics or IAP antagonists may avoid mutation-related illnesses that afflict

  11. Frequent mutations of genes encoding ubiquitin-mediated proteolysis pathway components in clear cell renal cell carcinoma

    DEFF Research Database (Denmark)

    Guo, Guangwu; Gui, Yaoting; Gao, Shengjie

    2012-01-01

    We sequenced whole exomes of ten clear cell renal cell carcinomas (ccRCCs) and performed a screen of similar to 1,100 genes in 88 additional ccRCCs, from which we discovered 12 previously unidentified genes mutated at elevated frequencies in ccRCC. Notably, we detected frequent mutations in the u...

  12. Mutational status of synchronous and metachronous tumor samples in patients with metastatic non-small-cell lung cancer

    International Nuclear Information System (INIS)

    Quéré, Gilles; Descourt, Renaud; Robinet, Gilles; Autret, Sandrine; Raguenes, Odile; Fercot, Brigitte; Alemany, Pierre; Uguen, Arnaud; Férec, Claude; Quintin-Roué, Isabelle; Le Gac, Gérald

    2016-01-01

    Despite reported discordance between the mutational status of primary lung cancers and their metastases, metastatic sites are rarely biopsied and targeted therapy is guided by genetic biomarkers detected in the primary tumor. This situation is mostly explained by the apparent stability of EGFR-activating mutations. Given the dramatic increase in the range of candidate drugs and high rates of drug resistance, rebiopsy or liquid biopsy may become widespread. The purpose of this study was to test genetic biomarkers used in clinical practice (EGFR, ALK) and candidate biomarkers identified by the French National Cancer Institute (KRAS, BRAF, PIK3CA, HER2) in patients with metastatic non-small-cell lung cancer for whom two tumor samples were available. A retrospective study identified 88 tumor samples collected synchronously or metachronously, from the same or two different sites, in 44 patients. Mutation analysis used SNaPshot (EGFR, KRAS, BRAF missense mutations), pyrosequencing (EGFR and PIK3CA missense mutations), sizing assays (EGFR and HER2 indels) and IHC and/or FISH (ALK rearrangements). About half the patients (52 %) harbored at least one mutation. Five patients had an activating mutation of EGFR in both the primary tumor and the metastasis. The T790M resistance mutation was detected in metastases in 3 patients with acquired resistance to EGFR tyrosine kinase inhibitors. FISH showed discordance in ALK status between a small biopsy sample and the surgical specimen. KRAS mutations were observed in 36 % of samples, six patients (14 %) having discordant genotypes; all discordances concerned sampling from different sites. Two patients (5 %) showed PI3KCA mutations. One metastasis harbored both PI3KCA and KRAS mutations, while the synchronously sampled primary tumor was mutation free. No mutations were detected in BRAF and HER2. This study highlighted noteworthy intra-individual discordance in KRAS mutational status, whereas EGFR status was stable. Intratumoral

  13. The Arctic Alzheimer mutation enhances sensitivity to toxic stress in human neuroblastoma cells

    DEFF Research Database (Denmark)

    Sennvik, Kristina; Nilsberth, Camilla; Stenh, Charlotte

    2002-01-01

    The E693G (Arctic) mutation of the amyloid precursor protein was recently found to lead to early-onset Alzheimer's disease in a Swedish family. In the present study, we report that the Arctic mutation decreases cell viability in human neuroblastoma cells. The cell viability, as measured by the MTT...... their secretion of beta-secretase cleaved amyloid precursor protein. The enhanced sensitivity to toxic stress in cells with the Arctic mutation most likely contributes to the pathogenic pathway leading to Alzheimer's disease....

  14. Dysfunctional telomeres in human BRCA2 mutated breast tumors and cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Bodvarsdottir, Sigridur K., E-mail: skb@hi.is [Cancer Research Laboratory, BioMedical Centre, Faculty of Medicine, University of Iceland, Vatnsmyrarvegi 16, 101 Reykjavik (Iceland); Steinarsdottir, Margret [Chromosome Laboratory, Department of Genetics and Molecular Medicine, Landspitali University Hospital, Reykjavik (Iceland); Bjarnason, Hordur; Eyfjord, Jorunn E. [Cancer Research Laboratory, BioMedical Centre, Faculty of Medicine, University of Iceland, Vatnsmyrarvegi 16, 101 Reykjavik (Iceland)

    2012-01-03

    In the present study the possible involvement of telomeres in chromosomal instability of breast tumors and cell lines from BRCA2 mutation carriers was examined. Breast tumors from BRCA2 mutation carriers showed significantly higher frequency of chromosome end-to-end fusions (CEFs) than tumors from non-carriers despite normal telomere DNA content. Frequent CEFs were also found in four different BRCA2 heterozygous breast epithelial cell lines, occasionally with telomere signal at the fusion point, indicating telomere capping defects. Extrachromosomal telomeric repeat (ECTR) DNA was frequently found scattered around metaphase chromosomes and interstitial telomere sequences (ITSs) were also common. Telomere sister chromatid exchanges (T-SCEs), characteristic of cells using alternative lengthening of telomeres (ALT), were frequently detected in all heterozygous BRCA2 cell lines as well as the two ALT positive cell lines tested. Even though T-SCE frequency was similar in BRCA2 heterozygous and ALT positive cell lines they differed in single telomere signal loss and ITSs. Chromatid type alterations were more prominent in the BRCA2 heterozygous cell lines that may have propensity for telomere based chromosome healing. Telomere dysfunction-induced foci (TIFs) formation, identified by co-localization of telomeres and {gamma}-H2AX, supported telomere associated DNA damage response in BRCA2 heterozygous cell lines. TIFs were found in interphase nuclei, at chromosome ends, ITSs and ECTR DNA. In conclusion, our results suggest that BRCA2 has an important role in telomere stabilization by repressing CEFs through telomere capping and the prevention of telomere loss by replication stabilization.

  15. Dysfunctional telomeres in human BRCA2 mutated breast tumors and cell lines

    International Nuclear Information System (INIS)

    Bodvarsdottir, Sigridur K.; Steinarsdottir, Margret; Bjarnason, Hordur; Eyfjord, Jorunn E.

    2012-01-01

    In the present study the possible involvement of telomeres in chromosomal instability of breast tumors and cell lines from BRCA2 mutation carriers was examined. Breast tumors from BRCA2 mutation carriers showed significantly higher frequency of chromosome end-to-end fusions (CEFs) than tumors from non-carriers despite normal telomere DNA content. Frequent CEFs were also found in four different BRCA2 heterozygous breast epithelial cell lines, occasionally with telomere signal at the fusion point, indicating telomere capping defects. Extrachromosomal telomeric repeat (ECTR) DNA was frequently found scattered around metaphase chromosomes and interstitial telomere sequences (ITSs) were also common. Telomere sister chromatid exchanges (T-SCEs), characteristic of cells using alternative lengthening of telomeres (ALT), were frequently detected in all heterozygous BRCA2 cell lines as well as the two ALT positive cell lines tested. Even though T-SCE frequency was similar in BRCA2 heterozygous and ALT positive cell lines they differed in single telomere signal loss and ITSs. Chromatid type alterations were more prominent in the BRCA2 heterozygous cell lines that may have propensity for telomere based chromosome healing. Telomere dysfunction-induced foci (TIFs) formation, identified by co-localization of telomeres and γ-H2AX, supported telomere associated DNA damage response in BRCA2 heterozygous cell lines. TIFs were found in interphase nuclei, at chromosome ends, ITSs and ECTR DNA. In conclusion, our results suggest that BRCA2 has an important role in telomere stabilization by repressing CEFs through telomere capping and the prevention of telomere loss by replication stabilization.

  16. Mutation Pattern of Paired Immunoglobulin Heavy and Light Variable Domains in Chronic Lymphocytic Leukemia B Cells

    Science.gov (United States)

    Ghiotto, Fabio; Marcatili, Paolo; Tenca, Claudya; Calevo, Maria Grazia; Yan, Xiao-Jie; Albesiano, Emilia; Bagnara, Davide; Colombo, Monica; Cutrona, Giovanna; Chu, Charles C; Morabito, Fortunato; Bruno, Silvia; Ferrarini, Manlio; Tramontano, Anna; Fais, Franco; Chiorazzi, Nicholas

    2011-01-01

    B-cell chronic lymphocytic leukemia (CLL) patients display leukemic clones bearing either germline or somatically mutated immunoglobulin heavy variable (IGHV ) genes. Most information on CLL immunoglobulins (Igs), such as the definition of stereotyped B-cell receptors (BCRs), was derived from germline unmutated Igs. In particular, detailed studies on the distribution and nature of mutations in paired heavy- and light-chain domains of CLL clones bearing mutated Igs are lacking. To address the somatic hyper-mutation dynamics of CLL Igs, we analyzed the mutation pattern of paired IGHV–diversity-joining (IGHV-D-J ) and immunoglobulin kappa/lambda variable-joining (IGK/LV-J ) rearrangements of 193 leukemic clones that displayed ≥2% mutations in at least one of the two immunoglobulin variable (IGV ) genes (IGHV and/or IGK/LV ). The relationship between the mutation frequency in IGHV and IGK/LV complementarity determining regions (CDRs) and framework regions (FRs) was evaluated by correlation analysis. Replacement (R) mutation frequency within IGK/LV chain CDRs correlated significantly with mutation frequency of paired IGHV CDRs in λ but not κ isotype CLL clones. CDRs of IGKV-J rearrangements displayed a lower percentage of R mutations than IGHVs. The frequency/pattern of mutations in kappa CLL Igs differed also from that in κ-expressing normal B cells described in the literature. Instead, the mutation frequency within the FRs of IGHV and either IGKV or IGLV was correlated. Notably, the amount of diversity introduced by replaced amino acids was comparable between IGHVs and IGKVs. The data indicate a different mutation pattern between κ and λ isotype CLL clones and suggest an antigenic selection that, in κ samples, operates against CDR variation. PMID:21785810

  17. Mutation Pattern of Paired Immunoglobulin Heavy and Light Variable Domains in Chronic Lymphocytic Leukemia B Cells

    KAUST Repository

    Ghiotto, Fabio; Marcatili, Paolo

    2011-01-01

    B-cell chronic lymphocytic leukemia (CLL) patients display leukemic clones bearing either germline or somatically mutated immunoglobulin heavy variable (IGHV ) genes. Most information on CLL immunoglobulins (Igs), such as the definition of stereotyped B-cell receptors (BCRs), was derived from germline unmutated Igs. In particular, detailed studies on the distribution and nature of mutations in paired heavy- and light-chain domains of CLL clones bearing mutated Igs are lacking. To address the somatic hyper-mutation dynamics of CLL Igs, we analyzed the mutation pattern of paired IGHV-diversity-joining (IGHV-D-J ) and immunoglobulin kappa/lambda variable-joining (IGK/LV-J ) rearrangements of 193 leukemic clones that displayed ≥ 2% mutations in at least one of the two immunoglobulin variable (IGV ) genes (IGHV and/or IGK/LV ). The relationship between the mutation frequency in IGHV and IGK/LV complementarity determining regions (CDRs) and framework regions (FRs) was evaluated by correlation analysis. Replacement (R) mutation frequency within IGK/LV chain CDRs correlated significantly with mutation frequency of paired IGHV CDRs in λ but not κ isotype CLL clones. CDRs of IGKV-J rearrangements displayed a lower percentage of R mutations than IGHVs. The frequency/pattern of mutations in kappa CLL Igs differed also from that in κ-expressing normal B cells described in the literature. Instead, the mutation frequency within the FRs of IGHV and either IGKV or IGLV was correlated. Notably, the amount of diversity introduced by replaced amino acids was comparable between IGHVs and IGKVs. The data indicate a different mutation pattern between κ and λ isotype CLL clones and suggest an antigenic selection that, in κ samples, operates against CDR variation.

  18. KRAS mutation testing of tumours in adults with metastatic colorectal cancer: a systematic review and cost-effectiveness analysis.

    Science.gov (United States)

    Westwood, Marie; van Asselt, Thea; Ramaekers, Bram; Whiting, Penny; Joore, Manuela; Armstrong, Nigel; Noake, Caro; Ross, Janine; Severens, Johan; Kleijnen, Jos

    2014-10-01

    Bowel cancer is the third most common cancer in the UK. Most bowel cancers are initially treated with surgery, but around 17% spread to the liver. When this happens, sometimes the liver tumour can be treated surgically, or chemotherapy may be used to shrink the tumour to make surgery possible. Kirsten rat sarcoma viral oncogene (KRAS) mutations make some tumours less responsive to treatment with biological therapies such as cetuximab. There are a variety of tests available to detect these mutations. These vary in the specific mutations that they detect, the amount of mutation they detect, the amount of tumour cells needed, the time to give a result, the error rate and cost. To compare the performance and cost-effectiveness of KRAS mutation tests in differentiating adults with metastatic colorectal cancer whose metastases are confined to the liver and are unresectable and who may benefit from first-line treatment with cetuximab in combination with standard chemotherapy from those who should receive standard chemotherapy alone. Thirteen databases, including MEDLINE and EMBASE, research registers and conference proceedings were searched to January 2013. Additional data were obtained from an online survey of laboratories participating in the UK National External Quality Assurance Scheme pilot for KRAS mutation testing. A systematic review of the evidence was carried out using standard methods. Randomised controlled trials were assessed for quality using the Cochrane risk of bias tool. Diagnostic accuracy studies were assessed using the QUADAS-2 tool. There were insufficient data for meta-analysis. For accuracy studies we calculated sensitivity and specificity together with 95% confidence intervals (CIs). Survival data were summarised as hazard ratios and tumour response data were summarised as relative risks, with 95% CIs. The health economic analysis considered the long-term costs and quality-adjusted life-years associated with different tests followed by treatment

  19. Mature Microsatellites: Mechanisms Underlying Dinucleotide Microsatellite Mutational Biases in Human Cells

    OpenAIRE

    Baptiste, Beverly A.; Ananda, Guruprasad; Strubczewski, Noelle; Lutzkanin, Andrew; Khoo, Su Jen; Srikanth, Abhinaya; Kim, Nari; Makova, Kateryna D.; Krasilnikova, Maria M.; Eckert, Kristin A.

    2013-01-01

    Dinucleotide microsatellites are dynamic DNA sequences that affect genome stability. Here, we focused on mature microsatellites, defined as pure repeats of lengths above the threshold and unlikely to mutate below it in a single mutational event. We investigated the prevalence and mutational behavior of these sequences by using human genome sequence data, human cells in culture, and purified DNA polymerases. Mature dinucleotides (?10 units) are present within exonic sequences of >350 genes, re...

  20. The complete linkage disequilibrium test: a test that points to causative mutations underlying quantitative traits

    Directory of Open Access Journals (Sweden)

    Uleberg Eivind

    2011-05-01

    Full Text Available Abstract Background Genetically, SNP that are in complete linkage disequilibrium with the causative SNP cannot be distinguished from the causative SNP. The Complete Linkage Disequilibrium (CLD test presented here tests whether a SNP is in complete LD with the causative mutation or not. The performance of the CLD test is evaluated in 1000 simulated datasets. Methods The CLD test consists of two steps i.e. analysis I and analysis II. Analysis I consists of an association analysis of the investigated region. The log-likelihood values from analysis I are next ranked in descending order and in analysis II the CLD test evaluates differences in log-likelihood ratios between the best and second best markers. Under the null-hypothesis distribution, the best SNP is in greater LD with the QTL than the second best, while under the alternative-CLD-hypothesis, the best SNP is alike-in-state with the QTL. To find a significance threshold, the test was also performed on data excluding the causative SNP. The 5th, 10th and 50th highest TCLD value from 1000 replicated analyses were used to control the type-I-error rate of the test at p = 0.005, p = 0.01 and p = 0.05, respectively. Results In a situation where the QTL explained 48% of the phenotypic variance analysis I detected a QTL in 994 replicates (p = 0.001, where 972 were positioned in the correct QTL position. When the causative SNP was excluded from the analysis, 714 replicates detected evidence of a QTL (p = 0.001. In analysis II, the CLD test confirmed 280 causative SNP from 1000 simulations (p = 0.05, i.e. power was 28%. When the effect of the QTL was reduced by doubling the error variance, the power of the test reduced relatively little to 23%. When sequence data were used, the power of the test reduced to 16%. All SNP that were confirmed by the CLD test were positioned in the correct QTL position. Conclusions The CLD test can provide evidence for a causative SNP, but its power may be low in situations

  1. [Views of Icelandic women towards genetic counseling - and testing of BRCA2 mutations].

    Science.gov (United States)

    Jonsdottir, Thordis; Valdimarsdottir, Heiddis; Tryggvadottir, Laufey; Lund, Sigrun Helga; Thordardottir, Marianna; Magnusson, Magnus Karl; Valdimarsdottir, Unnur

    2018-01-01

    Introduction The aim of this study was to explore the attitudes of Icelandic women towards existing genetic information, genetic counseling and genetic testing for BRCA mutations which dramatically increase risk for aggressive cancers. Materials and methods Women attending the cancer prevention clinic in Reykjavik, capital of Iceland, from October 12th until November 20th 2015 received an invitation to participate. Participation involved answering a short online questionnaire about background, family history of cancer as well as attitudes towards genetic counseling, BRCA testing and preventive use of such information. Descriptive statistics and chi-square tests were used to describe differences in attitudes towards those questions between subgroups of women. Results 1129 women (69% response rate) answered the questionnaire. Mean age was 47 years (span 21-76 years). Around half (47%) had heard fairly much about the mutations. Independent of family history of cancer, the majority of women were positive towards receiving genetic counseling (79%) and to undergo genetic testing (83%) for BRCA mutation with younger women being more interested than older women. On the other hand, only 4% of the women had already received genetic counseling and 7% undergone genetic testing. Women with family history of cancer were more knowledgeable about BRCA mutations (pcounseling and testing for BRCA mutations although half of them worry that a positive result might affect their health insurance. Nevertheless, almost all women believe that existing genetic information should be used to inform carriers for preventive purposes.

  2. Detection of EGFR mutations in plasma and biopsies from non-small cell lung cancer patients by allele-specific PCR assays

    DEFF Research Database (Denmark)

    Weber, Britta; Meldgaard, Peter; Hager, Henrik

    2014-01-01

    samples with allele-specific PCR assays. METHODS: Pairs of the diagnostic biopsy and plasma obtained just prior to start of erlotinib treatment were collected from 199 patients with adenocarcinoma of non-small-cell lung cancer. DNA from both sample types was isolated and examined for the presence...... of mutations in exons 18-21 of the EGFR gene, employing the cobas(®) EGFR Tissue Test and cobas(®) EGFR Blood Test (in development, Roche Molecular Systems, Inc., CA, USA). RESULTS: Test results were obtained in all 199 (100%) plasma samples and 196/199 (98%) of the biopsies. EGFR-activating mutations were...... identified in 24/199 (12%) plasma samples and 28/196 (14%) biopsy samples, and 17/196 (9%) matched pairs contained the same mutation. Six EGFR mutations were present only in plasma samples but not in the biopsy samples. The overall concordance of the EGFR gene mutations detected in plasma and biopsy tissue...

  3. Pathways to Tumorigenesis—Modeling Mutation Acquisition in Stem Cells and Their Progeny

    Directory of Open Access Journals (Sweden)

    Rina Ashkenazi

    2008-11-01

    Full Text Available Most adult tissues consist of stem cells, progenitors, and mature cells, and this hierarchical architecture may play an important role in the multistep process of carcinogenesis. Here, we develop and discuss the important predictions of a simple mathematical model of cancer initiation and early progression within a hierarchically structured tissue. This work presents a model that incorporates both the sequential acquisition of phenotype altering mutations and tissue hierarchy. The model simulates the progressive effect of accumulating mutations that lead to an increase in fitness or the induction of genetic instability. A novel aspect of the model is that symmetric self-renewal, asymmetric division, and differentiation are all incorporated, and this enables the quantitative study of the effect of mutations that deregulate the normal, homeostatic stem cell division pattern. The model is also capable of predicting changes in both tissue composition and in the progression of cells along their lineage at any given time and for various sequences of mutations. Simulations predict that the specific order in which mutations are acquired is crucial for determining the pace of cancer development. Interestingly, we find that the importance of genetic stability differs significantly depending on the physiological expression of mutations related to symmetric self-renewal and differentiation of stem and progenitor cells. In particular, mutations that lead to the alteration of the stem cell division pattern or the acquisition of some degree of immortality in committed progenitors lead to an early onset of cancer and diminish the impact of genetic instability.

  4. Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer

    NARCIS (Netherlands)

    Peifer, Martin; Fernandez-Cuesta, Lynnette; Sos, Martin L.; George, Julie; Seidel, Danila; Kasper, Lawryn H.; Plenker, Dennis; Leenders, Frauke; Sun, Ruping; Zander, Thomas; Menon, Roopika; Koker, Mirjam; Dahmen, Ilona; Mueller, Christian; Di Cerbo, Vincenzo; Schildhaus, Hans-Ulrich; Altmueller, Janine; Baessmann, Ingelore; Becker, Christian; de Wilde, Bram; Vandesompele, Jo; Boehm, Diana; Ansen, Sascha; Gabler, Franziska; Wilkening, Ines; Heynck, Stefanie; Heuckmann, Johannes M.; Lu, Xin; Carter, Scott L.; Cibulskis, Kristian; Banerji, Shantanu; Getz, Gad; Park, Kwon-Sik; Rauh, Daniel; Gruetter, Christian; Fischer, Matthias; Pasqualucci, Laura; Wright, Gavin; Wainer, Zoe; Russell, Prudence; Petersen, Iver; Chen, Yuan; Stoelben, Erich; Ludwig, Corinna; Schnabel, Philipp; Hoffmann, Hans; Muley, Thomas; Brockmann, Michael; Engel-Riedel, Walburga; Muscarella, Lucia A.; Fazio, Vito M.; Groen, Harry; Timens, Wim; Sietsma, Hannie; Thunnissen, Erik; Smit, Egbert; Heideman, Danielle A. M.; Snijders, Peter J. F.; Cappuzzo, Federico; Ligorio, Claudia; Damiani, Stefania; Field, John; Solberg, Steinar; Brustugun, Odd Terje; Lund-Iversen, Marius; Saenger, Joerg; Clement, Joachim H.; Soltermann, Alex; Moch, Holger; Weder, Walter; Solomon, Benjamin; Soria, Jean-Charles; Validire, Pierre; Besse, Benjamin; Brambilla, Elisabeth; Brambilla, Christian; Lantuejoul, Sylvie; Lorimier, Philippe; Schneider, Peter M.; Hallek, Michael; Pao, William; Meyerson, Matthew; Sage, Julien; Shendure, Jay; Schneider, Robert; Buettner, Reinhard; Wolf, Juergen; Nuernberg, Peter; Perner, Sven; Heukamp, Lukas C.; Brindle, Paul K.; Haas, Stefan; Thomas, Roman K.

    2012-01-01

    Small-cell lung cancer (SCLC) is an aggressive lung tumor subtype with poor prognosis(1-3). We sequenced 29 SCLC exomes, 2 genomes and 15 transcriptomes and found an extremely high mutation rate of 7.4 +/- 1 protein-changing mutations per million base pairs. Therefore, we conducted integrated

  5. 40 CFR 798.5300 - Detection of gene mutations in somatic cells in culture.

    Science.gov (United States)

    2010-07-01

    ... cells in culture. 798.5300 Section 798.5300 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY....5300 Detection of gene mutations in somatic cells in culture. (a) Purpose. Mammalian cell culture... selected by resistance to ouabain. (2) Description. Cells in suspension or monolayer culture are exposed to...

  6. Cis-regulatory somatic mutations and gene-expression alteration in B-cell lymphomas.

    Science.gov (United States)

    Mathelier, Anthony; Lefebvre, Calvin; Zhang, Allen W; Arenillas, David J; Ding, Jiarui; Wasserman, Wyeth W; Shah, Sohrab P

    2015-04-23

    With the rapid increase of whole-genome sequencing of human cancers, an important opportunity to analyze and characterize somatic mutations lying within cis-regulatory regions has emerged. A focus on protein-coding regions to identify nonsense or missense mutations disruptive to protein structure and/or function has led to important insights; however, the impact on gene expression of mutations lying within cis-regulatory regions remains under-explored. We analyzed somatic mutations from 84 matched tumor-normal whole genomes from B-cell lymphomas with accompanying gene expression measurements to elucidate the extent to which these cancers are disrupted by cis-regulatory mutations. We characterize mutations overlapping a high quality set of well-annotated transcription factor binding sites (TFBSs), covering a similar portion of the genome as protein-coding exons. Our results indicate that cis-regulatory mutations overlapping predicted TFBSs are enriched in promoter regions of genes involved in apoptosis or growth/proliferation. By integrating gene expression data with mutation data, our computational approach culminates with identification of cis-regulatory mutations most likely to participate in dysregulation of the gene expression program. The impact can be measured along with protein-coding mutations to highlight key mutations disrupting gene expression and pathways in cancer. Our study yields specific genes with disrupted expression triggered by genomic mutations in either the coding or the regulatory space. It implies that mutated regulatory components of the genome contribute substantially to cancer pathways. Our analyses demonstrate that identifying genomically altered cis-regulatory elements coupled with analysis of gene expression data will augment biological interpretation of mutational landscapes of cancers.

  7. Spectrum of EGFR gene mutations in Vietnamese patients with non-small cell lung cancer.

    Science.gov (United States)

    Vu, Hoang Anh; Xinh, Phan Thi; Ha, Hua Thi Ngoc; Hanh, Ngo Thi Tuyet; Bach, Nguyen Duc; Thao, Doan Thi Phuong; Dat, Ngo Quoc; Trung, Nguyen Sao

    2016-03-01

    Epidermal growth factor receptor (EGFR) mutational status is a crucial biomarker for prediction of response to tyrosine kinase inhibitors in patients with non-small cell lung cancer (NSCLC). Although these mutations have been well characterized in other countries, little is known about the frequency or spectrum of EGFR mutations in Vietnamese NSCLC patients. Using Sanger DNA sequencing, we investigated mutations in EGFR exons 18-21 from 332 patients diagnosed with NSCLC at University of Medicine and Pharmacy, Ho Chi Minh City, Vietnam. DNA was extracted from formalin-fixed, paraffin-embedded tissues, followed by PCR amplification and sequencing. EGFR mutations were detected in 135 samples (40.7%), of which eight samples carried double mutations. In total, 46 different types of EGFR mutations were found, including six novel mutations (p.K713E, p.K714R, p.P794S, p.R803W, p.P848S, and p.K867E). Among the four exons investigated, exon 19 was most frequently mutated (63 out of 332 patients, 19%), with the p.E746_A750del appearing in 43 samples. Exon 21 was mutated in 56 samples (16.9%), of which 47 were p.L858R. Each of exons 18 and 20 was mutated in 12 samples (3.6%). The frequency of EGFR mutations was higher in females than in males (48.9% vs 35%, P = 0.012), but not statistically different between adenocarcinomas and other histological types of NSCLC (41.3% vs 34.5%, P = 0.478). DNA sequencing detected EGFR mutations with high frequency and revealed a broad spectrum of mutation type in Vietnamese patients with NSCLC. © 2015 Wiley Publishing Asia Pty Ltd.

  8. Novel allelic mutations in murine Serca2 induce differential development of squamous cell tumors

    Energy Technology Data Exchange (ETDEWEB)

    Toki, Hideaki; Minowa, Osamu; Inoue, Maki; Motegi, Hiromi; Karashima, Yuko; Ikeda, Ami [Team for Advanced Development and Evaluation of Human Disease Models, Riken BioResource Center (BRC), Tsukuba, Ibaraki (Japan); Kaneda, Hideki [Technology and Development Team for Mouse Phenotype Analysis, Riken BRC, Tsukuba, Ibaraki (Japan); Sakuraba, Yoshiyuki [Mutagenesis and Genomics Team, Riken BRC, Tsukuba, Ibaraki (Japan); Saiki, Yuriko [Department of Molecular Pathology, Tohoku University Graduate School of Medicine, Sendai, Miyagi (Japan); Wakana, Shigeharu [Technology and Development Team for Mouse Phenotype Analysis, Riken BRC, Tsukuba, Ibaraki (Japan); Suzuki, Hiroshi [Department of Biochemistry, Asahikawa Medical University, Asahikawa, Hokkaido (Japan); Gondo, Yoichi [Mutagenesis and Genomics Team, Riken BRC, Tsukuba, Ibaraki (Japan); Shiroishi, Toshihiko [Mammalian Genetics Laboratory, National Institute of Genetics, Mishima, Shizuoka (Japan); Noda, Tetsuo, E-mail: tnoda@jfcr.or.jp [Team for Advanced Development and Evaluation of Human Disease Models, Riken BioResource Center (BRC), Tsukuba, Ibaraki (Japan); Department of Cell Biology, Cancer Institute, The Japanese Foundation for Cancer Research, Tokyo (Japan)

    2016-08-05

    Dominant mutations in the Serca2 gene, which encodes sarco(endo)plasmic reticulum calcium-ATPase, predispose mice to gastrointestinal epithelial carcinoma [1–4] and humans to Darier disease (DD) [14–17]. In this study, we generated mice harboring N-ethyl-N-nitrosourea (ENU)-induced allelic mutations in Serca2: three missense mutations and one nonsense mutation. Mice harboring these Serca2 mutations developed tumors that were categorized as either early onset squamous cell tumors (SCT), with development similar to null-type knockout mice [2,4] (aggressive form; M682, M814), or late onset tumors (mild form; M1049, M1162). Molecular analysis showed no aberration in Serca2 mRNA or protein expression levels in normal esophageal cells of any of the four mutant heterozygotes. There was no loss of heterozygosity at the Serca2 locus in the squamous cell carcinomas in any of the four lines. The effect of each mutation on Ca{sup 2+}-ATPase activity was predicted using atomic-structure models and accumulated mutated protein studies, suggesting that putative complete loss of Serca2 enzymatic activity may lead to early tumor onset, whereas mutations in which Serca2 retains residual enzymatic activity result in late onset. We propose that impaired Serca2 gene product activity has a long-term effect on squamous cell carcinogenesis from onset to the final carcinoma stage through an as-yet unrecognized but common regulatory pathway. -- Highlights: •Novel mutations in murine Serca2 caused early onset or late onset of tumorigenesis. •They also caused higher or lower incidence of Darier Disease phenotype. •3D structure model suggested the former mutations led to severer defect on ATPase. •Driver gene mutations via long-range effect on Ca2+ distributions are suggested.

  9. Cell killing and mutation induction on Chinese hamster cells by photoradiations

    International Nuclear Information System (INIS)

    Lam, C.K.C.

    1982-01-01

    The subject matter of this investigation concerns the killing and mutagenic effects induced by far-UV radiation and broad spectra of black, white and gold lights. Applying radiation directly on CHO (Chinese hamster ovary) cells, far-UV is more effective than black light, and black light is more effective than white light in inducing proliferative death and in inducing resistance to 6-thioguanine (6TG), ouabain and diptheria toxin (DT). Cells in the G1/early S boundary are the most sensitive to far-UV or unfiltered fluorescent lights. When synchronous cells are irradiated with moderate doses of far-UV or unfiltered broad spectra of black light, mutations to 6-TG and ouabain resistance are slightly higher in early S period than in the remaining parts of the cell cycle. Mutation induction of 6-TG, ouabain or DT resistance is increased in the split-dose samples of the asynchronous and synchronous CHO cells. CHO cells predominantly express an error-prone repair mechanism after photoirradiation

  10. Cumulative BRCA mutation analysis in the Greek population confirms that homogenous ethnic background facilitates genetic testing.

    Science.gov (United States)

    Tsigginou, Alexandra; Vlachopoulos, Fotios; Arzimanoglou, Iordanis; Zagouri, Flora; Dimitrakakis, Constantine

    2015-01-01

    Screening for BRCA 1 and BRCA 2 mutations has long moved from the research lab to the clinic as a routine clinical genetic testing. BRCA molecular alteration pattern varies among ethnic groups which makes it already a less straightforward process to select the appropriate mutations for routine genetic testing on the basis of known clinical significance. The present report comprises an in depth literature review of the so far reported BRCA 1 and BRCA 2 molecular alterations in Greek families. Our analysis of Greek cumulative BRCA 1 and 2 molecular data, produced by several independent groups, confirmed that six recurrent deleterious mutations account for almost 60 % and 70 % of all BRCA 1 and 2 and BRCA 1 mutations, respectively. As a result, it makes more sense to perform BRCA mutation analysis in the clinic in two sequential steps, first conventional analysis for the six most prevalent pathogenic mutations and if none identified, a second step of New Generation Sequencing-based whole genome or whole exome sequencing would follow. Our suggested approach would enable more clinically meaningful, considerably easier and less expensive BRCA analysis in the Greek population which is considered homogenous.

  11. Visualizing the origins of selfish de novo mutations in individual seminiferous tubules of human testes.

    Science.gov (United States)

    Maher, Geoffrey J; McGowan, Simon J; Giannoulatou, Eleni; Verrill, Clare; Goriely, Anne; Wilkie, Andrew O M

    2016-03-01

    De novo point mutations arise predominantly in the male germline and increase in frequency with age, but it has not previously been possible to locate specific, identifiable mutations directly within the seminiferous tubules of human testes. Using microdissection of tubules exhibiting altered expression of the spermatogonial markers MAGEA4, FGFR3, and phospho-AKT, whole genome amplification, and DNA sequencing, we establish an in situ strategy for discovery and analysis of pathogenic de novo mutations. In 14 testes from men aged 39-90 y, we identified 11 distinct gain-of-function mutations in five genes (fibroblast growth factor receptors FGFR2 and FGFR3, tyrosine phosphatase PTPN11, and RAS oncogene homologs HRAS and KRAS) from 16 of 22 tubules analyzed; all mutations have known associations with severe diseases, ranging from congenital or perinatal lethal disorders to somatically acquired cancers. These results support proposed selfish selection of spermatogonial mutations affecting growth factor receptor-RAS signaling, highlight its prevalence in older men, and enable direct visualization of the microscopic anatomy of elongated mutant clones.

  12. Leukemia-associated activating mutation of Flt3 expands dendritic cells and alters T cell responses.

    Science.gov (United States)

    Lau, Colleen M; Nish, Simone A; Yogev, Nir; Waisman, Ari; Reiner, Steven L; Reizis, Boris

    2016-03-07

    A common genetic alteration in acute myeloid leukemia is the internal tandem duplication (ITD) in FLT3, the receptor for cytokine FLT3 ligand (FLT3L). Constitutively active FLT3-ITD promotes the expansion of transformed progenitors, but also has pleiotropic effects on hematopoiesis. We analyzed the effect of FLT3-ITD on dendritic cells (DCs), which express FLT3 and can be expanded by FLT3L administration. Pre-leukemic mice with the Flt3(ITD) knock-in allele manifested an expansion of classical DCs (cDCs) and plasmacytoid DCs. The expansion originated in DC progenitors, was cell intrinsic, and was further enhanced in Flt3(ITD/ITD) mice. The mutation caused the down-regulation of Flt3 on the surface of DCs and reduced their responsiveness to Flt3L. Both canonical Batf3-dependent CD8(+) cDCs and noncanonical CD8(+) cDCs were expanded and showed specific alterations in their expression profiles. Flt3(ITD) mice showed enhanced capacity to support T cell proliferation, including a cell-extrinsic expansion of regulatory T (T reg) cells. Accordingly, these mice restricted alloreactive T cell responses during graft-versus-host reaction, but failed to control autoimmunity without T reg cells. Thus, the FLT3-ITD mutation directly affects DC development, indirectly modulating T cell homeostasis and supporting T reg cell expansion. We hypothesize that this effect of FLT3-ITD might subvert immunosurveillance and promote leukemogenesis in a cell-extrinsic manner. © 2016 Lau et al.

  13. Effects of track structure and cell inactivation on the calculation of heavy ion mutation rates in mammalian cells

    Science.gov (United States)

    Cucinotta, F. A.; Wilson, J. W.; Shavers, M. R.; Katz, R.

    1996-01-01

    It has long been suggested that inactivation severely effects the probability of mutation by heavy ions in mammalian cells. Heavy ions have observed cross sections of inactivation that approach and sometimes exceed the geometric size of the cell nucleus in mammalian cells. In the track structure model of Katz the inactivation cross section is found by summing an inactivation probability over all impact parameters from the ion to the sensitive sites within the cell nucleus. The inactivation probability is evaluated using the dose-response of the system to gamma-rays and the radial dose of the ions and may be equal to unity at small impact parameters for some ions. We show how the effects of inactivation may be taken into account in the evaluation of the mutation cross sections from heavy ions in the track structure model through correlation of sites for gene mutation and cell inactivation. The model is fit to available data for HPRT mutations in Chinese hamster cells and good agreement is found. The resulting calculations qualitatively show that mutation cross sections for heavy ions display minima at velocities where inactivation cross sections display maxima. Also, calculations show the high probability of mutation by relativistic heavy ions due to the radial extension of ions track from delta-rays in agreement with the microlesion concept. The effects of inactivation on mutations rates make it very unlikely that a single parameter such as LET or Z*2/beta(2) can be used to specify radiation quality for heavy ion bombardment.

  14. Spontaneous mutation rate in Chinese hamster cell clones differing in UV-sensitivity

    International Nuclear Information System (INIS)

    Manuilova, E.S.; Bagrova, A.M.; Moskovskij Gosudarstvennyj Univ.

    1983-01-01

    The spontaneous rate of appearance of mutations to 6-mercaptopurine (6 MP) resistence in the cells of CHR2 and CHs2 clones dofferent in sensitivity to lethal and matagenous effect of UV-rays, is investigated. Increased UV-sensitivity of CHs2 clone is caused by the violation of postreplicative DNA reparation. It is established that the purity of spontaneously occuring mutations in both clones turns out to be similar, i.e. (1.5-1.8)x10 -5 for the cell pergeneration. It is shown that the effect of postreplicative DNA reparation in the cells of chinese hamster is not connected with the increase of spontaneous mutation ability. The problem on the possible role of reparation in the mechanism of appearance of spontaneous and induced mutations in the cells of Chinese hamster with increased UV-sensitivity is discussed

  15. De novo activating epidermal growth factor mutations (EGFR) in small-cell lung cancer.

    Science.gov (United States)

    Thai, Alesha; Chia, Puey L; Russell, Prudence A; Do, Hongdo; Dobrovic, Alex; Mitchell, Paul; John, Thomas

    2017-09-01

    In Australia, mutations in epidermal growth factor mutations (EGFR) occur in 15% of patients diagnosed with non-small-cell lung cancer and are found with higher frequency in female, non-smokers of Asian ethnicity. Activating mutations in the EGFR gene are rarely described in SCLC. We present two cases of de novo EGFR mutations in patients with SCLC detected in tissue and in plasma cell free DNA, both of whom were of Asian ethnicity and never-smokers. These two cases add to the growing body of evidence suggesting that screening for EGFR mutations in SCLC should be considered in patients with specific clinical features. © 2017 Royal Australasian College of Physicians.

  16. [The quantitative testing of V617F mutation in gen JAK2 using pyrosequencing technique].

    Science.gov (United States)

    Dunaeva, E A; Mironov, K O; Dribnokhodova, T E; Subbotina, E E; Bashmakova; Ol'hovskiĭ, I A; Shipulin, G A

    2014-11-01

    The somatic mutation V617F in gen JAK2 is a frequent cause of chronic myeloprolific diseases not conditioned by BCR/ABL mutation. The quantitative testing of relative percentage of mutant allele can be used in establishing severity of disease and its prognosis and in prescription of remedy inhibiting activity of JAK2. To quantitatively test mutation the pyrosequencing technique was applied. The developed technique permits detecting and quantitatively, testing percentage of mutation fraction since 7%. The "gray zone" is presented by samples with percentage of mutant allele from 4% to 7%. The dependence of expected percentage of mutant fraction in analyzed sample from observed value of signal is described by equation of line with regression coefficients y = - 0.97, x = -1.32 and at that measurement uncertainty consists ± 0.7. The developed technique is approved officially on clinical material from 192 patients with main forms of myeloprolific diseases not conditioned by BCR/ABL mutation. It was detected 64 samples with mautant fraction percentage from 13% to 91%. The developed technique permits implementing monitoring of therapy of myeloprolific diseases and facilitates to optimize tactics of treatment.

  17. A study of dose effect response and the interrelationship between cell survival, mutation and chromosomal aberration

    International Nuclear Information System (INIS)

    Chadwick, K.H.; Leenhouts, H.P.; Werry, P.A.Th.J.; Ramulu, K.S.

    1980-01-01

    Research was carried out on four topics: generation of cell reproductive death and mutation induction in cell cultures of Haplopappus gracilis by ionizing radiation; radiation induced repair in Saintpaulia epidermis cells; analysis of the combined effect of radiation with other mutagens; and the effect of very low doses of radiation on the Oenothera model system. (Auth.)

  18. On the effect of certain mutations on the radiosensitivity of haploid and diploid yeast cells

    International Nuclear Information System (INIS)

    Sokurova, E.N.; Korogodin, V.I.

    1978-01-01

    Mutation ade 1-6 in haploid cell Saccharomyces cerevisiae increases half as much against radioresistance of cells. Diploid cells lacking in adenine, homozygous by ade 1-6 mutation, are nearly twice as radiosensitive as prototrophic cells. Hence ade 1-6 mutation increases radioresistance of haploid cells and decreases that of diplois. These changes in radioresistance are not connected with variations in the extrapolation number of survival curve, the ability of cells to recover from radiation damages upon cultivation in an innutrient medium, and with the inactivation form ratio. Lack of adenine influences the radioresistance of diploid yeast irrespective of whether it is or it is not affected by homo- or heterozygosity by the locus of mating type

  19. The p16INK4alpha/p19ARF gene mutations are infrequent and are mutually exclusive to p53 mutations in Indian oral squamous cell carcinomas.

    Science.gov (United States)

    Kannan, K; Munirajan, A K; Krishnamurthy, J; Bhuvarahamurthy, V; Mohanprasad, B K; Panishankar, K H; Tsuchida, N; Shanmugam, G

    2000-03-01

    Eighty-seven untreated primary oral squamous cell carcinomas (SCCs) associated with betel quid and tobacco chewing from Indian patients were analysed for the presence of mutations in the commonly shared exon 2 of p16INK4alpha/p19ARF genes. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and sequencing analysis were used to detect mutations. SSCP analysis indicated that only 9% (8/87) of the tumours had mutation in p16INK4alpha/p19ARF genes. Seventy-two tumours studied here were previously analysed for p53 mutations and 21% (15/72) of them were found to have mutations in p53 gene. Only one tumour was found to have mutation at both p53 and p16INK4alpha/p19ARF genes. Thus, the mutation rates observed were 21% for p53, 9% for p16INK4alpha/p19ARF, and 1% for both. Sequencing analysis revealed two types of mutations; i) G to C (GCAG to CCAG) transversion type mutation at intron 1-exon 2 splice junction and ii) another C to T transition type mutation resulting in CGA to TGA changing arginine to a termination codon at p16INK4alpha gene codon 80 and the same mutation will alter codon 94 of p19ARF gene from CCG to CTG (proline to leucine). These results suggest that p16INK4alpha/p19ARF mutations are less frequent than p53 mutations in Indian oral SCCs. The p53 and p16INK4alpha/p19ARF mutational events are independent and are mutually exclusive suggesting that mutational inactivation of either p53 or p16INK4alpha/p19ARF may alleviate the need for the inactivation of the other gene.

  20. Advantages of a single-cycle production assay to study cell culture-adaptive mutations of hepatitis C virus

    DEFF Research Database (Denmark)

    Russell, Rodney S; Meunier, Jean-Christophe; Takikawa, Shingo

    2008-01-01

    mutations that were selected during serial passage in Huh-7.5 cells were studied. Recombinant genomes containing all five mutations produced 3-4 logs more infectious virions than did wild type. Neither a coding mutation in NS5A nor a silent mutation in E2 was adaptive, whereas coding mutations in E2, p7......The JFH1 strain of hepatitis C virus (HCV) is unique among HCV isolates, in that the wild-type virus can traverse the entire replication cycle in cultured cells. However, without adaptive mutations, only low levels of infectious virus are produced. In the present study, the effects of five...

  1. Clinical, pathologic, and biologic features associated with BRAF mutations in non-small cell lung cancer.

    Science.gov (United States)

    Cardarella, Stephanie; Ogino, Atsuko; Nishino, Mizuki; Butaney, Mohit; Shen, Jeanne; Lydon, Christine; Yeap, Beow Y; Sholl, Lynette M; Johnson, Bruce E; Jänne, Pasi A

    2013-08-15

    BRAF mutations are found in a subset of non-small cell lung cancers (NSCLC). We examined the clinical characteristics and treatment outcomes of patients with NSCLC harboring BRAF mutations. Using DNA sequencing, we successfully screened 883 patients with NSCLC for BRAF mutations between July 1, 2009 and July 16, 2012. Baseline characteristics and treatment outcomes were compared between patients with and without BRAF mutations. Wild-type controls consisted of patients with NSCLC without a somatic alteration in BRAF, KRAS, EGFR, and ALK. In vitro studies assessed the biologic properties of selected non-V600E BRAF mutations identified from patients with NSCLC. Of 883 tumors screened, 36 (4%) harbored BRAF mutations (V600E, 18; non-V600E, 18) and 257 were wild-type for BRAF, EGFR, KRAS, and ALK negative. Twenty-nine of 36 patients with BRAF mutations were smokers. There were no distinguishing clinical features between BRAF-mutant and wild-type patients. Patients with advanced NSCLC with BRAF mutations and wild-type tumors showed similar response rates and progression-free survival (PFS) to platinum-based combination chemotherapy and no difference in overall survival. Within the BRAF cohort, patients with V600E-mutated tumors had a shorter PFS to platinum-based chemotherapy compared with those with non-V600E mutations, although this did not reach statistical significance (4.1 vs. 8.9 months; P = 0.297). We identified five BRAF mutations not previously reported in NSCLC; two of five were associated with increased BRAF kinase activity. BRAF mutations occur in 4% of NSCLCs and half are non-V600E. Prospective trials are ongoing to validate BRAF as a therapeutic target in NSCLC. ©2013 AACR.

  2. Gene mutation in ATM/PI3K region of nasopharyngeal carcinoma cell lines

    International Nuclear Information System (INIS)

    Wang Hongmei; Wu Xinyao; Xia Yunfei

    2002-01-01

    Objective: To define the correlation between nasopharyngeal carcinoma (NPC) cell radiosensitivity and gene mutation in the ATM/PI3K coding region. Methods: The gene mutation in the ATM/PI3K region of nasopharyngeal carcinoma cell lines which vary in radiosensitivity, was monitored by reverse transcription-polymerase chain reaction (RT-PCR) and fluorescence-marked ddNTP cycle sequencing technique. Results: No gene mutation was detected in the ATM/PI3K region of either CNE1 or CNE2. Conclusion: Disparity in intrinsic radiosensitivity between different NPC cell lines depends on some other factors and mechanism without being related to ATM/PI3K mutations

  3. Factors affecting the spontaneous mutational spectra in somatic mammalian cells

    Directory of Open Access Journals (Sweden)

    О.А. Ковальова

    2006-04-01

    Full Text Available  In our survey of references we are discussed the influence of factors biological origin on the spontaneous mutation specters in mammalian. Seasonal and age components influence on the frequence of cytogenetic anomalies. The immune and endocrinous systems are take part in control of the alteration of the spontaneous mutation specters. Genetical difference of sensibility in animal and human at the alteration of factors enviroment as and  genetical differences of repair systems activity are may influence on individual variation of spontaneous destabilization characters of chromosomal apparatus.

  4. Higher Quality of Molecular Testing, an Unfulfilled Priority: Results from External Quality Assessment for KRAS Mutation Testing in Colorectal Cancer

    NARCIS (Netherlands)

    Tembuyser, L.; Ligtenberg, M.J.L.; Normanno, N.; Delen, S.; Krieken, J.H.J.M. van; Dequeker, E.M.

    2014-01-01

    Precision medicine is now a key element in clinical oncology. RAS mutational status is a crucial predictor of responsiveness to anti-epidermal growth factor receptor agents in metastatic colorectal cancer. In an effort to guarantee high-quality testing services in molecular pathology, the European

  5. Mutagenicity of Flavonoids Assayed by Bacterial Reverse Mutation (Ames Test

    Directory of Open Access Journals (Sweden)

    Eliana Aparecida Varanda

    2012-05-01

    Full Text Available The mutagenicity of ten flavonoids was assayed by the Ames test, in Salmonella typhimurium strains TA98, TA100 and TA102, with the aim of establishing hydroxylation pattern-mutagenicity relationship profiles. The compounds assessed were: quercetin, kaempferol, luteolin, fisetin, chrysin, galangin, flavone, 3-hydroxyflavone, 5-hydroxyflavone and 7-hydroxyflavone. In the Ames assay, quercetin acted directly and its mutagenicity increased with metabolic activation. In the presence of S9 mix, kaempferol and galangin were mutagenic in the TA98 strain and kaempferol showed signs of mutagenicity in the other strains. The absence of hydroxyl groups, as in flavone, only signs of mutagenicity were shown in strain TA102, after metabolization and, among monohydroxylated flavones (3-hydroxyflavone, 5-hydroxyflavone and 7-hydroxyflavone, the presence of hydroxyl groups only resulted in minor changes. Luteolin and fisetin also showed signs of mutagenicity in strain TA102. Finally, chrysin, which has only two hydroxy groups, at the 5-OH and 7-OH positions, also did not induce mutagenic activity in any of the bacterial strains used, under either activation condition. All the flavonoids were tested at concentrations varying from 2.6 to 30.7 nmol/plate for galangin and 12.1 to 225.0 nmol/plate for other flavonoids. In light of the above, it is necessary to clarify the conditions and the mechanisms that mediate the biological effects of flavonoids before treating them as therapeutical agents, since some compounds can be biotransformed into more genotoxic products; as is the case for galangin, kaempferol and quercetin.

  6. Iodine Absorption Cells Purity Testing

    Directory of Open Access Journals (Sweden)

    Jan Hrabina

    2017-01-01

    Full Text Available This article deals with the evaluation of the chemical purity of iodine-filled absorption cells and the optical frequency references used for the frequency locking of laser standards. We summarize the recent trends and progress in absorption cell technology and we focus on methods for iodine cell purity testing. We compare two independent experimental systems based on the laser-induced fluorescence method, showing an improvement of measurement uncertainty by introducing a compensation system reducing unwanted influences. We show the advantages of this technique, which is relatively simple and does not require extensive hardware equipment. As an alternative to the traditionally used methods we propose an approach of hyperfine transitions’ spectral linewidth measurement. The key characteristic of this method is demonstrated on a set of testing iodine cells. The relationship between laser-induced fluorescence and transition linewidth methods will be presented as well as a summary of the advantages and disadvantages of the proposed technique (in comparison with traditional measurement approaches.

  7. Pitfalls in genetic testing : the story of missed SCN1A mutations

    NARCIS (Netherlands)

    Djémié, Tania; Weckhuysen, Sarah; von Spiczak, Sarah; Carvill, Gemma L; Jaehn, Johanna; Anttonen, Anna-Kaisa; Brilstra, Eva; Caglayan, Hande S; de Kovel, Carolien G; Depienne, Christel; Gaily, Eija; Gennaro, Elena; Giraldez, Beatriz G; Gormley, Padhraig; Guerrero-López, Rosa; Guerrini, Renzo; Hämäläinen, Eija; Hartmann, Corinna; Hernandez-Hernandez, Laura; Hjalgrim, Helle; Koeleman, Bobby P C; Leguern, Eric; Lehesjoki, Anna-Elina; Lemke, Johannes R; Leu, Costin; Marini, Carla; McMahon, Jacinta M; Mei, Davide; Møller, Rikke S; Muhle, Hiltrud; Myers, Candace T; Nava, Caroline; Serratosa, Jose M; Sisodiya, Sanjay M; Stephani, Ulrich; Striano, Pasquale; van Kempen, Marjan J A; Verbeek, Nienke E; Usluer, Sunay; Zara, Federico; Palotie, Aarno; Mefford, Heather C; Scheffer, Ingrid E; De Jonghe, Peter; Helbig, Ingo; Suls, Arvid

    BACKGROUND: Sanger sequencing, still the standard technique for genetic testing in most diagnostic laboratories and until recently widely used in research, is gradually being complemented by next-generation sequencing (NGS). No single mutation detection technique is however perfect in identifying

  8. Identification and Functional Testing of ERCC2 Mutations in a Multi-national Cohort of Patients with Familial Breast- and Ovarian Cancer.

    Directory of Open Access Journals (Sweden)

    Andreas Rump

    2016-08-01

    Full Text Available The increasing application of gene panels for familial cancer susceptibility disorders will probably lead to an increased proposal of susceptibility gene candidates. Using ERCC2 DNA repair gene as an example, we show that proof of a possible role in cancer susceptibility requires a detailed dissection and characterization of the underlying mutations for genes with diverse cellular functions (in this case mainly DNA repair and basic cellular transcription. In case of ERCC2, panel sequencing of 1345 index cases from 587 German, 405 Lithuanian and 353 Czech families with breast and ovarian cancer (BC/OC predisposition revealed 25 mutations (3 frameshift, 2 splice-affecting, 20 missense, all absent or very rare in the ExAC database. While 16 mutations were unique, 9 mutations showed up repeatedly with population-specific appearance. Ten out of eleven mutations that were tested exemplarily in cell-based functional assays exert diminished excision repair efficiency and/or decreased transcriptional activation capability. In order to provide evidence for BC/OC predisposition, we performed familial segregation analyses and screened ethnically matching controls. However, unlike the recently published RECQL example, none of our recurrent ERCC2 mutations showed convincing co-segregation with BC/OC or significant overrepresentation in the BC/OC cohort. Interestingly, we detected that some deleterious founder mutations had an unexpectedly high frequency of > 1% in the corresponding populations, suggesting that either homozygous carriers are not clinically recognized or homozygosity for these mutations is embryonically lethal. In conclusion, we provide a useful resource on the mutational landscape of ERCC2 mutations in hereditary BC/OC patients and, as our key finding, we demonstrate the complexity of correct interpretation for the discovery of "bonafide" breast cancer susceptibility genes.

  9. Mechanisms of Mutation in Non-Dividing Cells

    National Research Council Canada - National Science Library

    Petrosino, Joseph

    2002-01-01

    .... Previously, our laboratory discovered that RecA (an hRAD51 homolog) and RecBCD recombination repair proteins are necessary for the acquisition of 13-lactam drug-resistant mutations in the Escherichia coli chromosome during stationary-phase...

  10. FBXW7 and NOTCH1 mutations in childhood T cell acute lymphoblastic leukaemia and T cell non-Hodgkin lymphoma.

    Science.gov (United States)

    Park, Myoung-Ja; Taki, Tomohiko; Oda, Megumi; Watanabe, Tomoyuki; Yumura-Yagi, Keiko; Kobayashi, Ryoji; Suzuki, Nobuhiro; Hara, Junichi; Horibe, Keizo; Hayashi, Yasuhide

    2009-04-01

    Mutation analysis of FBXW7 and NOTCH1 genes was performed in 55 T cell acute lymphoblastic leukaemia (T-ALL) and 14 T cell non-Hodgkin lymphoma (T-NHL) patients who were treated on the Japan Association of Childhood Leukaemia Study (JACLS) protocols ALL-97 and NHL-98. FBXW7 and/or NOTCH1 mutations were found in 22 (40.0%) of 55 T-ALL and 7 (50.0%) of 14 T-NHL patients. FBXW7 mutations were found in 8 (14.6%) of 55 T-ALL and 3 (21.4%) of 14 T-NHL patients, and NOTCH1 mutations in 17 (30.9%) of 55 T-ALL and 6 (42.9%) of 14 T-NHL patients. Three (5.4%) T-ALL and two (1.4%) T-NHL patients had mutations in both FBXW7 and NOTCH1. FBXW7 mutations included one insertion, one deletion, one deletion/insertion and nine missense mutations. NOTCH1 mutations were detected in the heterodimerization domain (HD) in 15 cases, in the PEST domain in seven cases, and in both the HD and PEST domains in one case. Five-year event-free survival and overall survival for patients with FBXW7 and/or NOTCH1 mutations were 95.5% (95% CI, 71.9-99.4%) and 100% respectively, suggesting that T-ALL patients with FBXW7 and/or NOTCH1 mutation represent a good prognosis compared to those without FBXW7 and/or NOTCH1 mutations (63.6%, P = 0.007 and 78.8%, P = 0.023, respectively).

  11. The Number of Point Mutations in Induced Pluripotent Stem Cells and Nuclear Transfer Embryonic Stem Cells Depends on the Method and Somatic Cell Type Used for Their Generation.

    Science.gov (United States)

    Araki, Ryoko; Mizutani, Eiji; Hoki, Yuko; Sunayama, Misato; Wakayama, Sayaka; Nagatomo, Hiroaki; Kasama, Yasuji; Nakamura, Miki; Wakayama, Teruhiko; Abe, Masumi

    2017-05-01

    Induced pluripotent stem cells hold great promise for regenerative medicine but point mutations have been identified in these cells and have raised serious concerns about their safe use. We generated nuclear transfer embryonic stem cells (ntESCs) from both mouse embryonic fibroblasts (MEFs) and tail-tip fibroblasts (TTFs) and by whole genome sequencing found fewer mutations compared with iPSCs generated by retroviral gene transduction. Furthermore, TTF-derived ntESCs showed only a very small number of point mutations, approximately 80% less than the number observed in iPSCs generated using retrovirus. Base substitution profile analysis confirmed this greatly reduced number of point mutations. The point mutations in iPSCs are therefore not a Yamanaka factor-specific phenomenon but are intrinsic to genome reprogramming. Moreover, the dramatic reduction in point mutations in ntESCs suggests that most are not essential for genome reprogramming. Our results suggest that it is feasible to reduce the point mutation frequency in iPSCs by optimizing various genome reprogramming conditions. We conducted whole genome sequencing of ntES cells derived from MEFs or TTFs. We thereby succeeded in establishing TTF-derived ntES cell lines with far fewer point mutations. Base substitution profile analysis of these clones also indicated a reduced point mutation frequency, moving from a transversion-predominance to a transition-predominance. Stem Cells 2017;35:1189-1196. © 2017 AlphaMed Press.

  12. Effect of KCNJ5 Mutations on Gene Expression in Aldosterone-Producing Adenomas and Adrenocortical Cells

    Science.gov (United States)

    Monticone, Silvia; Hattangady, Namita G.; Nishimoto, Koshiro; Mantero, Franco; Rubin, Beatrice; Cicala, Maria Verena; Pezzani, Raffaele; Auchus, Richard J.; Ghayee, Hans K.; Shibata, Hirotaka; Kurihara, Isao; Williams, Tracy A.; Giri, Judith G.; Bollag, Roni J.; Edwards, Michael A.; Isales, Carlos M.

    2012-01-01

    Context: Primary aldosteronism is a heterogeneous disease that includes both sporadic and familial forms. A point mutation in the KCNJ5 gene is responsible for familial hyperaldosteronism type III. Somatic mutations in KCNJ5 also occur in sporadic aldosterone producing adenomas (APA). Objective: The objective of the study was to define the effect of the KCNJ5 mutations on gene expression and aldosterone production using APA tissue and human adrenocortical cells. Methods: A microarray analysis was used to compare the transcriptome profiles of female-derived APA samples with and without KCNJ5 mutations and HAC15 adrenal cells overexpressing either mutated or wild-type KCNJ5. Real-time PCR validated a set of differentially expressed genes. Immunohistochemical staining localized the KCNJ5 expression in normal adrenals and APA. Results: We report a 38% (18 of 47) prevalence of KCNJ5 mutations in APA. KCNJ5 immunostaining was highest in the zona glomerulosa of NA and heterogeneous in APA tissue, and KCNJ5 mRNA was 4-fold higher in APA compared with normal adrenals (P APA with and without KCNJ5 mutations displayed slightly different gene expression patterns, notably the aldosterone synthase gene (CYP11B2) was more highly expressed in APA with KCNJ5 mutations. Overexpression of KCNJ5 mutations in HAC15 increased aldosterone production and altered expression of 36 genes by greater than 2.5-fold (P APA, and our data suggest that these mutations increase expression of CYP11B2 and NR4A2, thus increasing aldosterone production. PMID:22628608

  13. Epidermal growth factor receptor (EGFR mutations and expression in squamous cell carcinoma of the esophagus in central Asia

    Directory of Open Access Journals (Sweden)

    Abedi-Ardekani Behnoush

    2012-12-01

    Full Text Available Abstract Background Esophageal squamous cell carcinoma (ESCC shows geographic variations in incidence, with high incidences (>50/105 person-years in central Asia, including North Eastern Iran (Golestan and Northern India (Kashmir. In contrast to Western countries, smoking does not appear to be a significant risk factor for ESCC in central Asia. In lung adenocarcinoma, activating mutations in the gene encoding epidermal growth factor receptor (EGFR are frequent in tumors of never smokers of Asian origin, predicting therapeutic sensitivity to Egfr-targeting drugs. Methods In this study 152 cases of histologically confirmed ESCC from Iran (Tehran and Golestan Province and North India (Kashmir Valley have been analyzed for EGFR mutation by direct sequencing of exons 18–21. Egfr protein expression was evaluated by immunohistochemistry in 34 samples from Tehran and HER2 mutations were analyzed in 54 cases from Kashmir. Results A total of 14 (9.2% EGFR variations were detected, including seven variations in exons. Among those, four (2.6% were already documented in lung cancers, two were reported as polymorphisms and one was a potentially new activating mutation. All but one variation in introns were previously identified as polymorphisms. Over-expression of Egfr was detected in 22/34 (65% of tested cases whereas no HER2 mutation was found in 54 cases from Kashmir. Conclusion Overall, EGFR mutations appear to be a rare event in ESCC in high incidence areas of central Asia, although a very small proportion of cases may harbor mutations predicting sensitivity to anti-Egfr drugs.

  14. Induction of mutations in Thai rice varieties and subsequent selection and testing of beneficial mutant lines

    Energy Technology Data Exchange (ETDEWEB)

    Dasananda, S; Khambanonda, P [Ministry of Agriculture, Bangkok (Thailand)

    1970-03-01

    Ionizing radiations were first used in the Thailand Rice Breeding Program in 1955 when seeds of two recommended varieties were sent to the United States of America for treatment. As a result, five promising mutant lines are at present in regional yield tests where they are being considered for recommendation to rice growers. During the period 1960-1961 an unsuccessful attempt was made to induce resistance to blast in three susceptible varieties by exposing seeds to a local source of ionizing radiation In 1964, after an elapse of about 4 years, another attempt was made to utilize ionizing radiations in the breeding program by treating seeds of two recommended varieties. In 1965, a co-ordinated rice mutation breeding program was initiated under the auspices of the Joint FAO/IAEA Division of Atomic Energy in Food and Agriculture which resulted in treating seeds of twelve different rice varieties with both ethyl methane sulphonate and gamma rays from a {sup 60}Co gamma cell. The results so far indicate that mutagenic agents have been successful in producing genetic variability. Differences in heading date, mature plant height and plant type are frequently observed in the M{sub 2} and M{sub 3} generations. Several lines obtained from two of the irradiated varieties have exhibited a higher degree of resistance to blast than the parental material. From 15-kR treatments of non-glutinous varieties, mutants with glutinous endosperm have been obtained. Not all varieties gave the same response to treatment. (author)

  15. MicroRNA-142 is mutated in about 20% of diffuse large B-cell lymphoma

    International Nuclear Information System (INIS)

    Kwanhian, Wiyada; Lenze, Dido; Alles, Julia; Motsch, Natalie; Barth, Stephanie; Döll, Celina; Imig, Jochen; Hummel, Michael; Tinguely, Marianne; Trivedi, Pankaj; Lulitanond, Viraphong; Meister, Gunter; Renner, Christoph; Grässer, Friedrich A

    2012-01-01

    MicroRNAs (miRNAs) are short 18–23 nucleotide long noncoding RNAs that posttranscriptionally regulate gene expression by binding to mRNA. Our previous miRNA profiling of diffuse large B-cell lymphoma (DLBCL) revealed a mutation in the seed sequence of miR-142-3p. Further analysis now showed that miR-142 was mutated in 11 (19.64%) of the 56 DLBCL cases. Of these, one case had a mutation in both alleles, with the remainder being heterozygous. Four mutations were found in the mature miR-142-5p, four in the mature miR-142-3p, and three mutations affected the miR-142 precursor. Two mutations in the seed sequence redirected miR-142-3p to the mRNA of the transcriptional repressor ZEB2 and one of them also targeted the ZEB1 mRNA. However, the other mutations in the mature miR-142-3p did not influence either the ZEB1 or ZEB2 3′ untranslated region (3′ UTR). On the other hand, the mutations affecting the seed sequence of miR-142-3p resulted in a loss of responsiveness in the 3′ UTR of the known miR-142-3p targets RAC1 and ADCY9. In contrast to the mouse p300 gene, the human p300 gene was not found to be a target for miR-142-5p. In one case with a mutation of the precursor, we observed aberrant processing of the miR-142-5p. Our data suggest that the mutations in miR-142 probably lead to a loss rather than a gain of function. This is the first report describing mutations of a miRNA gene in a large percentage of a distinct lymphoma subtype

  16. Functional Studies of Missense TREM2 Mutations in Human Stem Cell-Derived Microglia

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    Philip W. Brownjohn

    2018-04-01

    Full Text Available Summary: The derivation of microglia from human stem cells provides systems for understanding microglial biology and enables functional studies of disease-causing mutations. We describe a robust method for the derivation of human microglia from stem cells, which are phenotypically and functionally comparable with primary microglia. We used stem cell-derived microglia to study the consequences of missense mutations in the microglial-expressed protein triggering receptor expressed on myeloid cells 2 (TREM2, which are causal for frontotemporal dementia-like syndrome and Nasu-Hakola disease. We find that mutant TREM2 accumulates in its immature form, does not undergo typical proteolysis, and is not trafficked to the plasma membrane. However, in the absence of plasma membrane TREM2, microglia differentiate normally, respond to stimulation with lipopolysaccharide, and are phagocytically competent. These data indicate that dementia-associated TREM2 mutations have subtle effects on microglia biology, consistent with the adult onset of disease in individuals with these mutations. : Brownjohn and colleagues report methods to generate microglia from induced pluripotent human stem cells, which they demonstrate are highly similar to cultured primary human microglia. Microglia differentiated from patient-derived stem cells carrying neurological disease-causing mutations in the TREM2 receptor differentiate normally and respond appropriately to pathogenic stimuli, despite the absence of functional TREM2 receptor on the plasma membrane. Keywords: dementia, microglia, TREM2, Nasu-Hakola disease, frontotemporal dementia, iPSC-microglia, neuroinflammation

  17. Radioprotection of IDH1-Mutated Cancer Cells by the IDH1-Mutant Inhibitor AGI-5198.

    Science.gov (United States)

    Molenaar, Remco J; Botman, Dennis; Smits, Myrthe A; Hira, Vashendriya V; van Lith, Sanne A; Stap, Jan; Henneman, Peter; Khurshed, Mohammed; Lenting, Krissie; Mul, Adri N; Dimitrakopoulou, Dionysia; van Drunen, Cornelis M; Hoebe, Ron A; Radivoyevitch, Tomas; Wilmink, Johanna W; Maciejewski, Jaroslaw P; Vandertop, W Peter; Leenders, William P; Bleeker, Fonnet E; van Noorden, Cornelis J

    2015-11-15

    Isocitrate dehydrogenase 1 (IDH1) is mutated in various types of human cancer to IDH1(R132H), a structural alteration that leads to catalysis of α-ketoglutarate to the oncometabolite D-2-hydroxyglutarate. In this study, we present evidence that small-molecule inhibitors of IDH1(R132H) that are being developed for cancer therapy may pose risks with coadministration of radiotherapy. Cancer cells heterozygous for the IDH1(R132H) mutation exhibited less IDH-mediated production of NADPH, such that after exposure to ionizing radiation (IR), there were higher levels of reactive oxygen species, DNA double-strand breaks, and cell death compared with IDH1 wild-type cells. These effects were reversed by the IDH1(R132H) inhibitor AGI-5198. Exposure of IDH1 wild-type cells to D-2-hydroxyglutarate was sufficient to reduce IDH-mediated NADPH production and increase IR sensitivity. Mechanistic investigations revealed that the radiosensitivity of heterozygous cells was independent of the well-described DNA hypermethylation phenotype in IDH1-mutated cancers. Thus, our results argue that altered oxidative stress responses are a plausible mechanism to understand the radiosensitivity of IDH1-mutated cancer cells. Further, they offer an explanation for the relatively longer survival of patients with IDH1-mutated tumors, and they imply that administration of IDH1(R132H) inhibitors in these patients may limit irradiation efficacy in this setting. ©2015 American Association for Cancer Research.

  18. Competitive amplification of differentially melting amplicons (CADMA improves KRAS hotspot mutation testing in colorectal cancer

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    Kristensen Lasse

    2012-11-01

    Full Text Available Abstract Background Cancer is an extremely heterogeneous group of diseases traditionally categorized according to tissue of origin. However, even among patients with the same cancer subtype the cellular alterations at the molecular level are often very different. Several new therapies targeting specific molecular changes found in individual patients have initiated the era of personalized therapy and significantly improved patient care. In metastatic colorectal cancer (mCRC a selected group of patients with wild-type KRAS respond to antibodies against the epidermal growth factor receptor (EGFR. Testing for KRAS mutations is now required prior to anti-EGFR treatment, however, less sensitive methods based on conventional PCR regularly fail to detect KRAS mutations in clinical samples. Methods We have developed sensitive and specific assays for detection of the seven most common KRAS mutations based on a novel methodology named Competitive Amplification of Differentially Melting Amplicons (CADMA. The clinical applicability of these assays was assessed by analyzing 100 colorectal cancer samples, for which KRAS mutation status has been evaluated by the commercially available TheraScreen® KRAS mutation kit. Results The CADMA assays were sensitive to at least 0.5% mutant alleles in a wild-type background when using 50 nanograms of DNA in the reactions. Consensus between CADMA and the TheraScreen kit was observed in 96% of the colorectal cancer samples. In cases where disagreement was observed the CADMA result could be confirmed by a previously published assay based on TaqMan probes and by fast COLD-PCR followed by Sanger sequencing. Conclusions The high analytical sensitivity and specificity of CADMA may increase diagnostic sensitivity and specificity of KRAS mutation testing in mCRC patients.

  19. Determination of EGFR and KRAS mutational status in Greek non-small-cell lung cancer patients.

    Science.gov (United States)

    Papadopoulou, Eirini; Tsoulos, Nikolaos; Tsirigoti, Angeliki; Apessos, Angela; Agiannitopoulos, Konstantinos; Metaxa-Mariatou, Vasiliki; Zarogoulidis, Konstantinos; Zarogoulidis, Pavlos; Kasarakis, Dimitrios; Kakolyris, Stylianos; Dahabreh, Jubrail; Vlastos, Fotis; Zoublios, Charalampos; Rapti, Aggeliki; Papageorgiou, Niki Georgatou; Veldekis, Dimitrios; Gaga, Mina; Aravantinos, Gerasimos; Karavasilis, Vasileios; Karagiannidis, Napoleon; Nasioulas, George

    2015-10-01

    It has been reported that certain patients with non-small-cell lung cancer (NSCLC) that harbor activating somatic mutations within the tyrosine kinase domain of the epidermal growth factor receptor ( EGFR ) gene may be effectively treated using targeted therapy. The use of EGFR inhibitors in patient therapy has been demonstrated to improve response and survival rates; therefore, it was suggested that clinical screening for EGFR mutations should be performed for all patients. Numerous clinicopathological factors have been associated with EGFR and Kirsten-rat sarcoma oncogene homolog (KRAS) mutational status including gender, smoking history and histology. In addition, it was reported that EGFR mutation frequency in NSCLC patients was ethnicity-dependent, with an incidence rate of ~30% in Asian populations and ~15% in Caucasian populations. However, limited data has been reported on intra-ethnic differences throughout Europe. The present study aimed to investigate the frequency and spectrum of EGFR mutations in 1,472 Greek NSCLC patients. In addition, KRAS mutation analysis was performed in patients with known smoking history in order to determine the correlation of type and mutation frequency with smoking. High-resolution melting curve (HRM) analysis followed by Sanger sequencing was used to identify mutations in exons 18-21 of the EGFR gene and in exon 2 of the KRAS gene. A sensitive next-generation sequencing (NGS) technology was also employed to classify samples with equivocal results. The use of sensitive mutation detection techniques in a large study population of Greek NSCLC patients in routine diagnostic practice revealed an overall EGFR mutation frequency of 15.83%. This mutation frequency was comparable to that previously reported in other European populations. Of note, there was a 99.8% concordance between the HRM method and Sanger sequencing. NGS was found to be the most sensitive method. In addition, female non-smokers demonstrated a high prevalence of

  20. MASA syndrome is caused by mutations in the neural cell adhesion gene, L1CAM

    Energy Technology Data Exchange (ETDEWEB)

    Schwartz, C.E.; Wang, Y.; Schroer, R.J.; Stevenson, R.E. [Greenwood Genetic Center, SC (United States)

    1994-09-01

    The MASA syndrome is a recessive X-linked disorder characterized by Mental retardation, Adducted thumbs, Shuffling gait and Aphasia. Recently we found that MASA in one family was likely caused by a point mutation in exon 6 of the L1CAM gene. This gene has also been shown to be involved in X-linked hydrocephalus (HSAS). We have screened 60 patients with either sporadic HSAS or MASA as well as two additional families with MASA. For the screening, we initially utilized 3 cDNA probes for the L1CAM gene. In one of the MASA families, K8310, two affected males were found to have an altered BglII band. The band was present in their carrier mother but not in their normal brothers. This band was detected by the entire cDNA probe as well as the cDNA probe for 3{prime} end of the gene. Analysis of the L1CAM sequence indicated the altered BglII site is distal to the exon 28 but proximal to the punative poly A signal site. It is hypothesized that this point mutation alters the stability of the L1CAM mRNA. This is being tested using cell lines established from the two affected males.

  1. Biology of lung cancer: genetic mutation, epithelial-mesenchymal transition, and cancer stem cells.

    Science.gov (United States)

    Aoi, Takashi

    2016-09-01

    At present, most cases of unresectable cancer cannot be cured. Genetic mutations, EMT, and cancer stem cells are three major issues linked to poor prognosis in such cases, all connected by inter- and intra-tumor heterogeneity. Issues on inter-/intra-tumor heterogeneity of genetic mutation could be resolved with recent and future technologies of deep sequencers, whereas, regarding such issues as the "same genome, different epigenome/phenotype", we expect to solve many of these problems in the future through further research in stem cell biology. We herein review and discuss the three major issues in the biology of cancers, especially from the standpoint of stem cell biology.

  2. Rasfonin, a novel 2-pyrone derivative, induces ras-mutated Panc-1 pancreatic tumor cell death in nude mice.

    Science.gov (United States)

    Xiao, Z; Li, L; Li, Y; Zhou, W; Cheng, J; Liu, F; Zheng, P; Zhang, Y; Che, Y

    2014-05-22

    Rasfonin is a novel 2-pyrone derivative reported to induce apoptosis in ras-dependent cells. In this study, its effects on ras-mutated pancreatic cancer cells were investigated in vitro and in vivo. Two human pancreatic cancer cell lines Panc-1 (mutated K-ras) and BxPC-3 (wild-type K-ras) were selected to test the effects of rasfonin on cell proliferation, clone formation, migration and invasion in vitro. Immunoblotting was used to detect the expressions of EGFR-Ras-Raf-MEK-ERK signaling pathway proteins. Ras activity was measured using a pull-down ELISA kit and guanine exchange factor (GEF)/GTPase-activating proteins (GAP) activity was measured by [(3)H]-GDP radiometric ligand binding. For an in vivo study, CD1 nude mice bearing Panc-1 cells were treated with rasfonin or Salirasib (FTS). We found that rasfonin suppressed proliferation more strongly in Panc-1 cells (IC50=5.5 μM) than BxPC-3 cells (IC50=10 μM) in vitro. Clone formation, migration and invasion by Panc-1 cells were also reduced by rasfonin. Rasfonin had little effect on the farnesylation of Ras, but it strongly downregulated Ras activity and consequently phosphorylation of c-Raf/MEK/ERK. Further experiments indicated that rasfonin reduced Son of sevenless (Sos1) expression but did not alter GEF and GAP activities. The in vivo experiments also revealed that rasfonin (30 mg/kg) delayed the growth of xenograft tumors originating from Panc-1 cells. Tumor weight was ultimately decreased after 20 days of treatment of rasfonin. Rasfonin is a robust inhibitor of pancreatic cancers with the K-ras mutation. The reduction of Sos1 expression and the consequently depressed Ras-MAPK activity could be important in its anticancer activity.

  3. An integrated inspection of the somatic mutations in a lung squamous cell carcinoma using next-generation sequencing.

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    Lucy F Stead

    Full Text Available Squamous cell carcinoma (SCC of the lung kills over 350,000 people annually worldwide, and is the main lung cancer histotype with no targeted treatments. High-coverage whole-genome sequencing of the other main subtypes, small-cell and adenocarcinoma, gave insights into carcinogenic mechanisms and disease etiology. The genomic complexity within the lung SCC subtype, as revealed by The Cancer Genome Atlas, means this subtype is likely to benefit from a more integrated approach in which the transcriptional consequences of somatic mutations are simultaneously inspected. Here we present such an approach: the integrated analysis of deep sequencing data from both the whole genome and whole transcriptome (coding and non-coding of LUDLU-1, a SCC lung cell line. Our results show that LUDLU-1 lacks the mutational signature that has been previously associated with tobacco exposure in other lung cancer subtypes, and suggests that DNA-repair efficiency is adversely affected; LUDLU-1 contains somatic mutations in TP53 and BRCA2, allelic imbalance in the expression of two cancer-associated BRCA1 germline polymorphisms and reduced transcription of a potentially endogenous PARP2 inhibitor. Functional assays were performed and compared with a control lung cancer cell line. LUDLU-1 did not exhibit radiosensitisation or an increase in sensitivity to PARP inhibitors. However, LUDLU-1 did exhibit small but significant differences with respect to cisplatin sensitivity. Our research shows how integrated analyses of high-throughput data can generate hypotheses to be tested in the lab.

  4. X-ray-induced bystander response reduce spontaneous mutations in V79 cells

    International Nuclear Information System (INIS)

    Maeda, Munetoshi; Kobayashi, Katsumi; Matsumoto, Hideki; Usami, Noriko; Tomiya, Masanori

    2013-01-01

    The potential for carcinogenic risks is increased by radiation-induced bystander responses; these responses are the biological effects in unirradiated cells that receive signals from the neighboring irradiated cells. Bystander responses have attracted attention in modern radiobiology because they are characterized by non-linear responses to low-dose radiation. We used a synchrotron X-ray microbeam irradiation system developed at the Photon Factory, High Energy Accelerator Research Organization, KEK, and showed that nitric oxide (NO)-mediated bystander cell death increased biphasically in a dose-dependent manner. Here, we irradiated five cell nuclei using 10 × 10 µm 2 5.35 keV X-ray beams and then measured the mutation frequency at the hypoxanthine-guanosine phosphoribosyl transferase (HPRT) locus in bystander cells. The mutation frequency with the null radiation dose was 2.6 × 10 -5 (background level), and the frequency decreased to 5.3 × 10 -6 with a dose of approximately 1 Gy (absorbed dose in the nucleus of irradiated cells). At high doses, the mutation frequency returned to the background level. A similar biphasic dose-response effect was observed for bystander cell death. Furthermore, we found that incubation with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), a specific scavenger of NO, suppressed not only the biphasic increase in bystander cell death but also the biphasic reduction in mutation frequency of bystander cells. These results indicate that the increase in bystander cell death involves mechanisms that suppress mutagenesis. This study has thus shown that radiation-induced bystander responses could affect processes that protect the cell against naturally occurring alterations such as mutations. (author)

  5. TERT promoter hot spot mutations are frequent in Indian cervical and oral squamous cell carcinomas.

    Science.gov (United States)

    Vinothkumar, Vilvanathan; Arunkumar, Ganesan; Revathidevi, Sundaramoorthy; Arun, Kanagaraj; Manikandan, Mayakannan; Rao, Arunagiri Kuha Deva Magendhra; Rajkumar, Kottayasamy Seenivasagam; Ajay, Chandrasekar; Rajaraman, Ramamurthy; Ramani, Rajendren; Murugan, Avaniyapuram Kannan; Munirajan, Arasambattu Kannan

    2016-06-01

    Squamous cell carcinoma (SCC) of the uterine cervix and oral cavity are most common cancers in India. Telomerase reverse transcriptase (TERT) overexpression is one of the hallmarks for cancer, and activation through promoter mutation C228T and C250T has been reported in variety of tumors and often shown to be associated with aggressive tumors. In the present study, we analyzed these two hot spot mutations in 181 primary tumors of the uterine cervix and oral cavity by direct DNA sequencing and correlated with patient's clinicopathological characteristics. We found relatively high frequency of TERT hot spot mutations in both cervical [21.4 % (30/140)] and oral [31.7 % (13/41)] squamous cell carcinomas. In cervical cancer, TERT promoter mutations were more prevalent (25 %) in human papilloma virus (HPV)-negative cases compared to HPV-positive cases (20.6 %), and both TERT promoter mutation and HPV infection were more commonly observed in advanced stage tumors (77 %). Similarly, the poor and moderately differentiated tumors of the uterine cervix had both the TERT hot spot mutations and HPV (16 and 18) at higher frequency (95.7 %). Interestingly, we observed eight homozygous mutations (six 228TT and two 250TT) only in cervical tumors, and all of them were found to be positive for high-risk HPV. To the best of our knowledge, this is the first study from India reporting high prevalence of TERT promoter mutations in primary tumors of the uterine cervix and oral cavity. Our results suggest that TERT reactivation through promoter mutation either alone or in association with the HPV oncogenes (E6 and E7) could play an important role in the carcinogenesis of cervical and oral cancers.

  6. Novel Secondary Somatic Mutations in Ewing's Sarcoma and Desmoplastic Small Round Cell Tumors

    Science.gov (United States)

    Janku, Filip; Ludwig, Joseph A.; Naing, Aung; Benjamin, Robert S.; Brown, Robert E.; Anderson, Pete; Kurzrock, Razelle

    2014-01-01

    Background Ewing's sarcoma (ES) and desmoplastic small round cell tumors (DSRCT) are small round blue cell tumors driven by an N-terminal containing EWS translocation. Very few somatic mutations have been reported in ES, and none have been identified in DSRCT. The aim of this study is to explore potential actionable mutations in ES and DSRCT. Methodology Twenty eight patients with ES or DSRCT had tumor tissue available that could be analyzed by one of the following methods: 1) Next-generation exome sequencing platform; 2) Multiplex PCR/Mass Spectroscopy; 3) Polymerase chain reaction (PCR)-based single- gene mutation screening; 4) Sanger sequencing; 5) Morphoproteomics. Principal Findings Novel somatic mutations were identified in four out of 18 patients with advanced ES and two of 10 patients with advanced DSRCT (six out of 28 (21.4%));KRAS (n = 1), PTPRD (n = 1), GRB10 (n = 2), MET (n = 2) and PIK3CA (n = 1). One patient with both PTPRD and GRB10 mutations and one with a GRB10 mutation achieved a complete remission (CR) on an Insulin like growth factor 1 receptor (IGF1R) inhibitor based treatment. One patient, who achieved a partial remission (PR) with IGF1R inhibitor treatment, but later developed resistance, demonstrated a KRAS mutation in the post-treatment resistant tumor, but not in the pre-treatment tumor suggesting that the RAF/RAS/MEK pathway was activated with progression. Conclusions We have reported several different mutations in advanced ES and DSRCT that have direct implications for molecularly-directed targeted therapy. Our technology agnostic approach provides an initial mutational roadmap used in the path towards individualized combination therapy. PMID:25119929

  7. Sequential cancer mutations in cultured human intestinal stem cells

    NARCIS (Netherlands)

    Drost, Jarno; van Jaarsveld, Richard H.; Ponsioen, Bas; Zimberlin, Cheryl; van Boxtel, Ruben; Buijs, Arjan; Sachs, Norman; Overmeer, René M.; Offerhaus, G. Johan; Begthel, Harry; Korving, Jeroen; van de Wetering, Marc; Schwank, Gerald; Logtenberg, Meike; Cuppen, Edwin; Snippert, Hugo J.; Medema, Jan Paul; Kops, Geert J. P. L.; Clevers, Hans

    2015-01-01

    Crypt stem cells represent the cells of origin for intestinal neoplasia. Both mouse and human intestinal stem cells can be cultured in medium containing the stem-cell-niche factors WNT, R-spondin, epidermal growth factor (EGF) and noggin over long time periods as epithelial organoids that remain

  8. Effect of Hereditary Hemochromatosis Gene H63D and C282Y Mutations on Iron Overload in Sickle Cell Disease Patients

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    Yunus Kasım Terzi

    2016-12-01

    Full Text Available Objective: Hemochromatosis is an autosomal recessive disease that is one of the most important reasons for iron overload. Sickle cell disease is a hemoglobinopathy that occurs as a result of a homozygous mutation in the hemoglobin gene. Erythrocyte transfusion is frequently used in the treatment of this disease. Iron overload as a result of transfusion is important in the mortality and morbidity of sickle cell anemia patients as well as in other hemoglobinopathies. In this study, the effect of hemochromatosis gene (HFE p.H63D and p.C282Y mutations on transfusion-related cardiac and liver iron overload in sickle cell disease patients who carry homozygous hemoglobin S mutation has been investigated. Materials and Methods: This is a prospective single-center crosssectional study in patients with homozygous hemoglobin S mutation between the years 2008 and 2013. The patients were divided into two groups. The first group (group A, n=31 was receiving chelation therapy and the second group (group B, n=13 was not. Direct and indirect iron loads were analyzed by magnetic resonance imaging and biochemically, respectively. HFE gene mutations were analyzed by polymerase chain reaction-restriction fragment length polymorphism method. Statistical analyses were performed by independent samples t-test. Results: p.H63D mutation was detected in 10 (32.3% patients in group A and in only 1 patient (7.7% in group B. When the 2 groups were compared for iron overload, iron deposition in the liver was significantly higher in group B (p=0.046. In addition, in group A, iron deposition was significantly higher in HFE mutation carriers compared to patients without the mutation (p=0.05. Conclusion: Results of this study showed that HFE gene mutations are important in iron deposition in the liver in patients with sickle cell disease.

  9. Trichloroethylene exposure and somatic mutations of the VHL gene in patients with Renal Cell Carcinoma

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    Fevotte Joelle

    2007-11-01

    Full Text Available Abstract Background We investigated the association between exposure to trichloroethylene (TCE and mutations in the von Hippel-Lindau (VHL gene and the subsequent risk for renal cell carcinoma (RCC. Methods Cases were recruited from a case-control study previously carried out in France that suggested an association between exposures to high levels of TCE and increased risk of RCC. From 87 cases of RCC recruited for the epidemiological study, 69 were included in the present study. All samples were evaluated by a pathologist in order to identify the histological subtype and then be able to focus on clear cell RCC. The majority of the tumour samples were fixed either in formalin or Bouin's solutions. The majority of the tumours were of the clear cell RCC subtype (48 including 2 cystic RCC. Mutation screening of the 3 VHL coding exons was carried out. A descriptive analysis was performed to compare exposed and non exposed cases of clear cell RCC in terms of prevalence of mutations in both groups. Results In the 48 cases of RCC, four VHL mutations were detected: within exon 1 (c.332G>A, p.Ser111Asn, at the exon 2 splice site (c.463+1G>C and c.463+2T>C and within exon 3 (c.506T>C, p.Leu169Pro. No difference was observed regarding the frequency of mutations in exposed versus unexposed groups: among the clear cell RCC, 25 had been exposed to TCE and 23 had no history of occupational exposure to TCE. Two patients with a mutation were identified in each group. Conclusion This study does not confirm the association between the number and type of VHL gene mutations and exposure to TCE previously described.

  10. The prevalence of EGFR mutations in non-small cell lung cancer in an unselected Caucasian population

    DEFF Research Database (Denmark)

    Skov, Birgit G; Høgdall, Estrid; Clementsen, Paul

    2015-01-01

    in a well-defined Danish population were included. The type of the diagnostic material, and data on smoking were registered. The mutation analyses were investigated by Therascreen EGFR RGQ-PCR Kit or Sanger sequencing. A total of 658 men and 598 women were included. 6.2% were never smokers, 38.9% were ex-smokers.......0% of adenocarcinomas, and 1.9% of squamous cell carcinomas were mutated. 29.4%, 4.4% and 2.9% of never, ex- and current smokers were mutated (p ... EGFR mutation. Adenocarcinomas were mutated more often (8.0%) than squamous cell carcinomas (1.9%). Mutations were found in never smokers as well as in former and current smokers. No difference in gender and age regarding mutation status was observed. EGFR mutations analysis was possible in almost all...

  11. Genomic mutation study for long-term cells induced by carbon ions

    International Nuclear Information System (INIS)

    Wang, X.; Furusawa, Y.; Suzuki, M.; Hirayama, R.; Matsumoto, Y.; Qin, Y.

    2007-01-01

    Complete text of publication follows. Objective: Densely ionizing (high LET) radiation can increase the relative biological effectiveness of cell and tissue. Astronauts in the space exploration have the potential exposure of chronic low-dose radiations in the field of low-flux galactic cosmic rays (GCR) and the subsequent biological effects have become one of the major concerns of space science. Furthermore, Heavy ions also are used new radiation therapy owing increased lethal effectiveness of high LET radiation. During radiation therapy, normal tissues also are exposed to ionizing radiation. Radiation can induce genomic mutation and instability in descendants of irradiated cells. Induction of genomic instability can represent one of the initiating steps leading to malignant transformation. Higher frequencies of mutation can be expected to provide higher rates of carcinogenicity with human exposure. Therefore, the study of radiation induced genomic mutation and instability is relevant to the estimates of the risk of secondary malignancies associated with radiation therapy and the carcinogenic effects of space environmental radiation. The hypoxanthine-guanine phosphoribosyltransferase (hprt) locus has been the most commonly used as a target gene for mutation detection studies. In this study, we investigated the generation expression dependence of mutation induction on HPRT locus in CHO cells irradiated with carbon ions. Methods: Chinese hamster ovary (CHO) cells were irradiated with graded doses of carbon ions (290MeV/u, LET:13kev/um) accelerated with Heavy Ion Medical Accelerator in Chiba (HIMAC) at National Institute of Radiological Sciences(NIRS). The survival effect of cells plated immediately after irradiation was measured with cell colony formation assay. After irradiation, cells were continues reseeding and cultures for lone-term proliferation. Cell samples were collected at 6, 12, 18, 24, 30, 37 and 44 days post irradiation. Mutation induction of cell

  12. K-RAS and N-RAS mutations in testicular germ cell tumors

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    Bekir Muhammet Hacioglu

    2017-05-01

    Full Text Available Testicular cancer is a relatively rare tumor type, accounting for approximately 1% of all cancers in men. However, among men aged between 15 and 40 years, testicular cancer is the most commonly diagnosed malignancy. Testicular germ cell tumors (TGCTs are classified as seminoma and non-seminoma. The RAS oncogene controls several cellular functions, including cell proliferation, apoptosis, migration, and differentiation. Thus, RAS signaling is important for normal germ cell development. Mutations of the Kirsten RAS (K-RAS gene are present in over 20% of all cancers. RAS gene mutations have also been reported in TGCTs. We investigated K-RAS and N-RAS mutations in seminoma and non-seminoma TGCT patients. A total of 24 (55% pure seminoma cases and 19 (45% non-seminoma cases were included in the study. K-RAS and N-RAS analyses were performed in our molecular pathology laboratory, using K-RAS and N-RAS Pyro Kit 24 V1 (Qiagen. In total, a RAS mutation was present in 12 patients (27%: 7 seminoma (29% and 5 non-seminoma cases (26% [p = 0.55]. A K-RAS mutation was present in 4 pure seminoma tumors (16% and 3 non-seminoma tumors (15% [p = 0.63], and an N-RAS mutation was observed in 4 seminoma tumors (16% and 3 non-seminoma tumors (15% [p = 0.63]. Both, K-RAS and N-RAS mutations were present in two patients: one with seminoma tumor and the other with non-seminoma tumor. To date, no approved targeted therapy is available for the treatment of TGCTs. The analysis of K-RAS and N-RAS mutations in these tumors may provide more treatment options, especially in platinum-resistant tumors.

  13. Mutation Induction with UV- and X-radiations in spores and vegetative cells of Bacillus subtilis

    International Nuclear Information System (INIS)

    Tanooka, H.; Munakata, N.; Kitahara, S.

    1978-01-01

    Spores and vegetative cells of Bacillus subtilis strains with various defects in DNA-repair capacities (hcr - , ssp - , hcr - ssp - ) were irradiated with UV radiation or X-rays. Induced mutation frequency was determined from the observed frequency of prototrophic reversion of a suppressible auxotropic mutation. At equal physical dose, after either UV- or X-irradiation, spores were more resistant to mutations as well as to killing than were vegetative cells. However, quantitative comparison revealed that, at equally lethal doses, spores and vegetative cells were almost equally mutable by X-rays whereas spores were considerably less mutable by UV than were vegetative cells. Thus, as judged from their mutagenic efficiency relative to the lethality, X-ray-induced damage in the spore DNA and the vegetative DNA were equally mutagenic, while UV-induced DNA photoproducts in the spore were less mutagenic than those in vegetative cells. Post-treatment of UV-irradiated cells with caffeine decreased the survival and the induced mutation frequency for either spores or vegetative cells for all the strains. In X-irradiated spores however, a similar suppressing effect of caffeine was observed only for mutability of a strain lacking DNA polymerase I activity

  14. Combination therapy with vemurafenib (PLX4032/RG7204 and metformin in melanoma cell lines with distinct driver mutations

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    Recio Juan A

    2011-05-01

    Full Text Available Abstract Background A molecular linkage between the MAPK and the LKB1-AMPK energy sensor pathways suggests that combined MAPK oncogene inhibition and metabolic modulation of AMPK would be more effective than either manipulation alone in melanoma cell lines. Materials and methods The combination of the BRAF inhibitor vemurafenib (formerly PLX4032 and metformin were tested against a panel of human melanoma cell lines with defined BRAF and NRAS mutations for effects on viability, cell cycle and apoptosis. Signaling molecules in the MAPK, PI3K-AKT and LKB1-AMPK pathways were studied by Western blot. Results Single agent metformin inhibited proliferation in 12 out of 19 cell lines irrespective of the BRAF mutation status, but in one NRASQ61K mutant cell line it powerfully stimulated cell growth. Synergistic anti-proliferative effects of the combination of metformin with vemurafenib were observed in 6 out of 11 BRAFV600E mutants, including highly synergistic effects in two BRAFV600E mutant melanoma cell lines. Antagonistic effects were noted in some cell lines, in particular in BRAFV600E mutant cell lines resistant to single agent vemurafenib. Seven out of 8 BRAF wild type cell lines showed marginally synergistic anti-proliferative effects with the combination, and one cell line had highly antagonistic effects with the combination. The differential effects were not dependent on the sensitivity to each drug alone, effects on cell cycle or signaling pathways. Conclusions The combination of vemurafenib and metformin tended to have stronger anti-proliferative effects on BRAFV600E mutant cell lines. However, determinants of vemurafenib and metformin synergism or antagonism need to be understood with greater detail before any potential clinical utility of this combination.

  15. Clinical efficacy of first-generation EGFR-TKIs in patients with advanced non-small-cell lung cancer harboring EGFR exon 20 mutations

    Directory of Open Access Journals (Sweden)

    Chen D

    2016-07-01

    Full Text Available Dan Chen,1 Zhengbo Song,2 Guoping Cheng3 1Department of Cardiothoracic Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 2Department of Chemotherapy, 3Department of Pathology, Zhejiang Cancer Hospital, Hangzhou, People’s Republic of China Purpose: Subsets of non-small-cell lung cancer patients with epidermal growth factor receptor (EGFR mutations carry uncommon subtypes. We evaluated the efficacy of first-generation EGFR-tyrosine kinase inhibitors (TKIs; erlotinib, gefitinib, and icotinib in patients with non-small-cell lung cancer carrying insertions and T790M and S768I mutations in EGFR exon 20. Patients and methods: Patients carrying EGFR exon 20 insertion/T790M/S768I mutations and treated with EGFR-TKIs were evaluated from 2005 to 2014 in Zhejiang Cancer Hospital. The efficacy was evaluated using the Kaplan–Meier method and compared with the log-rank test. Results: Sixty-two patients with exon 20 insertion/T790M/S768I mutations were enrolled. Mutations including exon 20 insertions and T790M and S768I mutations were observed in 29, 23, and ten patients, respectively. In total, the response rate and median progression-free survival (PFS were 8.1% and 2.1 months, respectively. Patients with S768I mutation manifested the longest median PFS (2.7 months, followed by those with T790M (2.4 months and exon 20 insertions (1.9 months; P=0.022. Patients with complex mutations show a better PFS than those with single mutations (2.7 months vs 1.9 months; P=0.034. Conclusion: First-generation EGFR-TKIs are less effective in patients with exon 20 uncommon mutations than in those with common mutations. Patients with complex mutations benefited more from first-generation EGFR-TKIs than those with single mutations. Keywords: non-small cell lung cancer, epidermal growth factor receptor, EGFR mutations, exon 20, tyrosine kinase inhibitor

  16. Mutation formation and inactivation of mammalian cells by heavy ions. 3

    International Nuclear Information System (INIS)

    Obaturov, G.M.

    1984-01-01

    Using parameter values of equations for cell inactivation cross-sections, theoretical curves were calculated for cell radiosensitivity (α 1 ) in dependence on LET. There is good agreement between calculated and experimental values of α 1 . The yield of lethal mutations of 1st and 2nd types and their sum in dependence on LET was also calculated. It was shown that for low LET ions cell radiosensitivity is caused by the 2nd type of mutation, independent of LET, proportional to DNA mass and inversely proportional to repair rate. For high LET ions when lethal mutation number per one track is more than one, α 1 is caused by the 1st type of mutation, proportional to cell nuclear cross-section and inversely proportional to LET. For intermediate LET ions α 1 is a complicated function of LET. It is follows from the equations that more than 17% of double strand breaks (DSB) take part in lethal mutation formation. Others are either repaired or formed in the process of experimentally determined DSB yield. (author)

  17. A PLK4 mutation causing azoospermia in a man with Sertoli cell-only syndrome.

    Science.gov (United States)

    Miyamoto, T; Bando, Y; Koh, E; Tsujimura, A; Miyagawa, Y; Iijima, M; Namiki, M; Shiina, M; Ogata, K; Matsumoto, N; Sengoku, K

    2016-01-01

    About 15% of couples wishing to have children are infertile; approximately half these cases involve a male factor. Polo-like kinase 4 (PLK-4) is a member of the polo protein family and a key regulator of centriole duplication. Male mice with a point mutation in the Plk4 gene show azoospermia associated with germ cell loss. Mutational analysis of 81 patients with azoospermia and Sertoli cell-only syndrome (SCOS) identified one man with a heterozygous 13-bp deletion in the Ser/Thr kinase domain of PLK4. Division of centrioles occurred in wild-type PLK4-transfected cells, but was hampered in PLK-4-mutant transfectants, which also showed abnormal nuclei. Thus, this PLK4 mutation might be a cause of human SCOS and nonobstructive azoospermia. © 2015 American Society of Andrology and European Academy of Andrology.

  18. Engine Test Cell Aeroacoustics and Recommendations

    National Research Council Canada - National Science Library

    Tam, Christopher

    2007-01-01

    Ground testing of turbojet engines in test cells necessarily involves very high acoustic amplitudes, often enough and severe enough that testing is interrupted and facility hardware and test articles are damaged...

  19. Restricted ultraviolet mutational spectrum in a shuttle vector propagated in xeroderma pigmentosum cells

    International Nuclear Information System (INIS)

    Bredberg, A.; Kraemer, K.H.; Seidman, M.M.

    1986-01-01

    A shuttle vector plasmid, pZ189, carrying a bacterial suppressor tRNA marker gene, was treated with ultraviolet radiation and propagated in cultured skin cells from a patient with the skin-cancer-prone, DNA repair-deficient disease xeroderma pigmentosum and in repair-proficient cells. After replication in the human cells, progeny plasmids were purified. Plasmid survival and mutations inactivating the marker gene were scored by transforming an indicator strain of Escherichia coli carrying a suppressible amber mutation in the beta-galactosidase gene. Plasmid survival in the xeroderma pigmentosum cells was less than that of pZ189 harvested from repair-proficient human cells. The point-mutation frequency in the 150-base-pair tRNA marker gene increased up to 100-fold with ultraviolet dose. Sequence analysis of 150 mutant plasmids revealed that mutations were infrequent at potential thymine-thymine dimer sites. Ninety-three percent of the mutant plasmids from the xeroderma pigmentosum cells showed G X C----A X T transitions, compared to 73% in the normal cells (P less than 0.002). There were significantly fewer transversions (P less than 0.002) (especially G X C----T X A) and multiple base substitutions (P less than 0.00001) than when pZ189 was passaged in repair-proficient cells. The subset of mutational changes that are common to ultraviolet-treated plasmids propagated in both repair-proficient and xeroderma pigmentosum skin cells may be associated with the development of ultraviolet-induced skin cancer in humans

  20. Base substitutions, frameshifts, and small deletions constitute ionizing radiation-induced point mutations in mammalian cells

    International Nuclear Information System (INIS)

    Grosovsky, A.J.; de Boer, J.G.; de Jong, P.J.; Drobetsky, E.A.; Glickman, B.W.

    1988-01-01

    The relative role of point mutations and large genomic rearrangements in ionizing radiation-induced mutagenesis has been an issue of long-standing interest. Recent studies using Southern blotting analysis permit the partitioning of ionizing radiation-induced mutagenesis in mammalian cells into detectable deletions and major genomic rearrangements and into point mutations. The molecular nature of these point mutations has been left unresolved; they may include base substitutions as well as small deletions, insertions, and frame-shifts below the level of resolution of Southern blotting analysis. In this investigation, we have characterized a collection of ionizing radiation-induced point mutations at the endogenous adenine phosphoribosyltransferase (aprt) locus of Chinese hamster ovary cells at the DNA sequence level. Base substitutions represented approximately equal to 2/3 of the point mutations analyzed. Although the collection of mutants is relatively small, every possible type of base substitution event has been recovered. These mutations are well distributed throughout the coding sequence with only one multiple occurrence. Small deletions represented the remainder of characterized mutants; no insertions have been observed. Sequence-directed mechanisms mediated by direct repeats could account for some of the observed deletions, while others appear to be directly attributable to radiation-induced strand breakage

  1. On the dynamics of neutral mutations in a mathematical model for a homogeneous stem cell population.

    Science.gov (United States)

    Traulsen, Arne; Lenaerts, Tom; Pacheco, Jorge M; Dingli, David

    2013-02-01

    The theory of the clonal origin of cancer states that a tumour arises from one cell that acquires mutation(s) leading to the malignant phenotype. It is the current belief that many of these mutations give a fitness advantage to the mutant population allowing it to expand, eventually leading to disease. However, mutations that lead to such a clonal expansion need not give a fitness advantage and may in fact be neutral--or almost neutral--with respect to fitness. Such mutant clones can be eliminated or expand stochastically, leading to a malignant phenotype (disease). Mutations in haematopoietic stem cells give rise to diseases such as chronic myeloid leukaemia (CML) and paroxysmal nocturnal haemoglobinuria (PNH). Although neutral drift often leads to clonal extinction, disease is still possible, and in this case, it has important implications both for the incidence of disease and for therapy, as it may be more difficult to eliminate neutral mutations with therapy. We illustrate the consequences of such dynamics, using CML and PNH as examples. These considerations have implications for many other tumours as well.

  2. The value of KRAS mutation testing with CEA for the diagnosis of pancreatic mucinous cysts

    Science.gov (United States)

    Kadayifci, Abdurrahman; Al-Haddad, Mohammad; Atar, Mustafa; Dewitt, John M.; Forcione, David G.; Sherman, Stuart; Casey, Brenna W.; Fernandez-del Castillo, Carlos; Schmidt, C. Max; Pitman, Martha B.; Brugge, William R.

    2016-01-01

    Background and aims: Pancreatic cyst fluid (PCF) CEA has been shown to be the most accurate preoperative test for detection of cystic mucinous neoplasms (CMNs). This study aimed to assess the added value of PCF KRAS mutational analysis to CEA for diagnosis of CMNs. Patients and methods: This is a retrospective study of prospectively collected endoscopic ultrasonography (EUS) fine-needle aspiration (FNA) data. KRAS mutation was determined by direct sequencing or equivalent methods. Cysts were classified histologically (surgical cohort) or by clinical (EUS or FNA) findings (clinical cohort). Performance characteristics of KRAS, CEA and their combination for detection of a cystic mucinous neoplasm (CMN) and malignancy were calculated. Results: The study cohort consisted of 943 patients: 147 in the surgical cohort and 796 in the clinical cohort. Overall, KRAS and CEA each had high specificity (100 % and 93.2 %), but low sensitivity (48.3 % and 56.3 %) for the diagnosis of a CMN. The positivity of KRAS or CEA increased the diagnostic accuracy (80.8 %) and AUC (0.84) significantly compared to KRAS (65.3 % and 0.74) or CEA (65.8 % and 0.74) alone, but only in the clinical cohort (P < 0.0001 for both). KRAS mutation was significantly more frequent in malignant CMNs compared to histologically confirmed non-malignant CMNs (73 % vs. 37 %, P = 0.001). The negative predictive value of KRAS mutation was 77.6 % in differentiating non-malignant cysts. Conclusions: The detection of a KRAS mutation in PCF is a highly specific test for mucinous cysts. It outperforms CEA for sensitivity in mucinous cyst diagnosis, but the data does not support its routine use. PMID:27092317

  3. Impaired replication stress response in cells from immunodeficiency patients carrying Cernunnos/XLF mutations.

    Directory of Open Access Journals (Sweden)

    Michal Schwartz

    Full Text Available Non-Homologous End Joining (NHEJ is one of the two major pathways of DNA Double Strand Breaks (DSBs repair. Mutations in human NHEJ genes can lead to immunodeficiency due to its role in V(DJ recombination in the immune system. In addition, most patients carrying mutations in NHEJ genes display developmental anomalies which are likely the result of a general defect in repair of endogenously induced DSBs such as those arising during normal DNA replication. Cernunnos/XLF is a recently identified NHEJ gene which is mutated in immunodeficiency with microcephaly patients. Here we aimed to investigate whether Cernunnos/XLF mutations disrupt the ability of patient cells to respond to replication stress conditions. Our results demonstrate that Cernunnos/XLF mutated cells and cells downregulated for Cernunnos/XLF have increased sensitivity to conditions which perturb DNA replication. In addition, under replication stress, these cells exhibit impaired DSB repair and increased accumulation of cells in G2/M. Moreover Cernunnos/XLF mutated and down regulated cells display greater chromosomal instability, particularly at fragile sites, under replication stress conditions. These results provide evidence for the role of Cernunnos/XLF in repair of DSBs and maintenance of genomic stability under replication stress conditions. This is the first study of a NHEJ syndrome showing association with impaired cellular response to replication stress conditions. These findings may be related to the clinical features in these patients which are not due to the V(DJ recombination defect. Additionally, in light of the emerging important role of replication stress in the early stages of cancer development, our findings may provide a mechanism for the role of NHEJ in preventing tumorigenesis.

  4. Mutation analysis with random DNA identifiers (MARDI) catalogs Pig-a mutations in heterogeneous pools of CD48-deficient T cells derived from DMBA-treated rats.

    Science.gov (United States)

    Revollo, Javier R; Crabtree, Nathaniel M; Pearce, Mason G; Pacheco-Martinez, M Monserrat; Dobrovolsky, Vasily N

    2016-03-01

    Identification of mutations induced by xenotoxins is a common task in the field of genetic toxicology. Mutations are often detected by clonally expanding potential mutant cells and genotyping each viable clone by Sanger sequencing. Such a "clone-by-clone" approach requires significant time and effort, and sometimes is even impossible to implement. Alternative techniques for efficient mutation identification would greatly benefit both basic and regulatory genetic toxicology research. Here, we report the development of Mutation Analysis with Random DNA Identifiers (MARDI), a novel high-fidelity Next Generation Sequencing (NGS) approach that circumvents clonal expansion and directly catalogs mutations in pools of mutant cells. MARDI uses oligonucleotides carrying Random DNA Identifiers (RDIs) to tag progenitor DNA molecules before PCR amplification, enabling clustering of descendant DNA molecules and eliminating NGS- and PCR-induced sequencing artifacts. When applied to the Pig-a cDNA analysis of heterogeneous pools of CD48-deficient T cells derived from DMBA-treated rats, MARDI detected nearly all Pig-a mutations that were previously identified by conventional clone-by-clone analysis and discovered many additional ones consistent with DMBA exposure: mostly A to T transversions, with the mutated A located on the non-transcribed DNA strand. © 2015 Wiley Periodicals, Inc.

  5. Condensin II mutation causes T-cell lymphoma through tissue-specific genome instability

    Science.gov (United States)

    Woodward, Jessica; Taylor, Gillian C.; Soares, Dinesh C.; Boyle, Shelagh; Sie, Daoud; Read, David; Chathoth, Keerthi; Vukovic, Milica; Tarrats, Nuria; Jamieson, David; Campbell, Kirsteen J.; Blyth, Karen; Acosta, Juan Carlos; Ylstra, Bauke; Arends, Mark J.; Kranc, Kamil R.; Jackson, Andrew P.; Bickmore, Wendy A.

    2016-01-01

    Chromosomal instability is a hallmark of cancer, but mitotic regulators are rarely mutated in tumors. Mutations in the condensin complexes, which restructure chromosomes to facilitate segregation during mitosis, are significantly enriched in cancer genomes, but experimental evidence implicating condensin dysfunction in tumorigenesis is lacking. We report that mice inheriting missense mutations in a condensin II subunit (Caph2nes) develop T-cell lymphoma. Before tumors develop, we found that the same Caph2 mutation impairs ploidy maintenance to a different extent in different hematopoietic cell types, with ploidy most severely perturbed at the CD4+CD8+ T-cell stage from which tumors initiate. Premalignant CD4+CD8+ T cells show persistent catenations during chromosome segregation, triggering DNA damage in diploid daughter cells and elevated ploidy. Genome sequencing revealed that Caph2 single-mutant tumors are near diploid but carry deletions spanning tumor suppressor genes, whereas P53 inactivation allowed Caph2 mutant cells with whole-chromosome gains and structural rearrangements to form highly aggressive disease. Together, our data challenge the view that mitotic chromosome formation is an invariant process during development and provide evidence that defective mitotic chromosome structure can promote tumorigenesis. PMID:27737961

  6. Highly sensitive detection of ESR1 mutations in cell-free DNA from patients with metastatic breast cancer using molecular barcode sequencing.

    Science.gov (United States)

    Masunaga, Nanae; Kagara, Naofumi; Motooka, Daisuke; Nakamura, Shota; Miyake, Tomohiro; Tanei, Tomonori; Naoi, Yasuto; Shimoda, Masafumi; Shimazu, Kenzo; Kim, Seung Jin; Noguchi, Shinzaburo

    2018-01-01

    We aimed to develop a highly sensitive method to detect ESR1 mutations in cell-free DNA (cfDNA) using next-generation sequencing with molecular barcode (MB-NGS) targeting the hotspot segment (c.1600-1713). The sensitivity of MB-NGS was tested using serially diluted ESR1 mutant DNA and then cfDNA samples from 34 patients with metastatic breast cancer were analyzed with MB-NGS. The results of MB-NGS were validated in comparison with conventional NGS and droplet digital PCR (ddPCR). MB-NGS showed a higher sensitivity (0.1%) than NGS without barcode (1%) by reducing background errors. Of the cfDNA samples from 34 patients with metastatic breast cancer, NGS without barcode revealed seven mutations in six patients (17.6%) and MB-NGS revealed six additional mutations including three mutations not reported in the COSMIC database of breast cancer, resulting in total 13 ESR1 mutations in ten patients (29.4%). Regarding the three hotspot mutations, all the patients with mutations detected by MB-NGS had identical mutations detected by droplet digital PCR (ddPCR), and mutant allele frequency correlated very well between both (r = 0.850, p < 0.01). Moreover, all the patients without these mutations by MB-NGS were found to have no mutations by ddPCR. In conclusion, MB-NGS could successfully detect ESR1 mutations in cfDNA with a higher sensitivity of 0.1% than conventional NGS and was considered as clinically useful as ddPCR.

  7. Mutation of the planar cell polarity gene VANGL1 in adolescent idiopathic scoliosis

    DEFF Research Database (Denmark)

    Andersen, Malene Rask; Farooq, Muhammad; Koefoed, Karen

    2017-01-01

    STUDY DESIGN: Mutation analysis of a candidate disease gene in a cohort of patients with moderate to severe Adolescent idiopathic scoliosis (AIS). OBJECTIVE: To investigate if damaging mutations in the planar cell polarity gene VANGL1 could be identified in AIS patients. SUMMARY OF BACKGROUND DATA......: AIS is a spinal deformity which occurs in 1-3% of the population. The cause of AIS is often unknown, but genetic factors are important in the etiology. Rare variants in genes encoding regulators of WNT/planar cell polarity (PCP) signaling were recently identified in AIS patients. METHODS: We analyzed...

  8. Allogeneic stem cell transplantation for patients harboring T315I BCR-ABL mutated leukemias

    DEFF Research Database (Denmark)

    Nicolini, Franck Emmanuel; Basak, Grzegorz W; Soverini, Simona

    2011-01-01

    T315I(+) Philadelphia chromosome-positive leukemias are inherently resistant to all licensed tyrosine kinase inhibitors, and therapeutic options remain limited. We report the outcome of allogeneic stem cell transplantation in 64 patients with documented BCR-ABL(T315I) mutations. Median follow......) as unfavorable factors. We conclude that allogeneic stem cell transplantation represents a valuable therapeutic tool for eligible patients with BCR-ABL(T315I) mutation, a tool that may or may not be replaced by third-generation tyrosine kinase inhibitors....

  9. Analysis of the BRAFV600E mutation in 19 cases of Langerhans cell histiocytosis in Japan.

    Science.gov (United States)

    Sasaki, Yuya; Guo, Ying; Arakawa, Fumiko; Miyoshi, Hiroaki; Yoshida, Noriaki; Koga, Yuhki; Nakashima, Kazutaka; Kurita, Daisuke; Niino, Daisuke; Seto, Masao; Ohshima, Koichi

    2017-09-01

    Langerhans cell histiocytosis (LCH) is a rare disease characterized by clonal proliferation of CD1a- and CD207 (langerin)-positive dendritic cells. Mutated BRAF (p.V600E) is observed in histiocyte-related diseases and dendritic cell-related diseases, including LCH. BRAFV600E is observed in some LCH cases and is thought to be involved in maintaining MAPK activation. We retrospectively analyzed BRAFV600E in 19 patients diagnosed with LCH. In our study, direct sequencing for exon 15, a mutation hotspot, demonstrated that 4 out of the 19 patients (21%) harbored a GTG > GAG (valine > glutamic acid) base substitution, which encodes BRAFV600E. The clinical impact of BRAFV600E in such diseases is unclear. The frequency of BRAFV600E in our LCH patients from Japan was lower than that reported in the United States and in Germany. However, reports from Asia tend to show a lower rate of the BRAFV600E mutation. These results imply the possibility of different genetic backgrounds in the pathogenesis of LCH across various ethnicities. We also performed an immunohistochemical analysis to detect BRAFV600E using the mutation-specific monoclonal antibody. However, immunohistochemical analysis failed to detect any mutated protein in any of the 4 BRAFV600E-positive cases. This implies that at present, BRAFV600E should be assessed by direct sequencing. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  10. P53 Gene Mutation as Biomarker of Radiation Induced Cell Injury and Genomic Instability

    International Nuclear Information System (INIS)

    Mukh-Syaifudin

    2006-01-01

    Gene expression profiling and its mutation has become one of the most widely used approaches to identify genes and their functions in the context of identify and categorize genes to be used as radiation effect markers including cell and tissue sensitivities. Ionizing radiation produces genetic damage and changes in gene expression that may lead to cancer due to specific protein that controlling cell proliferation altered the function, its expression or both. P53 protein encoded by p53 gene plays an important role in protecting cell by inducing growth arrest and or cell suicide (apoptosis) after deoxyribonucleic acid (DNA) damage induced by mutagen such as ionizing radiation. The mutant and thereby dysfunctional of this gene was found in more than 50% of various human cancers, but it is as yet unclear how p53 mutations lead to neoplastic development. Wild-type p53 has been postulated to play a role in DNA repair, suggesting that expression of mutant forms of p53 might alter cellular resistance to the DNA damage caused by radiation. Moreover, p53 is thought to function as a cell cycle checkpoint after irradiation, also suggesting that mutant p53 might change the cellular proliferative response to radiation. P53 mutations affect the cellular response to DNA damage, either by increasing DNA repair processes or, possibly, by increasing cellular tolerance to DNA damage. The association of p53 mutations with increased radioresistance suggests that alterations in the p53 gene might lead to oncogenic transformation. Current attractive model of carcinogenesis also showed that p53 gene is the major target of radiation. The majority of p53 mutations found so far is single base pair changes ( point mutations), which result in amino acid substitutions or truncated forms of the p53 protein, and are widely distributed throughout the evolutionary conserved regions of the gene. Examination of p53 mutations in human cancer also shows an association between particular carcinogens and

  11. Exact, time-independent estimation of clone size distributions in normal and mutated cells.

    Science.gov (United States)

    Roshan, A; Jones, P H; Greenman, C D

    2014-10-06

    Biological tools such as genetic lineage tracing, three-dimensional confocal microscopy and next-generation DNA sequencing are providing new ways to quantify the distribution of clones of normal and mutated cells. Understanding population-wide clone size distributions in vivo is complicated by multiple cell types within observed tissues, and overlapping birth and death processes. This has led to the increased need for mathematically informed models to understand their biological significance. Standard approaches usually require knowledge of clonal age. We show that modelling on clone size independent of time is an alternative method that offers certain analytical advantages; it can help parametrize these models, and obtain distributions for counts of mutated or proliferating cells, for example. When applied to a general birth-death process common in epithelial progenitors, this takes the form of a gambler's ruin problem, the solution of which relates to counting Motzkin lattice paths. Applying this approach to mutational processes, alternative, exact, formulations of classic Luria-Delbrück-type problems emerge. This approach can be extended beyond neutral models of mutant clonal evolution. Applications of these approaches are twofold. First, we resolve the probability of progenitor cells generating proliferating or differentiating progeny in clonal lineage tracing experiments in vivo or cell culture assays where clone age is not known. Second, we model mutation frequency distributions that deep sequencing of subclonal samples produce.

  12. EZH2 and CD79B mutational status over time in B-cell non-Hodgkin lymphomas detected by high-throughput sequencing using minimal samples

    Science.gov (United States)

    Saieg, Mauro Ajaj; Geddie, William R; Boerner, Scott L; Bailey, Denis; Crump, Michael; da Cunha Santos, Gilda

    2013-01-01

    BACKGROUND: Numerous genomic abnormalities in B-cell non-Hodgkin lymphomas (NHLs) have been revealed by novel high-throughput technologies, including recurrent mutations in EZH2 (enhancer of zeste homolog 2) and CD79B (B cell antigen receptor complex-associated protein beta chain) genes. This study sought to determine the evolution of the mutational status of EZH2 and CD79B over time in different samples from the same patient in a cohort of B-cell NHLs, through use of a customized multiplex mutation assay. METHODS: DNA that was extracted from cytological material stored on FTA cards as well as from additional specimens, including archived frozen and formalin-fixed histological specimens, archived stained smears, and cytospin preparations, were submitted to a multiplex mutation assay specifically designed for the detection of point mutations involving EZH2 and CD79B, using MassARRAY spectrometry followed by Sanger sequencing. RESULTS: All 121 samples from 80 B-cell NHL cases were successfully analyzed. Mutations in EZH2 (Y646) and CD79B (Y196) were detected in 13.2% and 8% of the samples, respectively, almost exclusively in follicular lymphomas and diffuse large B-cell lymphomas. In one-third of the positive cases, a wild type was detected in a different sample from the same patient during follow-up. CONCLUSIONS: Testing multiple minimal tissue samples using a high-throughput multiplex platform exponentially increases tissue availability for molecular analysis and might facilitate future studies of tumor progression and the related molecular events. Mutational status of EZH2 and CD79B may vary in B-cell NHL samples over time and support the concept that individualized therapy should be based on molecular findings at the time of treatment, rather than on results obtained from previous specimens. Cancer (Cancer Cytopathol) 2013;121:377–386. © 2013 American Cancer Society. PMID:23361872

  13. Highly sensitive KRAS mutation detection from formalin-fixed paraffin-embedded biopsies and circulating tumour cells using wild-type blocking polymerase chain reaction and Sanger sequencing.

    Science.gov (United States)

    Huang, Meggie Mo Chao; Leong, Sai Mun; Chua, Hui Wen; Tucker, Steven; Cheong, Wai Chye; Chiu, Lily; Li, Mo-Huang; Koay, Evelyn Siew-Chuan

    2014-08-01

    Among patients with colorectal cancer (CRC), KRAS mutations were reported to occur in 30-51 % of all cases. CRC patients with KRAS mutations were reported to be non-responsive to anti-epidermal growth factor receptor (EGFR) monoclonal antibody (MoAb) treatment in many clinical trials. Hence, accurate detection of KRAS mutations would be critical in guiding the use of anti-EGFR MoAb therapies in CRC. In this study, we carried out a detailed investigation of the efficacy of a wild-type (WT) blocking real-time polymerase chain reaction (PCR), employing WT KRAS locked nucleic acid blockers, and Sanger sequencing, for KRAS mutation detection in rare cells. Analyses were first conducted on cell lines to optimize the assay protocol which was subsequently applied to peripheral blood and tissue samples from patients with CRC. The optimized assay provided a superior sensitivity enabling detection of as little as two cells with mutated KRAS in the background of 10(4) WT cells (0.02 %). The feasibility of this assay was further investigated to assess the KRAS status of 45 colorectal tissue samples, which had been tested previously, using a conventional PCR sequencing approach. The analysis showed a mutational discordance between these two methods in 4 of 18 WT cases. Our results present a simple, effective, and robust method for KRAS mutation detection in both paraffin embedded tissues and circulating tumour cells, at single-cell level. The method greatly enhances the detection sensitivity and alleviates the need of exhaustively removing co-enriched contaminating lymphocytes.

  14. The prognostic value of KRAS mutated plasma DNA in advanced non-small cell lung cancer

    DEFF Research Database (Denmark)

    Nygaard, Anneli Dowler; Garm Spindler, Karen-Lise; Pallisgaard, Niels

    2013-01-01

    BACKGROUND: Lung cancer is one of the most common malignant diseases worldwide and associated with considerable morbidity and mortality. New agents targeting the epidermal growth factor system are emerging, but only a subgroup of the patients will benefit from the therapy. Cell free DNA (cf......DNA) in the blood allows for tumour specific analyses, including KRAS-mutations, and the aim of the study was to investigate the possible prognostic value of plasma mutated KRAS (pmKRAS) in patients with non-small cell lung cancer (NSCLC). MATERIAL AND METHODS: Patients with newly diagnosed, advanced NSCLC eligible....... RESULTS: The study included 246 patients receiving a minimum of 1 treatment cycle, and all but four were evaluable for response according to RECIST. Forty-three patients (17.5%) presented with a KRAS mutation. OS was 8.9 months and PFS by intention to treat 5.4 months. Patients with a detectable plasma...

  15. Flow-cytometric measurements of somatic cell mutations in Thorotrast patients

    International Nuclear Information System (INIS)

    Umeki, Shigeko; Kyoizumi, Seishi; Kusunoki, Yoichiro; Nakamura, Nori; Sasaki, Masao; Mori, Takesaburo; Ishikawa, Yuichi; Cologne, J.B.; Akiyama, Mitoshi.

    1992-10-01

    Exposure to ionizing radiation is a well-recognized risk factor for cancer development. Because ionizing radiation can induce mutations, an accurate way of measuring somatic mutation frequencies could be a useful tool for evaluating cancer risk. In the present study, we have examined in vivo somatic mutation frequencies at the erythrocyte glycophorin A and T-cell receptor loci in 18 Thorotrast patients. These persons have been continuously irradiated with alpha particles emitted from the internal deposition of thorium dioxide and thus have increased risks of certain malignant tumors. When compared with controls, the Thorotrast patients showed a significantly higher frequency of mutants at the lymphocyte T-cell receptor loci but not at the erythrocyte glycophorin A loci. (author)

  16. An activating mutation of interferon regulatory factor 4 (IRF4) in adult T cell leukemia.

    Science.gov (United States)

    Cherian, Mathew A; Olson, Sydney; Sundaramoorthi, Hemalatha; Cates, Kitra; Cheng, Xiaogang; Harding, John; Martens, Andrew; Challen, Grant A; Tyagi, Manoj; Ratner, Lee; Rauch, Daniel

    2018-03-14

    The human T cell leukemia virus-1 (HTLV-1) oncoprotein Tax drives cell proliferation and resistance to apoptosis early in the pathogenesis of adult T-cell leukemia (ATL). Subsequently, likely as a result of specific immuno-editing, Tax expression is downregulated and functionally replaced by somatic driver mutations of the host genome. Both amplification and point mutations of interferon regulatory factor 4 (IRF4) have been previously detected in ATL, and the K59R mutation is the most common single-nucleotide variation in IRF4 and is found exclusively in ATL. Here high throughput whole-exome sequencing revealed recurrent activating genetic alterations in the T cell receptor, CD28, and NF-kB pathways. Moreover, we found that IRF4, which is transcriptionally activated downstream of these pathways, is frequently mutated in ATL. IRF4 RNA, protein, and IRF4 transcriptional targets are uniformly elevated in HTLV transformed cells and ATL cell lines, and IRF4 was bound to genomic regulatory DNA of many of these transcriptional targets in HTLV-1 transformed cell lines. We further noted that the K59R IRF4 mutant is expressed at higher levels in the nucleus than is wild-type IRF4, and is transcriptionally more active. Expression of both wild-type and the K59R mutant of IRF4 from a constitutive promoter in retrovirally transduced murine bone marrow cells increased the abundance of T lymphocytes but not myeloid cells or B lymphocytes in mice. IRF4 may represent a therapeutic target in ATL since ATL cells select for a mutant of IRF4 with higher nuclear expression and transcriptional activity, and over-expression of IRF4 induces the expansion of T lymphocytes in vivo. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Mutational analysis of circulating tumor cells from colorectal cancer patients and correlation with primary tumor tissue.

    Directory of Open Access Journals (Sweden)

    Anna Lyberopoulou

    Full Text Available Circulating tumor cells (CTCs provide a non-invasive accessible source of tumor material from patients with cancer. The cellular heterogeneity within CTC populations is of great clinical importance regarding the increasing number of adjuvant treatment options for patients with metastatic carcinomas, in order to eliminate residual disease. Moreover, the molecular profiling of these rare cells might lead to insight on disease progression and therapeutic strategies than simple CTCs counting. In the present study we investigated the feasibility to detect KRAS, BRAF, CD133 and Plastin3 (PLS3 mutations in an enriched CTCs cell suspension from patients with colorectal cancer, with the hypothesis that these genes` mutations are of great importance regarding the generation of CTCs subpopulations. Subsequently, we compared CTCs mutational status with that of the corresponding primary tumor, in order to access the possibility of tumor cells characterization without biopsy. CTCs were detected and isolated from blood drawn from 52 colorectal cancer (CRC patients using a quantum-dot-labelled magnetic immunoassay method. Mutations were detected by PCR-RFLP or allele-specific PCR and confirmed by direct sequencing. In 52 patients, discordance between primary tumor and CTCs was 5.77% for KRAS, 3.85% for BRAF, 11.54% for CD133 rs3130, 7.69% for CD133 rs2286455 and 11.54% for PLS3 rs6643869 mutations. Our results support that DNA mutational analysis of CTCs may enable non-invasive, specific biomarker diagnostics and expand the scope of personalized medicine for cancer patients.

  18. Genome-scale mutational signatures of aflatoxin in cells, mice, and human tumors

    Science.gov (United States)

    Huang, Mi Ni; Yu, Willie; Teoh, Wei Wei; Ardin, Maude; Jusakul, Apinya; Ng, Alvin Wei Tian; Boot, Arnoud; Abedi-Ardekani, Behnoush; Villar, Stephanie; Myint, Swe Swe; Othman, Rashidah; Poon, Song Ling; Heguy, Adriana; Olivier, Magali; Hollstein, Monica; Tan, Patrick; Teh, Bin Tean; Sabapathy, Kanaga; Zavadil, Jiri; Rozen, Steven G.

    2017-01-01

    Aflatoxin B1 (AFB1) is a mutagen and IARC (International Agency for Research on Cancer) Group 1 carcinogen that causes hepatocellular carcinoma (HCC). Here, we present the first whole-genome data on the mutational signatures of AFB1 exposure from a total of >40,000 mutations in four experimental systems: two different human cell lines, in liver tumors in wild-type mice, and in mice that carried a hepatitis B surface antigen transgene—this to model the multiplicative effects of aflatoxin exposure and hepatitis B in causing HCC. AFB1 mutational signatures from all four experimental systems were remarkably similar. We integrated the experimental mutational signatures with data from newly sequenced HCCs from Qidong County, China, a region of well-studied aflatoxin exposure. This indicated that COSMIC mutational signature 24, previously hypothesized to stem from aflatoxin exposure, indeed likely represents AFB1 exposure, possibly combined with other exposures. Among published somatic mutation data, we found evidence of AFB1 exposure in 0.7% of HCCs treated in North America, 1% of HCCs from Japan, but 16% of HCCs from Hong Kong. Thus, aflatoxin exposure apparently remains a substantial public health issue in some areas. This aspect of our study exemplifies the promise of future widespread resequencing of tumor genomes in providing new insights into the contribution of mutagenic exposures to cancer incidence. PMID:28739859

  19. Stability Test For Sorghum Mutant Lines Derived From Induced Mutations with Gamma-Ray Irradiation

    Directory of Open Access Journals (Sweden)

    S. Human

    2011-12-01

    Full Text Available Sorghum breeding program had been conducted at the Center for the Application of Isotopes and Radiation Technology, BATAN. Plant genetic variability was increased through induced mutations using gamma-ray irradiation. Through selection process in successive generations, some promising mutant lines had been identified to have good agronomic characteristics with high grain yield. These breeding lines were tested in multi location trials and information of the genotypic stability was obtained to meet the requirements for officially varietal release by the Ministry of Agriculture. A total of 11 sorghum lines and varieties consisting of 8 mutant lines derived from induced mutations (B-100, B-95, B-92, B-83, B-76, B-75, B-69 and Zh-30 and 3 control varieties (Durra, UPCA-S1 and Mandau were included in the experiment. All materials were grown in 10 agro-ecologically different locations namely Gunungkidul, Bantul, Citayam, Garut, Lampung, Bogor, Anyer, Karawaci, Cianjur and Subang. In each location, the local adaptability test was conducted by randomized block design with 3 replications. Data of grain yield was used for evaluating genotypic stability using AMMI approach. Results revealed that sorghum mutation breeding had generated 3 mutant lines (B-100, B-76 and Zh-30 exhibiting grain yield significantly higher than the control varieties. These mutant lines were genetically stable in all locations so that they would be recommended for official release as new sorghum varieties to the Ministry of Agriculture

  20. Analysis of genotoxic activity of ketamine and rocuronium bromide using the somatic mutation and recombination test in Drosophila melanogaster.

    Science.gov (United States)

    Koksal, Pakize Muge; Gürbüzel, Mehmet

    2015-03-01

    The present study evaluated the mutagenic and recombinogenic effects of two commonly used anesthetic agents, ketamine and rocuronium bromide, in medicine using the wing somatic mutation and recombination test (SMART) in Drosophila. The standard (ST) cross and the high-bioactivation (HB) cross with high sensitivity to procarcinogens and promutagens were used. The SMART test is based on the loss of heterozygosity, which occurs via various mechanisms, such as chromosome loss and deletion, half-translocation, mitotic recombination, mutation, and non-disjunction. Genetic alterations occurring in the somatic cells of the wing's imaginal discs result in mutant clones in the wing blade. Three-day-old trans-heterozygous larvae with two recessive markers, multiple wing hairs (mwh) and flare (flr(3)), were treated with ketamine and rocuronium bromide. Analysis of the ST cross indicated that ketamine exhibited genotoxicity activity and that this activity was particularly dependent on homologous mitotic recombination at concentrations of 250 μg/ml and above. Rocuronium bromide did not exert mutagenic and/or recombinogenic effects. In the HB cross, ketamine at a concentration of 1000 μg/ml and rocuronium bromide at all concentrations, with the exception of 250 μg/ml (inconclusive), exerted genotoxic effects, which could also be associated with the increase in mitotic recombination. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Interleukin-7 receptor-α gene mutations are not detected in adult T-cell acute lymphoblastic leukemia

    International Nuclear Information System (INIS)

    Rozovski, Uri; Li, Ping; Harris, David; Ohanian, Maro; Kantarjian, Hagop; Estrov, Zeev

    2014-01-01

    Somatic mutations in cancer cell genes are classified according to their functional significance. Those that provide the malignant cells with significant advantage are collectively referred to as driver mutations and those that do not, are the passenger mutations. Accordingly, analytical criteria to distinguish driver mutations from passenger mutations have been recently suggested. Recent studies revealed mutations in interleukin-7 receptor-α (IL7R) gene in 10% of pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients and in only a few cases of pediatric B-ALL. IL7R mutations are also frequently found in patients with lung cancer, but whereas in pediatric T-ALL IL7R mutations are “drivers” (consisting of gain-of-function mutations within a narrow 50-base pair interval at exon 6 that confer cytokine-independent cell growth and promote tumor transformation), in lung cancer, mutations are substitution mutations randomly distributed across the gene and are probably only “passenger” events. Because the treatment response of adult T-ALL is significantly poorer than that of childhood T-ALL and because exon 6 IL7R mutations play a role in the pathogenesis of childhood T-ALL, we sought to determine how the pattern of IL7R mutations varies between adult and childhood T-ALL. To that end, we sequenced the 50-base pair interval in exon 6 of the IL7R of DNA obtained from bone marrow samples of 35 randomly selected adult patients with T-ALL. Our analysis revealed that none of these 35 samples carried an IL7R mutation in exon 6. Whether differences in the genetic makeup of adult and childhood T-ALL explain the differential response to therapy remains to be determined

  2. Somatic mutation and cell differentiation in neoplastic transformation

    International Nuclear Information System (INIS)

    Huberman, E.; Collart, F.R.

    1987-01-01

    In brief, the authors suggest that tumor formation may result from continuous expression of growth facilitating genes that, as a result of irreversible changes during the initiation step, are placed under the control of genes expressed during normal differentiation. Thus, to understand carcinogenesis, we must decipher the processes that lead to the acquisition of a mature phenotype in both normal and tumor cells and characterize the growth dependency of tumor cells to inducers of cell differentiation. Furthermore, the growth of a variety of tumors may be controlled through the use of inducers of maturation that activate genes located beyond the gene that is altered during tumor initiation. 22 refs., 3 figs

  3. Extremely high Tp53 mutation load in esophageal squamous cell carcinoma in Golestan Province, Iran.

    Directory of Open Access Journals (Sweden)

    Behnoush Abedi-Ardekani

    Full Text Available BACKGROUND: Golestan Province in northeastern Iran has one of the highest incidences of esophageal squamous cell carcinoma (ESCC in the world with rates over 50 per 100,000 person-years in both sexes. We have analyzed TP53 mutation patterns in tumors from this high-risk geographic area in search of clues to the mutagenic processes involved in causing ESCC. METHODOLOGY/PRINCIPAL FINDINGS: Biopsies of 119 confirmed ESCC tumor tissue from subjects enrolled in a case-control study conducted in Golestan Province were analyzed by direct sequencing of TP53 exons 2 through 11. Immunohistochemical staining for p53 was carried out using two monoclonal antibodies, DO7 and 1801. A total of 120 TP53 mutations were detected in 107/119 cases (89.9%, including 11 patients with double or triple mutations. The mutation pattern was heterogeneous with infrequent mutations at common TP53 "hotspots" but frequent transversions potentially attributable to environmental carcinogens forming bulky DNA adducts, including 40% at bases known as site of mutagenesis by polycyclic aromatic hydrocarbons (PAHs. Mutations showed different patterns according to the reported temperature of tea consumption, but no variation was observed in relation to ethnicity, tobacco or opium use, and alcoholic beverage consumption or urban versus rural residence. CONCLUSION/SIGNIFICANCE: ESCC tumors in people from Golestan Province show the highest rate of TP53 mutations ever reported in any cancer anywhere. The heterogeneous mutation pattern is highly suggestive of a causative role for multiple environmental carcinogens, including PAHs. The temperature and composition of tea may also influence mutagenesis.

  4. Mutated N-ras does not induce p19 arf in CO25 cell line | Saleh ...

    African Journals Online (AJOL)

    The mouse cell line (CO25) used in this study was transfected with a glucocorticoid inducible mutated human N-ras oncogene under transcriptional control of the steroid-sensitive promoter of the mouse mammary tumors virus long terminal repeat MMTV-LTR. This study was aimed to investigate the expression of p19arf and ...

  5. BRD4-targeted therapy induces Myc-independent cytotoxicity in Gnaq/11-mutatant uveal melanoma cells.

    Science.gov (United States)

    Ambrosini, Grazia; Sawle, Ashley D; Musi, Elgilda; Schwartz, Gary K

    2015-10-20

    Uveal melanoma (UM) is an aggressive intraocular malignancy with limited therapeutic options. Both primary and metastatic UM are characterized by oncogenic mutations in the G-protein alpha subunit q and 11. Furthermore, nearly 40% of UM has amplification of the chromosomal arm 8q and monosomy of chromosome 3, with consequent anomalies of MYC copy number. Chromatin regulators have become attractive targets for cancer therapy. In particular, the bromodomain and extra-terminal (BET) inhibitor JQ1 has shown selective inhibition of c-Myc expression with antiproliferative activity in hematopoietic and solid tumors. Here we provide evidence that JQ1 had cytotoxic activity in UM cell lines carrying Gnaq/11 mutations, while in cells without the mutations had little effects. Using microarray analysis, we identified a large subset of genes modulated by JQ1 involved in the regulation of cell cycle, apoptosis and DNA repair. Further analysis of selected genes determined that the concomitant silencing of Bcl-xL and Rad51 represented the minimal requirement to mimic the apoptotic effects of JQ1 in the mutant cells, independently of c-Myc. In addition, administration of JQ1 to mouse xenograft models of Gnaq-mutant UM resulted in significant inhibition of tumor growth.Collectively, our results define BRD4 targeting as a novel therapeutic intervention against UM with Gnaq/Gna11 mutations.

  6. Heterozygote FANCD2 mutations associated with childhood T Cell ALL and testicular seminoma.

    Science.gov (United States)

    Smetsers, Stephanie; Muter, Joanne; Bristow, Claire; Patel, Leena; Chandler, Kate; Bonney, Denise; Wynn, Robert F; Whetton, Anthony D; Will, Andrew M; Rockx, Davy; Joenje, Hans; Strathdee, Gordon; Shanks, Jonathan; Klopocki, Eva; Gille, Johan J P; Dorsman, Josephine; Meyer, Stefan

    2012-12-01

    Fanconi anaemia (FA) is an inherited disease with congenital and developmental abnormalities characterised by cellular cross linker hypersensitivity. FA is caused by mutations in any of so far 15 identified FANC genes, which encode proteins that interact in a common DNA damage response (DDR) pathway. Individuals with FA have a high risk of developing acute myeloid leukaemia (AML) and squamous cell carcinoma. An increased cancer risk has been firmly established for carriers of mutations in FANCD1/BRCA2, FANCJ/BRIP1, FANCN/PALB2, RAD51C/FANCO and link the FA pathway to inherited breast and ovarian cancer. We describe a pedigree with FANCD2 mutations c.458T > C (p.Leu153Ser) and c.2715 + 1G > A (p.Glu906LeufsX4) with mild phenotype FA in the index case, T cell ALL in the Leu153Ser heterozygous brother and testicular seminoma in the p.Glu906LeufsX4 heterozygous father. Both FANCD2 alleles were present in the T Cell ALL and the seminoma. This links specific FANCD2 mutations to T cell ALL and seminoma without evidence of allelic loss in the tumour tissue.

  7. Analysis of P53 mutations and their expression in 56 colorectal cancer cell lines

    DEFF Research Database (Denmark)

    Liu, Ying; Bodmer, Walter F

    2006-01-01

    A comprehensive analysis of the TP53 gene and its protein status was carried out on a panel of 56 colorectal cancer cell lines. This analysis was based on a combination of denaturing HPLC mutation screening of all exons of the p53 gene, sequencing the cDNA, and assessing the function of the p53 p...

  8. A recurrent germline PAX5 mutation confers susceptibility to pre-B cell acute lymphoblastic leukemia

    NARCIS (Netherlands)

    Shah, S.; Schrader, K.A.; Waanders, E.; Timms, A.E.; Vijai, J.; Miething, C.; Wechsler, J.; Yang, J.; Hayes, J.; Klein, R.J.; Zhang, J.; Wei, L.; Wu, G.; Rusch, M.; Nagahawatte, P.; Ma, J; Chen, S.C.; Song, G.; Cheng, J.; Meyers, P.; Bhojwani, D.; Jhanwar, S.; Maslak, P.; Fleisher, M.; Littman, J.; Offit, L.; Rau-Murthy, R.; Fleischut, M.H.; Corines, M.; Murali, R.; Gao, X.; Manschreck, C.; Kitzing, T.; Murty, V.V.; Raimondi, S.C.; Kuiper, R.P.; Simons, A.; Schiffman, J.D.; Onel, K.; Plon, S.E.; Wheeler, D.A.; Ritter, D.; Ziegler, D.S.; Tucker, K.; Sutton, R.; Chenevix-Trench, G.; Li, J.; Huntsman, D.G.; Hansford, S.; Senz, J.; Walsh, T.; Lee (Helen Dowling Instituut), M. van der; Hahn, C.N.; Roberts, K.G.; King, M.C.; Lo, S.M.; Levine, R.L.; Viale, A.; Socci, N.D.; Nathanson, K.L.; Scott, H.S.; Daly, M.; Lipkin, S.M.; Lowe, S.W.; Downing, J.R.; Altshuler, D.; Sandlund, J.T.; Horwitz, M.S.; Mullighan, C.G.; Offit, K.

    2013-01-01

    Somatic alterations of the lymphoid transcription factor gene PAX5 (also known as BSAP) are a hallmark of B cell precursor acute lymphoblastic leukemia (B-ALL), but inherited mutations of PAX5 have not previously been described. Here we report a new heterozygous germline variant, c.547G>A

  9. MAP2K1 and MAP3K1 mutations in langerhans cell histiocytosis

    NARCIS (Netherlands)

    Nelson, David S.; van Halteren, Astrid; Quispel, Willemijn T.; van den Bos, Cor; Bovée, Judith V. M. G.; Patel, Bhumi; Badalian-Very, Gayane; van Hummelen, Paul; Ducar, Matthew; Lin, Ling; MacConaill, Laura E.; Egeler, R. Maarten; Rollins, Barrett J.

    2015-01-01

    Langerhans cell histiocytosis (LCH) is now understood to be a neoplastic disease in which over 50% of cases have somatic activating mutations of BRAF. However, the extracellular signal-related (ERK) pathway is activated in all cases including those with wild type BRAF alleles. Here, we applied a

  10. Cellular and molecular effects for mutation induction in normal human cells irradiated with accelerated neon ions

    International Nuclear Information System (INIS)

    Suzuki, Masao; Tsuruoka, Chizuru; Kanai, Tatsuaki; Kato, Takeshi; Yatagai, Fumio; Watanabe, Masami

    2006-01-01

    We investigated the linear energy transfer (LET) dependence of mutation induction on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in normal human fibroblast-like cells irradiated with accelerated neon-ion beams. The cells were irradiated with neon-ion beams at various LETs ranging from 63 to 335 keV/μm. Neon-ion beams were accelerated by the Riken Ring Cyclotron at the Institute of Physical and Chemical Research in Japan. Mutation induction at the HPRT locus was detected to measure 6-thioguanine-resistant clones. The mutation spectrum of the deletion pattern of exons of mutants was analyzed using the multiplex polymerase chain reaction (PCR). The dose-response curves increased steeply up to 0.5 Gy and leveled off or decreased between 0.5 and 1.0 Gy, compared to the response to 137 Cs γ-rays. The mutation frequency increased up to 105 keV/μm and then there was a downward trend with increasing LET values. The deletion pattern of exons was non-specific. About 75-100% of the mutants produced using LETs ranging from 63 to 335 keV/μm showed all or partial deletions of exons, while among γ-ray-induced mutants 30% showed no deletions, 30% partial deletions and 40% complete deletions. These results suggested that the dose-response curves of neon-ion-induced mutations were dependent upon LET values, but the deletion pattern of DNA was not

  11. Kolmogorov-Smirnov statistical test for analysis of ZAP-70 expression in B-CLL, compared with quantitative PCR and IgV(H) mutation status.

    Science.gov (United States)

    Van Bockstaele, Femke; Janssens, Ann; Piette, Anne; Callewaert, Filip; Pede, Valerie; Offner, Fritz; Verhasselt, Bruno; Philippé, Jan

    2006-07-15

    ZAP-70 has been proposed as a surrogate marker for immunoglobulin heavy-chain variable region (IgV(H)) mutation status, which is known as a prognostic marker in B-cell chronic lymphocytic leukemia (CLL). The flow cytometric analysis of ZAP-70 suffers from difficulties in standardization and interpretation. We applied the Kolmogorov-Smirnov (KS) statistical test to make analysis more straightforward. We examined ZAP-70 expression by flow cytometry in 53 patients with CLL. Analysis was performed as initially described by Crespo et al. (New England J Med 2003; 348:1764-1775) and alternatively by application of the KS statistical test comparing T cells with B cells. Receiver-operating-characteristics (ROC)-curve analyses were performed to determine the optimal cut-off values for ZAP-70 measured by the two approaches. ZAP-70 protein expression was compared with ZAP-70 mRNA expression measured by a quantitative PCR (qPCR) and with the IgV(H) mutation status. Both flow cytometric analyses correlated well with the molecular technique and proved to be of equal value in predicting the IgV(H) mutation status. Applying the KS test is reproducible, simple, straightforward, and overcomes a number of difficulties encountered in the Crespo-method. The KS statistical test is an essential part of the software delivered with modern routine analytical flow cytometers and is well suited for analysis of ZAP-70 expression in CLL. (c) 2006 International Society for Analytical Cytology.

  12. Tyrosine kinase domain mutations of EGFR gene in head and neck squamous cell carcinoma

    Directory of Open Access Journals (Sweden)

    Vatte C

    2017-03-01

    Full Text Available Chittibabu Vatte,1 Ali M Al Amri,2 Cyril Cyrus,1 Shahanas Chathoth,1 Sadananda Acharya,3 Tariq Mohammad Hashim,4 Zhara Al Ali,2 Saleh Tawfeeq Alshreadah,2 Ahmed Alsayyah,4 Amein K Al-Ali5 1Department of Genetic Research, Institute for Research and Medical Consultation, University of Dammam, Dammam, 2Department of Internal Medicine, King Fahd Hospital of the University, University of Dammam, Al-Khobar, 3Department of Stemcell Research, Institute for Research and Medical Consultation, 4Department of Pathology, King Fahd Hospital of the University, University of Dammam, Al-Khobar, 5Department of Biochemistry, College of Medicine, University of Dammam, Dammam, Kingdom of Saudi Arabia Background: Epidermal growth factor receptor (EGFR is a commonly altered gene that is identified in various cancers, including head and neck squamous cell carcinoma (HNSCC. Therefore, EGFR is a promising molecular marker targeted by monoclonal antibodies and small molecule inhibitors targeting the tyrosine kinase (TK domain. Objective: The objective of this study was to investigate the spectrum of mutations in exons 18, 19, 20, and 21 of the EGFR gene in HNSCC patients. Materials and methods: This retrospective study included 47 confirmed HNSCC cases. Mutations in the TK domain, exons 18, 19, 20, and 21 of the EGFR gene, were detected by Scorpion® chemistry and ARMS® technologies on Rotor-Gene Q real-time polymerase chain reaction.Results: The tumors exhibited EGFR-TK domain mutations in 57% of cases. Four cases of T790M mutations were reported for the first time among HNSCC patients. Out of the total mutations, L861Q (exon 21, exon 20 insertions and deletions of exon 19 accounted for the majority of mutations (21%, 19%, and 17%, respectively. EGFR mutation status was correlated with the higher grade (P=0.026 and advanced stage (P=0.034 of HNSCC tumors.Conclusion: Higher frequency of EGFR-TK domain mutations together with the presence of the T790M mutation suggests

  13. Elastatinal and leupeptin: effects on u.v.-induced mutation and sister-chromatid exchanges in Chinese hamster cells

    International Nuclear Information System (INIS)

    Paul, P.; Fujiwara, Y.

    1981-01-01

    Microbial protease inhibitors elastatinal and leupeptin were tested for cytotoxicity and for effects on spontaneous and u.v.-induced 6-thioguanine-resistant (6TGsup(r)) mutation and sister-chromatid exchange (SCE) in V79 Chinese hamster cells. Continuous treatment with elastatinal exhibited marked cytotoxicity, while leupeptin was almost non-cytotoxic. Elastatinal rapidly induced cytotoxic effects as a function of its concentration and time of exposure. Near maximum cytotoxicity was reached after exposures of 6-8 h and this was partially abolished by the presence of 2.5 μg cycloheximide per ml. Concentrations of either protease inhibitor which gave 60-80% survival had no appreciable effects on u.v. survival and frequencies of spontaneous and u.v.-induced 6TGsup(r) mutation and SCE. However, reconstruction experiments revealed that pretreatments of 6TGsup(r) and 6TGsup(s) (wild-type) cells with these inhibitors for 6 days tended to block metabolic co-operation in their co-cultures. Thus, elastatinal and leupeptin are neither clastogenic nor mutagenic by themselves, and do not alter mutation fixation and expression. (author)

  14. Elastatinal and leupeptin: effects on u.v.-induced mutation and sister-chromatid exchanges in Chinese hamster cells

    International Nuclear Information System (INIS)

    Paul, P.; Fujiwara, Y.

    1981-01-01

    Microbial protease inhibitors elastatinal and leupeptin were tested for cytotoxicity and for effects on spontaneous and u.v.-induced 6-thioguanine-resistant (6TGr) mutation and sister-chromatid exchange (SCE) in V79 Chinese hamster cells. Continuous treatment with elastatinal exhibited marked cytotoxicity, while leupeptin was almost non-cytotoxic. Elastatinal rapidly induced cytotoxic effects as a function of its concentration and time of exposure. Near maximum cytotoxicity was reached after exposure of 6-8 h and this was partially abolished by the presence of 2.5 micrograms cycloheximide per ml. Concentrations of either protease inhibitor which gave 60-80% survival had no appreciable effects on u.v. survival and frequencies of spontaneous and u.v.-induced 6TGr mutation and SCE. However, reconstruction experiments revealed that pretreatments of 6TGr and 6TGs (wild-type) cells with these inhibitors for 6 days tended to block metabolic co-operation in their co-cultures. Thus, elastatinal and leupeptin are neither clastogenic mutagenic by themselves, and do not alter mutation fixation and expression

  15. Cell surface fucosylation does not affect development of colon tumors in mice with germline Smad3 mutation

    Science.gov (United States)

    Domino, Steven E.; Karnak, David M.; Hurd, Elizabeth A.

    2006-01-01

    Background/Aims: Neoplasia-related alterations in cell surface α(1,2)fucosylated glycans have been reported in multiple tumors including colon, pancreas, endometrium, cervix, bladder, lung, and choriocarcinoma. Spontaneous colorectal tumors from mice with a germline null mutation of transforming growth factor-β signaling gene Smad3 (Madh3) were tested for α(1,2)fucosylated glycan expression. Methods: Ulex Europaeus Agglutinin-I lectin staining, fucosyltransferase gene northern blot analysis, and a cross of mutant mice with Fut2 and Smad3 germline mutations were performed. Results: Spontaneous colorectal tumors from Smad3 (-/-) homozygous null mice were found to express α(1,2)fucosylated glycans in an abnormal pattern compared to adjacent nonneoplastic colon. Northern blot analysis of α(1,2)fucosyltransferase genes Fut1 and Fut2 revealed that Fut2, but not Fut1, steady-state mRNA levels were significantly increased in tumors relative to adjacent normal colonic mucosa. Mutant mice with a Fut2-inactivating germline mutation were crossed with Smad3 targeted mice. In Smad3 (-/-)/Fut2 (-/-) double knock-out mice, UEA-I lectin staining was eliminated from colon and colon tumors, however, the number and size of tumors present by 24 weeks of age did not vary regardless of the Fut2 genotype. Conclusions: In this model of colorectal cancer, cell surface α(1,2)fucosylation does not affect development of colon tumors. PMID:17264540

  16. Mutator/hypermutable fetal/juvenile metakaryotic stem cells and human colorectal carcinogenesis.

    Directory of Open Access Journals (Sweden)

    Lohith G. Kini

    2013-10-01

    Full Text Available Adult age-specific colorectal cancer incidence rates increase exponentially from maturity, reach a maximum, then decline in extreme old age. Armitage and Doll (1957 postulated that the exponential increase resulted from n mutations occurring throughout adult life in normal cells at risk that initiated the growth of a preneoplastic colony in which subsequent m mutations promoted one of the preneoplastic cells at risk to form a lethal neoplasia. We have reported cytologic evidence that these cells at risk are fetal/juvenile organogenic, then preneoplastic metakaryotic stem cells. Metakaryotic cells display stem-like behaviors of both symmetric and asymmetric nuclear divisions and peculiarities such as bell shaped nuclei and amitotic nuclear fission that distinguish them from embryonic, eukaryotic stem cells. Analyses of mutant colony sizes and numbers in adult lung epithelia supported the inferences that the metakaryotic organogenic stem cells are constitutively mutator/hypermutable and that their contributions to cancer initiation are limited to the fetal/juvenile period. We have amended the two-stage model of Armitage and Doll and incorporated these several inferences in a computer program CancerFit v.5.0. We compared the expectations of the amended model to adult (15-104 yr age-specific colon cancer rates for European American males born 1890-99 and observed remarkable concordance. When estimates of normal colonic fetal/juvenile APC and OAT gene mutation rates (~2-5 x 10-5 per stem cell doubling and preneoplastic colonic gene loss rates (~ 8 x 10-3 were applied, the model was in accordance only for the values of n = 2 and m = 4 or 5.

  17. Rapid detection of RB1 recurrent mutations in retinoblastoma by ...

    Indian Academy of Sciences (India)

    In about half of the patients, one mutation is inherited via the germinal cells, while in the .... mutational hot spots in the RB1 gene, making genetic testing complex and challenging ... by direct sequencing. High normal background in sequenc-.

  18. The clinical spectrum of mutations in L1, a neuronal cell adhesion molecule

    Energy Technology Data Exchange (ETDEWEB)

    Fransen, E.; Vits, L.; Van Camp, G.; Willems, P.J. [Univ. of Antwerp (Belgium)

    1996-07-12

    Mutations in the gene encoding the neuronal cell adhesion molecule L1 are responsible for several syndromes with clinical overlap, including X-linked hydrocephalus (XLH, HSAS), MASA (mental retardation, aphasia, shuffling gait, adducted thumbs) syndrome, complicated X-linked spastic paraplegia (SP 1), X-linked mental retardation-clasped thumb (MR-CT) syndrome, and some forms of X-linked agenesis of the corpus callosum (ACC). We review 34 L1 mutations in patients with these phenotypes. 22 refs., 3 figs., 4 tabs.

  19. A magic bullet to specifically eliminate mutated mitochondrial genomes from patients' cells

    Science.gov (United States)

    Moraes, Carlos T

    2014-01-01

    When mitochondrial diseases result from mutations found in the mitochondrial DNA, engineered mitochondrial-targeted nucleases such as mitochondrial-targeted zinc finger nucleases are shown to specifically eliminate the mutated molecules, leaving the wild-type mitochondrial DNA intact to replicate and restore normal copy number. In this issue, Gammage and colleagues successfully apply this improved technology on patients' cells with two types of genetic alterations responsible for neuropathy ataxia and retinitis pigmentosa (NARP) syndrome and Kearns Sayre syndrome and progressive external ophthalmoplegia (PEO). PMID:24623377

  20. Mutational analysis of cutaneous squamous cell carcinomas and verrucal keratosis in patients taking BRAF inhibitors.

    Science.gov (United States)

    Anforth, Rachael; Tembe, Varsha; Blumetti, Tatiana; Fernandez-Peñas, Pablo

    2012-09-01

    B-RAF inhibitors (BRAFi) have been shown to improve rates of overall and progression-free survival in patients with stage IV metastatic melanoma positive for the BRAF V600E mutation. However, the main drawback is the development of verrucal keratosis (hyperkeratotic papules with verruca-like characteristics with benign histological findings) and cutaneous squamous cell carcinomas (cuSCC). We have found upstream mutations in RAS as well as PIK3CA in both verrucal keratosis and cuSCC. This suggests that verrucal keratosis is an early clinical presentation of cuSCC in patients on BRAFi. © 2012 John Wiley & Sons A/S.

  1. Sensitive detection of pre-existing BCR-ABL kinase domain mutations in CD34+ cells of newly diagnosed chronic-phase chronic myeloid leukemia patients is associated with imatinib resistance: implications in the post-imatinib era.

    Directory of Open Access Journals (Sweden)

    Zafar Iqbal

    leukemia along with imatinib, all of which vary in their effectiveness against mutated BCR-ABL forms, detection of pre-existing BCR-ABL mutations can help in selection of appropriate first-line drug therapy. Thus, mutation testing using CD34+ cells may facilitate improved, patient-tailored treatment.

  2. Accelerated stress testing of terrestrial solar cells

    Science.gov (United States)

    Lathrop, J. W.; Hawkins, D. C.; Prince, J. L.; Walker, H. A.

    1982-01-01

    The development of an accelerated test schedule for terrestrial solar cells is described. This schedule, based on anticipated failure modes deduced from a consideration of IC failure mechanisms, involves bias-temperature testing, humidity testing (including both 85-85 and pressure cooker stress), and thermal-cycle thermal-shock testing. Results are described for 12 different unencapsulated cell types. Both gradual electrical degradation and sudden catastrophic mechanical change were observed. These effects can be used to discriminate between cell types and technologies relative to their reliability attributes. Consideration is given to identifying laboratory failure modes which might lead to severe degradation in the field through second quadrant operation. Test results indicate that the ability of most cell types to withstand accelerated stress testing depends more on the manufacturer's design, processing, and worksmanship than on the particular metallization system. Preliminary tests comparing accelerated test results on encapsulated and unencapsulated cells are described.

  3. PHF6 mutations in T-cell acute lymphoblastic leukemia

    NARCIS (Netherlands)

    P. van Vlierberghe (Pieter); T. Palomero (Teresa); H. Khiabanian (Hossein); J. van der Meulen (Joni); M. Castillo (Mireia); N. van Roy (Nadine); B. de Moerloose (Barbara); J. Philippé (Jan); S. González-García (Sara); M.L. Toribio (María); T. Taghon (Tom); L.C. Zuurbier (Linda); B. Cauwelier (Barbara); C.J. Harrison (Christine); C. Schwab (Claire); M. Pisecker (Markus); S. Strehl; A.W. Langerak (Anton); J. Gecz (Jozef); E. Sonneveld (Edwin); R. Pieters (Rob); E. Paietta (Elisabeth); J. Rowe (Jacob); P.H. Wiernik (Peter); Y. Benoit (Yves); J. Soulier (Jean); B. Poppe (Bruce); X. Yao (Xiaopan); C. Cordon-Cardo (Carlos); J.P.P. Meijerink (Jules); R. Rabadan (Raul); F. Speleman (Franki); A.A. Ferrando (Adolfo)

    2010-01-01

    textabstractTumor suppressor genes on the X chromosome may skew the gender distribution of specific types of cancer. T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with an increased incidence in males. In this study, we report the identification of inactivating

  4. Single-Cell Analysis of Human Pancreas Reveals Transcriptional Signatures of Aging and Somatic Mutation Patterns.

    Science.gov (United States)

    Enge, Martin; Arda, H Efsun; Mignardi, Marco; Beausang, John; Bottino, Rita; Kim, Seung K; Quake, Stephen R

    2017-10-05

    As organisms age, cells accumulate genetic and epigenetic errors that eventually lead to impaired organ function or catastrophic transformation such as cancer. Because aging reflects a stochastic process of increasing disorder, cells in an organ will be individually affected in different ways, thus rendering bulk analyses of postmitotic adult cells difficult to interpret. Here, we directly measure the effects of aging in human tissue by performing single-cell transcriptome analysis of 2,544 human pancreas cells from eight donors spanning six decades of life. We find that islet endocrine cells from older donors display increased levels of transcriptional noise and potential fate drift. By determining the mutational history of individual cells, we uncover a novel mutational signature in healthy aging endocrine cells. Our results demonstrate the feasibility of using single-cell RNA sequencing (RNA-seq) data from primary cells to derive insights into genetic and transcriptional processes that operate on aging human tissue. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. CHARACTERIZATION OF ENU-INDUCED MUTATIONS IN RED BLOOD CELL STRUCTURAL PROTEINS

    Directory of Open Access Journals (Sweden)

    Katrina Kildey

    2013-03-01

    Full Text Available Murine models with modified gene function as a result of N-ethyl-N-nitrosourea (ENU mutagenesis have been used to study phenotypes resulting from genetic change. This study investigated genetic factors associated with red blood cell (RBC physiology and structural integrity that may impact on blood component storage and transfusion outcome. Forward and reverse genetic approaches were employed with pedigrees of ENU-treated mice using a homozygous recessive breeding strategy. In a “forward genetic” approach, pedigree selection was based upon identification of an altered phenotype followed by exome sequencing to identify a causative mutation. In a second strategy, a “reverse genetic” approach based on selection of pedigrees with mutations in genes of interest was utilised and, following breeding to homozygosity, phenotype assessed. Thirty-three pedigrees were screened by the forward genetic approach. One pedigree demonstrated reticulocytosis, microcytic anaemia and thrombocytosis. Exome sequencing revealed a novel single nucleotide variation (SNV in Ank1 encoding the RBC structural protein ankyrin-1 and the pedigree was designated Ank1EX34. The reticulocytosis and microcytic anaemia observed in the Ank1EX34 pedigree were similar to clinical features of hereditary spherocytosis in humans. For the reverse genetic approach three pedigrees with different point mutations in Spnb1 encoding RBC protein spectrin-1β, and one pedigree with a mutation in Epb4.1, encoding band 4.1 were selected for study. When bred to homozygosity two of the spectrin-1β pedigrees (a, b demonstrated increased RBC count, haemoglobin (Hb and haematocrit (HCT. The third Spnb1 mutation (spectrin-1β c and mutation in Epb4.1 (band 4.1 did not significantly affect the haematological phenotype, despite these two mutations having a PolyPhen score predicting the mutation may be damaging. Exome sequencing allows rapid identification of causative mutations and development of

  6. Detecting β-thalassaemia mutations from a single cell by PEP and RDB

    Institute of Scientific and Technical Information of China (English)

    YI Ping; LI Li; YAO Hong; ZHOU Yuan-guo; DENG Bing; CHEN Zhu-qin

    2006-01-01

    Objective:To evaluate the possibility of the technology involving PEP and RDB for detecting β-thalassaemia multipoint mutations from a single cell simultaneously. Methods: A set of allele specific oligonucleotide (ASO) probes used for detecting 8 familiar β-thalassaemia mutations (CD41-42, IVS- Ⅱ -654, CD17, TATA box nt-28, CD71-72, TATA box nt-29, CD26, IVS- Ⅰ -5) were immobilized on a strip of nylon membrane. The genome of a individual cell was amplified by primer extension preamplification (PEP) with the mixture of15-base random oligonucleotides. The aliquots from PEP were used to amplify the objective gene fractions of β-thalassaemia gene by nested or semi-nested PCR. The membrane was hybridized with the final amplified products and then treated with Streptavidin-HRP and color development.Results :Totally 30 lymphocytes were picked up from blood samples of 1 healthy female and 4 patients with known β-thalassaemia mutations respectively. Each single lymphocyte was lysed in the proteinase K buffer. The amplification efficacy was 94.0% and alle drop-out(ADO) rate was 8.0%. Revert dot blot (RDB) was applied to the final amplified products from the 5 participants. The results of diagnosis were the same to the expected, and their genotypes were N/N, CD17 (A→T)/N, IVS- Ⅱ -654(C→T)/CD17(A → T), CD41-42 (-CTTT)/N and TATA box nt-28 (A→G)/N, respectively. Conclusion: The technology involving PEP and RDB could detectmultiple β-thalassaemia mutations from a single cell simultaneously,and the research provides experimental evidences for the feasibility of applying PEP and DNA array technology to screening multiple genetic mutations from a single cell, and will be applied to preimplantation genetic diagnosis and non-invasive prenatal diagnosis for β-thalassaemia.

  7. Genetic Testing for TMEM154 Mutations Associated with Lentivirus Susceptibility in Sheep

    Science.gov (United States)

    Petrik, Dustin T.; Simpson, Barry; Kijas, James W.; Clawson, Michael L.; Chitko-McKown, Carol G.; Harhay, Gregory P.; Leymaster, Kreg A.

    2013-01-01

    In sheep, small ruminant lentiviruses cause an incurable, progressive, lymphoproliferative disease that affects millions of animals worldwide. Known as ovine progressive pneumonia virus (OPPV) in the U.S., and Visna/Maedi virus (VMV) elsewhere, these viruses reduce an animal’s health, productivity, and lifespan. Genetic variation in the ovine transmembrane protein 154 gene (TMEM154) has been previously associated with OPPV infection in U.S. sheep. Sheep with the ancestral TMEM154 haplotype encoding glutamate (E) at position 35, and either form of an N70I variant, were highly-susceptible compared to sheep homozygous for the K35 missense mutation. Our current overall aim was to characterize TMEM154 in sheep from around the world to develop an efficient genetic test for reduced susceptibility. The average frequency of TMEM154 E35 among 74 breeds was 0.51 and indicated that highly-susceptible alleles were present in most breeds around the world. Analysis of whole genome sequences from an international panel of 75 sheep revealed more than 1,300 previously unreported polymorphisms in a 62 kb region containing TMEM154 and confirmed that the most susceptible haplotypes were distributed worldwide. Novel missense mutations were discovered in the signal peptide (A13V) and the extracellular domains (E31Q, I74F, and I102T) of TMEM154. A matrix-assisted laser desorption/ionization–time-of flight mass spectrometry (MALDI-TOF MS) assay was developed to detect these and six previously reported missense and two deletion mutations in TMEM154. In blinded trials, the call rate for the eight most common coding polymorphisms was 99.4% for 499 sheep tested and 96.0% of the animals were assigned paired TMEM154 haplotypes (i.e., diplotypes). The widespread distribution of highly-susceptible TMEM154 alleles suggests that genetic testing and selection may improve the health and productivity of infected flocks. PMID:23408992

  8. Genetic testing for TMEM154 mutations associated with lentivirus susceptibility in sheep.

    Directory of Open Access Journals (Sweden)

    Michael P Heaton

    Full Text Available In sheep, small ruminant lentiviruses cause an incurable, progressive, lymphoproliferative disease that affects millions of animals worldwide. Known as ovine progressive pneumonia virus (OPPV in the U.S., and Visna/Maedi virus (VMV elsewhere, these viruses reduce an animal's health, productivity, and lifespan. Genetic variation in the ovine transmembrane protein 154 gene (TMEM154 has been previously associated with OPPV infection in U.S. sheep. Sheep with the ancestral TMEM154 haplotype encoding glutamate (E at position 35, and either form of an N70I variant, were highly-susceptible compared to sheep homozygous for the K35 missense mutation. Our current overall aim was to characterize TMEM154 in sheep from around the world to develop an efficient genetic test for reduced susceptibility. The average frequency of TMEM154 E35 among 74 breeds was 0.51 and indicated that highly-susceptible alleles were present in most breeds around the world. Analysis of whole genome sequences from an international panel of 75 sheep revealed more than 1,300 previously unreported polymorphisms in a 62 kb region containing TMEM154 and confirmed that the most susceptible haplotypes were distributed worldwide. Novel missense mutations were discovered in the signal peptide (A13V and the extracellular domains (E31Q, I74F, and I102T of TMEM154. A matrix-assisted laser desorption/ionization-time-of flight mass spectrometry (MALDI-TOF MS assay was developed to detect these and six previously reported missense and two deletion mutations in TMEM154. In blinded trials, the call rate for the eight most common coding polymorphisms was 99.4% for 499 sheep tested and 96.0% of the animals were assigned paired TMEM154 haplotypes (i.e., diplotypes. The widespread distribution of highly-susceptible TMEM154 alleles suggests that genetic testing and selection may improve the health and productivity of infected flocks.

  9. Nonsense and missense mutation of mitochondrial ND6 gene promotes cell migration and invasion in human lung adenocarcinoma

    International Nuclear Information System (INIS)

    Yuan, Yang; Wang, Weixing; Li, Huizhong; Yu, Yongwei; Tao, Jin; Huang, Shengdong; Zeng, Zhiyong

    2015-01-01

    Previous study showed that mitochondrial ND6 (mitND6) gene missense mutation resulted in NADH dehydrogenase deficiency and was associated with tumor metastasis in several mouse tumor cell lines. In the present study, we investigated the possible role of mitND6 gene nonsense and missense mutations in the metastasis of human lung adenocarcinoma. The presence of mitND6 gene mutations was screened by DNA sequencing of tumor tissues from 87 primary lung adenocarcinoma patients and the correlation of the mutations with the clinical features was analyzed. In addition, we constructed cytoplasmic hybrid cells with denucleared primary lung adenocarcinoma cell as the mitochondria donor and mitochondria depleted lung adenocarcinoma A549 cell as the nuclear donor. Using these cells, we studied the effects of mitND6 gene nonsense and missense mutations on cell migration and invasion through wounding healing and matrigel-coated transwell assay. The effects of mitND6 gene mutations on NADH dehydrogenase activity and ROS production were analyzed by spectrophotometry and flow cytometry. mitND6 gene nonsense and missense mutations were detected in 11 of 87 lung adenocarcinoma specimens and was correlated with the clinical features including age, pathological grade, tumor stage, lymph node metastasis and survival rate. Moreover, A549 cell containing mitND6 gene nonsense and missense mutation exhibited significantly lower activity of NADH dehydrogenase, higher level of ROS, higher capacity of cell migration and invasion, and higher pAKT and pERK1/ERK2 expression level than cells with the wild type mitND6 gene. In addition, NADH dehydrogenase inhibitor rotenone was found to significantly promote the migration and invasion of A549 cells. Our data suggest that mitND6 gene nonsense and missense mutation might promote cell migration and invasion in lung adenocarcinoma, probably by NADH dehydrogenase deficiency induced over-production of ROS

  10. The use of a mutationally unstable X-chromosome in Drosophila melanogaster for mutagenicity testing

    International Nuclear Information System (INIS)

    Rasmuson, B.; Svahlin, H.; Rasmuson, A.; Montell, I.; Olofsson, H.

    1978-01-01

    Somatic eye-colour mutations in an unstable genetic system, caused by a transposable element in the white locus of the X-chromosome in Drosophila melanogaster, is suggested as an assay system for mutagenicity testing. The system is evaluated by comparison with a corresponding system in a stable X-chromosome. Its sensitivity is confirmed with X-ray and EMS treatment, and it is found to be confined to the specific segment of the X-chromosome where the transposable element is localized. (Auth.)

  11. A New Generalizable Test for Detection of Mutations Affecting Tn10 Transposition

    OpenAIRE

    Huisman, Olivier; Kleckner, Nancy

    1987-01-01

    We describe here a new rapid screen that allows easy detection of transposon or host mutations that affect Tn10 transposition in Escherichia coli. This test involves a new Tn10 derivative called the "mini-lacZ-kanR fusion hopper" or mini-Tn10-LK for short. This element does not direct expression of β-galactosidase when present at its original starting location on a suitably engineered plasmid or phage genome because it lacks appropriate transcription and translation start signals. However, t...

  12. Mutations in the satellite cell gene MEGF10 cause a recessive congenital myopathy with minicores.

    Science.gov (United States)

    Boyden, Steven E; Mahoney, Lane J; Kawahara, Genri; Myers, Jennifer A; Mitsuhashi, Satomi; Estrella, Elicia A; Duncan, Anna R; Dey, Friederike; DeChene, Elizabeth T; Blasko-Goehringer, Jessica M; Bönnemann, Carsten G; Darras, Basil T; Mendell, Jerry R; Lidov, Hart G W; Nishino, Ichizo; Beggs, Alan H; Kunkel, Louis M; Kang, Peter B

    2012-05-01

    We ascertained a nuclear family in which three of four siblings were affected with an unclassified autosomal recessive myopathy characterized by severe weakness, respiratory impairment, scoliosis, joint contractures, and an unusual combination of dystrophic and myopathic features on muscle biopsy. Whole genome sequence from one affected subject was filtered using linkage data and variant databases. A single gene, MEGF10, contained nonsynonymous mutations that co-segregated with the phenotype. Affected subjects were compound heterozygous for missense mutations c.976T > C (p.C326R) and c.2320T > C (p.C774R). Screening the MEGF10 open reading frame in 190 patients with genetically unexplained myopathies revealed a heterozygous mutation, c.211C > T (p.R71W), in one additional subject with a similar clinical and histological presentation as the discovery family. All three mutations were absent from at least 645 genotyped unaffected control subjects. MEGF10 contains 17 atypical epidermal growth factor-like domains, each of which contains eight cysteine residues that likely form disulfide bonds. Both the p.C326R and p.C774R mutations alter one of these residues, which are completely conserved in vertebrates. Previous work showed that murine Megf10 is required for preserving the undifferentiated, proliferative potential of satellite cells, myogenic precursors that regenerate skeletal muscle in response to injury or disease. Here, knockdown of megf10 in zebrafish by four different morpholinos resulted in abnormal phenotypes including unhatched eggs, curved tails, impaired motility, and disorganized muscle tissue, corroborating the pathogenicity of the human mutations. Our data establish the importance of MEGF10 in human skeletal muscle and suggest satellite cell dysfunction as a novel myopathic mechanism.

  13. Frequency of p53 Gene Mutation and Protein Expression in Oral Squamous Cell Carcinoma

    International Nuclear Information System (INIS)

    Ara, N.; Atique, M.; Ahmed, S.; Bukhari, S. G. A.

    2014-01-01

    Objective: To determine the frequency of p53 gene mutation and protein expression in Oral Squamous Cell Carcinoma (OSCC) and to establish correlation between the two. Study Design: Analytical study. Place and Duration of Study: Histopathology Department and Molecular Biology Laboratory, Armed Forces Institute of Pathology (AFIP), Rawalpindi, from May 2010 to May 2011. Methodology: Thirty diagnosed cases of OSCC were selected by consecutive sampling. Seventeen were retrieved from the record files of the AFIP, and 13 fresh/frozen sections were selected from patients reporting to the Oral Surgery Department, Armed Forces Institute of Dentistry (AFID). Gene p53 mutation was analyzed in all the cases using PCRSSCP analysis. DNA was extracted from the formalin-fixed and paraffin-embedded tissue sections and fresh/frozen sections. DNA thus extracted was amplified by polymerase chain reaction. The amplified products were denatured and finally analyzed by gel electrophoresis. Gene mutation was detected as electrophoretic mobility shift. The immunohistochemical marker p53 was applied to the same 30 cases and overexpression of protein p53 was recorded. Results: Immunohistochemical expression of marker p53 was positive in 67% (95% Confidence Interval (CI) 48.7 - 80.9) of the cases. Mutations of the p53 gene were detected in 23% (95% CI 11.5 - 41.2) of the OSCC. No statistically significant correlation was found between p53 gene mutation and protein p53 expression (rs = - 0.057, p = 0.765). Conclusion: A substantial number of patients have p53 gene mutation (23%) and protein p53 expression (67%) in oral squamous cell carcinoma (OSCC). (author)

  14. The F309S mutation increases factor VIII secretion in human cell line

    Directory of Open Access Journals (Sweden)

    Daianne Maciely Carvalho Fantacini

    2016-06-01

    Full Text Available ABSTRACT OBJECTIVES: The capacity of a human cell line to secrete recombinant factor VIII with a F309S point mutation was investigated, as was the effect of the addition of chemical chaperones (betaine and sodium-4-phenylbutyrate on the secretion of factor VIII. METHODS: This work used a vector with a F309S mutation in the A1 domain to investigate FVIII production in the HEK 293 human cell line. Factor VIII activity was measured by chromogenic assay. Furthermore, the effects of chemical drugs on the culture were evaluated. RESULTS: The addition of the F309S mutation to a previously described FVIII variant increased FVIII secretion by 4.5 fold. Moreover, the addition of betaine or sodium-4-phenylbutyrate increased the secretion rate of FVIIIΔB proteins in HEK 293 cells, but the same effect was not seen for FVIIIΔB-F309S indicating that all the recombinant protein produced had been efficiently secreted. CONCLUSION: Bioengineering factor VIII expressed in human cells may lead to an efficient production of recombinant factor VIII and contribute toward low-cost coagulation factor replacement therapy for hemophilia A. FVIII-F309S produced in human cells can be effective in vivo.

  15. Simple PCR assays improve the sensitivity of HIV-1 subtype B drug resistance testing and allow linking of resistance mutations.

    Directory of Open Access Journals (Sweden)

    Jeffrey A Johnson

    Full Text Available BACKGROUND: The success of antiretroviral therapy is known to be compromised by drug-resistant HIV-1 at frequencies detectable by conventional bulk sequencing. Currently, there is a need to assess the clinical consequences of low-frequency drug resistant variants occurring below the detection limit of conventional genotyping. Sensitive detection of drug-resistant subpopulations, however, requires simple and practical methods for routine testing. METHODOLOGY: We developed highly-sensitive and simple real-time PCR assays for nine key drug resistance mutations and show that these tests overcome substantial sequence heterogeneity in HIV-1 clinical specimens. We specifically used early wildtype virus samples from the pre-antiretroviral drug era to measure background reactivity and were able to define highly-specific screening cut-offs that are up to 67-fold more sensitive than conventional genotyping. We also demonstrate that sequencing the mutation-specific PCR products provided a direct and novel strategy to further detect and link associated resistance mutations, allowing easy identification of multi-drug-resistant variants. Resistance mutation associations revealed in mutation-specific amplicon sequences were verified by clonal sequencing. SIGNIFICANCE: Combined, sensitive real-time PCR testing and mutation-specific amplicon sequencing provides a powerful and simple approach that allows for improved detection and evaluation of HIV-1 drug resistance mutations.

  16. Fuel Cell Stations Automate Processes, Catalyst Testing

    Science.gov (United States)

    2010-01-01

    Glenn Research Center looks for ways to improve fuel cells, which are an important source of power for space missions, as well as the equipment used to test fuel cells. With Small Business Innovation Research (SBIR) awards from Glenn, Lynntech Inc., of College Station, Texas, addressed a major limitation of fuel cell testing equipment. Five years later, the company obtained a patent and provided the equipment to the commercial world. Now offered through TesSol Inc., of Battle Ground, Washington, the technology is used for fuel cell work, catalyst testing, sensor testing, gas blending, and other applications. It can be found at universities, national laboratories, and businesses around the world.

  17. Effect of dedifferentiation on time to mutation acquisition in stem cell-driven cancers.

    Directory of Open Access Journals (Sweden)

    Alexandra Jilkine

    2014-03-01

    Full Text Available Accumulating evidence suggests that many tumors have a hierarchical organization, with the bulk of the tumor composed of relatively differentiated short-lived progenitor cells that are maintained by a small population of undifferentiated long-lived cancer stem cells. It is unclear, however, whether cancer stem cells originate from normal stem cells or from dedifferentiated progenitor cells. To address this, we mathematically modeled the effect of dedifferentiation on carcinogenesis. We considered a hybrid stochastic-deterministic model of mutation accumulation in both stem cells and progenitors, including dedifferentiation of progenitor cells to a stem cell-like state. We performed exact computer simulations of the emergence of tumor subpopulations with two mutations, and we derived semi-analytical estimates for the waiting time distribution to fixation. Our results suggest that dedifferentiation may play an important role in carcinogenesis, depending on how stem cell homeostasis is maintained. If the stem cell population size is held strictly constant (due to all divisions being asymmetric, we found that dedifferentiation acts like a positive selective force in the stem cell population and thus speeds carcinogenesis. If the stem cell population size is allowed to vary stochastically with density-dependent reproduction rates (allowing both symmetric and asymmetric divisions, we found that dedifferentiation beyond a critical threshold leads to exponential growth of the stem cell population. Thus, dedifferentiation may play a crucial role, the common modeling assumption of constant stem cell population size may not be adequate, and further progress in understanding carcinogenesis demands a more detailed mechanistic understanding of stem cell homeostasis.

  18. Effect of Dedifferentiation on Time to Mutation Acquisition in Stem Cell-Driven Cancers

    Science.gov (United States)

    Jilkine, Alexandra; Gutenkunst, Ryan N.

    2014-01-01

    Accumulating evidence suggests that many tumors have a hierarchical organization, with the bulk of the tumor composed of relatively differentiated short-lived progenitor cells that are maintained by a small population of undifferentiated long-lived cancer stem cells. It is unclear, however, whether cancer stem cells originate from normal stem cells or from dedifferentiated progenitor cells. To address this, we mathematically modeled the effect of dedifferentiation on carcinogenesis. We considered a hybrid stochastic-deterministic model of mutation accumulation in both stem cells and progenitors, including dedifferentiation of progenitor cells to a stem cell-like state. We performed exact computer simulations of the emergence of tumor subpopulations with two mutations, and we derived semi-analytical estimates for the waiting time distribution to fixation. Our results suggest that dedifferentiation may play an important role in carcinogenesis, depending on how stem cell homeostasis is maintained. If the stem cell population size is held strictly constant (due to all divisions being asymmetric), we found that dedifferentiation acts like a positive selective force in the stem cell population and thus speeds carcinogenesis. If the stem cell population size is allowed to vary stochastically with density-dependent reproduction rates (allowing both symmetric and asymmetric divisions), we found that dedifferentiation beyond a critical threshold leads to exponential growth of the stem cell population. Thus, dedifferentiation may play a crucial role, the common modeling assumption of constant stem cell population size may not be adequate, and further progress in understanding carcinogenesis demands a more detailed mechanistic understanding of stem cell homeostasis. PMID:24603301

  19. Mutations That Alter the Bacterial Cell Envelope Increase Lipid Production

    Energy Technology Data Exchange (ETDEWEB)

    Lemmer, Kimberly C.; Zhang, Weiping; Langer, Samantha J.; Dohnalkova, Alice; Hu, Dehong; Lemke, Rachelle A.; Piotrowski, Jeff S.; Orr, Galya; Noguera, Daniel R.; Donohue, Timothy J.

    2017-05-23

    ABSTRACT

    Lipids from microbes offer a promising source of renewable alternatives to petroleum-derived compounds. In particular, oleaginous microbes are of interest because they accumulate a large fraction of their biomass as lipids. In this study, we analyzed genetic changes that alter lipid accumulation inRhodobacter sphaeroides. By screening anR. sphaeroidesTn5mutant library for insertions that increased fatty acid content, we identified 10 high-lipid (HL) mutants for further characterization. These HL mutants exhibited increased sensitivity to drugs that target the bacterial cell envelope and changes in shape, and some had the ability to secrete lipids, with two HL mutants accumulating ~60% of their total lipids extracellularly. When one of the highest-lipid-secreting strains was grown in a fed-batch bioreactor, its lipid content was comparable to that of oleaginous microbes, with the majority of the lipids secreted into the medium. Based on the properties of these HL mutants, we conclude that alterations of the cell envelope are a previously unreported approach to increase microbial lipid production. We also propose that this approach may be combined with knowledge about biosynthetic pathways, in this or other microbes, to increase production of lipids and other chemicals.

    IMPORTANCEThis paper reports on experiments to understand how to increase microbial lipid production. Microbial lipids are often cited as one renewable replacement for petroleum-based fuels and chemicals, but strategies to increase the yield of these compounds are needed to achieve this goal. While lipid biosynthesis is often well understood, increasing yields of these compounds to industrially relevant levels is a challenge, especially since genetic, synthetic biology, or engineering approaches are not feasible in many microbes. We show that altering the bacterial cell envelope can be used to increase

  20. The glycophorin A assay for somatic cell mutations in humans

    International Nuclear Information System (INIS)

    Langlois, R.G.; Bigbee, W.L.; Jensen, R.H.

    1989-01-01

    In this report we briefly review our past experience and some new developments with the GPA assay. Particular emphasis will be placed on two areas that affect the utility of the GPA assay for human population monitoring. The first is our efforts to simplify the GPA assay to make it more generally available for large population studies. The second is to begin to understand some of the characteristics of human hemopoiesis which affect the accumulation and expression of mutant phenotype cells. 11 refs., 4 figs

  1. High Resolution Melting Analysis for Detecting p53 Gene Mutations in Patients with Non-small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Zhihong CHEN

    2011-10-01

    Full Text Available Background and objective It has been proven that p53 gene was related to many human cancers. The mutations in p53 gene play an important role in carcinogensis and mostly happened in exon 5-8. The aim of this study is to establish a high resolution melting (HRM assay to detect p53 mutations from patients with non-small cell lung cancer (NSCLC, to investigate the characteristics of p53 gene mutations, and to analyze the relationship between p53 mutations and evolution regularity of pathogenesis. Methods p53 mutations in exon 5-8 were detected by HRM assay on DNA insolated from 264 NSCLC samples derived from tumor tissues and 54 control samples from pericancerous pulmonary tissues. The mutation samples by the HRM assay were confirmed by sequencing technique. Samples which were positive by HRM but wild type by sequencing were further confirmed by sub-clone and sequencing. Results No mutation was found in 54 pericancerous pulmonary samples by HRM assay. 104 of the 264 tumor tissues demonstrated mutation curves by HRM assay, 102 samples were confirmed by sequencing, including 95 point mutations and 7 frame shift mutations by insertion or deletion. The mutation rate of p53 gene was 39.4%. The mutation rate from exon 5-8 were 11.7%, 8%, 12.5% and 10.6%, respectively and there was no statistically significant difference between them (P=0.35. p53 mutations were significantly more frequent in males than that in females, but not related to the other clinicopathologic characteristics. Conclusion The results indicate that HRM is a sensitive in-tube methodology to detect for mutations in clinical samples. The results suggest that the arising p53 mutations in NSCLC may be due to spontaneous error in DNA synthesis and repair.

  2. Nickel hydrogen battery cell storage matrix test

    Science.gov (United States)

    Wheeler, James R.; Dodson, Gary W.

    1993-01-01

    Test were conducted to evaluate post storage performance of nickel hydrogen cells with various design variables, the most significant being nickel precharge versus hydrogen precharge. Test procedures and results are presented in outline and graphic form.

  3. Targeted disruption of Ataxia-telangiectasia mutated gene in miniature pigs by somatic cell nuclear transfer

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Young June; Ahn, Kwang Sung; Kim, Minjeong; Kim, Min Ju; Park, Sang-Min; Ryu, Junghyun; Ahn, Jin Seop; Heo, Soon Young; Kang, Jee Hyun; Choi, You Jung [Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan (Korea, Republic of); Choi, Seong-Jun [Institute of Tissue Regeneration Engineering, Dankook University, Cheonan (Korea, Republic of); Shim, Hosup, E-mail: shim@dku.edu [Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan (Korea, Republic of); Institute of Tissue Regeneration Engineering, Dankook University, Cheonan (Korea, Republic of); Department of Physiology, Dankook University School of Medicine, Cheonan (Korea, Republic of)

    2014-10-03

    Highlights: • ATM gene-targeted pigs were produced by somatic cell nuclear transfer. • A novel large animal model for ataxia telangiectasia was developed. • The new model may provide an alternative to the mouse model. - Abstract: Ataxia telangiectasia (A-T) is a recessive autosomal disorder associated with pleiotropic phenotypes, including progressive cerebellar degeneration, gonad atrophy, and growth retardation. Even though A-T is known to be caused by the mutations in the Ataxia telangiectasia mutated (ATM) gene, the correlation between abnormal cellular physiology caused by ATM mutations and the multiple symptoms of A-T disease has not been clearly determined. None of the existing ATM mouse models properly reflects the extent to which neurological degeneration occurs in human. In an attempt to provide a large animal model for A-T, we produced gene-targeted pigs with mutations in the ATM gene by somatic cell nuclear transfer. The disrupted allele in the ATM gene of cloned piglets was confirmed via PCR and Southern blot analysis. The ATM gene-targeted pigs generated in the present study may provide an alternative to the current mouse model for the study of mechanisms underlying A-T disorder and for the development of new therapies.

  4. Targeted disruption of Ataxia-telangiectasia mutated gene in miniature pigs by somatic cell nuclear transfer

    International Nuclear Information System (INIS)

    Kim, Young June; Ahn, Kwang Sung; Kim, Minjeong; Kim, Min Ju; Park, Sang-Min; Ryu, Junghyun; Ahn, Jin Seop; Heo, Soon Young; Kang, Jee Hyun; Choi, You Jung; Choi, Seong-Jun; Shim, Hosup

    2014-01-01

    Highlights: • ATM gene-targeted pigs were produced by somatic cell nuclear transfer. • A novel large animal model for ataxia telangiectasia was developed. • The new model may provide an alternative to the mouse model. - Abstract: Ataxia telangiectasia (A-T) is a recessive autosomal disorder associated with pleiotropic phenotypes, including progressive cerebellar degeneration, gonad atrophy, and growth retardation. Even though A-T is known to be caused by the mutations in the Ataxia telangiectasia mutated (ATM) gene, the correlation between abnormal cellular physiology caused by ATM mutations and the multiple symptoms of A-T disease has not been clearly determined. None of the existing ATM mouse models properly reflects the extent to which neurological degeneration occurs in human. In an attempt to provide a large animal model for A-T, we produced gene-targeted pigs with mutations in the ATM gene by somatic cell nuclear transfer. The disrupted allele in the ATM gene of cloned piglets was confirmed via PCR and Southern blot analysis. The ATM gene-targeted pigs generated in the present study may provide an alternative to the current mouse model for the study of mechanisms underlying A-T disorder and for the development of new therapies

  5. Development of the adverse outcome pathway "alkylation of DNA in male premeiotic germ cells leading to heritable mutations" using the OECD's users' handbook supplement.

    Science.gov (United States)

    Yauk, Carole L; Lambert, Iain B; Meek, M E Bette; Douglas, George R; Marchetti, Francesco

    2015-12-01

    The Organisation for Economic Cooperation and Development's (OECD) Adverse Outcome Pathway (AOP) programme aims to develop a knowledgebase of all known pathways of toxicity that lead to adverse effects in humans and ecosystems. A Users' Handbook was recently released to provide supplementary guidance on AOP development. This article describes one AOP-alkylation of DNA in male premeiotic germ cells leading to heritable mutations. This outcome is an important regulatory endpoint. The AOP describes the biological plausibility and empirical evidence supporting that compounds capable of alkylating DNA cause germ cell mutations and subsequent mutations in the offspring of exposed males. Alkyl adducts are subject to DNA repair; however, at high doses the repair machinery becomes saturated. Lack of repair leads to replication of alkylated DNA and ensuing mutations in male premeiotic germ cells. Mutations that do not impair spermatogenesis persist and eventually are present in mature sperm. Thus, the mutations are transmitted to the offspring. Although there are some gaps in empirical support and evidence for essentiality of the key events for certain aspects of this AOP, the overall AOP is generally accepted as dogma and applies broadly to any species that produces sperm. The AOP was developed and used in an iterative process to test and refine the Users' Handbook, and is one of the first publicly available AOPs. It is our hope that this AOP will be leveraged to develop other AOPs in this field to advance method development, computational models to predict germ cell effects, and integrated testing strategies. © 2015 Her Majesty the Queen in Right of Canada.

  6. PMS2 gene mutation results in DNA mismatch repair system failure in a case of adult granulosa cell tumor

    OpenAIRE

    Wang, Wen-Chung; Lee, Ya-Ting; Lai, Yen-Chein

    2017-01-01

    Background Granulosa cell tumors are rare ovarian malignancies. Their characteristics include unpredictable indolent growth with malignant potential and late recurrence. Approximately 95% are of adult type. Recent molecular studies have characterized the FOXL2 402C?>?G mutation in adult granulosa cell tumor. Our previous case report showed that unique FOXL2 402C?>?G mutation and defective DNA mismatch repair system are associated with the development of adult granulosa cell tumor. Findings In...

  7. Aging-Induced Stem Cell Mutations as Drivers for Disease and Cancer

    Science.gov (United States)

    Adams, Peter D.; Jasper, Heinrich; Rudolph, K. Lenhard

    2015-01-01

    Aging is characterized by a decrease in genome integrity, impaired organ maintenance, and an increased risk of cancer, which coincide with clonal dominance of expanded mutant stem and progenitor cell populations in aging tissues, such as the intestinal epithelium, the hematopoietic system, and the male germline. Here we discuss possible explanations for age-associated increases in the initiation and/or progression of mutant stem/progenitor clones and highlight the roles of stem cell quiescence, replication-associated DNA damage, telomere shortening, epigenetic alterations, and metabolic challenges as determinants of stem cell mutations and clonal dominance in aging. PMID:26046760

  8. Mutation induction in a mouse lymphoma cell mutant sensitive to 4-nitroquinoline 1-oxide and ultraviolet radiation

    International Nuclear Information System (INIS)

    Sato, K.; Hieda, N.

    1980-01-01

    The mutant mouse lymphoma cell Q31, which is sensitive to 4-nitroquinoline 1-oxide and ultraviolet radiation (UV), was compared with the parental L5178Y cell for the effect of caffeine and mutation induction after UV irradiation. Caffeine potentiated the lethal effect of UV in both cell strains to a similar extent, indicating that the defective process in Q31 cells was caffeine-insensitive. UV-induced mutation to 6-thioguanine resistance was determined in L5178Y and Q31 cells. The maximal yield of mutants was obtained 7 days post-irradiation in L5178Y cells and 14 days in Q31 cells for higher UV doses. It appears that a much longer time is required for the mutant cells than for the parental cells for full expression of the resistance phenotype even at equitoxic UV doses. A substantially higher frequency in induced mutations was observed in Q31 cells than in L5178Y cells at a given dose of UV. A plot of induced mutation frequency as a function of logarithm of surviving fraction again indicates hypermutability of Q31 cells as compared with the parental strain. In contrast, X-rays induced a similar frequency of mutations to 6-thioguanine resistance in L5178Y and Q31 cells. (orig.)

  9. Targeted mutations induced by a single acetylaminofluorene DNA adduct in mammalian cells and bacteria

    International Nuclear Information System (INIS)

    Moryia, M.; Takeshita, M.; Johnson, F.; Peden, K.; Will, S.; Grollman, A.P.

    1988-01-01

    Mutagenic specificity of 2-acetylaminofluorene (AAF) has been established in mammalian cells and several strains of bacteria by using a shuttle plasmid vector containing a single N-(deoxyguanosin-8-yl)acetylaminofluorene (C8-dG-AAF) adduct. The nucleotide sequence of the gene conferring tetracycline resistance was modified by conservative codon replacement so as to accommodate the sequence d(CCTTCGCTAC) flanked by two restriction sites, Bsm I and Xho I. The corresponding synthetic oligodeoxynucleotide underwent reaction with 2-(N-acetoxy-N-acetylamino)-fluorene (AAAF), forming a single dG-AAF adduct. This modified oligodeoxynucleotide was hybridized to its complementary strand and ligated between the Bsm I and Xho I sites of the vector. Plasmids containing the C8-dG-AAF adduct were used to transfect simian virus 40-transformed simian kidney (COS-1) cells and to transform several AB strains of Escherichia coli. Colonies containing mutant plasmides were detected by hybridization to 32 P-labeled oligodeoxynucleotides. Presence of the single DNA adduct increased the mutation frequency by 8-fold in both COS cells and E. coli. Over 80% of mutations detected in both systems were targeted and represented G x C → C x G or G x C → T x A transversions or single nucleotide deletions. The authors conclude that modification of a deoxyguanosine residue with AAF preferentially induces mutations targeted at this site when a plasmid containing a single C8-dG-AAF adduct is introduced into mammalian cells or bacteria

  10. Theory of mutation induction by accelerated very heavy ions in cells

    International Nuclear Information System (INIS)

    Kozubek, S.; Ryznar, L.

    1995-01-01

    Stochastic effects of ionising radiation in humans are related to mutation induction in cells. Therefore experimental data on mutation induction represents one of the endpoints used for the estimation of risk in radiation protection. Only very rough estimates can be made owing to the fact that a suitable theoretical approach does not exist. A simple method is proposed for the evaluation of the efficiency of mutation induction by accelerated very heavy ions in mammalian cells. The approach is based on the calculation of the fraction of energy deposited by accelerated particles in indirect collisions (hits) in the cells. Two different modes of particle mutagenic action can be distinguished. δ ray mutagenesis is related to those particles that preferentially kill the cells in direct hits. Track-core mutagenesis arises from direct hits and is observed for lighter ions or ions with very high energy (LET ≤ 500 keV.μm -1 ). Available experimental data agree reasonably well with the results based on theory. (author)

  11. [Evaluation of the mutagenicity of detergents by tests on bacteria, plant cells and human leucocytes.].

    Science.gov (United States)

    Feretti, Donatella; Pedrazzani, Roberta; Ceretti, Elisabetta; Zerbini, Ilaria; Gozio, Eleonora; Belotti, Caterina; Alias, Carlotta; Donato, Francesco; Gelatti, Umberto

    2009-01-01

    The aim of this study was to evaluate the mutagenicity of several traditional detergents and that of newer more biodegradable detergents, by using a bacterial test (Ames test), a plant cell test (Allium cepa micronuclei test) and a human leucocyte test (Comet test). All tests were conducted using a wide range of doses (1-2000 mg/l). None of the examined detergents induced mutations in S.typhimurium. One traditional detergent showed a genotoxic effect with the A. cepa test, while all newer detergents and one traditional detergent were shown by the Comet test to be capable of inducing DNA damage.

  12. Customized treatment in non-small-cell lung cancer based on EGFR mutations and BRCA1 mRNA expression.

    Directory of Open Access Journals (Sweden)

    Rafael Rosell

    Full Text Available BACKGROUND: Median survival is 10 months and 2-year survival is 20% in metastatic non-small-cell lung cancer (NSCLC treated with platinum-based chemotherapy. A small fraction of non-squamous cell lung cancers harbor EGFR mutations, with improved outcome to gefitinib and erlotinib. Experimental evidence suggests that BRCA1 overexpression enhances sensitivity to docetaxel and resistance to cisplatin. RAP80 and Abraxas are interacting proteins that form complexes with BRCA1 and could modulate the effect of BRCA1. In order to further examine the effect of EGFR mutations and BRCA1 mRNA levels on outcome in advanced NSCLC, we performed a prospective non-randomized phase II clinical trial, testing the hypothesis that customized therapy would confer improved outcome over non-customized therapy. In an exploratory analysis, we also examined the effect of RAP80 and Abraxas mRNA levels. METHODOLOGY/PRINCIPAL FINDINGS: We treated 123 metastatic non-squamous cell lung carcinoma patients using a customized approach. RNA and DNA were isolated from microdissected specimens from paraffin-embedded tumor tissue. Patients with EGFR mutations received erlotinib, and those without EGFR mutations received chemotherapy with or without cisplatin based on their BRCA1 mRNA levels: low, cisplatin plus gemcitabine; intermediate, cisplatin plus docetaxel; high, docetaxel alone. An exploratory analysis examined RAP80 and Abraxas expression. Median survival exceeded 28 months for 12 patients with EGFR mutations, and was 11 months for 38 patients with low BRCA1, 9 months for 40 patients with intermediate BRCA1, and 11 months for 33 patients with high BRCA1. Two-year survival was 73.3%, 41.2%, 15.6% and 0%, respectively. Median survival was influenced by RAP80 expression in the three BRCA1 groups. For example, for patients with both low BRCA1 and low RAP80, median survival exceeded 26 months. RAP80 was a significant factor for survival in patients treated according to BRCA1

  13. Identification of novel driver mutations of the discoidin domain receptor 2 (DDR2) gene in squamous cell lung cancer of Chinese patients

    International Nuclear Information System (INIS)

    Miao, Liyun; Zhang, Deping; Liu, Hongbing; Song, Yong; Wang, Yongsheng; Zhu, Suhua; Shi, Minke; Li, Yan; Ding, Jingjing; Yang, Jun; Ye, Qing; Cai, Hourong

    2014-01-01

    Although many of the recently approved genomically targeted therapies have improved outcomes for patients in non–small-cell lung cancer (NSCLC) with lung adenocarcinoma, little is known about the genomic alterations that drive lung squamous cell cancer (SCC) and development of effective targeted therapies in lung SCC is a promising area to be further investigated. Discoidin domain receptor 2 (DDR2), is a novel receptor tyrosine kinases that respond to several collagens and involved in tissue repair, primary and metastatic cancer progression. Expression of DDR2 mRNA was analyzed in 54 lung SCC tissues by qRT-PCR. Over-expression approaches were used to investigate the biological functions of DDR2 and its’ mutations in lung SCC cells. Conventional Sanger sequencing was used to investigate the mutations of DDR2 gene in 86 samples. The effect of DDR2 and its’ mutations on proliferation was evaluated by MTT and colony formation assays; cell migration and invasion was evaluated by trasnwell assays. Lung SCC cells stably transfected with pEGFP-DDR2 WT, pEGFP-DDR2-S131C or empty vector were injection into nude mice to study the effect of DDR2 and its’ mutation on tumorigenesis in vivo. Protein and mRNA expression levels of E-cadherin and MMP2 were determined by qRT-PCR and western blot analysis. Differences between groups were tested for significance using Student’s t-test (two-tailed). In this study, we found that DDR2 mRNA levels were significantly decreased in 54 lung SCC tissues compared with normal lung tissues. Moreover, there were 3 novel DDR2 mutations (G531V, S131C, T681I) in 4 patients and provide the mutation rate of 4.6% in the 86 patients with lung SCC. The mutation of S131C in DDR2 could promote lung SCC cells proliferation, migration and invasion via inducing MMP-2, but reducing E-cadherin expression. These data indicated that the novel DDR2 mutation may contribute to the development and progression of lung SCC and this effect may be associated

  14. Propagation testing multi-cell batteries.

    Energy Technology Data Exchange (ETDEWEB)

    Orendorff, Christopher J. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Lamb, Joshua [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Steele, Leigh Anna Marie [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Spangler, Scott Wilmer [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

    2014-10-01

    Propagation of single point or single cell failures in multi-cell batteries is a significant concern as batteries increase in scale for a variety of civilian and military applications. This report describes the procedure for testing failure propagation along with some representative test results to highlight the potential outcomes for different battery types and designs.

  15. Novel somatic mutations in large granular lymphocytic leukemia affecting the STAT-pathway and T-cell activation

    International Nuclear Information System (INIS)

    Andersson, E I; Rajala, H L M; Eldfors, S; Ellonen, P; Olson, T; Jerez, A; Clemente, M J; Kallioniemi, O; Porkka, K; Heckman, C; Loughran, T P Jr; Maciejewski, J P; Mustjoki, S

    2013-01-01

    T-cell large granular lymphocytic (T-LGL) leukemia is a clonal disease characterized by the expansion of mature CD3+CD8+ cytotoxic T cells. It is often associated with autoimmune disorders and immune-mediated cytopenias. Our recent findings suggest that up to 40% of T-LGL patients harbor mutations in the STAT3 gene, whereas STAT5 mutations are present in 2% of patients. In order to identify putative disease-causing genetic alterations in the remaining T-LGL patients, we performed exome sequencing from three STAT mutation-negative patients and validated the findings in 113 large granular lymphocytic (LGL) leukemia patients. On average, 11 CD8+ LGL leukemia cell-specific high-confidence nonsynonymous somatic mutations were discovered in each patient. Interestingly, all patients had at least one mutation that affects either directly the STAT3-pathway (such as PTPRT) or T-cell activation (BCL11B, SLIT2 and NRP1). In all three patients, the STAT3 pathway was activated when studied by RNA expression or pSTAT3 analysis. Screening of the remaining 113 LGL leukemia patients did not reveal additional patients with same mutations. These novel mutations are potentially biologically relevant and represent rare genetic triggers for T-LGL leukemia, and are associated with similar disease phenotype as observed in patients with mutations in the STAT3 gene

  16. A recurrent germline BAP1 mutation and extension of the BAP1 tumor predisposition spectrum to include basal cell carcinoma

    DEFF Research Database (Denmark)

    Wadt, Karin Anna Wallentin; Aoude, L G; Johansson, P

    2015-01-01

    ) and mesothelioma, as previously reported for germline BAP1 mutations. However, mutation carriers from three new families, and one previously reported family, developed basal cell carcinoma (BCC), thus suggesting inclusion of BCC in the phenotypic spectrum of the BAP1 tumor syndrome. This notion is supported...

  17. TP53 mutation and human papilloma virus status of oral squamous cell carcinomas in young adult patients

    NARCIS (Netherlands)

    Braakhuis, B.J.M.; Rietbergen, M.M.; Buijze, M.; Snijders, P.J.F.; Bloemena, E.; Brakenhoff, R.H.; Leemans, C.R.

    2014-01-01

    Objective Little is known about the molecular carcinogenesis of oral squamous cell carcinoma (OSCC) in young adult patients. The aim of this study was to investigate the detailed TP53 mutation and human papilloma virus (HPV) status of OSCC in patients, younger than 45 years. Methods TP53 mutations

  18. Mutation of Chinese hamster cells by near-UV activation of promutagens

    International Nuclear Information System (INIS)

    Barnhart, B.J.; Cox, S.H.

    1980-01-01

    A tissue-culture assay for mutagenesis and cytotoxicity incorporating near ultraviolet (NUV) light activation of polyaromatic hydrocarbons (PAH) has been developed. Cultures of Chinese hamster cells (line CHO) growing in suspension culture were inoculated with benzo[a]pyrene (B[a]P), 7,12-dimethylbenzanthracene (DMBA) or shale-oil retort-water and exposed to light from a high-pressure mercury lamp fitted with a Corning NUV bandpass filter. This light source both permitted activation of PAH and the shale-oil water and precluded detectable damage to DNA. Neither the PAH nor the NUV alone had any effect on cell survival or mutation frequencies but the chemicals plus NUV were extremely effective in producing mutations to 6-thioguanine resistance (hgprt gene). (orig.)

  19. Rhabdomyosarcoma-associated renal cell carcinoma: a link with constitutional Tp53 mutation.

    LENUS (Irish Health Repository)

    Curry, Sarah

    2012-02-01

    The 2004 World Health Organization classification includes the new entity "neuroblastoma-associated renal cell carcinoma." The pathogenetic link between these entities is unknown as yet. The patient reported herein developed renal cell carcinoma after anaplastic embryonal rhabdomyosarcoma, a previously unknown association. The 2nd malignancy developed very soon after the 1st one, prompting concern for inherent cancer predisposition rather than a therapy-induced 2nd malignancy. A variety of features raised suspicion for Tp53 mutation, and indeed a pathogenic germline Tp53 mutation was identified in this child, despite a negative family history for Li-Fraumeni syndrome. Consideration of underlying predisposition is advocated in the context of rapid evolution of 2nd childhood malignancy.

  20. Concomitant BCORL1 and BRAF Mutations in Vemurafenib-Resistant Melanoma Cells

    OpenAIRE

    Luca Mologni; Mariantonia Costanza; Geeta Geeta Sharma; Michela Viltadi; Luca Massimino; Stefania Citterio; Stefania Purgante; Hima Raman; Alessandra Pirola; Massimo Zucchetti; Rocco Piazza; Carlo Gambacorti-Passerini

    2018-01-01

    BRAF is the most frequently mutated gene in melanoma. Constitutive activation of mutant BRAFV600E leads to aberrant Ras-independent MAPK signaling and cell transformation. Inhibition of mutant BRAF is a current frontline therapy for such cases, with improved survival compared with chemotherapy. Unfortunately, reactivation of MAPK signaling by several mechanisms has been shown to cause drug resistance and disease recurrence. In this work, we describe the co-occurrence of an in-frame deletion w...

  1. Optimal Therapeutic Strategy for Non-small Cell Lung Cancer with Mutated Epidermal Growth Factor Receptor

    Directory of Open Access Journals (Sweden)

    Zhong SHI

    2015-02-01

    Full Text Available Although epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs have been widely used in non-small cell lung cancer (NSCLC patients, it is still controversial about how to combine EGFR-TKI with chemotherapy and other targeted drugs. We have made a summary on the current therapeutic models of EGFR-TKI combined with chemotherapy/bevacizumab in this review and aimed to find the optimal therapeutic strategy for NSCLC patients with EGFR mutation.

  2. Optimised Pre-Analytical Methods Improve KRAS Mutation Detection in Circulating Tumour DNA (ctDNA) from Patients with Non-Small Cell Lung Cancer (NSCLC)

    Science.gov (United States)

    Sherwood, James L.; Corcoran, Claire; Brown, Helen; Sharpe, Alan D.; Musilova, Milena; Kohlmann, Alexander

    2016-01-01

    Introduction Non-invasive mutation testing using circulating tumour DNA (ctDNA) is an attractive premise. This could enable patients without available tumour sample to access more treatment options. Materials & Methods Peripheral blood and matched tumours were analysed from 45 NSCLC patients. We investigated the impact of pre-analytical variables on DNA yield and/or KRAS mutation detection: sample collection tube type, incubation time, centrifugation steps, plasma input volume and DNA extraction kits. Results 2 hr incubation time and double plasma centrifugation (2000 x g) reduced overall DNA yield resulting in lowered levels of contaminating genomic DNA (gDNA). Reduced “contamination” and increased KRAS mutation detection was observed using cell-free DNA Blood Collection Tubes (cfDNA BCT) (Streck), after 72 hrs following blood draw compared to EDTA tubes. Plasma input volume and use of different DNA extraction kits impacted DNA yield. Conclusion This study demonstrated that successful ctDNA recovery for mutation detection in NSCLC is dependent on pre-analytical steps. Development of standardised methods for the detection of KRAS mutations from ctDNA specimens is recommended to minimise the impact of pre-analytical steps on mutation detection rates. Where rapid sample processing is not possible the use of cfDNA BCT tubes would be advantageous. PMID:26918901

  3. Optimised Pre-Analytical Methods Improve KRAS Mutation Detection in Circulating Tumour DNA (ctDNA from Patients with Non-Small Cell Lung Cancer (NSCLC.

    Directory of Open Access Journals (Sweden)

    James L Sherwood

    Full Text Available Non-invasive mutation testing using circulating tumour DNA (ctDNA is an attractive premise. This could enable patients without available tumour sample to access more treatment options.Peripheral blood and matched tumours were analysed from 45 NSCLC patients. We investigated the impact of pre-analytical variables on DNA yield and/or KRAS mutation detection: sample collection tube type, incubation time, centrifugation steps, plasma input volume and DNA extraction kits.2 hr incubation time and double plasma centrifugation (2000 x g reduced overall DNA yield resulting in lowered levels of contaminating genomic DNA (gDNA. Reduced "contamination" and increased KRAS mutation detection was observed using cell-free DNA Blood Collection Tubes (cfDNA BCT (Streck, after 72 hrs following blood draw compared to EDTA tubes. Plasma input volume and use of different DNA extraction kits impacted DNA yield.This study demonstrated that successful ctDNA recovery for mutation detection in NSCLC is dependent on pre-analytical steps. Development of standardised methods for the detection of KRAS mutations from ctDNA specimens is recommended to minimise the impact of pre-analytical steps on mutation detection rates. Where rapid sample processing is not possible the use of cfDNA BCT tubes would be advantageous.

  4. Detection of EGFR somatic mutations in non-small cell lung cancer (NSCLC) using a novel mutant-enriched liquidchip (MEL) technology.

    Science.gov (United States)

    Zhang, Li; Yang, Huiyi; Zhao, Yanwei; Liu, Wenchao; Wu, Shiyang; He, Jiaying; Luo, Xiaodi; Zhu, Zeyao; Xu, Jiasen; Zhou, Qinghua; Ren-Heidenreich, Lifen

    2012-09-01

    We have developed and standardized a novel technology, mutant-enriched liquidchip (MEL), for clinical detection of EGFR mutations. The MEL integrates a mutant-enriched PCR procedure with liquidchip technology for detections of EGFR exon 19 deletions and L858R mutation on both formalin-fixed and paraffin-embedded (FFPE) slides and plasma samples from patients with non-small cell lung cancer (NSCLC). The detection sensitivity was 0.1% of mutant DNA in the presence of its wild-type DNA. The cross-reaction rate was lower than 5%. To evaluate the MEL platform, the EGFR mutation status of 59 patients with advanced NSCLC treated with EGFRTKIs (Tyrosine Kinase Inhibitors) were tested on their FFPE samples. EGFR exon 19 deletions and L858R were detected in 21 patients (21/59) and 76.2% (16/21) of them had partial response to the EGFR-TKIs, while by sequencing method, only 4 (4/59) mutations were detected. Plasma samples from 627 patients with various stages of NSCLC were examined with the MEL and 22% of EGFR exon 19 deletions and L858R were detected. Furthermore, in patients with advanced disease there are more mutations detected in plasma samples than in patients with less advanced disease. In conclusion, the MEL is a sensitive, stable, and robust technology for detecting EGFR DNA mutations from both FFPE and plasma samples from patients with NSCLC and is now routinely used for clinical diagnosis.

  5. Parametric Sensitivity Tests- European PEM Fuel Cell Stack Test Procedures

    DEFF Research Database (Denmark)

    Araya, Samuel Simon; Andreasen, Søren Juhl; Kær, Søren Knudsen

    2014-01-01

    performed based on test procedures proposed by a European project, Stack-Test. The sensitivity of a Nafion-based low temperature PEMFC stack’s performance to parametric changes was the main objective of the tests. Four crucial parameters for fuel cell operation were chosen; relative humidity, temperature......As fuel cells are increasingly commercialized for various applications, harmonized and industry-relevant test procedures are necessary to benchmark tests and to ensure comparability of stack performance results from different parties. This paper reports the results of parametric sensitivity tests......, pressure, and stoichiometry at varying current density. Furthermore, procedures for polarization curve recording were also tested both in ascending and descending current directions....

  6. Minisatellite mutations and retrospective biodosimetry of population living close to the Semipalatinsk nuclear test site

    International Nuclear Information System (INIS)

    Lindholm, C.; Bersimbaev, R.I.; Dubrova, Y.E. EI KAUP; EI MAATA

    2003-01-01

    During the period between 1949 and 1989 nuclear weapon testing carried out at the Semipalatinsk Nuclear Test Site (STS) resulted in local fallout affecting the residents of Semipalatinsk, East Kazakhstan and Pavlodar districts of Kazakhstan and Altai region of Russia. The Semipalatinsk nuclear polygon in Kazakhstan has been the site for 470 nuclear tests, including 26 tests performed on the ground and 87 in the atmosphere. More than 1.5 million people living in the vicinity of the test site were repeatedly exposed to ionizing radiation. The paper reviews the study where the main objectives are: (1) to establish a biosample database of blood samples of families in three generations living close to the STS and control families in three generations from clean areas, (2) to determine the minisatellite mutation rates in the three generations of exposed people and the control families of the same ethinic origin living in non-contaminated areas, and (3) to determine the chromosomal translocation frequencies by FISH chromosome painting in the lymphocytes of the exposed and the control people in order to determine the radiation exposure. The aim of the study was to select the population living near to the STS and subjected to the greatest radiation exposure. Of particular interest was the first test of 29th of August 1949, as this was reported to have caused heavy fallout along a narrow trajectory extending north-east from Polygon, also covering parts of the Altai region of Russia and parts of Pavlodar and Karaganda regions in Kazakhstan

  7. Cost effectiveness of population based BRCA1 founder mutation testing in Sephardi Jewish women.

    Science.gov (United States)

    Patel, Shreeya; Legood, Rosa; Evans, D Gareth; Turnbull, Clare; Antoniou, Antonis C; Menon, Usha; Jacobs, Ian; Manchanda, Ranjit

    2018-04-01

    Population-based BRCA1/BRCA2 founder-mutation testing has been demonstrated as cost effective compared with family history based testing in Ashkenazi Jewish women. However, only 1 of the 3 Ashkenazi Jewish BRCA1/BRCA2 founder mutations (185delAG[c.68_69delAG]), 5382insC[c.5266dupC]), and 6174delT[c.5946delT]) is found in the Sephardi Jewish population (185delAG[c.68_69delAG]), and the overall prevalence of BRCA mutations in the Sephardi Jewish population is accordingly lower (0.7% compared with 2.5% in the Ashkenazi Jewish population). Cost-effectiveness analyses of BRCA testing have not previously been performed at these lower BRCA prevalence levels seen in the Sephardi Jewish population. Here we present a cost-effectiveness analysis for UK and US populations comparing population testing with clinical criteria/family history-based testing in Sephardi Jewish women. A Markov model was built comparing the lifetime costs and effects of population-based BRCA1 testing, with testing using family history-based clinical criteria in Sephardi Jewish women aged ≥30 years. BRCA1 carriers identified were offered magnetic resonance imaging/mammograms and risk-reducing surgery. Costs are reported at 2015 prices. Outcomes include breast cancer, ovarian cancer, and excess deaths from heart disease. All costs and outcomes are discounted at 3.5%. The time horizon is lifetime, and perspective is payer. The incremental cost-effectiveness ratio per quality-adjusted life-year was calculated. Parameter uncertainty was evaluated through 1-way and probabilistic sensitivity analysis. Population testing resulted in gain in life expectancy of 12 months (quality-adjusted life-year = 1.00). The baseline discounted incremental cost-effectiveness ratio for UK population-based testing was £67.04/quality-adjusted life-year and for US population was $308.42/quality-adjusted life-year. Results were robust in the 1-way sensitivity analysis. The probabilistic sensitivity analysis showed 100% of

  8. Concomitant BCORL1 and BRAF Mutations in Vemurafenib-Resistant Melanoma Cells.

    Science.gov (United States)

    Mologni, Luca; Costanza, Mariantonia; Sharma, Geeta Geeta; Viltadi, Michela; Massimino, Luca; Citterio, Stefania; Purgante, Stefania; Raman, Hima; Pirola, Alessandra; Zucchetti, Massimo; Piazza, Rocco; Gambacorti-Passerini, Carlo

    2018-05-01

    BRAF is the most frequently mutated gene in melanoma. Constitutive activation of mutant BRAF V600E leads to aberrant Ras-independent MAPK signaling and cell transformation. Inhibition of mutant BRAF is a current frontline therapy for such cases, with improved survival compared with chemotherapy. Unfortunately, reactivation of MAPK signaling by several mechanisms has been shown to cause drug resistance and disease recurrence. In this work, we describe the co-occurrence of an in-frame deletion within an amplified BRAF V600E locus and a missense point mutation of the transcriptional repressor BCORL1 in vemurafenib-resistant A375 melanoma cells. Functional data confirmed that truncated p47BRAF V600E and mutant BCORL1 Q1076H both contribute to resistance. Interestingly, either endogenous BCORL1 silencing or ectopic BCORL1 Q1076H expression mimicked the effects of a CRISPR/Cas9-edited BCORL1 Q1076H locus, suggesting a complex mixture of loss- and gain-of-function effects caused by the mutation. Transcriptomic data confirmed this hypothesis. Finally, we show that the pan-RAF inhibitor sorafenib is not affected by expression of BRAF deletion variant and effectively synergizes with vemurafenib to block resistant cells, suggesting a possible intervention for this class of mutants. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Concomitant BCORL1 and BRAF Mutations in Vemurafenib-Resistant Melanoma Cells

    Directory of Open Access Journals (Sweden)

    Luca Mologni

    2018-05-01

    Full Text Available BRAF is the most frequently mutated gene in melanoma. Constitutive activation of mutant BRAFV600E leads to aberrant Ras-independent MAPK signaling and cell transformation. Inhibition of mutant BRAF is a current frontline therapy for such cases, with improved survival compared with chemotherapy. Unfortunately, reactivation of MAPK signaling by several mechanisms has been shown to cause drug resistance and disease recurrence. In this work, we describe the co-occurrence of an in-frame deletion within an amplified BRAFV600E locus and a missense point mutation of the transcriptional repressor BCORL1 in vemurafenib-resistant A375 melanoma cells. Functional data confirmed that truncated p47BRAFV600E and mutant BCORL1Q1076H both contribute to resistance. Interestingly, either endogenous BCORL1 silencing or ectopic BCORL1Q1076H expression mimicked the effects of a CRISPR/Cas9-edited BCORL1Q1076H locus, suggesting a complex mixture of loss- and gain-of-function effects caused by the mutation. Transcriptomic data confirmed this hypothesis. Finally, we show that the pan-RAF inhibitor sorafenib is not affected by expression of BRAF deletion variant and effectively synergizes with vemurafenib to block resistant cells, suggesting a possible intervention for this class of mutants.

  10. Effects of smoke and tea on radiation-induced bone marrow cell mutation and marrow inhibition

    International Nuclear Information System (INIS)

    Gao Yong; Zhang Weiguang

    2004-01-01

    Objective: To provide scientific information for the prevention and treatment of the radiation damage by analyzing the effects of smoke and tea on radiation-induced bone marrow cell mutation and marrow inhibition. Methods: 7 group mice were exposed to smoke and/or tea and/or radiation respectively. There were also b blank control group and a cyclophosphamide positive control group. The frequencies of micronucleated polychromatic erythrocytes (MPCE), the ratio of polychromatic erythrocytes (PCE) to mature erythrocytes (RBC) in marrow, and the count of peripheral blood hemoleukocyte were observed. Results: The frequencies of MPCE in the groups irradiated with γ-rays were significantly higher than that in the blank control group (P<0.05 or 0.01). The smoke + radiation group's frequency was significantly higher than single radiation group (P<0.05). The ratios of PCE to RBC in the groups irradiated were significantly lower than that in the blank control group (P<0.01). The counts of peripheral blood hemoleukocyte in the groups irradiated were significantly lower than the blank control group (P<0.01). Conclusion: Radiation were able to cause marrow cell mutation and induce marrow inhibition. Smoke increases the effect of radiation-induced marrow cell mutation. Tea and smoke could not affect radiation-induced bone marrow inhibition

  11. Radiation-induced cell mutations as a function of dose rate

    International Nuclear Information System (INIS)

    Kiefer, J.

    1987-01-01

    A brief review of the data in the literature is presented and forms the background of the experimental data given by the author obtained with exponential long-term cultures of V79 hamster cells exposed over a period of up to 35 days to different dose rates of gamma radiation. The experimental results show that at a dose rate of 40 mGy/hour the number of induced mutations is reduced, - which is in agreement with literature data - , but a dose rate of less than 30 mGy/hour makes the induced mutations leap to a value clearly higher than those induced by acute irradiation. As in addition to the mutations recombination is a significant factor of the radiation risk, experiments with a heterozygotic yeast strain have been made, as there is to date no reliable mammalian cell system available for this kind of research. Long-term radiation exposure of the yeast cells over a period of six weeks drastically increased the rate of recombinations, to a value higher by a factor of about 4 than that induced by acute irradiation. (orig.) [de

  12. MO-DE-207B-01: JACK FOWLER JUNIOR INVESTIGATOR COMPETITION WINNER: Between Somatic Mutations and PET-Based Radiomic Features in Non-Small Cell Lung Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Yip, S; Coroller, T; Rios Velazquez, E; Parmar, C; Mak, R; Aerts, H [Brigham and Women’s Hospital, Dana Farber Cancer Institute, and Harvard Medical School, Boston, MA (United States); Kim, J [Brigham and Women’s Hospital, Boston Children’s Hospital and Harvard Medical School, Boston, MA (United States)

    2016-06-15

    Purpose: Although PET-based radiomic features have been proposed to quantify tumor heterogeneity and shown promise in outcome prediction, little is known about their relationship with tumor genetics. This study assessed the association of [{sup 18}F]fluorodeoxyglucose (FDG)-PET-based radiomic features with non-small cell lung cancer (NSCLC) mutations. Methods: 348 NSCLC patients underwent FDG-PET/CT scans before treatment and were tested for genetic mutations. 13% (44/348) and 28% (96/348) patients were found to harbor EGFR (EGFR+) and KRAS (KRAS+) mutations, respectively. We evaluated nineteen PET-based radiomic features quantifying phenotypic traits, and compared them with conventional PET features (metabolic tumor volume (MTV) and maximum-SUV). The association between the feature values and mutation status was evaluated using the Wilcoxcon-rank-sum-test. The ability of each measure to predict mutations was assessed by the area under the receiver operating curve (AUC). Noether’s test was used to determine if the AUCs were significantly from random (AUC=0.50). All p-values were corrected for multiple testing by controlling the false discovery rate (FDR{sub Wilcoxon} and FDR{sub Noether}) of 10%. Results: Eight radiomic features, MTV, and maximum-SUV, were significantly associated with the EGFR mutation (FDR{sub Wilcoxon}=0.01–0.10). However, KRAS+ demonstrated no significantly distinctive imaging features compared to KRAS− (FDR{sub Wilcoxon}≥0.92). EGFR+ and EGFR− were significantly discriminated by conventional PET features (AUC=0.61, FDR{sub Noether}=0.04 for MTV and AUC=0.64, FDR{sub Noether}=0.01 for maximum-SUV). Eight radiomic features were significantly predictive for EGFR+ compared to EGFR− (AUC=0.59–0.67, FDR{sub Noether}=0.0032–0.09). Normalized-inverse-difference-moment outperformed all features in predicting EGFR mutation (AUC=0.67, FDR{sub Noether}=0.0032). Moreover, only the radiomic feature normalized-inverse-difference-moment could

  13. Differential effects of genistein on prostate cancer cells depend on mutational status of the androgen receptor.

    Directory of Open Access Journals (Sweden)

    Abeer M Mahmoud

    Full Text Available Blocking the androgen receptor (AR activity is the main goal of therapies for advanced prostate cancer (PCa. However, relapse with a more aggressive, hormone refractory PCa arises, which harbors restored AR activity. One mechanism of such reactivation occurs through acquisition of AR mutations that enable its activation by various steroidal and non-steroidal structures. Thus, natural and chemical compounds that contribute to inappropriate (androgen-independent activation of the AR become an area of intensive research. Here, we demonstrate that genistein, a soy phytoestrogen binds to both the wild and the Thr877Ala (T877A mutant types of AR competitively with androgen, nevertheless, it exerts a pleiotropic effect on PCa cell proliferation and AR activity depending on the mutational status of the AR. Genistein inhibited, in a dose-dependent way, cell proliferation and AR nuclear localization and expression in LAPC-4 cells that have wild AR. However, in LNCaP cells that express the T877A mutant AR, genistein induced a biphasic effect where physiological doses (0.5-5 µmol/L stimulated cell growth and increased AR expression and transcriptional activity, and higher doses induced inhibitory effects. Similar biphasic results were achieved in PC-3 cells transfected with AR mutants; T877A, W741C and H874Y. These findings suggest that genistein, at physiological concentrations, potentially act as an agonist and activate the mutant AR that can be present in advanced PCa after androgen ablation therapy.

  14. The occurrence and significance of V gene mutations in B cell-derived human malignancy.

    Science.gov (United States)

    Stevenson, F K; Sahota, S S; Ottensmeier, C H; Zhu, D; Forconi, F; Hamblin, T J

    2001-01-01

    The classification of B cell tumors has relevance for refining and improving clinical strategies. However, consensus has been difficult to establish, and although a scheme is now available, objective criteria are desirable. Genetic technology will underpin and extend current knowledge, and it is certain to reveal further subdivisions of current tumor categories. The Ig variable region genes of B cell tumors present a considerable asset for this area of investigation. The unique sequences carried in neoplastic B cells are easily isolated and sequenced. In addition to acting as clone-specific markers of each tumor, they indicate where the cell has come from and track its history following transformation. There is emerging clinical value in knowing whether the cell of origin has encountered antigen and has moved from the naive compartment to the germinal center, where somatic mutation is activated. This is amply illustrated by the subdivision of chronic lymphocytic leukemia into two subsets, unmutated or mutated, each with very different prognosis. Other tumors may be subdivided in a similar way. Microarray technology is developing rapidly to probe gene expression and to further divide tumor categories. All these genetic analyses will provide objective data to enhance both our understanding of B cell tumors and our ability to treat them.

  15. The role of IDH1 mutated tumour cells in secondary glioblastomas: an evolutionary game theoretical view

    Science.gov (United States)

    Basanta, David; Scott, Jacob G.; Rockne, Russ; Swanson, Kristin R.; Anderson, Alexander R. A.

    2011-02-01

    Recent advances in clinical medicine have elucidated two significantly different subtypes of glioblastoma which carry very different prognoses, both defined by mutations in isocitrate dehydrogenase-1 (IDH-1). The mechanistic consequences of this mutation have not yet been fully clarified, with conflicting opinions existing in the literature; however, IDH-1 mutation may be used as a surrogate marker to distinguish between primary and secondary glioblastoma multiforme (sGBM) from malignant progression of a lower grade glioma. We develop a mathematical model of IDH-1 mutated secondary glioblastoma using evolutionary game theory to investigate the interactions between four different phenotypic populations within the tumor: autonomous growth, invasive, glycolytic, and the hybrid invasive/glycolytic cells. Our model recapitulates glioblastoma behavior well and is able to reproduce two recent experimental findings, as well as make novel predictions concerning the rate of invasive growth as a function of vascularity, and fluctuations in the proportions of phenotypic populations that a glioblastoma will experience under different microenvironmental constraints.

  16. The role of IDH1 mutated tumour cells in secondary glioblastomas: an evolutionary game theoretical view

    International Nuclear Information System (INIS)

    Basanta, David; Scott, Jacob G; Anderson, Alexander R A; Rockne, Russ; Swanson, Kristin R

    2011-01-01

    Recent advances in clinical medicine have elucidated two significantly different subtypes of glioblastoma which carry very different prognoses, both defined by mutations in isocitrate dehydrogenase-1 (IDH-1). The mechanistic consequences of this mutation have not yet been fully clarified, with conflicting opinions existing in the literature; however, IDH-1 mutation may be used as a surrogate marker to distinguish between primary and secondary glioblastoma multiforme (sGBM) from malignant progression of a lower grade glioma. We develop a mathematical model of IDH-1 mutated secondary glioblastoma using evolutionary game theory to investigate the interactions between four different phenotypic populations within the tumor: autonomous growth, invasive, glycolytic, and the hybrid invasive/glycolytic cells. Our model recapitulates glioblastoma behavior well and is able to reproduce two recent experimental findings, as well as make novel predictions concerning the rate of invasive growth as a function of vascularity, and fluctuations in the proportions of phenotypic populations that a glioblastoma will experience under different microenvironmental constraints

  17. CP-31398 inhibits the growth of p53-mutated liver cancer cells in vitro and in vivo.

    Science.gov (United States)

    He, Xing-Xing; Zhang, Yu-Nan; Yan, Jun-Wei; Yan, Jing-Jun; Wu, Qian; Song, Yu-Hu

    2016-01-01

    The tumor suppressor p53 is one of the most frequently mutated genes in hepatocellular carcinoma (HCC). Previous studies demonstrated that CP-31398 restored the native conformation of mutant p53 and trans-activated p53 downstream genes in tumor cells. However, the research on the application of CP-31398 to liver cancer has not been reported. Here, we investigated the effects of CP-31398 on the phenotype of HCC cells carrying p53 mutation. The effects of CP-31398 on the characteristic of p53-mutated HCC cells were evaluated through analyzing cell cycle, cell apoptosis, cell proliferation, and the expression of p53 downstream genes. In tumor xenografts developed by PLC/PRF/5 cells, the inhibition of tumor growth by CP-31398 was analyzed through gross morphology, growth curve, and the expression of p53-related genes. Firstly, we demonstrated that CP-31398 inhibited the growth of p53-mutated liver cancer cells in a dose-dependent and p53-dependent manner. Then, further study showed that CP-31398 re-activated wild-type p53 function in p53-mutated HCC cells, which resulted in inhibitive response of cell proliferation and an induction of cell-cycle arrest and apoptosis. Finally, in vivo data confirmed that CP-31398 blocked the growth of xenografts tumors through transactivation of p53-responsive downstream molecules. Our results demonstrated that CP-31398 induced desired phenotypic change of p53-mutated HCC cells in vitro and in vivo, which revealed that CP-31398 would be developed as a therapeutic candidate for HCC carrying p53 mutation.

  18. IgV H mutations in blastoid mantle cell lymphoma characterize a subgroup with a tendency to more favourable clinical outcome.

    Science.gov (United States)

    Cogliatti, Sergio B; Bertoni, Francesco; Zimmermann, Dieter R; Henz, Samuel; Diss, Tim C; Ghielmini, Michele; Schmid, Ulrico

    2005-07-01

    Mantle cell lymphoma (MCL) is associated with a very unfavourable clinical course. This is particularly true for mantle cell lymphoma of the blastoid subtype (MCL-b). In order to define prognostic factors, we analysed the impact of immunoglobulin heavy chain variable (IgV H) gene somatic hypermutations on clinical outcome in a series of 21 cases of morphologically, phenotypically, and genotypically well-characterized MCL-b. Testing and estimation were performed using log-rank statistics and displayed on Kaplan-Meier graphs. Thirteen of 21 cases of MCL-b revealed a homology rate of > or = 99% compared to IgV H germ-line sequences in the databases and were scored as non-mutated. Eight of 21 cases (38%) of MCL-b were mutated. In MCL-b the mutation frequency was usually low and the mutation pattern was only rarely antigen-selected, in contrast to a control group of 11 cases with morphologically almost identical, but phenotypically and genotypically clearly distinguishable, diffuse large B cell lymphoma, derived, most likely, from germinal centre B cells. In our series of 21 MCL-b, positive IgV H mutational status, irrespective of varying homology thresholds, had no statistically significant prognostic impact on event-free or overall survival. However, mutated MCL-b tended to present more frequently at an earlier stage and without bone marrow involvement and to show lower rates of relapse and death, resulting in a more favourable clinical outcome. Copyright 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  19. Iodine Absorption Cells Purity Testing

    Czech Academy of Sciences Publication Activity Database

    Hrabina, Jan; Zucco, M.; Philippe, Ch.; Pham, Minh Tuan; Holá, Miroslava; Acef, O.; Lazar, Josef; Číp, Ondřej

    2017-01-01

    Roč. 17, č. 1 (2017), s. 1-13, č. článku 17010102. ISSN 1424-8220 R&D Projects: GA ČR GA15-18430S; GA MŠk(CZ) LO1212; GA MŠk ED0017/01/01 Institutional support: RVO:68081731 Keywords : iodine cells * absorption spectroscopy * laser spectroscopy * laser standards * frequency stability Subject RIV: BH - Optics, Masers, Lasers OBOR OECD: Optics (including laser optics and quantum optics) Impact factor: 2.677, year: 2016

  20. In utero exposure to nanosized carbon black (Printex90) does not induce tandem repeat mutations in female murine germ cells

    DEFF Research Database (Denmark)

    Boisen, Anne Mette Zenner; Shipley, Thomas; Jackson, Petra

    2013-01-01

    Inhalation of particles has been shown to induce mutations in the male germline in mice following both prenatal and adult exposures in several experiments. In contrast, the effects of particles on female germ cell mutagenesis are not well established. Germline mutations are induced during active...... cell division, which occurs during fetal development in females. We investigated the effects of prenatal exposure to carbon black nanoparticles (CB) on induction of mutations in the female mouse germline during fetal development, spanning the critical developmental stages of oogenesis. Pregnant C57BL/6...... mutation rates in the resulting F2 generation were determined from full pedigrees (mother, father, offspring) of F1 female mice (178 CB-exposed and 258 control F2 offspring). ESTR mutation rates in CB-exposed F2 female offspring were not statistically different from those of F2 female control offspring....

  1. Genetic Testing and Post-Testing Decision Making among BRCA-Positive Mutation Women: A Psychosocial Approach.

    Science.gov (United States)

    Hesse-Biber, Sharlene; An, Chen

    2016-10-01

    Through an analysis of an online survey of women who tested positive for the BRCA genetic mutation for breast cancer, this research uses a social constructionist and feminist standpoint lens to understand the decision-making process that leads BRCA-positive women to choose genetic testing. Additionally, this research examines how they socially construct and understand their risk for developing breast cancer, as well as which treatment options they undergo post-testing. BRCA-positive women re-frame their statistical medical risk for developing cancer and their post-testing treatment choices through a broad psychosocial context of engagement that also includes their social networks. Important psychosocial factors drive women's medical decisions, such as individual feelings of guilt and vulnerability, and the degree of perceived social support. Women who felt guilty and fearful that they might pass the BRCA gene to their children were more likely to undergo risk reducing surgery. Women with at least one daughter and women without children were more inclined toward the risk reducing surgery compared to those with only sons. These psychosocial factors and social network engagements serve as a "nexus of decision making" that does not, for the most part, mirror the medical assessments of statistical odds for hereditary cancer development, nor the specific treatment protocols outlined by the medical establishment.

  2. High-throughput in vivo genotoxicity testing: an automated readout system for the somatic mutation and recombination test (SMART.

    Directory of Open Access Journals (Sweden)

    Benoit Lombardot

    Full Text Available Genotoxicity testing is an important component of toxicity assessment. As illustrated by the European registration, evaluation, authorization, and restriction of chemicals (REACH directive, it concerns all the chemicals used in industry. The commonly used in vivo mammalian tests appear to be ill adapted to tackle the large compound sets involved, due to throughput, cost, and ethical issues. The somatic mutation and recombination test (SMART represents a more scalable alternative, since it uses Drosophila, which develops faster and requires less infrastructure. Despite these advantages, the manual scoring of the hairs on Drosophila wings required for the SMART limits its usage. To overcome this limitation, we have developed an automated SMART readout. It consists of automated imaging, followed by an image analysis pipeline that measures individual wing genotoxicity scores. Finally, we have developed a wing score-based dose-dependency approach that can provide genotoxicity profiles. We have validated our method using 6 compounds, obtaining profiles almost identical to those obtained from manual measures, even for low-genotoxicity compounds such as urethane. The automated SMART, with its faster and more reliable readout, fulfills the need for a high-throughput in vivo test. The flexible imaging strategy we describe and the analysis tools we provide should facilitate the optimization and dissemination of our methods.

  3. The effects of radiation on p53-mutated glioma cells using cDNA microarray technique

    International Nuclear Information System (INIS)

    Ngo, F.Q.H.; Hsiao, Y.-Y.H.

    2003-01-01

    Full text: In this study, we investigated the effects of 10-Gy irradiation on cell-cycle arrest, apoptosis and clonogenic death in the p53-mutated human U138MG (malignant glioblastoma) cell line. In order to evaluate time-dependent events in cellular responses to radiation, we did a time course study by incubating cells ranging from 0.5 to 48 hours after irradiation. Cell-cycle distribution and apoptosis were evaluated by flow cytometry using propidium iodide (PI) and annexin-V plus PI staining. Cell viability and proliferative capacity were studied by colony formation assay. Dual fluorescence cDNA microarray technique was used to examine the differential expression patterns of the irradiated cells. The cDNA microarray chips used contained DNA sequences corresponding to 12,814 human genes. From the flow cytometry data, it can be observed that radiation induced G2/M phase arrest and that late apoptosis was more evident following G2/M arrest. After 36 hours, some cells underwent senescence and the remains continued on with the cell cycle. Microarray analyses revealed changes in the expression of a small number of cell-cycle-related genes (p21, cyclin B1, etc.) and cell-death genes (tumor necrosis factors, DDB2, etc.) suggesting their involvement in radiation-induced cell-cycle arrest and apoptosis. In silico interpretations of the molecular mechanisms responsible for these radiation effects are in progress

  4. Postirradiation expression of lethal mutations in an immortalized human keratinocyte cell line

    International Nuclear Information System (INIS)

    O'Reilly, S.; Mothersill, C.; Seymour, C.B.

    1994-01-01

    The quantification of the extent of delayed cell death and the rate and pattern of its occurrence in relation to the cell division cycle is important in radiotherapy and also in radiation transformation studies related to protection and dose limits. Here the numbers of lethal mutations occurring over 45 population doublings (clonal expansion to about 10 13 cells per cell originally surviving irradiation) was measured in an HPV 16 immortalized human keratinocyte cell lines used for transformation studies. The results showed that when postirradiation (dose range 1-6 Gy) growth curves were constructed, the difference in slopes could be accounted for entirely by correcting for the non-clonogenic fraction in the cell count, excluding a longer cell generation time as an explanation. When the cell loss was examined over the entire growth period of 6 weeks (about 45 doublings of the cell population), it was found to be dose dependent for the first two passages, but then to become more independent of dose. The results allow a time/cell generation dependent factor to be derived for the cell line and used in survival curve equations where effects of radiation are being measured at times distant from the original exposure. (author)

  5. Role of [18F]FDG PET in prediction of KRAS and EGFR mutation status in patients with advanced non-small-cell lung cancer

    International Nuclear Information System (INIS)

    Caicedo, Carlos; Garcia-Velloso, Maria Jose; Vigil Diaz, Carmen; Richter Echevarria, Jose Angel; Lozano, Maria Dolores; Labiano, Tania; Lopez-Picazo, Jose Maria; Gurpide, Alfonso; Perez Gracia, Jose Luis; Zulueta, Javier

    2014-01-01

    The tumour molecular profile predicts the activity of epidermal growth factor receptor (EGFR) inhibitors in non-small-cell lung cancer (NSCLC). However, tissue availability and tumour heterogeneity limit its assessment. We evaluated whether [ 18 F]FDG PET might help predict KRAS and EFGR mutation status in NSCLC. Between January 2005 and October 2011, 340 NSCLC patients were tested for KRAS and EGFR mutation status. We identified patients with stage III and IV disease who had undergone [ 18 F]FDG PET/CT scanning for initial staging. SUVpeak, SUVmax and SUVmean of the single hottest tumour lesions were calculated, and their association with KRAS and EGFR mutation status was assessed. A receiver operator characteristic (ROC) curve analysis and a multivariate analysis (including SUVmean, gender, age and AJCC stage) were performed to identify the potential value of [ 18 F]FDG PET/CT for predicting KRAS mutation. From 102 patients staged using [ 18 F]FDG PET/CT, 28 (27 %) had KRAS mutation (KRAS+), 22 (22 %) had EGFR mutation (EGFR+) and 52 (51 %) had wild-type KRAS and EGFR profiles (WT). KRAS+ patients showed significantly higher [ 18 F]FDG uptake than EGFR+ and WT patients (SUVmean 9.5, 5.7 and 6.6, respectively; p 18 F]FDG uptake between EGFR+ patients and WT patients. ROC curve analysis for KRAS mutation status discrimination yielded an area under the curve of 0.740 for SUVmean (p 18 F]FDG uptake than WT patients, as assessed in terms of SUVpeak, SUVmax and SUVmean. A multivariate model based on age, gender, AJCC stage and SUVmean might be used as a predictive marker of KRAS mutation status in patients with stage III or IV NSCLC. (orig.)

  6. Predictive efficacy of low burden EGFR mutation detected by next-generation sequencing on response to EGFR tyrosine kinase inhibitors in non-small-cell lung carcinoma.

    Directory of Open Access Journals (Sweden)

    Hye Sook Kim

    Full Text Available Direct sequencing remains the most widely used method for the detection of epidermal growth factor receptor (EGFR mutations in lung cancer; however, its relatively low sensitivity limits its clinical use. The objective of this study was to investigate the sensitivity of detecting an epidermal growth factor receptor (EGFR mutation from peptide nucleic acid-locked nucleic acid polymerase chain reaction (PNA-LNA PCR clamp and Ion Torrent Personal Genome Machine (PGM techniques compared to that by direct sequencing. Furthermore, the predictive efficacy of EGFR mutations detected by PNA-LNA PCR clamp was evaluated. EGFR mutational status was assessed by direct sequencing, PNA-LNA PCR clamp, and Ion Torrent PGM in 57 patients with non-small cell lung cancer (NSCLC. We evaluated the predictive efficacy of PNA-LNA PCR clamp on the EGFR-TKI treatment in 36 patients with advanced NSCLC retrospectively. Compared to direct sequencing (16/57, 28.1%, PNA-LNA PCR clamp (27/57, 47.4% and Ion Torrent PGM (26/57, 45.6% detected more EGFR mutations. EGFR mutant patients had significantly longer progressive free survival (14.31 vs. 21.61 months, P = 0.003 than that of EGFR wild patients when tested with PNA-LNA PCR clamp. However, no difference in response rate to EGFR TKIs (75.0% vs. 82.4%, P = 0.195 or overall survival (34.39 vs. 44.10 months, P = 0.422 was observed between the EGFR mutations by direct sequencing or PNA-LNA PCR clamp. Our results demonstrate firstly that patients with EGFR mutations were detected more frequently by PNA-LNA PCR clamp and Ion Torrent PGM than those by direct sequencing. EGFR mutations detected by PNA-LNA PCR clamp may be as a predicative factor for EGFR TKI response in patients with NSCLC.

  7. The Oncogenic Roles of DICER1 RNase IIIb Domain Mutations in Ovarian Sertoli-Leydig Cell Tumors

    Directory of Open Access Journals (Sweden)

    Yemin Wang

    2015-08-01

    Full Text Available DICER1, an endoribonuclease required for microRNA (miRNA biogenesis, is essential for embryogenesis and the development of many organs including ovaries. We have recently identified somatic hotspot mutations in RNase IIIb domain of DICER1 in half of ovarian Sertoli-Leydig cell tumors, a rare class of sex-cord stromal cell tumors in young women. These hotspot mutations lost IIIb cleavage activity of DICER1 in vitro and failed to produce 5p-derived miRNAs in mouse Dicer1-null ES cells. However, the oncogenic potential of these hotspot DICER1 mutations has not been studied. Here, we further revealed that the global expression of 5p-derived miRNAs was dramatically reduced in ovarian Sertoli-Leydig cell tumors carrying DICER1 hotspot mutations compared with those without DICER1 hotspot mutation. The miRNA production defect was associated with the deregulation of genes controlling cell proliferation and the cell fate. Using an immortalized human granulosa cell line, SVOG3e, we determined that the D1709N-DICER1 hotspot mutation failed to produce 5p-derived miRNAs, deregulated the expression of several genes that control gonadal differentiation and cell proliferation, and promoted cell growth. Re-expression of let-7 significantly inhibited the growth of D1709N-DICER1 SVOG3e cells, accompanied by the suppression of key regulators of cell cycle control and ovarian gonad differentiation. Taken together, our data revealed that DICER1 hotspot mutations cause systemic loss of 5p-miRNAs that can both drive pseudodifferentiation of testicular elements and cause oncogenic transformation in the ovary.

  8. Insights Gained from Testing Alternate Cell Designs

    International Nuclear Information System (INIS)

    O'Brien, J.E.; Stoots, C.M.; Herring, J.S.; Housley, G.K.; Sohal, M.S.; Milobar, D.G.; Cable, Thomas

    2009-01-01

    The Idaho National Laboratory (INL) has been researching the application of solid-oxide electrolysis cell for large-scale hydrogen production from steam over a temperature range of 800 to 900 C. The INL has been testing various solid oxide cell designs to characterize their electrolytic performance operating in the electrolysis mode for hydrogen production. Some results presented in this report were obtained from cells, initially developed by the Forschungszentrum Juelich and now manufactured by the French ceramics firm St. Gobain. These cells have an active area of 16 cm2 per cell. They were initially developed as fuel cells, but are being tested as electrolytic cells in the INL test stands. The electrolysis cells are electrode-supported, with ∼10 (micro)m thick yttria-stabilized zirconia (YSZ) electrolytes, ∼1400 (micro)m thick nickel-YSZ steam-hydrogen electrodes, and manganite (LSM) air-oxygen electrodes. The experiments were performed over a range of steam inlet mole fractions (0.1 to 0.6), gas flow rates, and current densities (0 to 0.6 A/cm2). Steam consumption rates associated with electrolysis were measured directly using inlet and outlet dewpoint instrumentation. On a molar basis, the steam consumption rate is equal to the hydrogen production rate. Cell performance was evaluated by performing DC potential sweeps at 800, 850, and 900 C. The voltage-current characteristics are presented, along with values of area-specific resistance as a function of current density. Long-term cell performance is also assessed to evaluate cell degradation. Details of the custom single-cell test apparatus developed for these experiments are also presented. NASA, in conjunction with the University of Toledo, has developed another fuel cell concept with the goals of reduced weight and high power density. The NASA cell is structurally symmetrical, with both electrodes supporting the thin electrolyte and containing micro-channels for gas diffusion. This configuration is

  9. Different spectra of recurrent gene mutations in subsets of chronic lymphocytic leukemia harboring stereotyped B-cell receptors

    DEFF Research Database (Denmark)

    Sutton, Lesley-Ann; Young, Emma; Baliakas, Panagiotis

    2016-01-01

    We report on markedly different frequencies of genetic lesions within subsets of chronic lymphocytic leukemia patients carrying mutated or unmutated stereotyped B-cell receptor immunoglobulins in the largest cohort (n=565) studied for this purpose. By combining data on recurrent gene mutations...... subsets implies that the mechanisms underlying clinical aggressiveness are not uniform, but rather support the existence of distinct genetic pathways of clonal evolution governed by a particular stereotyped B-cell receptor selecting a certain molecular lesion(s)....

  10. The combination of heteroduplex analysis and protein truncation test for exact detection of the APC gene mutations

    International Nuclear Information System (INIS)

    Tomka, M.; Kirchhoff, T.; Stefurkova, V.; Zajac, V.; Kulcsar, L.

    1998-01-01

    Familial adenomatous polyposis (FAP) is usually associated with mutation in the adenomatous polyposis coli (APC) gene. To examine the occurrence of these mutations in the number of FAP suspected families from the whole Slovakia effectively, we have applied heteroduplex analysis (HDA) and protein truncation test (PTT) for the analyses of 2-5 base pair deletions and point mutations of the APC gene. In the analyzed exon 15 of the APC gene determined by the primers 15Efor-15Grev for HDA and 15ET7-15J3 for PTT more than 70% of mutations should be deletions [3, 12], which are detectable by HDA. In our collection of 5 FAP families mutations in the APC gene were found in families 10, 27 and 41 using HDA. By PTT test the formation of truncated APC protein in FAP families 2, 10, 16 and 27 were revealed. The necessity of combination of at least HDA and PTT techniques for exact detection of APC mutations in analyzed APC region is discussed. (authors)

  11. Tritiated uracil, tritiated thymidine, and bromodeoxyuridine induced mutations in eucaryotic cells

    International Nuclear Information System (INIS)

    Burki, H.J.; Moustacchi, E.; Cleaver, J.E.

    1979-02-01

    The induction of gene conversion at the ARG-4 locus in strain BZ34 of Saccharomyces cerevisiae was examined after the cells incorporated y- 3 H uracil under optimum growth conditions for 16 hours, and then received damage at 4 0 C from tritium decays at very low dose rates of 1.4 to 27.6 tritium decays per hour. The results were compared to the results of gene conversion induced by 60 Co. The induction of resistance to 6TG in Chinese hamster ovary (CHO) cells has been studied after incorporation of 3 H-methyl thymidine, 6- 3 H-thymidine, and bromodeoxyuridine under several experimental conditions. The induction of mutations by incorporated 6- 3 H-thymidine is about three times as effective as the induction of mutations by tritiated-methyl thymidine. These results suggest that the determination of the RBE for tritium decays in model eucaryotic systems like yeast and cultured Chinese hamster cells will be influenced by the precise experimental conditions employed. In particular, experiments with mammalian cells will be affected by hot times for mutagenesis in the cell cycle and hot positions within the DNA in the nucleus, and also by the position of tritium decay within the DNA-incorporated molecule

  12. Prospects for cellular mutational assays in human populations

    International Nuclear Information System (INIS)

    Mendelsohn, M.L.

    1984-01-01

    Practical, sensitive, and effective human cellular assays for detecting somatic and germinal mutations would have great value in environmental mutagenesis and carcinogenesis studies. Such assays would fill the void between human mutagenicity and the data that exist from short-term tests and from mutagenicity in other species. This paper discusses the following possible human cellular assays: (1) HPRT (hypoxanthine phosphoribosyltransferase) somatic cell mutation based on 6-thioguanine resistance; (2) hemoglobin somatic cell mutation assay; (3) glycophorin somatic cell mutation assay; and (4) LDH-X sperm cell mutation assay. 18 references

  13. Prospects for cellular mutational assays in human populations

    Energy Technology Data Exchange (ETDEWEB)

    Mendelsohn, M.L.

    1984-06-29

    Practical, sensitive, and effective human cellular assays for detecting somatic and germinal mutations would have great value in environmental mutagenesis and carcinogenesis studies. Such assays would fill the void between human mutagenicity and the data that exist from short-term tests and from mutagenicity in other species. This paper discusses the following possible human cellular assays: (1) HPRT (hypoxanthine phosphoribosyltransferase) somatic cell mutation based on 6-thioguanine resistance; (2) hemoglobin somatic cell mutation assay; (3) glycophorin somatic cell mutation assay; and (4) LDH-X sperm cell mutation assay. 18 references.

  14. Point mutations in EBV gH that abrogate or differentially affect B cell and epithelial cell fusion

    International Nuclear Information System (INIS)

    Wu Liguo; Hutt-Fletcher, Lindsey M.

    2007-01-01

    Cell fusion mediated by Epstein-Barr virus requires three conserved glycoproteins, gB and gHgL, but activation is cell type specific. B cell fusion requires interaction between MHC class II and a fourth virus glycoprotein, gp42, which complexes non-covalently with gHgL. Epithelial cell fusion requires interaction between gHgL and a novel epithelial cell coreceptor and is blocked by excess gp42. We show here that gp42 interacts directly with gH and that point mutations in the region of gH recognized by an antibody that differentially inhibits epithelial and B cell fusion significantly impact both the core fusion machinery and cell-specific events. Substitution of alanine for glycine at residue 594 completely abrogates fusion with either B cells or epithelial cells. Substitution of alanine for glutamic acid at residue 595 reduces fusion with epithelial cells, greatly enhances fusion with B cells and allows low levels of B cell fusion even in the absence of gL

  15. Tumor mutational load and immune parameters across metastatic Renal Cell Carcinoma (mRCC) risk groups

    Science.gov (United States)

    de Velasco, Guillermo; Miao, Diana; Voss, Martin H.; Hakimi, A. Ari; Hsieh, James J.; Tannir, Nizar M.; Tamboli, Pheroze; Appleman, Leonard J.; Rathmell, W. Kimryn; Van Allen, Eliezer M.; Choueiri, Toni K.

    2016-01-01

    Patients with metastatic renal cell carcinoma (mRCC) have better overall survival when treated with nivolumab, a cancer immunotherapy that targets the immune checkpoint inhibitor programmed cell death 1 (PD-1), rather than everolimus (a chemical inhibitor of mTOR and immunosuppressant). Poor-risk mRCC patients treated with nivolumab seemed to experience the greatest overall survival benefit, compared to patients with favorable or intermediate-risk, in an analysis of the CheckMate-025 trial subgroup of the Memorial Sloan Kettering Cancer Center (MSKCC) prognostic risk groups. Here we explore whether tumor mutational load and RNA expression of specific immune parameters could be segregated by prognostic MSKCC risk strata and explain the survival seen in the poor-risk group. We queried whole exome transcriptome data in RCC patients (n = 54) included in The Cancer Genome Atlas that ultimately developed metastatic disease or were diagnosed with metastatic disease at presentation and did not receive immune checkpoint inhibitors. Nonsynonymous mutational load did not differ significantly by MSKCC risk group, nor was the expression of cytolytic genes –granzyme A and perforin – or selected immune checkpoint molecules different across MSKCC risk groups. In conclusion, this analysis found that mutational load and expression of markers of an active tumor microenvironment did not correlate with MSKCC risk prognostic classification in mRCC. PMID:27538576

  16. Efficient CRISPR-Cas9-mediated generation of knockin human pluripotent stem cells lacking undesired mutations at the targeted locus.

    Science.gov (United States)

    Merkle, Florian T; Neuhausser, Werner M; Santos, David; Valen, Eivind; Gagnon, James A; Maas, Kristi; Sandoe, Jackson; Schier, Alexander F; Eggan, Kevin

    2015-05-12

    The CRISPR-Cas9 system has the potential to revolutionize genome editing in human pluripotent stem cells (hPSCs), but its advantages and pitfalls are still poorly understood. We systematically tested the ability of CRISPR-Cas9 to mediate reporter gene knockin at 16 distinct genomic sites in hPSCs. We observed efficient gene targeting but found that targeted clones carried an unexpectedly high frequency of insertion and deletion (indel) mutations at both alleles of the targeted gene. These indels were induced by Cas9 nuclease, as well as Cas9-D10A single or dual nickases, and often disrupted gene function. To overcome this problem, we designed strategies to physically destroy or separate CRISPR target sites at the targeted allele and developed a bioinformatic pipeline to identify and eliminate clones harboring deleterious indels at the other allele. This two-pronged approach enables the reliable generation of knockin hPSC reporter cell lines free of unwanted mutations at the targeted locus. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  17. Biochemical characterization of the deafness-associated mitochondrial tRNASer(UCN) A7445G mutation in osteosarcoma cell cybrids

    International Nuclear Information System (INIS)

    Li Xiaoming; Zhang, Linda S.; Fischel-Ghodsian, Nathan; Guan Minxin

    2005-01-01

    The deafness-associated A7445G mutation in the precursor of mitochondrial tRNA Ser(UCN) has been identified in several pedigrees from different ethnic backgrounds. To determine the role of nuclear background in the biochemical manifestation associated with the A7445G mutation, we performed a biochemical characterization of this mutation using cybrids constructed by transferring mitochondria from lymphoblastoid cell lines derived from a New Zealand family into human osteosarcoma mtDNA-less (ρ 0 ) cells. Compared with three control cybrids, three cybrids derived from an affected matrilineal relative carrying the homoplasmic A7445G mutation exhibited ∼38-57% decrease in the steady-state level of tRNA Ser(UCN) , which is less reduced levels than in lymphoblastoid cells in the previous study. Furthermore, ∼22% reduction in the level of aminoacylation of tRNA Ser(UCN) was observed in the mutant cybrid cells. Interestingly, ∼60-63% decrease of steady-state level of ND6 gene, which belongs to the same precursor as that of tRNA Ser(UCN) , in cybrid cell lines carrying the A7445G mutation, is more than that observed in lymphoblastoid cells. These observations strongly point out a mechanistic link between the processing defect of the tRNA Ser(UCN) precursor and decreased stability of ND6 mRNA precursor. These results also imply the influence of nuclear background on the biochemical phenotype associated with the A7445G mutation

  18. Risk scaling factors from inactivation to chromosome aberrations, mutations and oncogenic transformations in mammalian cells

    International Nuclear Information System (INIS)

    Alkaharam, A.S.; Watt, D.E.

    1997-01-01

    Analyses of bio-effect mechanisms of damage to mammalian cells in terms of the quality parameter 'mean free path for primary ionisation', for heavy charged particles, strongly suggests that there is a common mechanism for the biological endpoints of chromosome aberrations, mutations and oncogenic transformation. The lethal lesions are identified as unrepaired double-strand breaks in the intracellular DNA. As data for the various endpoints studied can be represented in a unified scheme, for any radiation type, it follows that radiation risk factors can be determined on the basis of simple ratios to the inactivation cross sections. There are intrinsic physical reasons why neutrons can never reach the saturation level of heavier particles for equal fluences. The probabilities of risk with respect to inactivation, for chromosome dicentrics, mutation of the HPRT gene and of oncogenic transformation are respectively 0.24, 5.8 x 10 -5 , and 4.1 x 10 -3 . (author)

  19. Evaluation of EGFR, KRAS and BRAF gene mutations in renal cell carcinoma

    Directory of Open Access Journals (Sweden)

    Omer Bayrak

    2014-08-01

    Full Text Available A subset of renal cell carcinoma (RCC patients has been shown to respond to anti-EGFR therapy. As KRAS and BRAF mutations are associated with poor response to anti-EGFR therapy in some cancers, it has been suggested that screening for KRAS and BRAF mutations in RCC may be a promising strategy to identify patients who might respond to EGFR-targeted therapy. The aim of this study was to investigate the mutation status of EGFR, KRAS and BRAF in RCC patients. Renal tumors and normal renal samples from forty-eight patients who underwent radical or partial nephrectomy for kidney cancer were used in this study. Histological classification of the tumors was performed according to International Union against Cancer (UICC / American Joint Committee on Cancer (AJCC classification. Seventeen patients (48% had clear-cell RCC, 7 (20% had chromophobe RCC, and 11 patients (32% had papillary RCC. DNA isolated from the samples was subjected to melting curve mutation analysis for EGFR, BRAF and KRAS using ABI-3130 DNA sequencer. DNA sequencing analysis of RCC samples, when compared with morphologically normal matched regions, did not show any exon mutations. Our results do not support the notion that EGFR, KRAS and BRAF might be mutated in RCC. Normal 0 false false false TR X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-ansi-language:TR; mso-fareast-language:EN-US;}

  20. Presenilin-1 mutations alter K+ currents in the human neuroblastoma cell line, SH-SY5Y

    DEFF Research Database (Denmark)

    Plant, Leigh D; Boyle, John P; Thomas, Natasha M

    2002-01-01

    Mutations in presenilin 1 (PS1) are the major cause of autosomal dominant Alzheimer's disease. We have measured the voltage-gated K+ current in the human neuroblastoma cell line SH-SY5Y using whole-cell patch-clamp. When cells were stably transfected to over-express PS1, no change in K+ current...

  1. Two novel mutations in seven Czech and Slovak kindreds with familial neurohypophyseal diabetes insipidus-benefit of genetic testing.

    Science.gov (United States)

    Hrčková, Gabriela; Jankó, Viktor; Kytnarová, Jitka; Čižmárová, Michaela; Tesařová, Markéta; Košťálová, Ľudmila; Virgová, Daniela; Dallos, Tomáš; Hána, Václav; Lebl, Jan; Zeman, Jiří; Kovács, László

    2016-09-01

    Familial neurohypophyseal diabetes insipidus (FNDI) is a rare hereditary disorder with unknown prevalence characterized by arginine-vasopressin hormone (AVP) deficiency resulting in polyuria and polydipsia from early childhood. We report the clinical manifestation and genetic test results in seven unrelated kindreds of Czech or Slovak origin with FNDI phenotype. The age of the sign outset ranged from 2 to 17 years with remarkable interfamilial and intrafamilial variability. Inconclusive result of the fluid deprivation test in three children aged 7 and 17 years old might cause misdiagnosis; however, the AVP gene analysis confirmed the FNDI. The seven families segregated together five different mutations, two of them were novel (c.164C > A, c.298G > C). In addition, DNA analysis proved mutation carrier status in one asymptomatic 1-year-old infant. The present study together with previously published data identified 38 individuals with FNDI in the studied population of 16 million which predicts a disease prevalence of 1:450,000 for the Central European region. The paper underscores that diagnostic water deprivation test may be inconclusive in polyuric children with partial diabetes insipidus and points to the clinical importance and feasibility of molecular genetic testing for AVP gene mutations in the proband and her/his first degree relatives. • At least 70 different mutations were reported to date in about 100 families with neurohypophyseal diabetes insipidus (FNDI), and new mutations appear sporadically. What is New: • Two novel mutations of the AVP gene are reported • The importance of molecular testing in children with polyuria and inconclusive water deprivation test is emphasized.

  2. Compound heterozygous TYK2 mutations underlie primary immunodeficiency with T-cell lymphopenia.

    Science.gov (United States)

    Nemoto, Michiko; Hattori, Hiroyoshi; Maeda, Naoko; Akita, Nobuhiro; Muramatsu, Hideki; Moritani, Suzuko; Kawasaki, Tomonori; Maejima, Masami; Ode, Hirotaka; Hachiya, Atsuko; Sugiura, Wataru; Yokomaku, Yoshiyuki; Horibe, Keizo; Iwatani, Yasumasa

    2018-05-03

    Complete tyrosine kinase 2 (TYK2) deficiency has been previously described in patients with primary immunodeficiency diseases. The patients were infected with various pathogens, including mycobacteria and/or viruses, and one of the patients developed hyper-IgE syndrome. A detailed immunological investigation of these patients revealed impaired responses to type I IFN, IL-10, IL-12 and IL-23, which are associated with increased susceptibility to mycobacterial and/or viral infections. Herein, we report a recessive partial TYK2 deficiency in two siblings who presented with T-cell lymphopenia characterized by low naïve CD4 + T-cell counts and who developed Epstein-Barr virus (EBV)-associated B-cell lymphoma. Targeted exome-sequencing of the siblings' genomes demonstrated that both patients carried novel compound heterozygous mutations (c.209_212delGCTT/c.691C > T, p.Cys70Serfs*21/p.Arg231Trp) in the TYK2. The TYK2 protein levels were reduced by 35% in the T cells of the patient. Unlike the response under complete TYK2 deficiency, the patient's T cells responded normally to type I IFN, IL-6, IL-10 and IL-12, whereas the cells displayed an impaired response to IL-23. Furthermore, the level of STAT1 was low in the cells of the patient. These studies reveal a new clinical entity of a primary immunodeficiency with T-cell lymphopenia that is associated with compound heterozygous TYK2 mutations in the patients.

  3. Genetic Testing for Wolfram Syndrome Mutations in a Sample of 71 Patients with Hereditary Optic Neuropathy and Negative Genetic Test Results for OPA1/OPA3/LHON.

    Science.gov (United States)

    Galvez-Ruiz, Alberto; Galindo-Ferreiro, Alicia; Schatz, Patrik

    2018-04-01

    In this study, the authors present a sample of 71 patients with hereditary optic neuropathy and negative genetic test results for OPA1/OPA3/LHON. All of these patients later underwent genetic testing to rule out WFS. As a result, 53 patients (74.7%) were negative and 18 patients (25.3%) were positive for some type of mutation or variation in the WFS gene. The authors believe that this study is interesting because it shows that a sizeable percentage (25.3%) of patients with hereditary optic 25 neuropathy and negative genetic test results for OPA1/OPA3/LHON had WFS mutations or variants.

  4. FCTESTNET - Testing fuel cells for transportation

    NARCIS (Netherlands)

    Winkel, R.G.; Foster, D.L.; Smokers, R.T.M.

    2006-01-01

    FCTESTNET (Fuel Cell Testing and Standardization Network) is an ongoing European network project within Framework Program 5. It is a three-year project that commenced January 2003, with 55 partners from European research centers, universities, and industry, working in the field of fuel cell R and D.

  5. Adaptation of cancer cells from different entities to the MDM2 inhibitor nutlin-3 results in the emergence of p53-mutated multi-drug-resistant cancer cells

    NARCIS (Netherlands)

    Michaelis, M.; Rothweiler, F.; Barth, S.; Cinatl, J.; van Rikxoort, M.; Loeschmann, N.; Voges, Y.; Breitling, R.; von Deimling, A.; Roedel, F.; Weber, K.; Fehse, B.; Mack, E.; Stiewe, T.; Doerr, H. W.; Speidel, D.; Cinatl, J.; Cinatl jr., J.; Stephanou, A.

    2011-01-01

    Six p53 wild-type cancer cell lines from infrequently p53-mutated entities (neuroblastoma, rhabdomyosarcoma, and melanoma) were continuously exposed to increasing concentrations of the murine double minute 2 inhibitor nutlin-3, resulting in the emergence of nutlin-3-resistant, p53-mutated sublines

  6. A comparison of EGFR mutation testing methods in lung carcinoma: direct sequencing, real-time PCR and immunohistochemistry.

    Directory of Open Access Journals (Sweden)

    Bárbara Angulo

    Full Text Available The objective of this study is to compare two EGFR testing methodologies (a commercial real-time PCR kit and a specific EGFR mutant immunohistochemistry, with direct sequencing and to investigate the limit of detection (LOD of both PCR-based methods. We identified EGFR mutations in 21 (16% of the 136 tumours analyzed by direct sequencing. Interestingly, the Therascreen EGFR Mutation Test kit was able to characterize as wild-type one tumour that could not be analyzed by direct sequencing of the PCR product. We then compared the LOD of the kit and that of direct sequencing using the available mutant tumours. The kit was able to detect the presence of a mutation in a 1% dilution of the total DNA in nine of the 18 tumours (50%, which tested positive with the real-time quantitative PCR method. In all cases, EGFR mutation was identified at a dilution of 5%. Where the mutant DNA represented 30% of the total DNA, sequencing was able to detect mutations in 12 out of 19 cases (63%. Additional experiments with genetically defined standards (EGFR ΔE746-A750/+ and EGFR L858R/+ yielded similar results. Immunohistochemistry (IHC staining with exon 19-specific antibody was seen in eight out of nine cases with E746-A750del detected by direct sequencing. Neither of the two tumours with complex deletions were positive. Of the five L858R-mutated tumours detected by the PCR methods, only two were positive for the exon 21-specific antibody. The specificity was 100% for both antibodies. The LOD of the real-time PCR method was lower than that of direct sequencing. The mutation specific IHC produced excellent specificity.

  7. Conditional mutation of Smc5 in mouse embryonic stem cells perturbs condensin localization and mitotic progression.

    Science.gov (United States)

    Pryzhkova, Marina V; Jordan, Philip W

    2016-04-15

    Correct duplication of stem cell genetic material and its appropriate segregation into daughter cells are requisites for tissue, organ and organism homeostasis. Disruption of stem cell genomic integrity can lead to developmental abnormalities and cancer. Roles of the Smc5/6 structural maintenance of chromosomes complex in pluripotent stem cell genome maintenance have not been investigated, despite its important roles in DNA synthesis, DNA repair and chromosome segregation as evaluated in other model systems. Using mouse embryonic stem cells (mESCs) with a conditional knockout allele of Smc5, we showed that Smc5 protein depletion resulted in destabilization of the Smc5/6 complex, accumulation of cells in G2 phase of the cell cycle and apoptosis. Detailed assessment of mitotic mESCs revealed abnormal condensin distribution and perturbed chromosome segregation, accompanied by irregular spindle morphology, lagging chromosomes and DNA bridges. Mutation of Smc5 resulted in retention of Aurora B kinase and enrichment of condensin on chromosome arms. Furthermore, we observed reduced levels of Polo-like kinase 1 at kinetochores during mitosis. Our study reveals crucial requirements of the Smc5/6 complex during cell cycle progression and for stem cell genome maintenance. © 2016. Published by The Company of Biologists Ltd.

  8. Evaluating the genotoxic effects of workers exposed to lead using micronucleus assay, comet assay and TCR gene mutation test

    International Nuclear Information System (INIS)

    Chen Zhijian; Lou Jianlin; Chen Shijie; Zheng Wei; Wu Wei; Jin Lifen; Deng Hongping; He Jiliang

    2006-01-01

    To evaluate the genotoxic effects of lead (Pb) exposure, 25 workers in a workplace producing storage battery were monitored for three genetic end-points using micronucleus (MN) assay, comet assay and TCR gene mutation test. Twenty-five controls were matched with workers according to age, gender and smoking. The air Pb concentration in the workplace was 1.26 mg/m 3 . All subjects were measured for Pb concentration of blood by atom absorption spectrophotometry. The mean Pb concentration of blood in workers (0.32 mg/l) was significantly higher than that in controls (0.02 mg/l). The results of MN test showed that the mean micronuclei rate (MNR) and mean micronucleated cells rate (MCR) in workers were 9.04 ± 1.51 per mille and 7.76 ± 1.23 per mille , respectively, which were significantly higher than those (2.36 ± 0.42 per mille and 1.92 ± 0.31 per mille ) in controls (P -4 and 1.74 ± 0.17 x 10 -4 , respectively, there was no significant difference between workers and controls (P > 0.05). The results of our study indicated that the genetic damage was detectable in 25 workers occupationally exposed to lead

  9. Human Papillomavirus 16 Infection and TP53 Mutation: Two Distinct Pathogeneses for Oropharyngeal Squamous Cell Carcinoma in an Eastern Chinese Population

    OpenAIRE

    Wang, Zhen; Xia, Rong-Hui; Ye, Dong-Xia; Li, Jiang

    2016-01-01

    Objectives To investigate the clinicopathological characteristics, human papillomavirus (HPV) infection, p53 expression, and TP53 mutations in oropharyngeal squamous cell carcinoma (OPSCC) and determine their utility as prognostic predictors in a primarily eastern Chinese population. Methods The HPV infection status was tested via p16INK4A immunohistochemistry and validated using PCR, reverse blot hybridization and in situ hybridization (ISH) in 188 OPSCC samples. p53 expression levels and TP...

  10. HIV-1 replication in cell lines harboring INI1/hSNF5 mutations.

    Science.gov (United States)

    Sorin, Masha; Yung, Eric; Wu, Xuhong; Kalpana, Ganjam V

    2006-08-31

    INI1/hSNF5 is a cellular protein that directly interacts with HIV-1 integrase (IN). It is specifically incorporated into HIV-1 virions. A dominant negative mutant derived from INI1 inhibits HIV-1 replication. Recent studies indicate that INI1 is associated with pre-integration and reverse transcription complexes that are formed upon viral entry into the target cells. INI1 also is a tumor suppressor, biallelically deleted/mutated in malignant rhabdoid tumors. We have utilized cell lines derived from the rhabdoid tumors, MON and STA-WT1, that harbor either null or truncating mutations of INI1 respectively, to assess the effect of INI1 on HIV-1 replication. We found that while HIV-1 virions produced in 293T cells efficiently transduced MON and STA-WT1 cells, HIV-1 particle production was severely reduced in both of these cells. Reintroduction of INI1 into MON and STA-WT1 significantly enhanced the particle production in both cell lines. HIV-1 particles produced in MON cells were reduced for infectivity, while those produced in STA-WT1 were not. Further analysis indicated the presence of INI1 in those virions produced from STA-WT1 but not from those produced from MON cells. HIV-1 produced in MON cells were defective for synthesis of early and late reverse transcription products in the target cells. Furthermore, virions produced in MON cells were defective for exogenous reverse transcriptase activity carried out using exogenous template, primer and substrate. Our results suggest that INI1-deficient cells exhibit reduced particle production that can be partly enhanced by re-introduction of INI1. Infectivity of HIV-1 produced in some but not all INI1 defective cells, is affected and this defect may correlate to the lack of INI1 and/or some other proteins in these virions. The block in early events of virion produced from MON cells appears to be at the stage of reverse transcription. These studies suggest that presence of INI1 or some other host factor in virions and

  11. HIV-1 replication in cell lines harboring INI1/hSNF5 mutations

    Directory of Open Access Journals (Sweden)

    Wu Xuhong

    2006-08-01

    Full Text Available Abstract Background INI1/hSNF5 is a cellular protein that directly interacts with HIV-1 integrase (IN. It is specifically incorporated into HIV-1 virions. A dominant negative mutant derived from INI1 inhibits HIV-1 replication. Recent studies indicate that INI1 is associated with pre-integration and reverse transcription complexes that are formed upon viral entry into the target cells. INI1 also is a tumor suppressor, biallelically deleted/mutated in malignant rhabdoid tumors. We have utilized cell lines derived from the rhabdoid tumors, MON and STA-WT1, that harbor either null or truncating mutations of INI1 respectively, to assess the effect of INI1 on HIV-1 replication. Results We found that while HIV-1 virions produced in 293T cells efficiently transduced MON and STA-WT1 cells, HIV-1 particle production was severely reduced in both of these cells. Reintroduction of INI1 into MON and STA-WT1 significantly enhanced the particle production in both cell lines. HIV-1 particles produced in MON cells were reduced for infectivity, while those produced in STA-WT1 were not. Further analysis indicated the presence of INI1 in those virions produced from STA-WT1 but not from those produced from MON cells. HIV-1 produced in MON cells were defective for synthesis of early and late reverse transcription products in the target cells. Furthermore, virions produced in MON cells were defective for exogenous reverse transcriptase activity carried out using exogenous template, primer and substrate. Conclusion Our results suggest that INI1-deficient cells exhibit reduced particle production that can be partly enhanced by re-introduction of INI1. Infectivity of HIV-1 produced in some but not all INI1 defective cells, is affected and this defect may correlate to the lack of INI1 and/or some other proteins in these virions. The block in early events of virion produced from MON cells appears to be at the stage of reverse transcription. These studies suggest that

  12. Frequency of Somatic TP53 Mutations in Combination with Known Pathogenic Mutations in Colon Adenocarcinoma, Non–Small Cell Lung Carcinoma, and Gliomas as Identified by Next-Generation Sequencing

    Directory of Open Access Journals (Sweden)

    Zahra Shajani-Yi

    2018-03-01

    Full Text Available The tumor suppressor gene TP53 is the most frequently mutated gene in human cancer. It encodes p53, a DNA-binding transcription factor that regulates multiple genes involved in DNA repair, metabolism, cell cycle arrest, apoptosis, and senescence. TP53 is associated with human cancer by mutations that lead to a loss of wild-type p53 function as well as mutations that confer alternate oncogenic functions that enable them to promote invasion, metastasis, proliferation, and cell survival. Identifying the discrete TP53 mutations in tumor cells may help direct therapies that are more effective. In this study, we identified the frequency of individual TP53 mutations in patients with colon adenocarcinoma (48%, non–small cell lung carcinoma (NSCLC (36%, and glioma/glioblastoma (28% at our institution using next-generation sequencing. We also identified the occurrence of somatic mutations in numerous actionable genes including BRAF, EGFR, KRAS, IDH1, and PIK3CA that occurred concurrently with these TP53 mutations. Of the 480 tumors examined that contained one or more mutations in the TP53 gene, 219 were colon adenocarcinomas, 215 were NSCLCs, and 46 were gliomas/glioblastomas. Among the patients positive for TP53 mutations diagnosed with colon adenocarcinoma, 50% also showed at least one mutation in pathogenic genes of which 14% were BRAF, 33% were KRAS, and 3% were NRAS. Forty-seven percent of NSCLC patients harboring TP53 mutations also had a mutation in at least one actionable pathogenic variant with the following frequencies: BRAF: 4%, EGFR: 10%, KRAS: 28%, and PIK3CA: 4%. Fifty-two percent of patients diagnosed with glioma/glioblastoma with a positive TP53 mutation had at least one concurrent mutation in a known pathogenic gene of which 9% were CDKN2A, 41% were IDH1, and 11% were PIK3CA.

  13. Assessment of real-time PCR method for detection of EGFR mutation using both supernatant and cell pellet of malignant pleural effusion samples from non-small-cell lung cancer patients.

    Science.gov (United States)

    Shin, Saeam; Kim, Juwon; Kim, Yoonjung; Cho, Sun-Mi; Lee, Kyung-A

    2017-10-26

    EGFR mutation is an emerging biomarker for treatment selection in non-small-cell lung cancer (NSCLC) patients. However, optimal mutation detection is hindered by complications associated with the biopsy procedure, tumor heterogeneity and limited sensitivity of test methodology. In this study, we evaluated the diagnostic utility of real-time PCR using malignant pleural effusion samples. A total of 77 pleural fluid samples from 77 NSCLC patients were tested using the cobas EGFR mutation test (Roche Molecular Systems). Pleural fluid was centrifuged, and separated cell pellets and supernatants were tested in parallel. Results were compared with Sanger sequencing and/or peptide nucleic acid (PNA)-mediated PCR clamping of matched tumor tissue or pleural fluid samples. All samples showed valid real-time PCR results in one or more DNA samples extracted from cell pellets and supernatants. Compared with other molecular methods, the sensitivity of real-time PCR method was 100%. Concordance rate of real-time PCR and Sanger sequencing plus PNA-mediated PCR clamping was 98.7%. We have confirmed that real-time PCR using pleural fluid had a high concordance rate compared to conventional methods, with no failed samples. Our data demonstrated that the parallel real-time PCR testing using supernatant and cell pellet could offer reliable and robust surrogate strategy when tissue is not available.

  14. Using iron studies to predict HFE mutations in New Zealand: implications for laboratory testing.

    Science.gov (United States)

    O'Toole, Rebecca; Romeril, Kenneth; Bromhead, Collette

    2017-04-01

    The diagnosis of hereditary haemochromatosis (HH) is not straightforward because symptoms are often absent or non-specific. Biochemical markers of iron-overloading may be affected by other conditions. To measure the correlation between iron studies and HFE genotype to inform evidence-based recommendations for laboratory testing in New Zealand. Results from 2388 patients genotyped for C282Y, H63D and S65C in Wellington, New Zealand from 2007 to 2013 were compared with their biochemical phenotype as quantified by serum ferritin (SF), transferrin saturation (TS), serum iron (SI) and serum transferrin (ST). The predictive power of these markers was evaluated by receiver operator characteristic (ROC) curve analysis, and if a statistically significant association for a variable was seen, sensitivity, specificity and predictive values were calculated. Test ordering patterns showed that 62% of HFE genotyping tests were ordered because of an elevated SF alone and only 11% of these had a C-reactive protein test to rule out an acute phase reaction. The association between SF and significant HFE genotypes SF was low. However, TS values ≥45% predicted HH mutations with the highest sensitivity and specificity. A SF of >1000 µg/L was found in one at-risk patient (C282Y homozygote) who had a TS <45%. Our analysis highlights the need for clear guidelines for investigation of hyperferritinaemia and HH in New Zealand. Using our findings, we developed an evidence-based laboratory testing algorithm based on a TS ≥45%, a SF ≥1000 µg/L and/or a family history of HH which identified all C282Y homozygotes in this study. © 2016 Royal Australasian College of Physicians.

  15. Potential use of gradient denaturing gel electrophoresis in obtaining mutational spectra from human cells

    International Nuclear Information System (INIS)

    Thilly, W.G.

    1985-01-01

    A method is described to isolate mutations in DNA in human cells. When a double-stranded DNA migrates through an electric field on an electrophoretic gel, it is compact hydrodynamic structure relative to the same material in a melted form. Normally the solution in electrophoretic gels is uniform, but a way has been devised to set up a stable gradient of increasing solute concentration in the direction of DNA motion. Thus, as a double-stranded DNA molecule is drawn by the electric field into higher and higher concentrations of urea/formamide, it will eventually reach a point at which the concentration is high enough to melt the lower-melting-point region. The melting results in an essentially immobile structure within the gel so that the position at which the DNA molecule stops on the gradient gel is determined by its melting point, which is uniquely determined by its nucleotide sequence. A single base pair substitution within a low melting point sequence of some 100 base pairs changed the expected melting point by 0.4 0 C and resulted in about a 2-cm displacement under appropriate denaturing gel conditions. This expectation leads to the idea that if a mixture of DNA sequences derived from point mutations within the same restriction fragment were permitted to anneal with a complementary wild-type sequence, the melting point of each type of heteroduplex would differ depending on the kind and position of each mutation

  16. Heterozygosity for a Bub1 mutation causes female-specific germ cell aneuploidy in mice

    Energy Technology Data Exchange (ETDEWEB)

    Leland, Shawn; Nagarajan, Prabakaran; Polyzos, Aris; Thomas, Sharon; Samaan, George; Donnell, Robert; Marchetti, Francesco; Venkatachalam, Sundaresan

    2009-06-24

    Aneuploidy, the most common chromosomal abnormality at birth and the main ascertained cause of pregnancy loss in humans, originates primarily from chromosome segregation errors during oogenesis. Here we report that heterozygosity for a mutation in the mitotic checkpoint kinase gene, Bub1, induces aneuploidy in female germ cells of mice, and that the effect increases with advancing maternal age. Analysis of Bub1 heterozygous oocytes showed that aneuploidy occurred primarily during the first meiotic division and involved premature sister chromatid separation. Furthermore, aneuploidy was inherited in zygotes and resulted in the loss of embryos after implantation. The incidence of aneuploidy in zygotes was sufficient to explain the reduced litter size in matings with Bub1 heterozygous females. No effects were seen in germ cells from heterozygous males. These findings show that Bub1 dysfunction is linked to inherited aneuploidy in female germ cells and may contribute to the maternal age-related increase in aneuploidy and pregnancy loss.

  17. The use of a cloned bacterial gene to study mutation in mammalian cells

    International Nuclear Information System (INIS)

    Thacker, J.; Debenham, P.G.; Stretch, A.; Webb, M.B.T.

    1983-01-01

    The recombinant DNA molecule pSV2-gpt, which contains the bacterial gene coding for xanthine-guanine phosphoribosyl transferase (XGPRT) activity, was introduced into a hamster cell line lacking the equivalent mammalian enzyme (HGPRT). Hamster cell sublines were found with stable expression of XGPRT activity and were used to study mutation of the integrated pSV2-gpt DNA sequence. Mutants were selected by their resistance to 6-thioguanine (TG) under optimal conditions which were found to be very similar to those for selection of HGPRT-deficient mutants of mammalian cells. The frequency of XGPRT-deficient mutants was increased by treatment with X-rays, ethyl methanesulphonate and ethyl nitrosourea. X-Ray induction of mutants increased approximately linearly with dose up to about 500 rad, but the frequency of mutants per rad was very much higher than that usually found for 'native' mammalian genes. (orig./AJ)

  18. Identification of novel mutations in X-linked retinitis pigmentosa families and implications for diagnostic testing

    Science.gov (United States)

    Glaus, Esther; Lorenz, Birgit; Netzer, Christian; Li, Yün; Schambeck, Maria; Wittmer, Mariana; Feil, Silke; Kirschner-Schwabe, Renate; Rosenberg, Thomas; Cremers, Frans P.M.; Bergen, Arthur A.B.; Barthelmes, Daniel; Baraki, Husnia; Schmid, Fabian; Tanner, Gaby; Fleischhauer, Johannes; Orth, Ulrike; Becker, Christian; Wegscheider, Erika; Nürnberg, Gudrun; Nürnberg, Peter; Bolz, Hanno Jörn; Gal, Andreas; Berger, Wolfgang

    2008-01-01

    Purpose The goal of this study was to identify mutations in X-chromosomal genes associated with retinitis pigmentosa (RP) in patients from Germany, The Netherlands, Denmark, and Switzerland. Methods In addition to all coding exons of RP2, exons 1 through 15, 9a, ORF15, 15a and 15b of RPGR were screened for mutations. PCR products were amplified from genomic DNA extracted from blood samples and analyzed by direct sequencing. In one family with apparently dominant inheritance of RP, linkage analysis identified an interval on the X chromosome containing RPGR, and mutation screening revealed a pathogenic variant in this gene. Patients of this family were examined clinically and by X-inactivation studies. Results This study included 141 RP families with possible X-chromosomal inheritance. In total, we identified 46 families with pathogenic sequence alterations in RPGR and RP2, of which 17 mutations have not been described previously. Two of the novel mutations represent the most 3’-terminal pathogenic sequence variants in RPGR and RP2 reported to date. In exon ORF15 of RPGR, we found eight novel and 14 known mutations. All lead to a disruption of open reading frame. Of the families with suggested X-chromosomal inheritance, 35% showed mutations in ORF15. In addition, we found five novel mutations in other exons of RPGR and four in RP2. Deletions in ORF15 of RPGR were identified in three families in which female carriers showed variable manifestation of the phenotype. Furthermore, an ORF15 mutation was found in an RP patient who additionally carries a 6.4 kbp deletion downstream of the coding region of exon ORF15. We did not identify mutations in 39 sporadic male cases from Switzerland. Conclusions RPGR mutations were confirmed to be the most frequent cause of RP in families with an X-chromosomal inheritance pattern. We propose a screening strategy to provide molecular diagnostics in these families. PMID:18552978

  19. Somatic mosaicism containing double mutations in PTCH1 revealed by generation of induced pluripotent stem cells from nevoid basal cell carcinoma syndrome.

    Science.gov (United States)

    Ikemoto, Yu; Takayama, Yoshinaga; Fujii, Katsunori; Masuda, Mokuri; Kato, Chise; Hatsuse, Hiromi; Fujitani, Kazuko; Nagao, Kazuaki; Kameyama, Kohzoh; Ikehara, Hajime; Toyoda, Masashi; Umezawa, Akihiro; Miyashita, Toshiyuki

    2017-08-01

    Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterised by developmental defects and tumorigenesis, such as medulloblastomas and basal cell carcinomas, caused by mutations of the patched-1 ( PTCH1 ) gene. In this article, we seek to demonstrate a mosaicism containing double mutations in PTCH1 in an individual with NBCCS. A de novo germline mutation of PTCH1 (c.272delG) was detected in a 31-year-old woman with NBCCS. Gene analysis of two out of four induced pluripotent stem cell (iPSC) clones established from the patient unexpectedly revealed an additional mutation, c.274delT. Deep sequencing confirmed a low-prevalence somatic mutation (5.5%-15.6% depending on the tissue) identical to the one found in iPSC clones. This is the first case of mosaicism unequivocally demonstrated in NBCCS. Furthermore, the mosaicism is unique in that the patient carries one normal and two mutant alleles. Because these mutations are located in close proximity, reversion error is likely to be involved in this event rather than a spontaneous mutation. In addition, this study indicates that gene analysis of iPSC clones can contribute to the detection of mosaicism containing a minor population carrying a second mutation. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  20. High purity microfluidic sorting and analysis of circulating tumor cells: towards routine mutation detection.

    Science.gov (United States)

    Autebert, Julien; Coudert, Benoit; Champ, Jérôme; Saias, Laure; Guneri, Ezgi Tulukcuoglu; Lebofsky, Ronald; Bidard, François-Clément; Pierga, Jean-Yves; Farace, Françoise; Descroix, Stéphanie; Malaquin, Laurent; Viovy, Jean-Louis

    2015-05-07

    A new generation of the Ephesia cell capture technology optimized for CTC capture and genetic analysis is presented, characterized in depth and compared with the CellSearch system as a reference. This technology uses magnetic particles bearing tumour-cell specific EpCAM antibodies, self-assembled in a regular array in a microfluidic flow cell. 48,000 high aspect-ratio columns are generated using a magnetic field in a high throughput (>3 ml h(-1)) device and act as sieves to specifically capture the cells of interest through antibody-antigen interactions. Using this device optimized for CTC capture and analysis, we demonstrated the capture of epithelial cells with capture efficiency above 90% for concentrations as low as a few cells per ml. We showed the high specificity of capture with only 0.26% of non-epithelial cells captured for concentrations above 10 million cells per ml. We investigated the capture behavior of cells in the device, and correlated the cell attachment rate with the EpCAM expression on the cell membranes for six different cell lines. We developed and characterized a two-step blood processing method to allow for rapid processing of 10 ml blood tubes in less than 4 hours, and showed a capture rate of 70% for as low as 25 cells spiked in 10 ml blood tubes, with less than 100 contaminating hematopoietic cells. Using this device and procedure, we validated our system on patient samples using an automated cell immunostaining procedure and a semi-automated cell counting method. Our device captured CTCs in 75% of metastatic prostate cancer patients and 80% of metastatic breast cancer patients, and showed similar or better results than the CellSearch device in 10 out of 13 samples. Finally, we demonstrated the possibility of detecting cancer-related PIK3CA gene mutation in 20 cells captured in the chip with a good correlation between the cell count and the quantitation value Cq of the post-capture qPCR.

  1. Nitric Oxide Synthesis Is Increased in Cybrid Cells with m.3243A>G Mutation

    Directory of Open Access Journals (Sweden)

    Juliana Gamba

    2012-12-01

    Full Text Available Nitric oxide (NO is a free radical and a signaling molecule in several pathways, produced by nitric oxide synthase (NOS from the conversion of L-arginine to citrulline. Supplementation of L-arginine has been used to treat MELAS (mitochondrial encephalopathy with lactic acidosis and stroke like syndrome, a mitochondrial disease caused by the m.3243A>G mutation. Low levels of serum arginine and endothelium dysfunction have been reported in MELAS and this treatment may increase NO in endothelial cells and promote vasodilation, decreasing cerebral ischemia and strokes. Although clinical benefits have been reported, little is known about NO synthesis in MELAS. In this study we found that osteosarcoma derived cybrid cells with high levels of m.3243A>G had increased nitrite, an NO metabolite, and increased intracellular NO, demonstrated by an NO fluorescent probe (DAF-FM. Muscle vessels from patients with the same mutation had increased staining in NADPH diaphorase, suggestive of increased NOS. These results indicate increased production of NO in cells harboring the m.3243A>G, however no nitrated protein was detected by Western blotting. Further studies are necessary to clarify the exact mechanisms of L-arginine effect to determine the appropriate clinical use of this drug therapy.

  2. Pancreatic α-cell hyperplasia and hyperglucagonemia due to a glucagon receptor splice mutation

    Directory of Open Access Journals (Sweden)

    Etienne Larger

    2016-11-01

    Full Text Available Glucagon stimulates hepatic glucose production by activating specific glucagon receptors in the liver, which in turn increase hepatic glycogenolysis as well as gluconeogenesis and ureagenesis from amino acids. Conversely, glucagon secretion is regulated by concentrations of glucose and amino acids. Disruption of glucagon signaling in rodents results in grossly elevated circulating glucagon levels but no hypoglycemia. Here, we describe a patient carrying a homozygous G to A substitution in the invariant AG dinucleotide found in a 3′ mRNA splice junction of the glucagon receptor gene. Loss of the splice site acceptor consensus sequence results in the deletion of 70 nucleotides encoded by exon 9, which introduces a frame shift and an early termination signal in the receptor mRNA sequence. The mutated receptor neither bound 125I-labeled glucagon nor induced cAMP production upon stimulation with up to 1 μM glucagon. Despite the mutation, the only obvious pathophysiological trait was hyperglucagonemia, hyperaminoacidemia and massive hyperplasia of the pancreatic α-cells assessed by histology. Our case supports the notion of a hepato–pancreatic feedback system, which upon disruption leads to hyperglucagonemia and α-cell hyperplasia, as well as elevated plasma amino acid levels. Together with the glucagon-induced hypoaminoacidemia in glucagonoma patients, our case supports recent suggestions that amino acids may provide the feedback link between the liver and the pancreatic α-cells.

  3. IL-7 Receptor Mutations and Steroid Resistance in Pediatric T cell Acute Lymphoblastic Leukemia: A Genome Sequencing Study.

    Directory of Open Access Journals (Sweden)

    Yunlei Li

    2016-12-01

    Full Text Available Pediatric acute lymphoblastic leukemia (ALL is the most common childhood cancer and the leading cause of cancer-related mortality in children. T cell ALL (T-ALL represents about 15% of pediatric ALL cases and is considered a high-risk disease. T-ALL is often associated with resistance to treatment, including steroids, which are currently the cornerstone for treating ALL; moreover, initial steroid response strongly predicts survival and cure. However, the cellular mechanisms underlying steroid resistance in T-ALL patients are poorly understood. In this study, we combined various genomic datasets in order to identify candidate genetic mechanisms underlying steroid resistance in children undergoing T-ALL treatment.We performed whole genome sequencing on paired pre-treatment (diagnostic and post-treatment (remission samples from 13 patients, and targeted exome sequencing of pre-treatment samples from 69 additional T-ALL patients. We then integrated mutation data with copy number data for 151 mutated genes, and this integrated dataset was tested for associations of mutations with clinical outcomes and in vitro drug response. Our analysis revealed that mutations in JAK1 and KRAS, two genes encoding components of the interleukin 7 receptor (IL7R signaling pathway, were associated with steroid resistance and poor outcome. We then sequenced JAK1, KRAS, and other genes in this pathway, including IL7R, JAK3, NF1, NRAS, and AKT, in these 69 T-ALL patients and a further 77 T-ALL patients. We identified mutations in 32% (47/146 of patients, the majority of whom had a specific T-ALL subtype (early thymic progenitor ALL or TLX. Based on the outcomes of these patients and their prednisolone responsiveness measured in vitro, we then confirmed that these mutations were associated with both steroid resistance and poor outcome. To explore how these mutations in IL7R signaling pathway genes cause steroid resistance and subsequent poor outcome, we expressed wild

  4. IL-7 Receptor Mutations and Steroid Resistance in Pediatric T cell Acute Lymphoblastic Leukemia: A Genome Sequencing Study

    Science.gov (United States)

    Stubbs, Andrew P.; Vroegindeweij, Eric M.; Smits, Willem K.; van Marion, Ronald; Dinjens, Winand N. M.; Horstmann, Martin; Kuiper, Roland P.; Zaman, Guido J. R.; van der Spek, Peter J.; Pieters, Rob; Meijerink, Jules P. P.

    2016-01-01

    Background Pediatric acute lymphoblastic leukemia (ALL) is the most common childhood cancer and the leading cause of cancer-related mortality in children. T cell ALL (T-ALL) represents about 15% of pediatric ALL cases and is considered a high-risk disease. T-ALL is often associated with resistance to treatment, including steroids, which are currently the cornerstone for treating ALL; moreover, initial steroid response strongly predicts survival and cure. However, the cellular mechanisms underlying steroid resistance in T-ALL patients are poorly understood. In this study, we combined various genomic datasets in order to identify candidate genetic mechanisms underlying steroid resistance in children undergoing T-ALL treatment. Methods and Findings We performed whole genome sequencing on paired pre-treatment (diagnostic) and post-treatment (remission) samples from 13 patients, and targeted exome sequencing of pre-treatment samples from 69 additional T-ALL patients. We then integrated mutation data with copy number data for 151 mutated genes, and this integrated dataset was tested for associations of mutations with clinical outcomes and in vitro drug response. Our analysis revealed that mutations in JAK1 and KRAS, two genes encoding components of the interleukin 7 receptor (IL7R) signaling pathway, were associated with steroid resistance and poor outcome. We then sequenced JAK1, KRAS, and other genes in this pathway, including IL7R, JAK3, NF1, NRAS, and AKT, in these 69 T-ALL patients and a further 77 T-ALL patients. We identified mutations in 32% (47/146) of patients, the majority of whom had a specific T-ALL subtype (early thymic progenitor ALL or TLX). Based on the outcomes of these patients and their prednisolone responsiveness measured in vitro, we then confirmed that these mutations were associated with both steroid resistance and poor outcome. To explore how these mutations in IL7R signaling pathway genes cause steroid resistance and subsequent poor outcome, we

  5. Diffuse large B-cell lymphoma with combined TP53 mutation and MIR34A> methylation

    DEFF Research Database (Denmark)

    Asmar, Fazila; Hother, Christoffer; Kulosman, Gorjan

    2014-01-01

    and MIR34A methylation ("double hit") and these patients have an exceedingly poor prognosis with a median survival of 9.4 months (Phit") influence on survival. The TP53/MIR34A "double-hit" is an independent...... negative prognostic factor for survival (P=0.0002). In 2 DLBCL-cell lines with both TP53 mutation and promoter methylation of MIR34A, miR34A-5p is upregulated by 5-aza-2'deoxycytidine. Thus, the TP53/MIR34A "double hit" characterizes a very aggressive subgroup of DLBCL, which may be treatable...

  6. Frequent mutations of chromatin remodeling genes in transitional cell carcinoma of the bladder

    DEFF Research Database (Denmark)

    Gui, Yaoting; Guo, Guangwu; Huang, Yi

    2011-01-01

    frequently in tumors of low stages and grades, highlighting its potential role in the classification and diagnosis of bladder cancer. Our results provide an overview of the genetic basis of TCC and suggest that aberration of chromatin regulation might be a hallmark of bladder cancer.......Transitional cell carcinoma (TCC) is the most common type of bladder cancer. Here we sequenced the exomes of nine individuals with TCC and screened all the somatically mutated genes in a prevalence set of 88 additional individuals with TCC with different tumor stages and grades. In our study, we...

  7. Mast Cell Leukaemia: c-KIT Mutations Are Not Always Positive

    Directory of Open Access Journals (Sweden)

    Magalie Joris

    2012-01-01

    Full Text Available Mast cell leukemia (MCL is a rare and aggressive disease with poor prognosis and short survival time. D816V c-KIT mutation is the most frequent molecular abnormality and plays a crucial role in the pathogenesis and development of the disease. Thus, comprehensive diagnostic investigations and molecular studies should be carefully carried out to facilitate the therapeutic choice. A MCL patient’s case with rare phenotypic and genotypic characteristics is described with review of major clinical biological and therapeutic approaches in MCL.

  8. Stability of transmembrane amyloid β-peptide and membrane integrity tested by molecular modeling of site-specific Aβ42 mutations.

    Directory of Open Access Journals (Sweden)

    Chetan Poojari

    Full Text Available Interactions of the amyloid β-protein (Aβ with neuronal cell membranes, leading to the disruption of membrane integrity, are considered to play a key role in the development of Alzheimer's disease. Natural mutations in Aβ42, such as the Arctic mutation (E22G have been shown to increase Aβ42 aggregation and neurotoxicity, leading to the early-onset of Alzheimer's disease. A correlation between the propensity of Aβ42 to form protofibrils and its effect on neuronal dysfunction and degeneration has been established. Using rational mutagenesis of the Aβ42 peptide it was further revealed that the aggregation of different Aβ42 mutants in lipid membranes results in a variety of polymorphic aggregates in a mutation dependent manner. The mutant peptides also have a variable ability to disrupt bilayer integrity. To further test the connection between Aβ42 mutation and peptide-membrane interactions, we perform molecular dynamics simulations of membrane-inserted Aβ42 variants (wild-type and E22G, D23G, E22G/D23G, K16M/K28M and K16M/E22G/D23G/K28M mutants as β-sheet monomers and tetramers. The effects of charged residues on transmembrane Aβ42 stability and membrane integrity are analyzed at atomistic level. We observe an increased stability for the E22G Aβ42 peptide and a decreased stability for D23G compared to wild-type Aβ42, while D23G has the largest membrane-disruptive effect. These results support the experimental observation that the altered toxicity arising from mutations in Aβ is not only a result of the altered aggregation propensity, but also originates from modified Aβ interactions with neuronal membranes.

  9. High-incidence of PTEN mutations in Chinese patients with primary small cell carcinoma of the esophagus

    International Nuclear Information System (INIS)

    Zhang, Zhimin; Wang, Ge; Xiao, Hualiang; Xie, Fei; Zhang, Hui; Chen, Chuan; Xiao, He; Yang, Zhenzhou; Wang, Dong; Li, Zengpeng

    2014-01-01

    Primary small cell carcinoma of the esophagus (PSCCE) is a rare and aggressive tumor with poor prognosis. The aim of this study was to investigate the existence of EGFR, KRAS, PIK3CA and PTEN mutations in PSCCE. Clinical–pathological data and paraffin-embedded specimens were collected from 38 patients. Exons 18 to 21 of EGFR, KRAS and PIK3CA status were analyzed by real-time PCR based on ARMS and Scorpion technology in all patients, and the PTEN gene was also screened using real-time PCR and high-resolution melting curve analysis (HRMA). Only 1 (2.63%) out of 38 patients had EGFR mutations in L858R missense, and KRAS and PIK3CA were not found in the mutational spot in all patients. However, PTEN mutations presented in 14 (36.84%) out of 38 patients, including exon 5 coding for PTEN missense mutation (n =4, 10.53%), exon 6 (n =7, 18.42%), concurrent exon 5 and exon 6 (n =2, 5.26%), and exon 8 (n =1, 2.63%). Concurrent mutations of these genes were not detected in all samples. No statistically significant associations were found between the clinicopathological features and the mutation status of PTEN. The incidence of PTEN mutations in Chinese patients with PSCCE was higher than that of previous reports in other histological subtypes of esophageal cancer

  10. Gene Mutation Profiles in Primary Diffuse Large B Cell Lymphoma of Central Nervous System: Next Generation Sequencing Analyses

    Science.gov (United States)

    Todorovic Balint, Milena; Jelicic, Jelena; Mihaljevic, Biljana; Kostic, Jelena; Stanic, Bojana; Balint, Bela; Pejanovic, Nadja; Lucic, Bojana; Tosic, Natasa; Marjanovic, Irena; Stojiljkovic, Maja; Karan-Djurasevic, Teodora; Perisic, Ognjen; Rakocevic, Goran; Popovic, Milos; Raicevic, Sava; Bila, Jelena; Antic, Darko; Andjelic, Bosko; Pavlovic, Sonja

    2016-01-01

    The existence of a potential primary central nervous system lymphoma-specific genomic signature that differs from the systemic form of diffuse large B cell lymphoma (DLBCL) has been suggested, but is still controversial. We investigated 19 patients with primary DLBCL of central nervous system (DLBCL CNS) using the TruSeq Amplicon Cancer Panel (TSACP) for 48 cancer-related genes. Next generation sequencing (NGS) analyses have revealed that over 80% of potentially protein-changing mutations were located in eight genes (CTNNB1, PIK3CA, PTEN, ATM, KRAS, PTPN11, TP53 and JAK3), pointing to the potential role of these genes in lymphomagenesis. TP53 was the only gene harboring mutations in all 19 patients. In addition, the presence of mutated TP53 and ATM genes correlated with a higher total number of mutations in other analyzed genes. Furthermore, the presence of mutated ATM correlated with poorer event-free survival (EFS) (p = 0.036). The presence of the mutated SMO gene correlated with earlier disease relapse (p = 0.023), inferior event-free survival (p = 0.011) and overall survival (OS) (p = 0.017), while mutations in the PTEN gene were associated with inferior OS (p = 0.048). Our findings suggest that the TP53 and ATM genes could be involved in the molecular pathophysiology of primary DLBCL CNS, whereas mutations in the PTEN and SMO genes could affect survival regardless of the initial treatment approach. PMID:27164089

  11. Optimal selection for BRCA1 and BRCA2 mutation testing using a combination of ' easy to apply ' probability models

    NARCIS (Netherlands)

    Bodmer, D.; Ligtenberg, M. J. L.; van der Hout, A. H.; Gloudemans, S.; Ansink, K.; Oosterwijk, J. C.; Hoogerbrugge, N.

    2006-01-01

    To establish an efficient, reliable and easy to apply risk assessment tool to select families with breast and/or ovarian cancer patients for BRCA mutation testing, using available probability models. In a retrospective study of 263 families with breast and/or ovarian cancer patients, the utility of

  12. KRAS mutation testing in colorectal cancer: comparison of the results obtained using 3 different methods for the analysis of codons G12 and G13.

    Science.gov (United States)

    Bihl, Michel P; Hoeller, Sylvia; Andreozzi, Maria Carla; Foerster, Anja; Rufle, Alexander; Tornillo, Luigi; Terracciano, Luigi

    2012-03-01

    Targeting the epidermal growth factor receptor (EGFR) is a new therapeutic option for patients with metastatic colorectal or lung carcinoma. However, the therapy efficiency highly depends on the KRAS mutation status in the given tumour. Therefore a reliable and secure KRAS mutation testing is crucial. Here we investigated 100 colorectal carcinoma samples with known KRAS mutation status (62 mutated cases and 38 wild type cases) in a comparative manner with three different KRAS mutation testing techniques (Pyrosequencing, Dideoxysequencing and INFINITI) in order to test their reliability and sensitivity. For the large majority of samples (96/100, 96%), the KRAS mutation status obtained by all three methods was the same. Only two cases with clear discrepancies were observed. One case was reported as wild type by the INFINITI method while the two other methods detected a G13C mutation. In the second case the mutation could be detected by the Pyrosequencing and INFINITI method (15% and 15%), while no signal for mutation could be observed with the Dideoxysequencing method. Additional two unclear results were due to a detection of a G12V with the INFINITI method, which was below cut-off when repeated and which was not detectable by the other two methods and very weak signals in a G12V mutated case with the Dideoxy- and Pyroseqencing method compared to the INFINITI method, respectively. In summary all three methods are reliable and robust methods in detecting KRAS mutations. INFINITI, however seems to be slightly more sensitive compared to Dideoxy- and Pyrosequencing.

  13. Isolation of chromosomal mutations in Drosophila melanogaster by the nondisjunction test

    International Nuclear Information System (INIS)

    Chadov, B.F.; Ivanov, Yu.N.; Artemova, E.V.

    1986-01-01

    The females carrying the combination of metacentric autosome 2 with two acrocentrics, F(2L) and F(2R), produce aneuploid egg cells 2/F(2L) and F(2R)(2-F(2L) nondisjunction) as well as egg cells 2/F(2R) and F(2L) (2-F(2R) nondisjunction). The frequency of nondisjunction 2-F(2L) sharply increases in the presence of inversion in the left arm of metacentric 2, and the frequency of nondisjunction 2-F(2R) increases when the right arm of the metacentric carries the inversion. The presence of the rearrangement in the left arm can be detected by the unusually high progeny size in the cross between 2/F(2L);F(2R) females, and C(2L);F(2R)/F(2R) males, and the presence of rearrangement in the right arm of the metacentric is inferred from a large progeny in the cross of similar females with F(2L)/F(2L);C(2R) males. The SPx 2 /F(2L);F(2R) daughters were obtained from SPx 2 /+ males irradiated with the dose of 3000R. Out of these, 972 daughters were individually crossed with F(2L)/F(2L); C(2R) males. Fifty-two cultures with large progeny size were identified by visual observation. Preparations of polytene chromsomes were made from the larvae. Twenty-six cultures had arrangements in 2 R: eight carried rearrangement In (2R), six In(2LR), ten T(2R; 3) and T(2R; X), and two Tp (2). The method can be used effective to detect chromosomal mutations, especially inversions in large samples

  14. DNA replication in necessary for fixing induced mutations to streptomycin-resistance in UV-irradiated Escherichia coli cells

    Energy Technology Data Exchange (ETDEWEB)

    Dubinin, N P; Filippov, V D

    1986-01-01

    A suspension of E.coli cells has been subjected to UV radiation, then it has been incubated in the growth medium for 15 min. After that one of the portions was incubated with nalidixic acid (NA), and the other one without it in the presence of an antibiotic. Frequency of mutations depending on or irrespective of photoactivation, has been determined. Dependence of Str mutation fixing, induced by low UV radiation doses, on DNA synthesis is determined. Results indicate that both photoreactivation of mutations and its senstivity to mfd system are simultaneously lost.

  15. Gene mutation, quantitative mutagenesis, and mutagen screening in mammalian cells: study with the CHO/HGPRT system

    International Nuclear Information System (INIS)

    Hsie, A.W.

    1980-01-01

    We have employed CHO cells to develop and define a set of stringent conditions for studying mutation induction to TG resistance. Several lines of evidence support the CHO/HGPRT system as a specific-locus mutational assay. The system permits quantification of mutation at the HGPRT locus induced by various physical and chemical mutagens. The quantitative nature of the system provides a basis for the study of structure-function relationships of various classes of chemical mutagens. The intra- and interlaboratory reproducibility of this system suggests its potential for screening environmental agents for mutagenic activity

  16. A common mutation in the methylenetetrahydrofolate reductase gene is associated with an accumulation of formylated tetrahydrofolates in red blood cells

    Science.gov (United States)

    Bagley, Pamela J.; Selhub, Jacob

    1998-01-01

    A common mutation (C677T) in the gene encoding for methylenetetrahydrofolate reductase (MTHFR) (5-methyltetrahydrofolate:(acceptor) oxidoreductase, EC 1.7.99.5), a key regulatory enzyme in one-carbon metabolism, results in a thermolabile variant of the MTHFR enzyme with reduced activity in vitro. In the present study we used a chromatographic method for folate analysis to test the hypothesis that this mutation would be associated with altered distribution of red blood cell (RBC) folates. An alteration was found as manifested by the presence of formylated tetrahydrofolate polyglutamates in addition to methylated derivatives in the RBCs from homozygous mutant individuals. 5-Methyltetrahydrofolate polyglutamates were the only folate form found in RBCs from individuals with the wild-type genotype. Existence of formylated folates in RBCs only from individuals with the thermolabile MTHFR is consistent with the hypothesis that there is in vivo impairment in the activity of the thermolabile variant of MTHFR and that this impairment results in an altered distribution of RBC folates. PMID:9789068

  17. Discovery and prioritization of somatic mutations in diffuse large B-cell lymphoma (DLBCL) by whole-exome sequencing

    OpenAIRE

    Lohr, Jens G.; Stojanov, Petar; Lawrence, Michael S.; Auclair, Daniel; Chapuy, Bjoern; Sougnez, Carrie; Cruz-Gordillo, Peter; Knoechel, Birgit; Asmann, Yan W.; Slager, Susan L.; Novak, Anne J.; Dogan, Ahmet; Ansell, Stephen M.; Link, Brian K.; Zou, Lihua

    2012-01-01

    To gain insight into the genomic basis of diffuse large B-cell lymphoma (DLBCL), we performed massively parallel whole-exome sequencing of 55 primary tumor samples from patients with DLBCL and matched normal tissue. We identified recurrent mutations in genes that are well known to be functionally relevant in DLBCL, including MYD88, CARD11, EZH2, and CREBBP. We also identified somatic mutations in genes for which a functional role in DLBCL has not been previously suspected. These genes include...

  18. High LET radiation enhances apoptosis in mutated p53 cancer cells through Caspase-9 activation

    International Nuclear Information System (INIS)

    Yamakawa, Nobuhiro; Takahashi, Akihisa; Mori, Eiichiro; Imai, Yuichiro; Ohnishi, Ken; Kirita, Tadaaki; Ohnishi, Takeo; Furusawa, Yoshiya

    2008-01-01

    Although mutations in the p53 gene can lead to resistance to radiotherapy, chemotherapy and thermotherapy, high linear energy transfer (LET) radiation induces apoptosis regardless of p53 gene status in cancer cells. The aim of this study was to clarify the mechanisms involved in high LET radiation-induced apoptosis. Human gingival cancer cells (Ca9-22 cells) containing a mutated p53 (mp53) gene were irradiated with X-rays, C-ion (13-100 KeV/μm), or Fe-ion beams (200 KeV/μm). Cellular sensitivities were determined using colony forming assays. Apoptosis was detected and quantified with Hoechst 33342 staining. The activity of Caspase-3 was analyzed with Western blotting and flow cytometry. Cells irradiated with high LET radiation showed a high sensitivity with a high frequency of apoptosis induction. The relative biological effectiveness (RBE) values for the surviving fraction and apoptosis induction increased in a LET-dependent manner. Both RBE curves reached a peak at 100 KeV/μm, and then decreased at values over 100 KeV/μm. When cells were irradiated with high LET radiation, Caspase-3 was cleaved and activated, leading to poly (ADP-ribose) polymerase (PARP) cleavage. In addition, Caspase-9 inhibitor suppressed Caspase-3 activation and apoptosis induction resulting from high LET radiation to a greater extent than Caspase-8 inhibitor. These results suggest that high LET radiation enhances apoptosis by activation of Caspase-3 through Caspase-9, even in the presence of mp53. (author)

  19. Gene mutations, chromosome aberrations and survival after X-ray irradiation of cultured Chinese hamster cells at cysteamine protection

    International Nuclear Information System (INIS)

    Elisova, I.V.; Feoktistova, I.P.

    1983-01-01

    The culture of Chinese hamster cells (clone 431) has been used to study cysteamine action on mutagenous effect of X-rays, determined by the induction of resistance of gene mutations to 6-thioguanine and chromosomal abberations, as well as on the reproductive form of death of irradiated cells. Dose--- effect curves are obtained under conditions of irradiation with and without protector. The factor of dose alteration is 2.0 for chromosomal aberrations and cell survival, and 2.8 for gene mutations. It is sUpposed that cysteamine affects the general mechanisms, which take part in the realis zation of injuries that bring about gene mutations, chromosomal aberrations and cell lethality

  20. Ultraviolet-irradiated simian virus 40 activates a mutator function in rat cells under conditions preventing viral DNA replication

    Energy Technology Data Exchange (ETDEWEB)

    Cornelis, J.; Su, Z.Z.; Dinsart, C.; Rommelaere, J. (Universite libre de Bruxelles, Rhode St Genese (Belgium))

    The UV-irradiated temperature-sensitive early SV40 mutant tsA209 is able to activate at the nonpermissive temperature the expression of mutator and recovery functions in rat cells. Unirradiated SV40 activates these functions only to a low extent. The expression of these mutator and recovery functions in SV40-infected cells was detected using the single-stranded DNA parvovirus H-1 as a probe. Because early SV40 mutants are defective in the initiation of viral DNA synthesis at the nonpermissive temperature, these results suggest that replication of UV-damaged DNA is not a prerequisite for the activation of mutator and recovery functions in mammalian cells. The expression of the mutator function is dose-dependent, i.e., the absolute number of UV-irradiated SV40 virions introduced per cell determines its level. Implications for the interpretation of mutation induction curves in the progeny of UV-irradiated SV40 in permissive host cells are discussed.

  1. Towards Measuring the Abstractness of State Machines based on Mutation Testing

    Directory of Open Access Journals (Sweden)

    Thomas Baar

    2017-01-01

    Full Text Available Abstract. The notation of state machines is widely adopted as a formalism to describe the behaviour of systems. Usually, multiple state machine models can be developed for the very same software system. Some of these models might turn out to be equivalent, but, in many cases, different state machines describing the same system also differ in their level of abstraction. In this paper, we present an approach to actually measure the abstractness level of state machines w.r.t. a given implemented software system. A state machine is considered to be less abstract when it is conceptionally closer to the implemented system. In our approach, this distance between state machine and implementation is measured by applying coverage criteria known from software mutation testing. Abstractness of state machines can be considered as a new metric. As for other metrics as well, a known value for the abstractness of a given state machine allows to assess its quality in terms of a simple number. In model-based software development projects, the abstract metric can help to prevent model degradation since it can actually measure the semantic distance from the behavioural specification of a system in form of a state machine to the current implementation of the system. In contrast to other metrics for state machines, the abstractness cannot be statically computed based on the state machine’s structure, but requires to execute both state machine and corresponding system implementation. The article is published in the author’s wording. 

  2. Proliferation and survival molecules implicated in the inhibition of BRAF pathway in thyroid cancer cells harbouring different genetic mutations

    International Nuclear Information System (INIS)

    Preto, Ana; Soares, Paula; Sobrinho-Simões, Manuel; Gonçalves, Joana; Rebocho, Ana P; Figueiredo, Joana; Meireles, Ana M; Rocha, Ana S; Vasconcelos, Helena M; Seca, Hugo; Seruca, Raquel

    2009-01-01

    Thyroid carcinomas show a high prevalence of mutations in the oncogene BRAF which are inversely associated with RAS or RET/PTC oncogenic activation. The possibility of using inhibitors on the BRAF pathway as became an interesting therapeutic approach. In thyroid cancer cells the target molecules, implicated on the cellular effects, mediated by inhibition of BRAF are not well established. In order to fill this lack of knowledge we studied the proliferation and survival pathways and associated molecules induced by BRAF inhibition in thyroid carcinoma cell lines harbouring distinct genetic backgrounds. Suppression of BRAF pathway in thyroid cancer cell lines (8505C, TPC1 and C643) was achieved using RNA interference (RNAi) for BRAF and the kinase inhibitor, sorafenib. Proliferation analysis was performed by BrdU incorporation and apoptosis was accessed by TUNEL assay. Levels of protein expression were analysed by western-blot. Both BRAF RNAi and sorafenib inhibited proliferation in all the cell lines independently of the genetic background, mostly in cells with BRAF V600E mutation. In BRAF V600E mutated cells inhibition of BRAF pathway lead to a decrease in ERK1/2 phosphorylation and cyclin D1 levels and an increase in p27 Kip1 . Specific inhibition of BRAF by RNAi in cells with BRAF V600E mutation had no effect on apoptosis. In the case of sorafenib treatment, cells harbouring BRAF V600E mutation showed increase levels of apoptosis due to a balance of the anti-apoptotic proteins Mcl-1 and Bcl-2. Our results in thyroid cancer cells, namely those harbouring BRAF V600E mutation showed that BRAF signalling pathway provides important proliferation signals. We have shown that in thyroid cancer cells sorafenib induces apoptosis by affecting Mcl-1 and Bcl-2 in BRAF V600E mutated cells which was independent of BRAF. These results suggest that sorafenib may prove useful in the treatment of thyroid carcinomas, particularly those refractory to conventional treatment and

  3. Myogenic Differentiation from MYOGENIN-Mutated Human iPS Cells by CRISPR/Cas9

    Directory of Open Access Journals (Sweden)

    Koki Higashioka

    2017-01-01

    Full Text Available It is well known that myogenic regulatory factors encoded by the Myod1 family of genes have pivotal roles in myogenesis, with partially overlapping functions, as demonstrated for the mouse embryo. Myogenin-mutant mice, however, exhibit severe myogenic defects without compensation by other myogenic factors. MYOGENIN might be expected to have an analogous function in human myogenic cells. To verify this hypothesis, we generated MYOGENIN-mutated human iPS cells by using CRISPR/Cas9 genome-editing technology. Our results suggest that MYOD1-independent or MYOD1-dependent mechanisms can compensate for the loss of MYOGENIN and that these mechanisms are likely to be crucial for regulating skeletal muscle differentiation and formation.

  4. k-RAS mutations in non-small cell lung cancer patients treated with TKIs among smokers and non-smokers: a meta-analysis

    Directory of Open Access Journals (Sweden)

    Ai-Gui Jiang

    2016-06-01

    Full Text Available Aim of the study : Recent studies have suggested that k-RAS mutations are related to the response to epidermal growth factor receptor (EGFR tyrosine-kinase inhibitions (TKIs in advanced non-small cell lung cancer (NSCLC treatment. The aim of this meta-analysis was to assess the relationship between smoking history and k-RAS mutations in NSCLC treated with TKIs. Material and methods : We searched MEDLINE and Web of Science up to 15 March 2014. The pooled relative risk (RR was estimated by using fixed effect model or random effect model, according to heterogeneity between studies. We also carried out power analyses. Results : We identified 12 studies with 1193 patients, including 196 patients (16.4% with k-RAS mutations. The pooled k-RAS mutations incidence was 22.8% (174/764 in patients with smoke expose vs. 5.4% (23/429 in those with no smoke exposure. The pooled RR was 2.991 (95% CI: 1.884–4.746; Z = 4.65, p = 0.000. No publication bias was found (Begg’s test: z = 1.09, p = 0.274 and Egger’s test: t = 1.38, p = 0.201. In subgroup analyses, the pooled RR was 3.336 (95% CI: 1.925–5.779; Z = 4.30, p = 0.000 in the Caucasian subgroup, while in the Asian subgroup the pooled RR was 2.093 (95% CI: 0.909–4.822; Z = 1.73, p = 0.083, but the sample size was underpowered (0.465. Conclusions : The current meta-analysis found that smoking was related to increased incidence of k-RAS mutations in non-small cell lung cancer treated with TKIs. This may be further evidence that smoking will lead to a worse prognosis in NSCLC patients treated with TKIs.

  5. Mutational analysis of the RNA-binding domain of the Prunus necrotic ringspot virus (PNRSV) movement protein reveals its requirement for cell-to-cell movement

    OpenAIRE

    Herranz, M. Carmen; Sánchez Navarro, Jesús A.; Saurí Peris, Ana; Mingarro Muñoz, Ismael; Pallás Benet, Vicente

    2005-01-01

    The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is required for cell-to-cell movement. MP subcellular localization studies using a GFP fusion protein revealed highly punctate structures between neighboring cells, believed to represent plasmodesmata. Deletion of the RNA-binding domain (RBD) of PNRSV MP abolishes the cell-to-cell movement. A mutational analysis on this RBD was performed in order to identify in vivo the features that govern viral transport. Loss of positive c...

  6. A novel natural killer cell line (KHYG-1) from a patient with aggressive natural killer cell leukemia carrying a p53 point mutation.

    Science.gov (United States)

    Yagita, M; Huang, C L; Umehara, H; Matsuo, Y; Tabata, R; Miyake, M; Konaka, Y; Takatsuki, K

    2000-05-01

    We present the establishment of a natural killer (NK) leukemia cell line, designated KHYG-1, from the blood of a patient with aggressive NK leukemia, which both possessed the same p53 point mutation. The immunophenotype of the primary leukemia cells was CD2+, surface CD3-, cytoplasmic CD3epsilon+, CD7+, CD8alphaalpha+, CD16+, CD56+, CD57+ and HLA-DR+. A new cell line (KHYG-1) was established by culturing peripheral leukemia cells with 100 units of recombinant interleukin (IL)-2. The KHYG-1 cells showed LGL morphology with a large nucleus, coarse chromatin, conspicuous nucleoli, and abundant basophilic cytoplasm with many azurophilic granules. The immunophenotype of KHYG-1 cells was CD1-, CD2+, surface CD3-, cytoplasmic CD3epsilon+, CD7+, CD8alphaalpha+, CD16-, CD25-, CD33+, CD34-, CD56+, CD57-, CD122+, CD132+, and TdT-. Southern blot analysis of these cells revealed a normal germline configuration for the beta, delta, and gamma chains of the T cell receptor and the immunoglobulin heavy-chain genes. Moreover, the KHYG-1 cells displayed NK cell activity and IL-2-dependent proliferation in vitro, suggesting that they are of NK cell origin. Epstein-Barr virus (EBV) DNA was not detected in KHYG-1 cells by Southern blot analysis with a terminal repeat probe from an EBV genome. A point mutation in exon 7 of the p53 gene was detected in the KHYG-1 cells by PCR/SSCP analysis, and direct sequencing revealed the conversion of C to T at nucleotide 877 in codon 248. The primary leukemia cells also carried the same point mutation. Although the precise role of the p53 point mutation in leukemogenesis remains to be clarified, the establishment of an NK leukemia cell line with a p53 point mutation could be valuable in the study of leukemogenesis.

  7. Fuel Cell Development and Test Laboratory | Energy Systems Integration

    Science.gov (United States)

    Facility | NREL Fuel Cell Development and Test Laboratory Fuel Cell Development and Test Laboratory The Energy System Integration Facility's Fuel Cell Development and Test Laboratory supports fuel cell research and development projects through in-situ fuel cell testing. Photo of a researcher running

  8. Induction of apoptosis by tomato using space mutation breeding in human colon cancer SW480 and HT-29 cells.

    Science.gov (United States)

    Shi, Jiahui; Yang, Bin; Feng, Pan; Li, Duo; Zhu, Jiajin

    2010-03-15

    As far as we know, there have been no reports concerning the functional characteristics of tomatoes using space mutation breeding. The aim of this study was to evaluate the anti-colon cancer effect of tomatoes M1 and M2 using space mutation breeding. In the present study, obvious anti-cancer activity was shown with tomato juice of M1 and M2 and their parent CK treatment in colon cancer cell lines SW480 and HT-29 in cell growth inhibition. In addition, SW480 cells were more sensitive to M1 and M2 than HT-29 cells in cell apoptosis. Furthermore, M1 and M2 induced cell cycle arrest both in G0-G1 and G2/M phases. These data suggest that consumption of tomato using space mutation breeding may provide benefits to inhibit growth of colon cancer cells. Therefore, tomato production using space mutation breeding may be a good candidate for development as a dietary supplement in drug therapy for colon cancer.

  9. Pitfalls in genetic testing: the story of missed SCN1A mutations

    OpenAIRE

    Jennings, Lawrence J.; Kirschmann, Dawn

    2016-01-01

    Investigators from the EuroEPINOMICS rare epilepsy syndromes Dravet working group performed whole-exome sequencing on 31 trios that had been reported negative for SCN1A mutations by Sanger sequencing.

  10. A neurodegenerative disease mutation that accelerates the clearance of apoptotic cells.

    Science.gov (United States)

    Kao, Aimee W; Eisenhut, Robin J; Martens, Lauren Herl; Nakamura, Ayumi; Huang, Anne; Bagley, Josh A; Zhou, Ping; de Luis, Alberto; Neukomm, Lukas J; Cabello, Juan; Farese, Robert V; Kenyon, Cynthia

    2011-03-15

    Frontotemporal lobar degeneration is a progressive neurodegenerative syndrome that is the second most common cause of early-onset dementia. Mutations in the progranulin gene are a major cause of familial frontotemporal lobar degeneration [Baker M, et al. (2006) Nature 442:916-919 and Cruts M, et al. (2006) Nature 442:920-924]. Although progranulin is involved in wound healing, inflammation, and tumor growth, its role in the nervous system and the mechanism by which insufficient levels result in neurodegeneration are poorly understood [Eriksen and Mackenzie (2008) J Neurochem 104:287-297]. We have characterized the normal function of progranulin in the nematode Caenorhabditis elegans. We found that mutants lacking pgrn-1 appear grossly normal, but exhibit fewer apoptotic cell corpses during development. This reduction in corpse number is not caused by reduced apoptosis, but instead by more rapid clearance of dying cells. Likewise, we found that macrophages cultured from progranulin KO mice displayed enhanced rates of apoptotic-cell phagocytosis. Although most neurodegenerative diseases are thought to be caused by the toxic effects of aggregated proteins, our findings suggest that susceptibility to neurodegeneration may be increased by a change in the kinetics of programmed cell death. We propose that cells that might otherwise recover from damage or injury are destroyed in progranulin mutants, which in turn facilitates disease progression.

  11. Curcumin induces apoptosis and cell cycle arrest via the activation of reactive oxygen species-independent mitochondrial apoptotic pathway in Smad4 and p53 mutated colon adenocarcinoma HT29 cells.

    Science.gov (United States)

    Agarwal, Ayushi; Kasinathan, Akiladdevi; Ganesan, Ramamoorthi; Balasubramanian, Akhila; Bhaskaran, Jahnavi; Suresh, Samyuktha; Srinivasan, Revanth; Aravind, K B; Sivalingam, Nageswaran

    2018-03-01

    Curcumin is a natural dietary polyphenol compound that has various pharmacological activities such as antiproliferative and cancer-preventive activities on tumor cells. Indeed, the role reactive oxygen species (ROS) generated by curcumin on cell death and cell proliferation inhibition in colon cancer is poorly understood. In the present study, we hypothesized that curcumin-induced ROS may promote apoptosis and cell cycle arrest in colon cancer. To test this hypothesis, the apoptosis-inducing potential and cell cycle inhibition effect of ROS induced by curcumin was investigated in Smd4 and p53 mutated HT-29 colon adenocarcinoma cells. We found that curcumin treatment significantly increased the level of ROS in HT-29 cells in a dose- and time-dependent manner. Furthermore, curcumin treatment markedly decreased the cell viability and proliferation potential of HT-29 cells in a dose- and time-dependent manner. Conversely, generation of ROS and inhibitory effect of curcumin on HT-29 cells were abrogated by N-acetylcysteine treatment. In addition, curcumin treatment did not show any cytotoxic effects on HT-29 cells. Furthermore, curcumin-induced ROS generation caused the DNA fragmentation, chromatin condensation, and cell nuclear shrinkage and significantly increased apoptotic cells in a dose- and time-dependent manner in HT-29 cells. However, pretreatment of N-acetylcysteine inhibited the apoptosis-triggering effect of curcumin-induced ROS in HT-29 cells. In addition, curcumin-induced ROS effectively mediated cell cycle inhibition in HT-29 cells. In conclusion, our data provide the first evidence that curcumin induces ROS independent apoptosis and cell cycle arrest in colon cancer cells that carry mutation on Smad4 and p53. Copyright © 2018. Published by Elsevier Inc.

  12. The identification of point mutations in Duchenne muscular dystrophy patients by using reverse-transcription PCR and the protein truncation test

    Energy Technology Data Exchange (ETDEWEB)

    Gardner, R.J.; Bobrow, M.; Roberts, R.G. [St. Thomas`s Hospitals, London (United Kingdom)

    1995-08-01

    The protein truncation test (PTT) is a mutation-detection method that monitors the integrity of the open reading frame (ORF). More than 60% of cases of Duchenne muscular dystrophy (DMD) result from gross frameshifting deletions in the dystrophin gene that are detectable by multiplex PCR system. It has become apparent that virtually all of the remaining DMD mutations also disrupt the translational reading frame, making the PTT a logical next step toward a comprehensive strategy for the identification of all DMD mutations. We report here a pilot study involving 22 patients and describe the mutations characterized. These constitute 12 point mutations or small insertions/deletions and 4 gross rearrangements. We also have a remaining five patients in whom there does not appear to be mutation in the ORF. We believe that reverse-transcription-PCR/PTT is an efficient method by which to screen for small mutations in DMD patients with no deletion. 29 refs., 2 figs., 3 tabs.

  13. Analysis of mutant frequencies and mutation spectra in hMTH1 knockdown TK6 cells exposed to UV radiation

    Energy Technology Data Exchange (ETDEWEB)

    Fotouhi, Asal [Center for Radiation Protection Research, Department of Molecular Biosciences, Wenner-Gren Institute, Stockholm University (Sweden); Hagos, Winta Woldai [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Ilic, Marina; Wojcik, Andrzej; Harms-Ringdahl, Mats [Center for Radiation Protection Research, Department of Molecular Biosciences, Wenner-Gren Institute, Stockholm University (Sweden); Gruijl, Frank de [Department of Dermatology, Leiden University Medical Center, Leiden (Netherlands); Mullenders, Leon; Jansen, Jacob G. [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Haghdoost, Siamak, E-mail: Siamak.Haghdoost@su.se [Center for Radiation Protection Research, Department of Molecular Biosciences, Wenner-Gren Institute, Stockholm University (Sweden)

    2013-11-15

    Highlights: • hMTH1 protects cells from mutagenesis induced by UVA and UVB, but not UVC. • No protective role of hMTH1 in cell survival post UVB and UVC irradiation. • hMTH1 prevents induction of transition-type mutations at AT and GC post UVA irradiation. • 2-OH-dATP rather than 8-oxo-dGTP in the nucleotide pool likely contributes in UVA-induced mutations. - Abstract: Ultraviolet radiation is a highly mutagenic agent that damages the DNA by the formation of mutagenic photoproducts at dipyrimidine sites and by oxidative DNA damages via reactive oxygen species (ROS). ROS can also give rise to mutations via oxidation of dNTPs in the nucleotide pool, e.g. 8-oxo-dGTP and 2-OH-dATP and subsequent incorporation during DNA replication. Here we show that expression of human MutT homolog 1 (hMTH1) which sanitizes the nucleotide pool by dephosphorylating oxidized dNTPs, protects against mutagenesis induced by long wave UVA light and by UVB light but not by short wave UVC light. Mutational spectra analyses of UVA-induced mutations at the endogenous Thymidine kinase gene in human lymphoblastoid cells revealed that hMTH1 mainly protects cells from transitions at GC and AT base pairs.

  14. Role of [{sup 18}F]FDG PET in prediction of KRAS and EGFR mutation status in patients with advanced non-small-cell lung cancer

    Energy Technology Data Exchange (ETDEWEB)

    Caicedo, Carlos; Garcia-Velloso, Maria Jose; Vigil Diaz, Carmen; Richter Echevarria, Jose Angel [University of Navarra, Nuclear Medicine Department, University Clinic of Navarra, Pamplona (Spain); Lozano, Maria Dolores; Labiano, Tania [University of Navarra, Pathology Department, University Clinic of Navarra, Pamplona (Spain); Lopez-Picazo, Jose Maria; Gurpide, Alfonso; Perez Gracia, Jose Luis [University of Navarra, Oncology Department, University Clinic of Navarra, Pamplona (Spain); Zulueta, Javier [University of Navarra, Pulmonology Department, University Clinic of Navarra, Pamplona (Spain)

    2014-11-15

    The tumour molecular profile predicts the activity of epidermal growth factor receptor (EGFR) inhibitors in non-small-cell lung cancer (NSCLC). However, tissue availability and tumour heterogeneity limit its assessment. We evaluated whether [{sup 18}F]FDG PET might help predict KRAS and EFGR mutation status in NSCLC. Between January 2005 and October 2011, 340 NSCLC patients were tested for KRAS and EGFR mutation status. We identified patients with stage III and IV disease who had undergone [{sup 18}F]FDG PET/CT scanning for initial staging. SUVpeak, SUVmax and SUVmean of the single hottest tumour lesions were calculated, and their association with KRAS and EGFR mutation status was assessed. A receiver operator characteristic (ROC) curve analysis and a multivariate analysis (including SUVmean, gender, age and AJCC stage) were performed to identify the potential value of [{sup 18}F]FDG PET/CT for predicting KRAS mutation. From 102 patients staged using [{sup 18}F]FDG PET/CT, 28 (27 %) had KRAS mutation (KRAS+), 22 (22 %) had EGFR mutation (EGFR+) and 52 (51 %) had wild-type KRAS and EGFR profiles (WT). KRAS+ patients showed significantly higher [{sup 18}F]FDG uptake than EGFR+ and WT patients (SUVmean 9.5, 5.7 and 6.6, respectively; p < 0.001). No significant differences were observed in [{sup 18}F]FDG uptake between EGFR+ patients and WT patients. ROC curve analysis for KRAS mutation status discrimination yielded an area under the curve of 0.740 for SUVmean (p < 0.001). The multivariate analysis showed a sensitivity and specificity of 78.6 % and 62.2 %, respectively, and the AUC was 0.773. NSCLC patients with tumours harbouring KRAS mutations showed significantly higher [{sup 18}F]FDG uptake than WT patients, as assessed in terms of SUVpeak, SUVmax and SUVmean. A multivariate model based on age, gender, AJCC stage and SUVmean might be used as a predictive marker of KRAS mutation status in patients with stage III or IV NSCLC. (orig.)

  15. SNPase-ARMS qPCR: Ultrasensitive Mutation-Based Detection of Cell-Free Tumor DNA in Melanoma Patients.

    Directory of Open Access Journals (Sweden)

    Julia Stadler

    Full Text Available Cell-free circulating tumor DNA in the plasma of cancer patients has become a common point of interest as indicator of therapy options and treatment response in clinical cancer research. Especially patient- and tumor-specific single nucleotide variants that accurately distinguish tumor DNA from wild type DNA are promising targets. The reliable detection and quantification of these single-base DNA variants is technically challenging. Currently, a variety of techniques is applied, with no apparent "gold standard". Here we present a novel qPCR protocol that meets the conditions of extreme sensitivity and specificity that are required for detection and quantification of tumor DNA. By consecutive application of two polymerases, one of them designed for extreme base-specificity, the method reaches unprecedented sensitivity and specificity. Three qPCR assays were tested with spike-in experiments, specific for point mutations BRAF V600E, PTEN T167A and NRAS Q61L of melanoma cell lines. It was possible to detect down to one copy of tumor DNA per reaction (Poisson distribution, at a background of up to 200 000 wild type DNAs. To prove its clinical applicability, the method was successfully tested on a small cohort of BRAF V600E positive melanoma patients.

  16. CP-31398 prevents the growth of p53-mutated colorectal cancer cells in vitro and in vivo.

    Science.gov (United States)

    He, Xingxing; Kong, Xinjuan; Yan, Junwei; Yan, Jingjun; Zhang, Yunan; Wu, Qian; Chang, Ying; Shang, Haitao; Dou, Qian; Song, Yuhu; Liu, Fang

    2015-03-01

    Rescuing the function of mutant p53 protein is an attractive cancer therapeutic strategy. Small molecule CP-31398 was shown to restore mutant p53 tumor suppressor functions in cancer cells. Here, we determined the effects of CP-31398 on the growth of p53-mutated colorectal cancer (CRC) cells in vitro and in vivo. CRC cells which carry p53 mutation in codon 273 were treated with CP-31398 and the control, and the effects of CP-31398 on cell cycle, cell apoptosis, and proliferation were determined. The expression of p53-responsive downstream genes was evaluated by quantitative reverse transcriptase PCR (RT-PCR) and Western blot. CP-31398 was administrated into xenograft tumors created by the inoculation of HT-29 cells, and then the effect of CP-31398 on the growth of xenograft tumors was examined. CP-31398 induced p53 downstream target molecules in cultured HT-29 cells, which resulted in the inhibition of CRC cell growth assessed by the determination of cell cycle, apoptosis, and cell proliferation. In xenograft tumors, CP-31398 modulated the expression of Bax, Bcl-2, caspase 3, cyclin D, and Mdm2 and then blocked the growth of xenograft tumors. CP-31398 would be developed as a therapeutic candidate for p53-mutated CRC due to the restoration of mutant p53 tumor suppressor functions.

  17. Development and applications of Bacillus subtilis test systems for mutagens, involving DNA-repair deficiency and suppressible auxotrophic mutations

    International Nuclear Information System (INIS)

    Tanooka, H.

    1977-01-01

    A mutagen-tester of Bacillus subtilis was constructed and tested with known carcinogens. The parental strain HA101 of Okubo and Yanagida carrying suppressible nonsense mutations in his and met genes was transformed to carry an excision-repair deficiency mutation. The constructed strain TKJ5211 showed a 20-30-fold higher sensitivity for His + reversion than the parental strain when treated with UV and UV-mimetic chemicals but unchanged mutation frequency with X-rays and methyl methanesulfonate. The tester strain was used in a spot test of 30 selected chemicals and also for testing with liver homogenate activation. The results showed an almost equivalent but somewhat broader detection spectrum than the Salmonella typhimurium TA100 system. Another test method used a pair of B. subtilis strains differing in their DNA-repair capacity, i.e. the most UV-sensitive mutant HJ-15 and a wild-type strain, to detect repair-dependent DNA damage produced by chemicals. Spores could be used in either test

  18. A comparative investigation of DNA strand breaks, sister chromatid exchanges and K-ras gene mutations induced by cadmium salts in cultured human cells

    International Nuclear Information System (INIS)

    Mouron, Silvana Andrea; Grillo, Claudia Alejandra; Dulout, Fernando Noel; Golijow, Carlos Daniel

    2004-01-01

    Cadmium (Cd) is a toxic heavy metal of continuing occupational and environmental concern with a wide variety of adverse effects. Several studies have shown that cadmium produces DNA strand breaks, DNA-protein cross-links, oxidative DNA damage, chromosomal aberrations, dysregulation of gene expression resulting in enhanced proliferation, depressed apoptosis and/or altered DNA repair. This study was undertaken to investigate the ability of cadmium chloride (CdCl 2 ) and cadmium sulphate (CdSO 4 ) to induce point mutations in codon 12 of the K-ras protooncogene assessed by polymerase chain reaction-single strand conformation polymorphisms (PCR-SSCP) and RFLP-enriched PCR methods. Also their genotoxic effects were analyzed by the comet assay and sister chromatid exchanges test. The human lung fibroblast cell line MRC-5 was used for the experiments. Sister chromatid exchanges assay (SCEs) frequencies were significantly increased in cells exposed to cadmium salts in relation to controls (p < 0.001). Despite the slow increment observed in the three comet parameters considered when cells were treated with cadmium chloride, significant differences between groups were only found in the variable comet moment (CM) (p < 0.005). On the other hand, when cells were exposed to cadmium sulphate, the Kruskal-Wallis test showed highly significant differences between groups for migration, tail moment and comet moment parameters (p < 0.001). Nevertheless, a null or weak point mutation induction in K-ras protooncogene was detected using polymerase chain reaction-low ionic strength-single strand conformation polymorphisms (PCR-LIS-SSCP) and RFLP-enriched PCR methods when cells were treated with cadmium salts. Thus, inorganic cadmium produces genotoxicity in human lung fibroblast MRC-5 cells, in the absence of significant point mutation of the K-ras gene

  19. FLT3 mutations in early T-cell precursor ALL characterize a stem cell like leukemia and imply the clinical use of tyrosine kinase inhibitors.

    Directory of Open Access Journals (Sweden)

    Martin Neumann

    Full Text Available Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL has been identified as high-risk subgroup of acute T-lymphoblastic leukemia (T-ALL with a high rate of FLT3-mutations in adults. To unravel the underlying pathomechanisms and the clinical course we assessed molecular alterations and clinical characteristics in a large cohort of ETP-ALL (n = 68 in comparison to non-ETP T-ALL adult patients. Interestingly, we found a high rate of FLT3-mutations in ETP-ALL samples (n = 24, 35%. Furthermore, FLT3 mutated ETP-ALL was characterized by a specific immunophenotype (CD2+/CD5-/CD13+/CD33-, a distinct gene expression pattern (aberrant expression of IGFBP7, WT1, GATA3 and mutational status (absence of NOTCH1 mutations and a low frequency, 21%, of clonal TCR rearrangements. The observed low GATA3 expression and high WT1 expression in combination with lack of NOTCH1 mutations and a low rate of TCR rearrangements point to a leukemic transformation at the pluripotent prothymocyte stage in FLT3 mutated ETP-ALL. The clinical outcome in ETP-ALL patients was poor, but encouraging in those patients with allogeneic stem cell transplantation (3-year OS: 74%. To further explore the efficacy of targeted therapies, we demonstrate that T-ALL cell lines transfected with FLT3 expression constructs were particularly sensitive to tyrosine kinase inhibitors. In conclusion, FLT3 mutated ETP-ALL defines a molecular distinct stem cell like leukemic subtype. These data warrant clinical studies with the implementation of FLT3 inhibitors in addition to early allogeneic stem cell transplantation for this high risk subgroup.

  20. Overview of the IFMIF test cell design

    International Nuclear Information System (INIS)

    Moeslang, A.; Daum, E.; Jitsukawa, S.; Noda, K.; Viola, R.

    1996-01-01

    The Conceptual Design Activity (CDA) for the International Fusion Materials Irradiation Facility (IFMIF) has entered its second and final year, and an outline design has been developed. Initial evaluations of the potential of this high flux, high intensity D-Li source have shown that the main materials testing needs can be fulfilled. According to these needs, Vertical Test Assemblies will accommodate test modules for the high flux (0.5 liter, 20 dpa/a, 250-1000 C), the medium flux (6 liter, 1-20 dpa/a, 250-1000 C), the low flux (7.5 liter, 0.1-1 dpa/a), and the very low flux (> 100 liter, 0.01-0.1 dpa/a) regions. Detailed test matrices have been defined for the high and medium flux regions, showing that on the basis of small specimen test technologies, a database for an engineering design of an advanced fusion reactor (DEMO) can be established for a variety of structural materials and ceramic breeders. The design concepts for the Test Cell, including test assemblies, remote handling equipment and Hot Cell Facilities with capacity for investigating all irradiation specimens at the IFMIF site are described

  1. The SSC full cell prototype string test

    International Nuclear Information System (INIS)

    Kraushaar, P.; Burgett, W.; Cromer, L.

    1994-11-01

    At the conclusion of the SSC half cell magnet string testing program. In February, 1993, the preliminary data analysis revealed that several substantive technical questions remained unresolved. These questions were: (1) could the high voltages to ground (>2 kV) measured during fault (quench) conditions be substantially reduced, (2) could the number of magnetic elements that became resistive (quenched) be controlled and (3) did the cryostats of the magnetic elements provide adequate insulation and isolation to meet designed refrigeration loads. To address these and other existing question a prototypical full cell of collider magnets (ten dipoles and two quadrupoles) was assembled and tested. At the conclusion of this testing there were definitive answers to most of the questions with numerical substantiation, the notable exception being the beat leak question. These answers and other results and issues are presented in this paper

  2. The SSC full cell prototype string test

    International Nuclear Information System (INIS)

    McInturff, A.D.; Kraushaar, P.; Burgett, W.; Cromer, L.

    1994-01-01

    At the conclusion of the SSC half cell magnet string testing program in February, 1993, the preliminary data analysis revealed that several substantive technical questions remained unresolved. These questions were: (1) could the high voltages to ground (>2 kV) measured during fault (quench) conditions be substantially reduced, (2) could the number of magnetic elements that became resistive (quenched) be controlled and 3) did the cryostats of the magnetic elements provide adequate insulation and isolation to meet designed refrigeration loads. To address these and other existing questions, a prototypical fall cell of collider magnets (ten dipoles and two quadrupoles) was assembled and tested. At the conclusion of this testing there were definitive answers to most of the questions with numerical substantiation, the notable exception being the beat leak question. These answers and other results and issues are presented in this paper

  3. Investigating the Consequences of Interference between Multiple CD8+ T Cell Escape Mutations in Early HIV Infection.

    Directory of Open Access Journals (Sweden)

    Victor Garcia

    2016-02-01

    Full Text Available During early human immunodeficiency virus (HIV infection multiple CD8+ T cell responses are elicited almost simultaneously. These responses exert strong selective pressures on different parts of HIV's genome, and select for mutations that escape recognition and are thus beneficial to the virus. Some studies reveal that the later these escape mutations emerge, the more slowly they go to fixation. This pattern of escape rate decrease(ERD can arise by distinct mechanisms. In particular, in large populations with high beneficial mutation rates interference among different escape strains--an effect that can emerge in evolution with asexual reproduction and results in delayed fixation times of beneficial mutations compared to sexual reproduction--could significantly impact the escape rates of mutations. In this paper, we investigated how interference between these concurrent escape mutations affects their escape rates in systems with multiple epitopes, and whether it could be a source of the ERD pattern. To address these issues, we developed a multilocus Wright-Fisher model of HIV dynamics with selection, mutation and recombination, serving as a null-model for interference. We also derived an interference-free null model assuming initial neutral evolution before immune response elicitation. We found that interference between several equally selectively advantageous mutations can generate the observed ERD pattern. We also found that the number of loci, as well as recombination rates substantially affect ERD. These effects can be explained by the underexponential decline of escape rates over time. Lastly, we found that the observed ERD pattern in HIV infected individuals is consistent with both independent, interference-free mutations as well as interference effects. Our results confirm that interference effects should be considered when analyzing HIV escape mutations. The challenge in estimating escape rates and mutation-associated selective

  4. Identifying activating mutations in the EGFR gene: prognostic and therapeutic implications in non-small cell lung cancer.

    Science.gov (United States)

    Lopes, Gabriel Lima; Vattimo, Edoardo Filippo de Queiroz; Castro Junior, Gilberto de

    2015-01-01

    Lung cancer is the leading cause of cancer-related deaths worldwide. Promising new therapies have recently emerged from the development of molecular targeted drugs; particularly promising are those blocking the signal transduction machinery of cancer cells. One of the most widely studied cell signaling pathways is that of EGFR, which leads to uncontrolled cell proliferation, increased cell angiogenesis, and greater cell invasiveness. Activating mutations in the EGFR gene (deletions in exon 19 and mutation L858R in exon 21), first described in 2004, have been detected in approximately 10% of all non-squamous non-small cell lung cancer (NSCLC) patients in Western countries and are the most important predictors of a response to EGFR tyrosine-kinase inhibitors (EGFR-TKIs). Studies of the EGFR-TKIs gefitinib, erlotinib, and afatinib, in comparison with platinum-based regimens, as first-line treatments in chemotherapy-naïve patients have shown that the EGFR-TKIs produce gains in progression-free survival and overall response rates, although only in patients whose tumors harbor activating mutations in the EGFR gene. Clinical trials have also shown EGFR-TKIs to be effective as second- and third-line therapies in advanced NSCLC. Here, we review the main aspects of EGFR pathway activation in NSCLC, underscore the importance of correctly identifying activating mutations in the EGFR gene, and discuss the main outcomes of EGFR-TKI treatment in NSCLC.

  5. Identifying activating mutations in the EGFR gene: prognostic and therapeutic implications in non-small cell lung cancer

    Directory of Open Access Journals (Sweden)

    Gabriel Lima Lopes

    2015-08-01

    Full Text Available AbstractLung cancer is the leading cause of cancer-related deaths worldwide. Promising new therapies have recently emerged from the development of molecular targeted drugs; particularly promising are those blocking the signal transduction machinery of cancer cells. One of the most widely studied cell signaling pathways is that of EGFR, which leads to uncontrolled cell proliferation, increased cell angiogenesis, and greater cell invasiveness. Activating mutations in the EGFR gene (deletions in exon 19 and mutation L858R in exon 21, first described in 2004, have been detected in approximately 10% of all non-squamous non-small cell lung cancer (NSCLC patients in Western countries and are the most important predictors of a response to EGFR tyrosine-kinase inhibitors (EGFR-TKIs. Studies of the EGFR-TKIs gefitinib, erlotinib, and afatinib, in comparison with platinum-based regimens, as first-line treatments in chemotherapy-naïve patients have shown that the EGFR-TKIs produce gains in progression-free survival and overall response rates, although only in patients whose tumors harbor activating mutations in the EGFR gene. Clinical trials have also shown EGFR-TKIs to be effective as second- and third-line therapies in advanced NSCLC. Here, we review the main aspects of EGFR pathway activation in NSCLC, underscore the importance of correctly identifying activating mutations in the EGFR gene, and discuss the main outcomes of EGFR-TKI treatment in NSCLC.

  6. Identifying activating mutations in the EGFR gene: prognostic and therapeutic implications in non-small cell lung cancer *

    Science.gov (United States)

    Lopes, Gabriel Lima; Vattimo, Edoardo Filippo de Queiroz; de Castro, Gilberto

    2015-01-01

    Abstract Lung cancer is the leading cause of cancer-related deaths worldwide. Promising new therapies have recently emerged from the development of molecular targeted drugs; particularly promising are those blocking the signal transduction machinery of cancer cells. One of the most widely studied cell signaling pathways is that of EGFR, which leads to uncontrolled cell proliferation, increased cell angiogenesis, and greater cell invasiveness. Activating mutations in the EGFR gene (deletions in exon 19 and mutation L858R in exon 21), first described in 2004, have been detected in approximately 10% of all non-squamous non-small cell lung cancer (NSCLC) patients in Western countries and are the most important predictors of a response to EGFR tyrosine-kinase inhibitors (EGFR-TKIs). Studies of the EGFR-TKIs gefitinib, erlotinib, and afatinib, in comparison with platinum-based regimens, as first-line treatments in chemotherapy-naïve patients have shown that the EGFR-TKIs produce gains in progression-free survival and overall response rates, although only in patients whose tumors harbor activating mutations in the EGFR gene. Clinical trials have also shown EGFR-TKIs to be effective as second- and third-line therapies in advanced NSCLC. Here, we review the main aspects of EGFR pathway activation in NSCLC, underscore the importance of correctly identifying activating mutations in the EGFR gene, and discuss the main outcomes of EGFR-TKI treatment in NSCLC. PMID:26398757

  7. Generation of KCL025 research grade human embryonic stem cell line carrying a mutation in NF1 gene

    Directory of Open Access Journals (Sweden)

    Heema Hewitson

    2016-03-01

    Full Text Available The KCL025 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation in the NF1 gene encoding neurofibromin (c.3739–3742 ΔTTTG. Mutations in this gene have been linked to neurofibromatosis type 1, juvenile myelomonocytic leukemia and Watson syndrome. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays.

  8. Discovery and prioritization of somatic mutations in diffuse large B-cell lymphoma (DLBCL) by whole-exome sequencing

    Science.gov (United States)

    Lohr, Jens G.; Stojanov, Petar; Lawrence, Michael S.; Auclair, Daniel; Chapuy, Bjoern; Sougnez, Carrie; Cruz-Gordillo, Peter; Knoechel, Birgit; Asmann, Yan W.; Slager, Susan L.; Novak, Anne J.; Dogan, Ahmet; Ansell, Stephen M.; Zou, Lihua; Gould, Joshua; Saksena, Gordon; Stransky, Nicolas; Rangel-Escareño, Claudia; Fernandez-Lopez, Juan Carlos; Hidalgo-Miranda, Alfredo; Melendez-Zajgla, Jorge; Hernández-Lemus, Enrique; Schwarz-Cruz y Celis, Angela; Imaz-Rosshandler, Ivan; Ojesina, Akinyemi I.; Jung, Joonil; Pedamallu, Chandra S.; Lander, Eric S.; Habermann, Thomas M.; Cerhan, James R.; Shipp, Margaret A.; Getz, Gad; Golub, Todd R.

    2012-01-01

    To gain insight into the genomic basis of diffuse large B-cell lymphoma (DLBCL), we performed massively parallel whole-exome sequencing of 55 primary tumor samples from patients with DLBCL and matched normal tissue. We identified recurrent mutations in genes that are well known to be functionally relevant in DLBCL, including MYD88, CARD11, EZH2, and CREBBP. We also identified somatic mutations in genes for which a functional role in DLBCL has not been previously suspected. These genes include MEF2B, MLL2, BTG1, GNA13, ACTB, P2RY8, PCLO, and TNFRSF14. Further, we show that BCL2 mutations commonly occur in patients with BCL2/IgH rearrangements as a result of somatic hypermutation normally occurring at the IgH locus. The BCL2 point mutations are primarily synonymous, and likely caused by activation-induced cytidine deaminase–mediated somatic hypermutation, as shown by comprehensive analysis of enrichment of mutations in WRCY target motifs. Those nonsynonymous mutations that are observed tend to be found outside of the functionally important BH domains of the protein, suggesting that strong negative selection against BCL2 loss-of-function mutations is at play. Last, by using an algorithm designed to identify likely functionally relevant but infrequent mutations, we identify KRAS, BRAF, and NOTCH1 as likely drivers of DLBCL pathogenesis in some patients. Our data provide an unbiased view of the landscape of mutations in DLBCL, and this in turn may point toward new therapeutic strategies for the disease. PMID:22343534

  9. Exosomes isolated from cancer patients' sera transfer malignant traits and confer the same phenotype of primary tumors to oncosuppressor-mutated cells.

    Science.gov (United States)

    Abdouh, Mohamed; Hamam, Dana; Gao, Zu-Hua; Arena, Vincenzo; Arena, Manuel; Arena, Goffredo Orazio

    2017-08-30

    Horizontal transfer of malignant traits from the primary tumor to distant organs, through blood circulating factors, has recently become a thoroughly studied metastatic pathway to explain cancer dissemination. Recently, we reported that oncosuppressor gene-mutated human cells undergo malignant transformation when exposed to cancer patients' sera. We also observed that oncosuppressor mutated cells would show an increased uptake of cancer-derived exosomes and we suggested that oncosuppressor genes might protect the integrity of the cell genome by blocking integration of cancer-derived exosomes. In the present study, we tested the hypothesis that cancer patients' sera-derived exosomes might be responsible for the malignant transformation of target cells and that oncosuppressor mutation would promote their increased uptake. We also sought to unveil the mechanisms behind the hypothesized phenomena. We used human BRCA1 knockout (BRCA1-KO) fibroblasts as target cells. Cells were treated in vitro with cancer patients' sera or cancer patients' sera-derived exosomes. Treated cells were injected into NOD-SCID mice. Immunohistochemical analyses were performed to determine the differentiation state of the xenotransplants. Mass spectrometry analyses of proteins from cancer exosomes and the BRCA1-KO fibroblasts' membrane were performed to investigate possible de novo expression of molecules involved in vesicles uptake. Blocking of the identified molecules in vitro was performed and in vivo experiments were conducted to confirm the role of these molecules in the malignant transformation carried out by cancer-derived exosomes. Cells treated with exosomes isolated from cancer patients' sera underwent malignant transformation and formed tumors when transplanted into immunodeficient mice. Histological analyses showed that the tumors were carcinomas that differentiated into the same lineage of the primary tumors of blood donors. Oncosuppressor mutation promoted the de novo expression

  10. A Comparative Study for Detection of EGFR Mutations in Plasma Cell-Free DNA in Korean Clinical Diagnostic Laboratories

    Directory of Open Access Journals (Sweden)

    Yoonjung Kim

    2018-01-01

    Full Text Available Liquid biopsies to genotype the epidermal growth factor receptor (EGFR for targeted therapy have been implemented in clinical decision-making in the field of lung cancer, but harmonization of detection methods is still scarce among clinical laboratories. We performed a pilot external quality assurance (EQA scheme to harmonize circulating tumor DNA testing among laboratories. For EQA, we created materials containing different levels of spiked cell-free DNA (cfDNA in normal plasma. The limit of detection (LOD of the cobas® EGFR Mutation Test v2 (Roche Molecular Systems was also evaluated. From November 2016 to June 2017, seven clinical diagnostic laboratories participated in the EQA program. The majority (98.94% of results obtained using the cobas assay and next-generation sequencing (NGS were acceptable. Quantitative results from the cobas assay were positively correlated with allele frequencies derived from digital droplet PCR measurements and showed good reproducibility among laboratories. The LOD of the cobas assay was 5~27 copies/mL for p.E746_A750del (exon 19 deletion, 35~70 copies/mL for p.L858R, 18~36 copies/mL for p.T790M, and 15~31 copies/mL for p.A767_V769dup (exon 20 insertion. Deep sequencing of materials (>100,000X depth of coverage resulted in detection of low-level targets present at frequencies of 0.06~0.13%. Our results indicate that the cobas assay is a reliable and rapid method for detecting EGFR mutations in plasma cfDNA. Careful interpretation is particularly important for p.T790M detection in the setting of relapse. Individual laboratories should optimize NGS performance to maximize clinical utility.

  11. Second-line combination therapies in nonsmall cell lung cancer without known driver mutations

    Directory of Open Access Journals (Sweden)

    Maria-Virginia Bluthgen

    2015-12-01

    Full Text Available In advanced nonsmall cell lung cancer (NSCLC patients, platinum-based combination chemotherapy is standard treatment in the first-line setting; however, the large majority of patients ultimately progress. For more than a decade, single-agent therapy with docetaxel, pemetrexed or erlotinib has been the standard of care after failure with platinum salts, showing some benefit over best supportive care. Nonetheless, prognosis remains poor and new second-line strategies are urgently needed. Combinations of cytotoxic agents, including rechallenge with platinum salts, do not offer clear benefit over single-agent therapy for the majority of patients. In patients without a known tumoural oncogenic driver mutation, regimens based on combinations of targeted agents have shown promising results; however, a clear role in therapeutic management is yet to be established. Some success has been reported in recent research combining a cytotoxic agent with targeted therapies. In this review, we summarise published data for the various strategies evaluated over the past decade in second-line treatment of NSCLC patients without a known driver mutation. We focus on combination treatments and consider future perspectives, including the need to identify predictive markers to support personalised therapeutic strategies.

  12. Quantification and analysis of reverse mutations at the hgprt locus in Chinese hamster ovary cells

    Energy Technology Data Exchange (ETDEWEB)

    Fuscoe, J.C.; O' Neill, J.P.; Machanoff, R.; Hsie, A.W.

    1982-01-01

    An assay is described for the quantification of reverse mutations at the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus in Chinese hamster ovary cells utilizing the selective agent L-azaserine (AS). Conditions are defined in terms of optimal AS concentration, cell density, and phenotypic expression time. After treatment, replicate cultures of 10/sup 6/ cells are allowed a 48-h phenotypic expression time in 100-mm plates. AS (10..mu..M) is then added directly to the growing culture and AS-resistant (AS/sup r/) cells form visible colonies. This assay is used to quantify ICR-191-, ICR-170-, and N-ethyl-N-nitrosourea-induced reversion of independently isolated HGPRT/sup -/ clones. The AS/sup r/ phenotype is characterized both physiologically and biochemically. All AS/sup r/ clones isolated are stably resistant to AS and aminopterin but sensitive to 6-thioguanine. They also have re-expressed HGPRT enzyme. In addition, several revertants are shown to contain altered HGPRT.

  13. WNT Inhibitory Activity of Malus Pumila miller cv Annurca and Malus domestica cv Limoncella Apple Extracts on Human Colon-Rectal Cells Carrying Familial Adenomatous Polyposis Mutations.

    Science.gov (United States)

    Riccio, Gennaro; Maisto, Maria; Bottone, Sara; Badolati, Nadia; Rossi, Giovanni Battista; Tenore, Gian Carlo; Stornaiuolo, Mariano; Novellino, Ettore

    2017-11-18

    Inhibitors of the Wingless-related Integration site (WNT)/β-catenin pathway have recently been under consideration as potential chemopreventive agents against Familial Adenomatous Polyposis (FAP). This autosomal-dominant syndrome is caused by germline mutations in the gene coding for the protein APC and leads to hyperactivation of the WNT/β-catenin signaling pathway, uncontrolled intestinal cell proliferation and formation of adenocarcinomas. The aim of the present work was to: (i) test, on in vitro cultures of cells carrying FAP mutations and on ex vivo biopsies of FAP patients, the WNT inhibitory activity of extracts from two common southern Italian apples, Malus pumila Miller cv. 'Annurca' and Malus domestica cv 'Limoncella'; (ii) identify the mechanisms underpinning their activities and; (iii) evaluate their potency upon gastrointestinal digestion. We here show that both Annurca and Limoncella apple extracts act as WNT inhibitors, mostly thanks to their polyphenolic contents. They inhibit the pathway in colon cells carrying FAP mutations with active dilutions falling in ranges close to consumer-relevant concentrations. Food-grade manufacturing of apple extracts increases their WNT inhibitory activity as result of the conversion of quercetin glycosides into the aglycone quercetin, a potent WNT inhibitor absent in the fresh fruit extract. However, in vitro simulated gastrointestinal digestion severely affected WNT inhibitory activity of apple extracts, as result of a loss of polyphenols. In conclusion, our results show that apple extracts inhibit the WNT pathway in colon cells carrying FAP mutations and represent a potential nutraceutical alternative for the treatment of this pathology. Enteric coating is advisable to preserve the activity of the extracts in the colon-rectal section of the digestive tract.

  14. WNT Inhibitory Activity of Malus Pumila miller cv Annurca and Malus domestica cv Limoncella Apple Extracts on Human Colon-Rectal Cells Carrying Familial Adenomatous Polyposis Mutations

    Directory of Open Access Journals (Sweden)

    Gennaro Riccio

    2017-11-01

    Full Text Available Inhibitors of the Wingless-related Integration site (WNT/β-catenin pathway have recently been under consideration as potential chemopreventive agents against Familial Adenomatous Polyposis (FAP. This autosomal-dominant syndrome is caused by germline mutations in the gene coding for the protein APC and leads to hyperactivation of the WNT/β-catenin signaling pathway, uncontrolled intestinal cell proliferation and formation of adenocarcinomas. The aim of the present work was to: (i test, on in vitro cultures of cells carrying FAP mutations and on ex vivo biopsies of FAP patients, the WNT inhibitory activity of extracts from two common southern Italian apples, Malus pumila Miller cv. ‘Annurca’ and Malus domestica cv ‘Limoncella’; (ii identify the mechanisms underpinning their activities and; (iii evaluate their potency upon gastrointestinal digestion. We here show that both Annurca and Limoncella apple extracts act as WNT inhibitors, mostly thanks to their polyphenolic contents. They inhibit the pathway in colon cells carrying FAP mutations with active dilutions falling in ranges close to consumer-relevant concentrations. Food-grade manufacturing of apple extracts increases their WNT inhibitory activity as result of the conversion of quercetin glycosides into the aglycone quercetin, a potent WNT inhibitor absent in the fresh fruit extract. However, in vitro simulated gastrointestinal digestion severely affected WNT inhibitory activity of apple extracts, as result of a loss of polyphenols. In conclusion, our results show that apple extracts inhibit the WNT pathway in colon cells carrying FAP mutations and represent a potential nutraceutical alternative for the treatment of this pathology. Enteric coating is advisable to preserve the activity of the extracts in the colon-rectal section of the digestive tract.

  15. The uranyl influence on a mutation process in germ and somatic cells of mice

    International Nuclear Information System (INIS)

    Kostrova, L.N.; Mosseh, I.B.; Molofej, V.P.

    2008-01-01

    The mutagenic effect of uranyl was revealed by the chromosome rearrangement test in germ and somatic cells of mice. The effect value depended on duration of substance administration into organism. (authors)

  16. Comprehensive genomic profiling reveals inactivating SMARCA4 mutations and low tumor mutational burden in small cell carcinoma of the ovary, hypercalcemic-type.

    Science.gov (United States)

    Lin, Douglas I; Chudnovsky, Yakov; Duggan, Bridget; Zajchowski, Deborah; Greenbowe, Joel; Ross, Jeffrey S; Gay, Laurie M; Ali, Siraj M; Elvin, Julia A

    2017-12-01

    Small cell carcinoma of the ovary, hypercalcemic-type (SCCOHT) is a rare, extremely aggressive neoplasm that usually occurs in young women and is characterized by deleterious germline or somatic SMARCA4 mutations. We performed comprehensive genomic profiling (CGP) to potentially identify additional clinically and pathophysiologically relevant genomic alterations in SCCOHT. CGP assessment of all classes of coding alterations in up to 406 genes commonly altered in cancer and intronic regions for up to 31 genes commonly rearranged in cancer was performed on 18 SCCOHT cases (16 exhibiting classic morphology and 2 cases exhibiting exclusive a large cell variant morphology). In addition, a retrospective database search for clinically advanced ovarian tumors with genomic profiles similar to SCCOHT yielded 3 additional cases originally diagnosed as non-SCCOHT. CGP revealed inactivating SMARCA4 alterations and low tumor mutational burden (TMB) (<6mutations/Mb) in 94% (15/16) of SCCOHT with classic morphology. In contrast, both (2/2) cases exhibiting only large cell variant morphology were hypermutated (TMB scores of 90 and 360mut/Mb) and were wildtype for SMARCA4. In our retrospective search, an index ovarian cancer patient harboring inactivating SMARCA4 alterations, initially diagnosed as endometrioid carcinoma, was re-classified as SCCOHT and responded to an SCCOHT chemotherapy regimen. The vast majority of SCCOHT demonstrate genomic SMARCA4 loss with only rare co-occurring alterations. Our data support a role for CGP in the diagnosis and management of SCCOHT and of other lesions with overlapping histological and clinical features, since identifying the former by genomic profile suggests benefit from an appropriate regimen and treatment decisions, as illustrated by an index patient. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. AT-101 enhances gefitinib sensitivity in non-small cell lung cancer with EGFR T790M mutations

    International Nuclear Information System (INIS)

    Zhao, Ren; Zhou, Shun; Xia, Bing; Zhang, Cui-ying; Hai, Ping; Zhe, Hong; Wang, Yan-yang

    2016-01-01

    Although epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) have become the standard care of patients with advanced EGFR-mutant non-small cell lung cancer (NSCLC), development of acquired resistance is inevitable. A secondary mutation of threonine 790 (T790M) is associated with approximately half of the cases of acquired resistance. Strategies or agents to overcome this type of resistance are still limited. In this study, enhanced antitumor effect of AT-101, a-pan-Bcl-2 inhibitor, on gefitinib was explored in NSCLC with T790M mutation. The effect of cotreatment with AT-101 and gefitinib on the viability of NSCLC cell lines harboring acquired T790M mutation was investigated using the MTT assay. The cellular apoptosis of NSCLC cells after cotreatment with AT-101 and gefitinib was assessed by FITC-annexin V/PI assay and Western blots analysis. The potential underlying mechanisms of the enhanced therapeutic effect for AT-101 was also studied using Western blots analysis. The in vivo anti-cancer efficacy of the combination with AT-101 and gefitinib was examined in a mouse xenograft model. In this study, we found that treatment with AT-101 in combination with gefitinib significantly inhibited cell proliferation, as well as promoted apoptosis of EGFR TKIs resistant lung cancer cells. The apoptotic effects of the use of AT-101 was related to the blocking of antiapoptotic protein: Bcl-2, Bcl-xl, and Mcl-1 and downregrulation of the molecules in EGFR pathway. The observed enhancements of tumor growth suppression in xenografts supported the reverse effect of AT-101 in NSCLC with T790M mutation, which has been found in in vitro studies before. AT-101 enhances gefitinib sensitivity in NSCLC with EGFR T790M mutations. The addition of AT-101 to gefitinib is a promising strategy to overcome EGFR TKIs resistance in NSCLC with EGFR T790M mutations

  18. Mutational landscape of MCPyV-positive and MCPyV-negative Merkel cell carcinomas with implications for immunotherapy.

    Science.gov (United States)

    Goh, Gerald; Walradt, Trent; Markarov, Vladimir; Blom, Astrid; Riaz, Nadeem; Doumani, Ryan; Stafstrom, Krista; Moshiri, Ata; Yelistratova, Lola; Levinsohn, Jonathan; Chan, Timothy A; Nghiem, Paul; Lifton, Richard P; Choi, Jaehyuk

    2016-01-19

    Merkel cell carcinoma (MCC) is a rare but highly aggressive cutaneous neuroendocrine carcinoma, associated with the Merkel cell polyomavirus (MCPyV) in 80% of cases. To define the genetic basis of MCCs, we performed exome sequencing of 49 MCCs. We show that MCPyV-negative MCCs have a high mutation burden (median of 1121 somatic single nucleotide variants (SSNVs) per-exome with frequent mutations in RB1 and TP53 and additional damaging mutations in genes in the chromatin modification (ASXL1, MLL2, and MLL3), JNK (MAP3K1 and TRAF7), and DNA-damage pathways (ATM, MSH2, and BRCA1). In contrast, MCPyV-positive MCCs harbor few SSNVs (median of 12.5 SSNVs/tumor) with none in the genes listed above. In both subgroups, there are rare cancer-promoting mutations predicted to activate the PI3K pathway (HRAS, KRAS, PIK3CA, PTEN, and TSC1) and to inactivate the Notch pathway (Notch1 and Notch2). TP53 mutations appear to be clinically relevant in virus-negative MCCs as 37% of these tumors harbor potentially targetable gain-of-function mutations in TP53 at p.R248 and p.P278. Moreover, TP53 mutational status predicts death in early stage MCC (5-year survival in TP53 mutant vs wild-type stage I and II MCCs is 20% vs. 92%, respectively; P = 0.0036). Lastly, we identified the tumor neoantigens in MCPyV-negative and MCPyV-positive MCCs. We found that virus-negative MCCs harbor more tumor neoantigens than melanomas or non-small cell lung cancers (median of 173, 65, and 111 neoantigens/sample, respectively), two cancers for which immune checkpoint blockade can produce durable clinical responses. Collectively, these data support the use of immunotherapies for virus-negative MCCs.

  19. Testing system for a fuel cells stack

    International Nuclear Information System (INIS)

    Culcer, Mihai; Iliescu, Mariana; Stefanescu, Ioan; Raceanu, Mircea; Enache, Adrian; Lazar, Roxana Elena

    2006-01-01

    Hydrogen and electricity together represent one of the most promising ways to realize sustainable energy, whilst fuel cells provide the most efficient conversion devices for converting hydrogen and possibly other fuels into electricity. Thus, the development of fuel cell technology is currently being actively pursued worldwide. Due to its simple operation and other fair characteristics, the Proton Exchange Membrane Fuel Cell (PEMFC) is especially suitable as a replacement for the internal combustion engine. The PEMFC is also being developed for decentralized electricity and heat generation in buildings and mobile applications. Starting with 2001 the Institute of Research - Development for Cryogenics and Isotopic Technologies - ICIT - Rm. Valcea developed research activities supported by the Romanian Ministry of Education and Research within the National Research Program in order to bridge the gap to European competencies in the area of hydrogen and fuel cells. The paper deals with the testing system designed and developed in ICIT Rm. Valcea as a flexible and versatile tool allowing a large scale of parameter settings and measurements on a single cell or on a fuel cells stack onto a wind range of output power values. (authors)

  20. Role of KEAP1/NRF2 and TP53 mutations in lung squamous cell carcinoma development and radiotherapy response prediction

    Science.gov (United States)

    Jeong, Youngtae; Hoang, Ngoc T.; Lovejoy, Alexander; Stehr, Henning; Newman, Aaron M.; Gentles, Andrew J.; Kong, William; Truong, Diana; Martin, Shanique; Chaudhuri, Aadel; Heiser, Diane; Zhou, Li; Say, Carmen; Carter, Justin N.; Hiniker, Susan M.; Loo, Billy W.; West, Robert B.; Beachy, Philip; Alizadeh, Ash A.; Diehn, Maximilian

    2016-01-01

    Lung squamous cell carcinomas (LSCC) pathogenesis remains incompletely understood and biomarkers predicting treatment response remain lacking. Here we describe novel murine LSCC models driven by loss of Trp53 and Keap1, both of which are frequently mutated in human LSCCs. Homozygous inactivation of Keap1 or Trp53 promoted airway basal stem cell (ABSC) self-renewal, suggesting that mutations in these genes lead to expansion of mutant stem cell clones. Deletion of Trp53 and Keap1 in ABSCs, but not more differentiated tracheal cells, produced tumors recapitulating histological and molecular features of human LSCCs, indicating that they represent the likely cell of origin in this model. Deletion of Keap1 promoted tumor aggressiveness, metastasis, and resistance to oxidative stress and radiotherapy (RT). KEAP1/NRF2 mutation status predicted risk of local recurrence after RT in non-small lung cancer (NSCLC) patients and could be non-invasively identified in circulating tumor DNA. Thus, KEAP1/NRF2 mutations could serve as predictive biomarkers for personalization of therapeutic strategies for NSCLCs. PMID:27663899

  1. Inactivation and mutation of cultured mammalian cells by aluminium characteristic ultrasoft X-rays

    International Nuclear Information System (INIS)

    Goodhead, D.T.

    1977-01-01

    Microdosimetric distributions for aluminium K characteristic ultrasoft X-rays and 4 He ion track intersections have been calculated and used to analyse recent biological results obtained with these radiations. Results on inactivation and mutation-induction to thioguanine resistance of both V79 Chinese hamster cells and HF19 human diploid fibroblasts in vitro were analysed in terms of the Kellerer-Rossi 'theory of dual radiation action'. The small quantum energy of the aluminium X-ray photons and the very short length of the secondary electrons which they produce highlight the inadequacy of the model. It has been shown that the model predicted r.b.e. values in conflict with those observed unless an additional variable was introduced, but that the introduction of such a variable created mathematical inconsistencies. The experimental evidence is contrary to the conventional usage and basis of the model. (author)

  2. Altered Mycobacterium tuberculosis Cell Wall Metabolism and Physiology Associated With RpoB Mutation H526D

    Directory of Open Access Journals (Sweden)

    Victoria L. Campodónico

    2018-03-01

    Full Text Available Background:Mycobacterium tuberculosis (Mtb rpoB mutations are associated with global metabolic remodeling. However, the net effects of rpoB mutations on Mtb physiology, metabolism and function are not completely understood. Based on previous work, we hypothesized that changes in the expression of cell wall molecules in Mtb mutant RpoB 526D lead to changes in cell wall permeability and to altered resistance to environmental stresses and drugs.Methods: The phenotypes of a fully drug-susceptible clinical strain of Mtb and its paired rifampin-monoresistant, RpoB H526D mutant progeny strain were compared.Results: The rpoB mutant showed altered colony morphology, bacillary length and cell wall thickness, which were associated with increased cell wall permeability and susceptibility to the cell wall detergent sodium dodecyl sulfate (SDS after exposure to nutrient starvation. Relative to the isogenic rifampin-susceptible strain, the RpoB H526D mutant showed altered bacterial cellular metabolic activity and an eightfold increase in susceptibility to the cell-wall acting drug vancomycin.Conclusion: Our data suggest that RpoB mutation H526D is associated with altered cell wall physiology and resistance to cell wall-related stress. These findings are expected to contribute to an improved understanding of the pathogenesis of drug-resistant M. tuberculosis infections.

  3. The TMEM43 Newfoundland mutation p.S358L causing ARVC-5 was imported from Europe and increases the stiffness of the cell nucleus

    DEFF Research Database (Denmark)

    Milting, Hendrik; Klauke, Bärbel; Christensen, Alex Hoerby

    2014-01-01

    atomic force microscopy and revealed that the cell nuclei exhibit an increased stiffness compared with TMEM43 wild-type controls. CONCLUSION: The German family is not affected by a de novo TMEM43 mutation. It is therefore expected that an unknown number of European families may be affected by the TMEM43...... without mutations in desmosomal genes and identified the TMEM43-p.S358L mutation in a German ARVC family. We excluded TMEM43-p.S358L in 22 unrelated patients with dilated cardiomyopathy. The German family shares a common haplotype with those from Newfoundland, USA, and Denmark, suggesting...... that the mutation originated from a common founder. Examination of 40 control chromosomes revealed an estimated age of 1300-1500 years for the mutation, which proves the European origin of the Newfoundland mutation. Skin fibroblasts from a female and two male mutation carriers were analysed in cell culture using...

  4. Progress in hprt mutation assay and its application in radiation biology

    International Nuclear Information System (INIS)

    He Jing; Li Qiang

    2008-01-01

    hprt gene is an X-linked locus that has been well studied and widely used as a bio-marker in mutation detection, hprt mutation assay is a gene mutation test system in mammalian cells in vitro which has been used as a biological dosimeter. In this paper, the biological characteristics of hprt gene, hprt mutation detection methodology and the application of hprt mutation assay in radiation biology are comprehensively reviewed. (authors)

  5. Nicotinamide starvation and inhibition of poly(ADP-Ribose) synthesis enhance the induced mutation in Chinese hamster V79 cells

    International Nuclear Information System (INIS)

    Okada, Gensaku; Kaneko, Ichiro; Mitsui, Hideki.

    1987-01-01

    The effects of nicotinamide (NA) deficiency and added NA and 3-aminobenzamide (3AB) on the cytotoxicity and the induction of mutations in Chinese hamster V79-14 cells were investigated. In NA deficiency the addition of NA (up to 4 mM) and 3AB (up to 7.5 mM) was not cytotoxic. The presence of NA prior to exposure to mitomycin C (MMC) or γ-rays produced a dose-dependent increase in the relative cloning ability of DNA-damaged cells. The lethality of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was significantly potentiated by pre-treatment with 5 mM 3AB, but no potentiation by 3AB was observed for MMC, ultraviolet (UV)-B light, or γ-rays. Among cells pre-cultured in NA-free medium there were increased frequencies of mutations at both the hypoxanthineguanine phosphoribosyltransferase (HGPRT) and the adenine phosphoribosyltransferase (APRT) loci following DNA damage. The enhancing effect by NA deficiency was time-dependent. Incubation with NA prior to DNA damage produced a significant reduction in the frequency of mutations. The addition of 3AB to the nicotinamide adenine dinucleotide (NAD + )-depleted cell cultures before or after the DNA damage also strongly increased the frequency of induced mutations, with increasing concentrations of 3AB up to 5 mM, but the frequency was reduced at higher concentrations. The interaction between NA deficiency and the addition of 3AB appears to act synergistically on mutation induction. A correlation was observed between the potential of inhibiting poly (ADP-ribose) polymerase and the enhancement of mutation frequency. (author)

  6. Association of mutator activity with UV sensitivity in an aphidicolin-resistant mutant of Chinese hamster V79 cells

    International Nuclear Information System (INIS)

    Liu, P.K.; Chang, C.; Trosko, J.E.

    1982-01-01

    The spontaneous mutation rates of an ultraviolet light (UV)-sensitive aphidicolin-resistant mutant (aphsup(r)-4-2) and its revertants have been determined by 2 techniques. By using the fluctuation analysis, the mutant and its thymidine (TdR)-prototrophic 'revertant' were found to exhibit elevated spontaneous mutation rates at the 6-thioguanine- and diphtheria-toxin-resistant loci. In contrast, the TdR-auxotrophic 'revertant' did not show this property. Similar results were obtained by the multiple replating technique. From these comparative studies and other previous characterizations, it appears that a single gene mutation is responsible for the following pleiotropic phenotype: slow growth, UV sensitivity, high UV-induced mutability, high frequency of site-specific bromodeoxyuridine (BrdU)-dependent chromosome breaks and enhanced spontaneous mutation rate. Recent studies indicate that the mutation may be on the gene for DNA polymerase α. The results further indicate that thymidine auxotrophy or imbalance in nucleotide pools is not necessarily associated with the mutator activity in mammalian cells. (orig.)

  7. TP53 mutation and human papilloma virus status of oral squamous cell carcinomas in young adult patients.

    Science.gov (United States)

    Braakhuis, B J M; Rietbergen, M M; Buijze, M; Snijders, P J F; Bloemena, E; Brakenhoff, R H; Leemans, C R

    2014-09-01

    Little is known about the molecular carcinogenesis of oral squamous cell carcinoma (OSCC) in young adult patients. The aim of this study was to investigate the detailed TP53 mutation and human papilloma virus (HPV) status of OSCC in patients, younger than 45 years. TP53 mutations were determined with direct sequencing on paraffin-embedded carcinoma tissue from 31 young patients and compared with two older age OSCC reference groups: one from the same institute (N = 87) and an independent one (N = 675). Biologically active tumour HPV was detected by p16-immunohistochemistry followed by a HPV-DNA GP5 + /6 + -PCR. HPV16 was present in one OSCC (3%). TP53 mutations were found in 14 (45%) OSCC: five were missense and nine resulted in a truncated protein. Six of these latter were insertions or deletions of one or more nucleotides leading to frameshift, one was at a splice site and two resulted in a stop codon. The percentage of truncating mutations (64% of all mutations) was higher than that observed in the institute's reference group (44%, P = 0.23) and in the independent reference group (24%, P = 0.002). This study shows that TP53 mutations are common in OSCC of young adult patients; infection with biologically active HPV is rare. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  8. Ovarian Sertoli-Leydig Cell Tumor with Elevated Inhibin B as a Cause of Secondary Amenorrhea in an Adolescent with Germ Line DICER1 Mutation.

    Science.gov (United States)

    Luke, Amy M; Moroney, John W; Snitchler, Andrea; Whiteway, Susan L

    2017-10-01

    Ovarian tumors, although uncommon in children, can retain endocrine function that disrupts normal feedback mechanisms leading to amenorrhea. Inheritance of germline DICER1 mutations can lead to increased risk for development of ovarian Sertoli-Leydig cell tumors (SLCTs). We report, to our knowledge, the first case of secondary amenorrhea due to elevated inhibin B levels in a female adolescent with an ovarian SLCT. Ovarian tumors should be included in the differential diagnosis for pediatric patients who present with menstrual irregularities. Early evaluation of the hypothalamic-pituitary-ovarian axis and inhibin levels is appropriate. Our case also emphasizes the need for testing for DICER1 mutations in pediatric patients with ovarian SLCTs. Published by Elsevier Inc.

  9. Coexistence of reduced function of natural killer cells and osteoclasts in two distinct osteopetrotic mutations in the rat

    International Nuclear Information System (INIS)

    Popoff, S.N.; Jackson, M.E.; Koevary, S.B.; Marks, S.C. Jr.

    1991-01-01

    Recent evidence suggesting that immune cells and their products (cytokines) play an important role in the regulation of skeletal development and function, particularly of the osteoclast, implies that immune cell dysfunction may be involved in the pathogenesis of certain skeletal disorders. The mammalian osteopetroses are a pathogenetically heterogeneous group of skeletal disorders characterized by skeletal sclerosis resulting from reduced osteoclast-mediated bone resorption. Using a 51 Cr release microcytotoxicity assay we demonstrated that splenic natural killer (NK) cell activity was significantly reduced in two distinctly different osteopetrotic mutations in the rat, osteopetrosis (op) and toothless (tl). To determine whether this reduction in NK cell-mediated cytotoxicity is caused by decreased cell number and/or function in these osteopetrotic mutants, we quantitated NK cells by analyzing mononuclear cell suspensions labeled for two-color fluorescence with OX8 and OX19 monoclonal antibodies in a fluorescence-activated cell sorter. Flow cytometry of these double-labeled cells revealed that the percentage of NK cells (OX8+/OX19- subset) in op and tl spleens was not significantly different from that of normal spleens. These results suggest that NK cells in these osteopetrotic mutants are functionally defective. Thus aberrations in osteoclast and NK cell function coexist in these mutations, and their developmental relationships deserve further study

  10. Distinct pattern of TP53 mutations in human immunodeficiency virus-related head and neck squamous cell carcinoma.

    Science.gov (United States)

    Gleber-Netto, Frederico O; Zhao, Mei; Trivedi, Sanchit; Wang, Jiping; Jasser, Samar; McDowell, Christina; Kadara, Humam; Zhang, Jiexin; Wang, Jing; William, William N; Lee, J Jack; Nguyen, Minh Ly; Pai, Sara I; Walline, Heather M; Shin, Dong M; Ferris, Robert L; Carey, Thomas E; Myers, Jeffrey N; Pickering, Curtis R

    2018-01-01

    Human immunodeficiency virus-infected individuals (HIVIIs) have a higher incidence of head and neck squamous cell carcinoma (HNSCC), and clinical and histopathological differences have been observed in their tumors in comparison with those of HNSCC patients without a human immunodeficiency virus (HIV) infection. The reasons for these differences are not clear, and molecular differences between HIV-related HNSCC and non-HIV-related HNSCC may exist. This study compared the mutational patterns of HIV-related HNSCC and non-HIV-related HNSCC. The DNA of 20 samples of HIV-related HNSCCs and 32 samples of non-HIV-related HNSCCs was sequenced. DNA libraries covering exons of 18 genes frequently mutated in HNSCC (AJUBA, CASP8, CCND1, CDKN2A, EGFR, FAT1, FBXW7, HLA-A, HRAS, KEAP1, NFE2L2, NOTCH1, NOTCH2, NSD1, PIK3CA, TGFBR2, TP53, and TP63) were prepared and sequenced on an Ion Personal Genome Machine sequencer. DNA sequencing data were analyzed with Ion Reporter software. The human papillomavirus (HPV) status of the tumor samples was assessed with in situ hybridization, the MassARRAY HPV multiplex polymerase chain reaction assay, and p16 immunostaining. Mutation calls were compared among the studied groups. HIV-related HNSCC revealed a distinct pattern of mutations in comparison with non-HIV-related HNSCC. TP53 mutation frequencies were significantly lower in HIV-related HNSCC. Mutations in HIV+ patients tended to be TpC>T nucleotide changes for all mutated genes but especially for TP53. HNSCC in HIVIIs presents a distinct pattern of genetic mutations, particularly in the TP53 gene. HIV-related HNSCC may have a distinct biology, and an effect of the HIV virus on the pathogenesis of these tumors should not be ruled out. Cancer 2018;124:84-94. © 2017 American Cancer Society. © 2017 American Cancer Society.

  11. Novel glucokinase gene mutation in the first Macedonian family tested for MODY.

    Science.gov (United States)

    Kocova, M; Elblova, L; Pruhova, S; Lebl, J; Dusatkova, P

    2017-08-01

    We present a boy with mild hyperglycemia detected during an upper respiratory infection. Novel splicing mutation in the intron 1 of the GCK gene (c.45+1G>A) was detected, and was subsequently confirmed in his father. This is the first case of genetically confirmed Macedonian family with MODY. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Shared Decision Making in women testing for a BRCA 1/2 mutation.

    NARCIS (Netherlands)

    Roosmalen, M.S van

    2005-01-01

    Women with a BRCA1/2 mutation have a high genetic risk of developing breast and ovarian cancer. They face the difficult choice between screening and prophylactic surgery for the breasts and ovaries. We have developed a shared decision making program to prepare these women for decision making. The

  13. Sequential treatment of tyrosine kinase inhibitors and chemotherapy for EGFR-mutated non-small cell lung cancer: a meta-analysis of Phase III trials

    Directory of Open Access Journals (Sweden)

    Zhang Y

    2013-11-01

    chemotherapy could be employed initially if mutation testing results are unavailable. Keywords: EGFR mutation, tyrosine kinase inhibitor, chemotherapy, non-small cell lung cancer, clinical trial

  14. Sapanisertib and Osimertinib in Treating Patients With Stage IV EGFR Mutation Positive Non-small Cell Lung Cancer After Progression on a Previous EGFR Tyrosine Kinase Inhibitor

    Science.gov (United States)

    2018-04-25

    EGFR Activating Mutation; EGFR Exon 19 Deletion Mutation; EGFR NP_005219.2:p.G719X; EGFR NP_005219.2:p.L858R; EGFR NP_005219.2:p.L861Q; EGFR T790M Mutation Negative; Recurrent Non-Small Cell Lung Carcinoma; Stage III Non-Small Cell Lung Cancer AJCC v7; Stage IIIA Non-Small Cell Lung Cancer AJCC v7; Stage IIIB Non-Small Cell Lung Cancer AJCC v7; Stage IV Non-Small Cell Lung Cancer AJCC v7

  15. Mutation spectrum in FE1-MUTA(TM) Mouse lung epithelial cells exposed to nanoparticulate carbon black

    DEFF Research Database (Denmark)

    Jacobsen, Nicklas Raun; White, Paul A; Gingerich, John

    2011-01-01

    It has been shown previously that carbon black (CB), Printex 90 exposure induces cII and lacZ mutants in the FE1-Muta(TM) Mouse lung epithelial cell line and causes oxidatively damaged DNA and the production of reactive oxygen species (ROS). The purpose of this study was to determine the mutation...

  16. Factors affecting mutational specificity in mammalian cells: Informal [technical] progress report, February 1, 1987-January 31, 1988

    International Nuclear Information System (INIS)

    Strauss, B.S.

    1986-01-01

    We analyzed the different c-H-ras mutations produced in cells after treatment with chemical carcinogens. The overall goal of this work is an understanding of the changes produced by environmental mutagens and carcinogens to learn how closely the results obtained with bacterial and cellular assay systems apply to the in vivo situation

  17. Mechanisms of mtDNA segregation and mitochondrial signalling in cells with the pathogenic A3243G mutation

    NARCIS (Netherlands)

    Jahangir Tafrechi, Roshan Sakineh

    2008-01-01

    Using newly developed single cell A3243G mutation load assays a novel mechanism of mtDNA segregation was identified in which the multi-copy mtDNA nucleoid takes a central position. Furthermore, likely due to low level changes in gene expression, no genes or gene sets could be identified with gene

  18. A novel splice mutation in the TP53 gene associated with Leydig cell tumor and primitive neuroectodermal tumor

    DEFF Research Database (Denmark)

    Stecher, Chalotte Willemann; Grønbaek, Kirsten; Hasle, Henrik

    2008-01-01

    A 20-month-old boy presented with precocious puberty due to a Leydig cell tumor, and at the age of 6 years with a primitive neuroectodermal brain-tumor (PNET). A novel splice site mutation of the TP53-gene, likely to be associated with a nonfunctional protein, was found in the proband, his father...

  19. "Sickle Cell Anemia: Tracking down a Mutation": An Interactive Learning Laboratory That Communicates Basic Principles of Genetics and Cellular Biology

    Science.gov (United States)

    Jarrett, Kevin; Williams, Mary; Horn, Spencer; Radford, David; Wyss, J. Michael

    2016-01-01

    "Sickle cell anemia: tracking down a mutation" is a full-day, inquiry-based, biology experience for high school students enrolled in genetics or advanced biology courses. In the experience, students use restriction endonuclease digestion, cellulose acetate gel electrophoresis, and microscopy to discover which of three putative patients…

  20. Clinical and Biologic Significance of MYC Genetic Mutations in De Novo Diffuse Large B-cell Lymphoma

    NARCIS (Netherlands)

    Xu-Monette, Z.Y.; Deng, Q.; Manyam, G.C.; Tzankov, A.; Li, L; Xia, Y.; Wang, X.X.; Zou, D.; Visco, C.; Dybkaer, K.; Li, J.; Zhang, L.; Liang, H.; Montes-Moreno, S.; Chiu, A.; Orazi, A.; Zu, Y.; Bhagat, G.; Richards, K.L.; Hsi, E.D.; Choi, W.W.; Krieken, J.H.J.M. van; Huh, J.; Ponzoni, M.; Ferreri, A.J.; Parsons, B.M.; Moller, M.B.; Wang, S.A.; Miranda, R.N.; Piris, M.A.; Winter, J.N.; Medeiros, L.J.; Li, Y.; Young, K.H.

    2016-01-01

    PURPOSE: MYC is a critical driver oncogene in many cancers, and its deregulation in the forms of translocation and overexpression has been implicated in lymphomagenesis and progression of diffuse large B-cell lymphoma (DLBCL). The MYC mutational profile and its roles in DLBCL are unknown. This study

  1. A Phex mutation in a murine model of X-linked hypophosphatemia alters phosphate responsiveness of bone cells.

    Science.gov (United States)

    Ichikawa, Shoji; Austin, Anthony M; Gray, Amie K; Econs, Michael J

    2012-02-01

    Mutations in the PHEX gene cause X-linked hypophosphatemia (XLH). Hypophosphatemia in XLH results from increased circulating levels of a phosphaturic hormone, fibroblast growth factor 23 (FGF23), which inhibits renal phosphate reabsorption and 1,25-dihydroxyvitamin D (calcitriol) synthesis. The current standard therapy for XLH--high-dose phosphate and calcitriol--further increases FGF23 concentrations, suggesting that patients with XLH may have an altered response to extracellular phosphate. To test for the presence of abnormal phosphate responsiveness, we compared serum biochemistries and femoral Fgf23 mRNA expression between wild-type mice, murine models of XLH (Phex(K496X)) and hyperphosphatemic tumoral calcinosis (Galnt3(-/-)), and Galnt3/Phex double-mutant mice. Phex mutant mice had not only increased Fgf23 expression but also reduced proteolytic cleavage of intact Fgf23 protein, resulting in markedly elevated intact Fgf23 levels and consequent hypophosphatemia. In contrast, despite markedly increased Fgf23 expression, Galnt3 knockout mice had significantly high proteolytic cleavage of Fgf23 protein, leading to low intact Fgf23 concentrations and hyperphosphatemia. Galnt3/Phex double-mutant mice had an intermediate biochemical phenotype between wild-type and Phex mutant mice, including slightly elevated intact Fgf23 concentrations with milder hypophosphatemia. Despite the hypophosphatemia, double-mutant mice attempted to reduce serum phosphate back to the level of Phex mutant mice by upregulating Fgf23 expression as much as 24-fold higher than Phex mutant mice. These data suggest that Phex mutations alter the responsiveness of bone cells to extracellular phosphate concentrations and may create a lower set point for "normal" phosphate levels.

  2. Stable cytotoxic T cell escape mutation in hepatitis C virus is linked to maintenance of viral fitness.

    Directory of Open Access Journals (Sweden)

    Luke Uebelhoer

    2008-09-01

    Full Text Available Mechanisms by which hepatitis C virus (HCV evades cellular immunity to establish persistence in chronically infected individuals are not clear. Mutations in human leukocyte antigen (HLA class I-restricted epitopes targeted by CD8(+ T cells are associated with persistence, but the extent to which these mutations affect viral fitness is not fully understood. Previous work showed that the HCV quasispecies in a persistently infected chimpanzee accumulated multiple mutations in numerous class I epitopes over a period of 7 years. During the acute phase of infection, one representative epitope in the C-terminal region of the NS3/4A helicase, NS3(1629-1637, displayed multiple serial amino acid substitutions in major histocompatibility complex (MHC anchor and T cell receptor (TCR contact residues. Only one of these amino acid substitutions at position 9 (P9 of the epitope was stable in the quasispecies. We therefore assessed the effect of each mutation observed during in vivo infection on viral fitness and T cell responses using an HCV subgenomic replicon system and a recently developed in vitro infectious virus cell culture model. Mutation of a position 7 (P7 TCR-contact residue, I1635T, expectedly ablated the T cell response without affecting viral RNA replication or virion production. In contrast, two mutations at the P9 MHC-anchor residue abrogated antigen-specific T cell responses, but additionally decreased viral RNA replication and virion production. The first escape mutation, L1637P, detected in vivo only transiently at 3 mo after infection, decreased viral production, and reverted to the parental sequence in vitro. The second P9 variant, L1637S, which was stable in vivo through 7 years of follow-up, evaded the antigen-specific T cell response and did not revert in vitro despite being less optimal in virion production compared to the parental virus. These studies suggest that HCV escape mutants emerging early in infection are not necessarily

  3. Antigen-specific primed cytotoxic T cells eliminate tumour cells in vivo and prevent tumour development, regardless of the presence of anti-apoptotic mutations conferring drug resistance.

    Science.gov (United States)

    Jaime-Sánchez, Paula; Catalán, Elena; Uranga-Murillo, Iratxe; Aguiló, Nacho; Santiago, Llipsy; M Lanuza, Pilar; de Miguel, Diego; A Arias, Maykel; Pardo, Julián

    2018-05-09

    Cytotoxic CD8 + T (Tc) cells are the main executors of transformed and cancer cells during cancer immunotherapy. The latest clinical results evidence a high efficacy of novel immunotherapy agents that modulate Tc cell activity against bad prognosis cancers. However, it has not been determined yet whether the efficacy of these treatments can be affected by selection of tumoural cells with mutations in the cell death machinery, known to promote drug resistance and cancer recurrence. Here, using a model of prophylactic tumour vaccination based on the LCMV-gp33 antigen and the mouse EL4 T lymphoma, we analysed the molecular mechanism employed by Tc cells to eliminate cancer cells in vivo and the impact of mutations in the apoptotic machinery on tumour development. First of all, we found that Tc cells, and perf and gzmB are required to efficiently eliminate EL4.gp33 cells after LCMV immunisation during short-term assays (1-4 h), and to prevent tumour development in the long term. Furthermore, we show that antigen-pulsed chemoresistant EL4 cells overexpressing Bcl-X L or a dominant negative form of caspase-3 are specifically eliminated from the peritoneum of infected animals, as fast as parental EL4 cells. Notably, antigen-specific Tc cells control the tumour growth of the mutated cells, as efficiently as in the case of parental cells. Altogether, expression of the anti-apoptotic mutations does not confer any advantage for tumour cells neither in the short-term survival nor in long-term tumour formation. Although the mechanism involved in the elimination of the apoptosis-resistant tumour cells is not completely elucidated, neither necroptosis nor pyroptosis seem to be involved. Our results provide the first experimental proof that chemoresistant cancer cells with mutations in the main cell death pathways are efficiently eliminated by Ag-specific Tc cells in vivo during immunotherapy and, thus, provide the molecular basis to treat chemoresistant cancer cells with CD8 Tc

  4. Preselection of EGFR mutations in non-small-cell lung cancer patients by immunohistochemistry: comparison with DNA-sequencing, EGFR wild-type expression, gene copy number gain and clinicopathological data.

    Science.gov (United States)

    Gaber, Rania; Watermann, Iris; Kugler, Christian; Vollmer, Ekkehard; Perner, Sven; Reck, Martin; Goldmann, Torsten

    2017-01-01

    Targeting epidermal growth factor receptor (EGFR) in patients with non-small-cell lung cancer (NSCLC) having EGFR mutations is associated with an improved overall survival. The aim of this study is to verify, if EGFR mutations detected by immunohistochemistry (IHC) is a convincing way to preselect patients for DNA-sequencing and to figure out, the statistical association between EGFR mutation, wild-type EGFR overexpression, gene copy number gain, which are the main factors inducing EGFR tumorigenic activity and the clinicopathological data. Two hundred sixteen tumor tissue samples of primarily chemotherapeutic naïve NSCLC patients were analyzed for EGFR mutations E746-A750del and L858R and correlated with DNA-sequencing. Two hundred six of which were assessed by IHC, using 6B6 and 43B2 specific antibodies followed by DNA-sequencing of positive cases and 10 already genotyped tumor tissues were also included to investigate debugging accuracy of IHC. In addition, EGFR wild-type overexpression was IHC evaluated and EGFR gene copy number determination was performed by fluorescence in situ hybridization (FISH). Forty-one÷206 (19.9%) cases were positive for mutated EGFR by IHC. Eight of them had EGFR mutations of exons 18-21 by DNA-sequencing. Hit rate of 10 already genotyped NSCLC mutated cases was 90% by IHC. Positive association was found between EGFR mutations determined by IHC and both EGFR overexpression and increased gene copy number (p=0.002 and p<0.001, respectively). Additionally, positive association was detected between EGFR mutations, high tumor grade and clinical stage (p<0.001). IHC staining with mutation specific antibodies was demonstrated as a possible useful screening test to preselect patients for DNA-sequencing.

  5. A telomerase immortalized human proximal tubule cell line with a truncation mutation (Q4004X in polycystin-1.

    Directory of Open Access Journals (Sweden)

    Brittney-Shea Herbert

    Full Text Available Autosomal dominant polycystic kidney disease (ADPKD is associated with a variety of cellular phenotypes in renal epithelial cells. Cystic epithelia are secretory as opposed to absorptive, have higher proliferation rates in cell culture and have some characteristics of epithelial to mesenchymal transitions. In this communication we describe a telomerase immortalized cell line that expresses proximal tubule markers and is derived from renal cysts of an ADPKD kidney. These cells have a single detectable truncating mutation (Q4004X in polycystin-1. These cells make normal appearing but shorter cilia and fail to assemble polycystin-1 in the cilia, and less uncleaved polycystin-1 in membrane fractions. This cell line has been maintained in continuous passage for over 35 passages without going into senescence. Nephron segment specific markers suggest a proximal tubule origin for these cells and the cell line will be useful to study mechanistic details of cyst formation in proximal tubule cells.

  6. Cell killing and mutation induction on Chinese hamster cells by photoradiations

    International Nuclear Information System (INIS)

    Lam, C.K.C.

    1982-11-01

    Applying radiation directly on cells, far-uv is more effective than black light, and black light is more effective than white light in inducing proliferative death and in inducing resistance to 6-thioguanine (6-TG), ouabain and diptheria toxin (DT). Gold light has no killing and mutagenic effects on CHO (Chinese hamster ovary) cells. Use of filters showed that a small percentage of shorter wavelengths in the far-uv region is responsible for most of the killing and mutagenic effects in the unfiltered broad spectra of black and white light

  7. Cell killing and mutation induction on Chinese hamster cells by photoradiations

    Energy Technology Data Exchange (ETDEWEB)

    Lam, C.K.C.

    1982-11-01

    Applying radiation directly on cells, far-uv is more effective than black light, and black light is more effective than white light in inducing proliferative death and in inducing resistance to 6-thioguanine (6-TG), ouabain and diptheria toxin (DT). Gold light has no killing and mutagenic effects on CHO (Chinese hamster ovary) cells. Use of filters showed that a small percentage of shorter wavelengths in the far-uv region is responsible for most of the killing and mutagenic effects in the unfiltered broad spectra of black and white light.

  8. Analytic performance studies and clinical reproducibility of a real-time PCR assay for the detection of epidermal growth factor receptor gene mutations in formalin-fixed paraffin-embedded tissue specimens of non-small cell lung cancer

    International Nuclear Information System (INIS)

    O’Donnell, Patrick; Shieh, Felice; Wei, Wen; Lawrence, H Jeffrey; Wu, Lin; Schilling, Robert; Bloom, Kenneth; Maltzman, Warren; Anderson, Steven; Soviero, Stephen; Ferguson, Jane; Shyu, Johnny; Current, Robert; Rehage, Taraneh; Tsai, Julie; Christensen, Mari; Tran, Ha Bich; Chien, Sean Shih-Chang

    2013-01-01

    Epidermal growth factor receptor (EGFR) gene mutations identify patients with non-small cell lung cancer (NSCLC) who have a high likelihood of benefiting from treatment with anti-EGFR tyrosine kinase inhibitors. Sanger sequencing is widely used for mutation detection but can be technically challenging, resulting in longer turn-around-time, with limited sensitivity for low levels of mutations. This manuscript details the technical performance verification studies and external clinical reproducibility studies of the cobas EGFR Mutation Test, a rapid multiplex real-time PCR assay designed to detect 41 mutations in exons 18, 19, 20 and 21. The assay’s limit of detection was determined using 25 formalin-fixed paraffin-embedded tissue (FFPET)-derived and plasmid DNA blends. Assay performance for a panel of 201 specimens was compared against Sanger sequencing with resolution of discordant specimens by quantitative massively parallel pyrosequencing (MPP). Internal and external reproducibility was assessed using specimens tested in duplicate by different operators, using different reagent lots, instruments and at different sites. The effects on the performance of the cobas EGFR test of endogenous substances and nine therapeutic drugs were evaluated in ten FFPET specimens. Other tests included an evaluation of the effects of necrosis, micro-organisms and homologous DNA sequences on assay performance, and the inclusivity of the assay for less frequent mutations. A >95% hit rate was obtained in blends with >5% mutant alleles, as determined by MPP analysis, at a total DNA input of 150 ng. The overall percent agreement between Sanger sequencing and the cobas test was 96.7% (negative percent agreement 97.5%; positive percent agreement 95.8%). Assay repeatability was 98% when tested with two operators, instruments, and reagent lots. In the external reproducibility study, the agreement was > 99% across all sites, all operators and all reagent lots for 11/12 tumors tested. Test

  9. EGFR T790M mutation after chemotherapy for small cell lung cancer transformation of EGFR-positive non-small cell lung cancer

    Directory of Open Access Journals (Sweden)

    Tomoaki Sonoda

    Full Text Available In non-small cell lung cancer (NSCLC with an epidermal growth factor receptor (EGFR mutation, 50%–65% of cases acquire resistance after treatment with EGFR-tyrosine kinase inhibitors (EGFR-TKIs because of an EGFR T790M point mutation and 3%–14% of these cases transformed to small cell lung cancer (SCLC. Generally, the EGFR T790M secondary mutation develops with ongoing ATP competitive inhibition. We present a case of a 76-year-old woman with lung adenocarcinoma harboring an EGFR-L858R mutation who received first-line gefitinib and developed SCLC transformation. She was administered several chemotherapy agents, including a platinum doublet. The primary lesion that showed SCLC transformation had reconverted to adenocarcinoma with EGFR L858R and T790M mutations at the time of a second re-biopsy. Therefore, she was administered osimertinib, which resulted in clinical remission. This case suggested that serial biopsies are necessary even after SCLC transformation. Keywords: NSCLC, EGFR mutation, SCLC transformation, T790M, Osimertinib

  10. Recurrent mutation testing of BRCA1 and BRCA2 in Asian breast cancer patients identify carriers in those with presumed low risk by family history.

    Science.gov (United States)

    Kang, Peter Choon Eng; Phuah, Sze Yee; Sivanandan, Kavitta; Kang, In Nee; Thirthagiri, Eswary; Liu, Jian Jun; Hassan, Norhashimah; Yoon, Sook-Yee; Thong, Meow Keong; Hui, Miao; Hartman, Mikael; Yip, Cheng Har; Mohd Taib, Nur Aishah; Teo, Soo Hwang

    2014-04-01

    Although the breast cancer predisposition genes BRCA1 and BRCA2 were discovered more than 20 years ago, there remains a gap in the availability of genetic counselling and genetic testing in Asian countries because of cost, access and inaccurate reporting of family history of cancer. In order to improve access to testing, we developed a rapid test for recurrent mutations in our Asian populations. In this study, we designed a genotyping assay with 55 BRCA1 and 44 BRCA2 mutations previously identified in Asian studies, and validated this assay in 267 individuals who had previously been tested by full sequencing. We tested the prevalence of these mutations in additional breast cancer cases. Using this genotyping approach, we analysed recurrent mutations in 533 Malaysian breast cancer cases with Malays, 3 BRCA1 and 2 BRCA2 mutations in Chinese and 1 BRCA1 mutation in Indians account for 60, 24 and 20 % of carrier families, respectively. By contrast, haplotype analyses suggest that a recurrent BRCA2 mutation (c.262_263delCT) found in 5 unrelated Malay families has at least 3 distinct haplotypes. Taken together, our data suggests that panel testing may help to identify carriers, particularly Asian BRCA2 carriers, who do not present with a priori strong family history characteristics.

  11. Generation of KCL035 research grade human embryonic stem cell line carrying a mutation in HBB gene

    Directory of Open Access Journals (Sweden)

    Heema Hewitson

    2016-03-01

    Full Text Available The KCL035 human embryonic stem cell line was derived from an embryo donated for research that carried a mutation in the HBB gene, which is linked to the β-thalassemia syndrome. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays.

  12. Selection and Neutral Mutations Drive Pervasive Mutability Losses in Long-Lived Anti-HIV B-Cell Lineages

    Science.gov (United States)

    Vieira, Marcos C; Zinder, Daniel; Cobey, Sarah

    2018-01-01

    Abstract High-affinity antibodies arise within weeks of infection from the evolution of B-cell receptors under selection to improve antigen recognition. This rapid adaptation is enabled by the distribution of highly mutable “hotspot” motifs in B-cell receptor genes. High mutability in antigen-binding regions (complementarity determining regions [CDRs]) creates variation in binding affinity, whereas low mutability in structurally important regions (framework regions [FRs]) may reduce the frequency of destabilizing mutations. During the response, loss of mutational hotspots and changes in their distribution across CDRs and FRs are predicted to compromise the adaptability of B-cell receptors, yet the contributions of different mechanisms to gains and losses of hotspots remain unclear. We reconstructed changes in anti-HIV B-cell receptor sequences and show that mutability losses were ∼56% more frequent than gains in both CDRs and FRs, with the higher relative mutability of CDRs maintained throughout the response. At least 21% of the total mutability loss was caused by synonymous mutations. However, nonsynonymous substitutions caused most (79%) of the mutability loss in CDRs. Because CDRs also show strong positive selection, this result suggests that selection for mutations that increase binding affinity contributed to loss of mutability in antigen-binding regions. Although recurrent adaptation to evolving viruses could indirectly select for high mutation rates, we found no evidence of indirect selection to increase or retain hotspots. Our results suggest mutability losses are intrinsic to both the neutral and adaptive evolution of B-cell populations and might constrain their adaptation to rapidly evolving pathogens such as HIV and influenza. PMID:29688540

  13. Mutations in the estrogen receptor alpha hormone binding domain promote stem cell phenotype through notch activation in breast cancer cell lines.

    Science.gov (United States)

    Gelsomino, L; Panza, S; Giordano, C; Barone, I; Gu, G; Spina, E; Catalano, S; Fuqua, S; Andò, S

    2018-04-24

    The detection of recurrent mutations affecting the hormone binding domain (HBD) of estrogen receptor alpha (ERα/ESR1) in endocrine therapy-resistant and metastatic breast cancers has prompted interest in functional characterization of these genetic alterations. Here, we explored the role of HBD-ESR1 mutations in influencing the behavior of breast cancer stem cells (BCSCs), using various BC cell lines stably expressing wild-type or mutant (Y537 N, Y537S, D538G) ERα. Compared to WT-ERα clones, mutant cells showed increased CD44 + /CD24 - ratio, mRNA levels of stemness genes, Mammosphere Forming Efficiency (MFE), Self-Renewal and migratory capabilities. Mutant clones exhibited high expression of NOTCH receptors/ligands/target genes and blockade of NOTCH signaling reduced MFE and migratory potential. Mutant BCSC activity was dependent on ERα phosphorylation at serine 118, since its inhibition decreased MFE and NOTCH4 activation only in mutant cells. Collectively, we demonstrate that the expression of HBD-ESR1 mutations may drive BC cells to acquire stem cell traits through ER/NOTCH4 interplay. We propose the early detection of HBD-ESR1 mutations as a challenge in precision medicine strategy, suggesting the development of tailored-approaches (i.e. NOTCH inhibitors) to prevent disease development and metastatic spread in BC mutant-positive patients. Copyright © 2018 Elsevier B.V. All rights reserved.

  14. A Biofunctional Molecular Beacon for Detecting Single Base Mutations in Cancer Cells

    Directory of Open Access Journals (Sweden)

    Haiyan Dong

    2016-01-01

    Full Text Available The development of a convenient and sensitive biosensing system to detect specific DNA sequences is an important issue in the field of genetic disease therapy. As a classic DNA detection technique, molecular beacon (MB is often used in the biosensing system. However, it has intrinsic drawbacks, including high assay cost, complicated chemical modification, and operational complexity. In this study, we developed a simple and cost-effective label-free multifunctional MB (LMMB by integrating elements of polymerization primer, template, target recognition, and G-quadruplex into one entity to detect target DNA. The core technique was accomplished by introducing a G-hairpin that features fragments of both G-quadruplex and target DNA recognition in the G-hairpin stem. Hybridization between LMMB and target DNA triggered conformational change between the G-hairpin and the common C-hairpin, resulting in significant SYBR-green signal amplification. The hybridization continues to the isothermal circular strand-displacement polymerization and accumulation of the double-stranded fragments, causing the uninterrupted extension of the LMMB without a need of chemical modification and other assistant DNA sequences. The novel and programmable LMMB could detect target DNA with sensitivity at 250 pmol/l with a linear range from 2 to 100 nmol/l and the relative standard deviation of 7.98%. The LMMB could sense a single base mutation from the normal DNA, and polymerase chain reaction (PCR amplicons of the mutant-type cell line from the wild-type one. The total time required for preparation and assaying was only 25 minutes. Apparently, the LMMB shows great potential for detecting DNA and its mutations in biosamples, and therefore it opens up a new prospect for genetic disease therapy.

  15. Prevalence of EGFR mutations in newly diagnosed locally advanced or metastatic non-small cell lung cancer Spanish patients and its association with histological subtypes and clinical features: The Spanish REASON study.

    Science.gov (United States)

    Esteban, E; Majem, M; Martinez Aguillo, M; Martinez Banaclocha, N; Dómine, M; Gómez Aldaravi, L; Juan, O; Cajal, R; Gonzalez Arenas, M C; Provencio, M

    2015-06-01

    The aim of the REASON study is to determine the frequency of EGFR mutation in advanced non-small cell lung cancer (aNSCLC) patients in Spain (all histologies), and to better understand the clinical factors (gender, smoking habits and histological subtypes) that may be associated with EGFR mutations, in an unselected sample of aNSCLC patients. All newly diagnosed aNSCLC patients from 40 selected centers in Spain were prospectively included for a 6-month period. Patient characteristics were obtained from clinical records. Mutation testing was performed on available tumor samples. Exploratory analyses were performed to characterize the clinico-pathological factors associated with presence of EGFR mutations. From March 2010 to March 2011, 1113 patients were included in the study, of which 1009 patients provided sample for EGFR mutation analysis (90.7%). Mutation analysis was not feasible in 146/1113 patients (13.1%) due to either sample unavailability (79/1113; 7.1%) or sample inadequacy (67/1113; 6.0%). Twenty-five out of 1113 patients (2.3%) were excluded due to unavailable information. Most patients (99.5%) were Caucasian, 74.5% were male, and predominantly were current (38.1%) or former smokers (44.0%). Median age was 66 years (range 25-90) and 70.7% of patients had non-squamous histology (57.8% adenocarcinoma, 1.8% bronchoalveolar, 11.1% large-cell carcinoma). Exon 19 deletions and the exon 21 L858R point mutation were analyzed in 942/1009 (93.4%) samples. Mutation rate was 11.6% (82.6% exon 19 dels and 17.4% L858R). To be never smoker (38.1%), female (25.4%), with bronchioloalveolar carcinoma (22.2%) or adenocarcinoma (15.4%) histology was associated with a higher prevalence of EGFR mutations. Exons 18, 20 and 21 (excluding L858R) were analyzed in 505/942 samples, and EGFR mutations were found in 22/505 samples (4.4%). The estimated prevalence of sensitizing EGFR mutations (exon 19 del, exon 21 L858R) in an unselected samples of newly diagnosed aNSCLC patients in

  16. EGFR Mutations in Surgically Resected Fresh Specimens from 697 Consecutive Chinese Patients with Non-Small Cell Lung Cancer and Their Relationships with Clinical Features

    Directory of Open Access Journals (Sweden)

    Yuanyang Lai

    2013-12-01

    Full Text Available We aimed to reveal the true status of epidermal growth factor receptor (EGFR mutations in Chinese patients with non-small cell lung cancer (NSCLC after lung resections. EGFR mutations of surgically resected fresh tumor samples from 697 Chinese NSCLC patients were analyzed by Amplification Refractory Mutation System (ARMS. Correlations between EGFR mutation hotspots and clinical features were also explored. Of the 697 NSCLC patients, 235 (33.7% patients had tyrosine kinase inhibitor (TKIs sensitive EGFR mutations in 41 (14.5% of the 282 squamous carcinomas, 155 (52.9% of the 293 adenocarcinomas, 34 (39.5% of the 86 adenosquamous carcinomas, one (9.1% of the 11 large-cell carcinomas, 2 (11.1% of the 18 sarcomatoid carcinomas, and 2 (28.6% of the 7 mucoepidermoid carcinomas. TKIs sensitive EGFR mutations were more frequently found in female patients (p < 0.001, non-smokers (p = 0.047 and adenocarcinomas (p < 0.001. The rates of exon 19 deletion mutation (19-del, exon 21 L858R point mutation (L858R, exon 21 L861Q point mutation (L861Q, exon 18 G719X point mutations (G719X, including G719C, G719S, G719A were 43.4%, 48.1%, 1.7% and 6.8%, respectively. Exon 20 T790M point mutation (T790M was detected in 3 squamous carcinomas and 3 adenocarcinomas and exon 20 insertion mutation (20-ins was detected in 2 patients with adenocarcinoma. Our results show the rates of EGFR mutations are higher in all types of NSCLC in Chinese patients. 19-del and L858R are two of the more frequent mutations. EGFR mutation detection should be performed as a routine postoperative examination in Chinese NSCLC patients.

  17. The TMEM43 Newfoundland mutation p.S358L causing ARVC-5 was imported from Europe and increases the stiffness of the cell nucleus.

    Science.gov (United States)

    Milting, Hendrik; Klauke, Bärbel; Christensen, Alex Hoerby; Müsebeck, Jörg; Walhorn, Volker; Grannemann, Sören; Münnich, Tamara; Šarić, Tomo; Rasmussen, Torsten Bloch; Jensen, Henrik Kjærulf; Mogensen, Jens; Baecker, Carolin; Romaker, Elena; Laser, Kai Thorsten; zu Knyphausen, Edzard; Kassner, Astrid; Gummert, Jan; Judge, Daniel P; Connors, Sean; Hodgkinson, Kathy; Young, Terry-L; van der Zwaag, Paul A; van Tintelen, J Peter; Anselmetti, Dario

    2015-04-07

    Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a rare genetic condition caused predominantly by mutations within desmosomal genes. The mutation leading to ARVC-5 was recently identified on the island of Newfoundland and caused by the fully penetrant missense mutation p.S358L in TMEM43. Although TMEM43-p.S358L mutation carriers were also found in the USA, Germany, and Denmark, the genetic relationship between North American and European patients and the disease mechanism of this mutation remained to be clarified. We screened 22 unrelated ARVC patients without mutations in desmosomal genes and identified the TMEM43-p.S358L mutation in a German ARVC family. We excluded TMEM43-p.S358L in 22 unrelated patients with dilated cardiomyopathy. The German family shares a common haplotype with those from Newfoundland, USA, and Denmark, suggesting that the mutation originated from a common founder. Examination of 40 control chromosomes revealed an estimated age of 1300-1500 years for the mutation, which proves the European origin of the Newfoundland mutation. Skin fibroblasts from a female and two male mutation carriers were analysed in cell culture using atomic force microscopy and revealed that the cell nuclei exhibit an increased stiffness compared with TMEM43 wild-type controls. The German family is not affected by a de novo TMEM43 mutation. It is therefore expected that an unknown number of European families may be affected by the TMEM43-p.S358L founder mutation. Due to its deleterious clinical phenotype, this mutation should be checked in any case of ARVC-related genotyping. It appears that the increased stiffness of the cell nucleus might be related to the massive loss of cardiomyocytes, which is typically found in ventricles of ARVC hearts. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.

  18. TP53 germline mutation testing in 180 families suspected of Li-Fraumeni syndrome: mutation detection rate and relative frequency of cancers in different familial phenotypes

    NARCIS (Netherlands)

    Ruijs, M.W.G.; Verhoef, S.; Rookus, M.A.; Pruntel, R.; van der Hout, A.H.; Hogervorst, F.B.L.; Kluijt, I.; Sijmons, R.H.; Aalfs, C.M.; Wagner, A.; Ausems, M.G.E.M.; Hoogerbrugge, N.; van Asperen, C.J.; Gómez García, E.B.; Meijers-Heijboer, H.; ten Kate, L.P.; Menko, F.H.; van 't Veer, L.J.

    2010-01-01

    Background Li-Fraumeni syndrome (LFS) is a rare autosomal dominant cancer predisposition syndrome. Most families fulfilling the classical diagnostic criteria harbour TP53 germline mutations. However, TP53 germline mutations may also occur in less obvious phenotypes. As a result, different criteria

  19. Protective action of DNA preparations on the survival of cells and yield of 8-azaguanine resistant mutations in X-irradiated cell culture of chinese hamsters

    International Nuclear Information System (INIS)

    Kuznetsova, N.N.; Feoktistova, T.P.

    1976-01-01

    A DNA preparation (molecular weight 19.6-21.0x1O 6 daltons) administered to cell culture of Chinese hamsters in concentrations of 100 to 122 μg/ml 60 minutes before and in the course of 3 days after X-irradiation (600 R) decreased the lethality of irradiated cells and reduced induction of 8-azaguanine resistant genic mutations. DNA preparations with the concentrations under study had no toxic action on cells and were not mutagenous

  20. Establishment of induced pluripotent stem cell line (ZZUi010-A from an Alzheimer's disease patient carrying an APP gene mutation

    Directory of Open Access Journals (Sweden)

    Zhilei Wang

    2017-12-01

    Full Text Available Alzheimer's disease (AD is one of the most common neurodegenerative disorders. Previous studies have identified mutations in several genes, such as amyloid precursor protein (APP, presenilin-1 (PSEN1, and presenilin-2 (PSEN2, in patients with early-onset (<65 years familial AD. Recently, a patient with an APP gene mutation was identified; the dermal fibroblasts of the patient were obtained and a line of induced pluripotent stem cells (iPSCs was successfully generated using the Sendai-virus (SeV delivery system. The iPSC line will be useful for further study of the pathomechanism and drug screening for AD.

  1. Modification of structural chromosome mutations by zinc ions at wavelike kinetics of radiation mutagenesis in Crepis Capillaris seed cells

    International Nuclear Information System (INIS)

    Mustafaev, Kh. B.; Pomanov, V.P.

    1979-01-01

    The resting seeds Cr. capillaris have been irradiated by gamma rays in the 4 kR dose. Immediately after irradiation and within different terms of storage the seeds have been grown in the 3.5x10 -5 M solution ZnCl 2 and in the distilled water. Chromosome structural mutations in the K-mitosis of the first cell cycle have been studied. The frequency modification of chromosomal rearrangement by zinc ions at the waveline kinetics of the radiation mutagenesis is revealed as follows: zinc ions increase the mutation frequency at the points of waveline kinetics maximum and exert no influence at minimum points

  2. Use of Mycobacterium smegmatis deficient in ADP-ribosyltransferase as surrogate for Mycobacterium tuberculosis in drug testing and mutation analysis.

    Science.gov (United States)

    Agrawal, Priyanka; Miryala, Sandeep; Varshney, Umesh

    2015-01-01

    Rifampicin (Rif) is a first line drug used for tuberculosis treatment. However, the emergence of drug resistant strains has necessitated synthesis and testing of newer analogs of Rif. Mycobacterium smegmatis is often used as a surrogate for M. tuberculosis. However, the presence of an ADP ribosyltransferase (Arr) in M. smegmatis inactivates Rif, rendering it impractical for screening of Rif analogs or other compounds when used in conjunction with them (Rif/Rif analogs). Rifampicin is also used in studying the role of various DNA repair enzymes by analyzing mutations in RpoB (a subunit of RNA polymerase) causing Rif resistance. These analyses use high concentrations of Rif when M. smegmatis is used as model. Here, we have generated M. smegmatis strains by deleting arr (Δarr). The M. smegmatis Δarr strains show minimum inhibitory concentration (MIC) for Rif which is similar to that for M. tuberculosis. The MICs for isoniazid, pyrazinamide, ethambutol, ciprofloxacin and streptomycin were essentially unaltered for M. smegmatis Δarr. The growth profiles and mutation spectrum of Δarr and, Δarr combined with ΔudgB (udgB encodes a DNA repair enzyme that excises uracil) strains were similar to their counterparts wild-type for arr. However, the mutation spectrum of ΔfpgΔarr strain differed somewhat from that of the Δfpg strain (fpg encodes a DNA repair enzyme that excises 8-oxo-G). Our studies suggest M. smegmatis Δarr strain as an ideal model system in drug testing and mutation spectrum determination in DNA repair studies.

  3. Use of Mycobacterium smegmatis deficient in ADP-ribosyltransferase as surrogate for Mycobacterium tuberculosis in drug testing and mutation analysis.

    Directory of Open Access Journals (Sweden)

    Priyanka Agrawal

    Full Text Available Rifampicin (Rif is a first line drug used for tuberculosis treatment. However, the emergence of drug resistant strains has necessitated synthesis and testing of newer analogs of Rif. Mycobacterium smegmatis is often used as a surrogate for M. tuberculosis. However, the presence of an ADP ribosyltransferase (Arr in M. smegmatis inactivates Rif, rendering it impractical for screening of Rif analogs or other compounds when used in conjunction with them (Rif/Rif analogs. Rifampicin is also used in studying the role of various DNA repair enzymes by analyzing mutations in RpoB (a subunit of RNA polymerase causing Rif resistance. These analyses use high concentrations of Rif when M. smegmatis is used as model. Here, we have generated M. smegmatis strains by deleting arr (Δarr. The M. smegmatis Δarr strains show minimum inhibitory concentration (MIC for Rif which is similar to that for M. tuberculosis. The MICs for isoniazid, pyrazinamide, ethambutol, ciprofloxacin and streptomycin were essentially unaltered for M. smegmatis Δarr. The growth profiles and mutation spectrum of Δarr and, Δarr combined with ΔudgB (udgB encodes a DNA repair enzyme that excises uracil strains were similar to their counterparts wild-type for arr. However, the mutation spectrum of ΔfpgΔarr strain differed somewhat from that of the Δfpg strain (fpg encodes a DNA repair enzyme that excises 8-oxo-G. Our studies suggest M. smegmatis Δarr strain as an ideal model system in drug testing and mutation spectrum determination in DNA repair studies.

  4. The Human Gene Mutation Database: building a comprehensive mutation repository for clinical and molecular genetics, diagnostic testing and personalized genomic medicine.

    Science.gov (United States)

    Stenson, Peter D; Mort, Matthew; Ball, Edward V; Shaw, Katy; Phillips, Andrew; Cooper, David N

    2014-01-01

    The Human Gene Mutation Database (HGMD®) is a comprehensive collection of germline mutations in nuclear genes that underlie, or are associated with, human inherited disease. By June 2013, the database contained over 141,000 different lesions detected in over 5,700 different genes, with new mutation entries currently accumulating at a rate exceeding 10,000 per annum. HGMD was originally established in 1996 for the scientific study of mutational mechanisms in human genes. However, it has since acquired a much broader utility as a central unified disease-oriented mutation repository utilized by human molecular geneticists, genome scientists, molecular biologists, clinicians and genetic counsellors as well as by those specializing in biopharmaceuticals, bioinformatics and personalized genomics. The public version of HGMD (http://www.hgmd.org) is freely available to registered users from academic institutions/non-profit organizations whilst the subscription version (HGMD Professional) is available to academic, clinical and commercial users under license via BIOBASE GmbH.

  5. Effects of diurnal temperature difference and gamma radiation on the frequency of somatic cell mutations in the stamen hairs

    International Nuclear Information System (INIS)

    Kim, Jin Kyu; Kim, Won Rok; Kim, Jae Sung; Shin, Hae Shick; Lee, Jeong Joo

    1998-01-01

    This study deals with the effects of diurnal temperature difference (DTD) on somatic cell mutation frequencies in Tradescantia stamen hairs irradiated with radiation. Potted plants of Tradescantia 4430 were irradiated with 0.3, 0.5, 1.0 and 2.0 Gy of gamma radiation. The irradiated plants were maintained under two different experimental conditions; at constant temperature of 20 degree C (DTD0) and at 28 degree C for 14-h day and 8 degree C for 10-h night (DTD20). The somatic cell mutation rate in 0.5 Gy irradiated group showed a big increase on the 6th day and reached a maximum value on the 10th day after irradiation while the rate in the experimental group under the condition of DTD20 started to increase on the 8th day and got to a maximal value on the 14th day postirradiation. In both of the two experiments, the dose-response relationships were clearly linear. The slope of the DTD20 dose-response curve was much steeper than that of the DTD0 one. In conclusion, a great DTD, as one of environmental stresses, enhanced the effectiveness of radiation in the induction of somatic cell mutations and caused a shift of the peak interval of radiation-induced mutations in Tradescantia stamen hairs

  6. Cytotoxicity and genotoxicity of UVA irradiation in Chinese hamster ovary cells measured by specific locus mutations, sister chromatid exchanges and chromosome aberrations

    International Nuclear Information System (INIS)

    Lundgren, Karsten; Wulf, H.C.

    1988-01-01

    The increasing use of artificial UVA (320-400 nm) suntanning devices has brought attention to possible hazardous effects of UVA. In contrast with earlier studies, several groups recently have described that UVA possibly is mutagenic. We evaluate the genotoxic properties of broad band UVA using CHO cells and three different assays: specific locus (HGPRT) mutations, chromosome aberrations, and sister chromatid exchanges (SCEs). The UVA-source was an UVASUN 2000 S (Mutzhas), emitting UVA above 340 nm. The survival curve of the cells exhibited a shoulder up to 200 kJ/m 2 , that was followed by exponential killing at higher fluences. Mutations were induced linearly in the fluence range of 0-200 kJ/m 2 to a level seven fold higher than the spontaneous, followed by a decrease at fluences above 300 kJ/m 2 . Over the total range of tested fluences (0-300 kJ/m 2 ) a linear dose-response relationship was observed for UVA-induced SCEs. A significantly higher percentage of the cells showed chromosomes with aberrations at the higher levels of exposure (200, 300 and 400 kJ/m 2 ), but no dose response was demonstrated. Our results confirm recent findings showing that UVA is mutagenic in mammalian cells and suggest that UVA exposure may contribute to the total burden of genetic damage caused by exposure to ultraviolet light. (author)

  7. Effects of icotinib, a novel epidermal growth factor receptor tyrosine kinase inhibitor, in EGFR-mutated non-small cell lung cancer.

    Science.gov (United States)

    Yang, Guangdie; Yao, Yinan; Zhou, Jianya; Zhao, Qiong

    2012-06-01

    Epidermal growth factor receptor (EGFR) is one of the most promising targets for non-small cell lung cancer (NSCLC). Our study demonstrated the antitumor effects of icotinib hydrochloride, a highly selective epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI), in two EGFR-mutated lung cancer cell lines compared to A549, a cell line without EGFR mutations. We incubated PC-9 and HCC827 human lung cancer cell lines both with (E746-A750) mutations with various concentrations of icotinib and gefitinib for 48 h. Cell proliferation and migration were determined using a real-time cell invasion and migration assay and cytotoxicity assay. Apoptosis was assessed by measuring Annexin V staining using flow cytometry. The antitumor effects of icotinib compared to gefitinib were similar and were most effective in reducing the proliferation of EGFR-mutated cells compared to non-mutated controls. Our results suggest the possibility of icotinib as a new therapeutic agent of EGFR-mutated cancer cells, which has the potential to be used in the first-line treatment of EGFR-mutated NSCLC.

  8. Mir-34a mimics are potential therapeutic agents for p53-mutated and chemo-resistant brain tumour cells.

    Directory of Open Access Journals (Sweden)

    Yuen Ngan Fan

    Full Text Available Chemotherapeutic drug resistance and relapse remains a major challenge for paediatric (medulloblastoma and adult (glioblastoma brain tumour treatment. Medulloblastoma tumours and cell lines with mutations in the p53 signalling pathway have been shown to be specifically insensitive to DNA damaging agents. The aim of this study was to investigate the potential of triggering cell death in p53 mutated medulloblastoma cells by a direct activation of pro-death signalling downstream of p53 activation. Since non-coding microRNAs (miRNAs have the ability to fine tune the expression of a variety of target genes, orchestrating multiple downstream effects, we hypothesised that triggering the expression of a p53 target miRNA could induce cell death in chemo-resistant cells. Treatment with etoposide, increased miR-34a levels in a p53-dependent fashion and the level of miR-34a transcription was correlated with the cell sensitivity to etoposide. miR-34a activity was validated by measuring the expression levels of one of its well described target: the NADH dependent sirtuin1 (SIRT1. Whilst drugs directly targeting SIRT1, were potent to trigger cell death at high concentrations only, introduction of synthetic miR-34a mimics was able to induce cell death in p53 mutated medulloblastoma and glioblastoma cell lines. Our results show that the need of a functional p53 signaling pathway can be bypassed by direct activation of miR-34a in brain tumour cells.

  9. Somatic mosaicism in families with hemophilia B: 11% of germline mutations originate within a few cell divisions post-fertilization

    Energy Technology Data Exchange (ETDEWEB)

    Knoell, A.; Ketterling, R.P.; Vielhaber, E. [Mayo Clinic/Foundation, Rochester, MN (United States)] [and others

    1994-09-01

    Previous molecular estimates of mosaicism in the dystrophin and other genes generally have focused on the transmission of the mutated allele to two or more children by an individual without the mutation in leukocyte DNA. We have analyzed 414 families with hemophilia B by direct genomic sequencing and haplotype analysis, and have deduced the origin of mutation in 56 families. There was no origin individual who transmitted a mutant allele to more than one child. However, somatic mosaicism was detected by sequence analysis of four origin individuals (3{female} and 1{male}). The sensitivity of this analysis is typically one part in ten. In one additional female who had close to a 50:50 ratio of mutant to normal alleles, three of four noncarrier daughters inherited the haplotype associated with the mutant allele. This highlights a caveat in molecular analysis: a presumptive carrier in a family with sporadic disease does not necessarily have a 50% probability of transmitting the mutant allele to her offspring. After eliminating those families in which mosaicism could not be detected because of a total gene deletion or absence of DNA from a deduced origin individual, 5 of 43 origin individuals exhibited somatic mosaicism at a level that reflects a mutation within the first few cell divisions after fertilization. In one patient, analysis of cervical scrapings and buccal mucosa confirm the generalized distribution of somatic mutation. Are the first few cell divisions post-fertilization highly mutagenic, or do mutations at later divisions also give rise to somatic mosaicism? To address this question, DNA from origin individuals are being analyzed to detect somatic mosaicism at a sensitivity of 1:1000. Single nucleotide primer extension (SNuPE) has been utilized in eight families to date and no mosaicism has been detected. When the remaining 30 samples are analyzed, it will be possible to compare the frequency of somatic mosaicism at 0.1-10% with that of {ge}10%.

  10. CRISPR Correction of a Homozygous Low-Density Lipoprotein Receptor Mutation in Familial Hypercholesterolemia Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Omer, Linda; Hudson, Elizabeth A; Zheng, Shirong; Hoying, James B; Shan, Yuan; Boyd, Nolan L

    2017-11-01

    Familial hypercholesterolemia (FH) is a hereditary disease primarily due to mutations in the low-density lipoprotein receptor (LDLR) that lead to elevated cholesterol and premature development of cardiovascular disease. Homozygous FH patients (HoFH) with two dysfunctional LDLR alleles are not as successfully treated with standard hypercholesterol therapies, and more aggressive therapeutic approaches to control cholesterol levels must be considered. Liver transplant can resolve HoFH, and hepatocyte transplantation has shown promising results in animals and humans. However, demand for donated livers and high-quality hepatocytes overwhelm the supply. Human pluripotent stem cells can differentiate to hepatocyte-like cells (HLCs) with the potential for experimental and clinical use. To be of future clinical use as autologous cells, LDLR genetic mutations in derived FH-HLCs need to be corrected. Genome editing technology clustered-regularly-interspaced-short-palindromic-repeats/CRISPR-associated 9 (CRISPR/Cas9) can repair pathologic genetic mutations in human induced pluripotent stem cells. We used CRISPR/Cas9 genome editing to permanently correct a 3-base pair homozygous deletion in LDLR exon 4 of patient-derived HoFH induced pluripotent stem cells. The genetic correction restored LDLR-mediated endocytosis in FH-HLCs and demonstrates the proof-of-principle that CRISPR-mediated genetic modification can be successfully used to normalize HoFH cholesterol metabolism deficiency at the cellular level.

  11. Experiences from treatment-predictive KRAS testing; high mutation frequency in rectal cancers from females and concurrent mutations in the same tumor

    DEFF Research Database (Denmark)

    Jönsson, Mats; Ekstrand, Anna; Edekling, Thomas

    2009-01-01

    . METHODS: We used a real-time PCR based method to determine KRAS mutations in 136 colorectal cancers with mutations identified in 53 (39%) tumors. RESULTS: KRAS mutations were significantly more often found in rectal cancer (21/38, 55%) than in colon cancer (32/98, 33%) (P = 0.02). This finding...... was explained by marked differences mutation rates in female patients who showed mutations in 33% of the colon cancers and in 67% of the rectal cancers (P = 0.01). Concurrent KRAS mutations were identified in three tumors; two colorectal cancers harbored Gly12Asp/Gly13Asp and Gly12Cys/Gly13Asp and a third tumor...... carried Gly12Cys/Gly12Asp in an adenomatous component and additionally acquired Gly12Val in the invasive component. CONCLUSION: The demonstration of a particularly high KRAS mutation frequency among female rectal cancer patients suggests that this subset is the least likely to respond to anti...

  12. A point mutation in the polymerase protein PB2 allows a reassortant H9N2 influenza isolate of wild-bird origin to replicate in human cells.

    Science.gov (United States)

    Hussein, Islam T.M.; Ma, Eric J.; Meixell, Brandt; Hill, Nichola J.; Lindberg, Mark S.; Albrecht , Randy A.; Bahl, Justin; Runstadler, Jonathan A.

    2016-01-01

    H9N2 influenza A viruses are on the list of potentially pandemic subtypes. Therefore, it is important to understand how genomic reassortment and genetic polymorphisms affect phenotypes of H9N2 viruses circulating in the wild bird reservoir. A comparative genetic analysis of North American H9N2 isolates of wild bird origin identified a naturally occurring reassortant virus containing gene segments derived from both North American and Eurasian lineage ancestors. The PB2 segment of this virus encodes 10 amino acid changes that distinguish it from other H9 strains circulating in North America. G590S, one of the 10 amino acid substitutions observed, was present in ~ 12% of H9 viruses worldwide. This mutation combined with R591 has been reported as a marker of pathogenicity for human pandemic 2009 H1N1 viruses. Screening by polymerase reporter assay of all the natural polymorphisms at these two positions identified G590/K591 and S590/K591 as the most active, with the highest polymerase activity recorded for the SK polymorphism. Rescued viruses containing these two polymorphic combinations replicated more efficiently in MDCK cells and they were the only ones tested that were capable of establishing productive infection in NHBE cells. A global analysis of all PB2 sequences identified the K591 signature in six viral HA/NA subtypes isolated from several hosts in seven geographic locations. Interestingly, introducing the K591 mutation into the PB2 of a human-adapted H3N2 virus did not affect its polymerase activity. Our findings demonstrate that a single point mutation in the PB2 of a low pathogenic H9N2 isolate could have a significant effect on viral phenotype and increase its propensity to infect mammals. However, this effect is not universal, warranting caution in interpreting point mutations without considering protein sequence context.

  13. Reliability of kinetic visual field testing in children with mutation-proven retinal dystrophies: Implications for therapeutic clinical trials.

    Science.gov (United States)

    Dedania, Vaidehi S; Liu, Jerry Y; Schlegel, Dana; Andrews, Chris A; Branham, Kari; Khan, Naheed W; Musch, David C; Heckenlively, John R; Jayasundera, K Thiran

    2018-01-01

    Kinetic visual field testing is used to monitor disease course in retinal dystrophy clinical care and treatment response in treatment trials, which are increasingly recruiting children. This study investigates Goldmann visual field (GVF) changes in young children with mutation-proven retinal dystrophies as they age and with progression of the retinal degeneration. Retrospective review of children ≤ 17 years old with a mutation-proven retinal dystrophy. Objective clinical disease activity was assessed by a retinal degeneration specialist masked to GVF results. Digital quantification of GVF area was performed. Twenty-nine children (58 eyes), ages 5-16, were identified. GVF area increased with age despite progression in 20 children and clinical stability in nine children. Mean ± standard error increase in GVF area/year was 333 ± 130 mm 2 (I4e, p = 0.012), 720 ± 155 mm 2 (III4e, p children with mutation-proven retinal dystrophies, there is a significant increase in GVF area with age, particularly those children with retinal dystrophies can be an unreliable measure of response to treatment and on which to base appropriate counseling about visual impairment.

  14. Next generation sequencing of Cytokeratin 20-negative Merkel cell carcinoma reveals ultraviolet-signature mutations and recurrent TP53 and RB1 inactivation.

    Science.gov (United States)

    Harms, Paul W; Collie, Angela M B; Hovelson, Daniel H; Cani, Andi K; Verhaegen, Monique E; Patel, Rajiv M; Fullen, Douglas R; Omata, Kei; Dlugosz, Andrzej A; Tomlins, Scott A; Billings, Steven D

    2016-03-01

    Merkel cell carcinoma is a rare but highly aggressive cutaneous neuroendocrine carcinoma. Cytokeratin 20 (CK20) is expressed in ~95% of Merkel cell carcinomas and is useful for distinction from morphologically similar entities including metastatic small-cell lung carcinoma. Lack of CK20 expression may make diagnosis of Merkel cell carcinoma more challenging, and has unknown biological significance. Approximately 80% of CK20-positive Merkel cell carcinomas are associated with the oncogenic Merkel cell polyomavirus. Merkel cell carcinomas lacking Merkel cell polyomavirus display distinct genetic changes from Merkel cell polyomavirus-positive Merkel cell carcinoma, including RB1 inactivating mutations. Unlike CK20-positive Merkel cell carcinoma, the majority of CK20-negative Merkel cell carcinomas are Merkel cell polyomavirus-negative, suggesting CK20-negative Merkel cell carcinomas predominantly arise through virus-independent pathway(s) and may harbor additional genetic differences from conventional Merkel cell carcinoma. Hence, we analyzed 15 CK20-negative Merkel cell carcinoma tumors (10 Merkel cell polyomavirus-negative, four Merkel cell polyomavirus-positive, and one undetermined) using the Ion Ampliseq Comprehensive Cancer Panel, which assesses copy number alterations and mutations in 409 cancer-relevant genes. Twelve tumors displayed prioritized high-level chromosomal gains or losses (average 1.9 per tumor). Non-synonymous high-confidence somatic mutations were detected in 14 tumors (average 11.9 per tumor). Assessing all somatic coding mutations, an ultraviolet-signature mutational profile was present, and more prevalent in Merkel cell polyomavirus-negative tumors. Recurrent deleterious tumor suppressor mutations affected TP53 (9/15, 60%), RB1 (3/15, 20%), and BAP1 (2/15, 13%). Oncogenic activating mutations included PIK3CA (3/15, 20%), AKT1 (1/15, 7%) and EZH2 (1/15, 7%). In conclusion, CK20-negative Merkel cell carcinoma display overlapping genetic changes

  15. Next Generation Sequencing of Cytokeratin 20-Negative Merkel Cell Carcinoma Reveals Ultraviolet Signature Mutations and Recurrent TP53 and RB1 Inactivation

    Science.gov (United States)

    Harms, Paul W.; Collie, Angela M. B.; Hovelson, Daniel H.; Cani, Andi K.; Verhaegen, Monique E.; Patel, Rajiv M.; Fullen, Douglas R.; Omata, Kei; Dlugosz, Andrzej A.; Tomlins, Scott A.; Billings, Steven D.

    2016-01-01

    Merkel cell carcinoma is a rare but highly aggressive cutaneous neuroendocrine carcinoma. Cytokeratin-20 (CK20) is expressed in approximately 95% of Merkel cell carcinomas and is useful for distinction from morphologically similar entities including metastatic small cell lung carcinoma. Lack of CK20 expression may make diagnosis of Merkel cell carcinoma more challenging, and has unknown biological significance. Approximately 80% of CK20-positive Merkel cell carcinomas are associated with the oncogenic Merkel cell polyomavirus. Merkel cell carcinomas lacking Merkel cell polyomavirus display distinct genetic changes from Merkel cell polyomavirus-positive Merkel cell carcinoma, including RB1 inactivating mutations. Unlike CK20-positive Merkel cell carcinoma, the majority of CK20-negative Merkel cell carcinomas are Merkel cell polyomavirus-negative, suggesting CK20-negative Merkel cell carcinomas predominantly arise through virus-independent pathway(s) and may harbor additional genetic differences from conventional Merkel cell carcinoma. Hence, we analyzed 15 CK20-negative Merkel cell carcinoma tumors (ten Merkel cell polyomavirus-negative, four Merkel cell polyomavirus-positive, and one undetermined) using the Ion Ampliseq Comprehensive Cancer Panel, which assesses copy number alterations and mutations in 409 cancer-relevant genes. Twelve tumors displayed prioritized high-level chromosomal gains or losses (average 1.9 per tumor). Non-synonymous high confidence somatic mutations were detected in 14 tumors (average 11.9 per tumor). Assessing all somatic coding mutations, an ultraviolet-signature mutational profile was present, and more prevalent in Merkel cell polyomavirus-negative tumors. Recurrent deleterious tumor suppressor mutations affected TP53 (9/15, 60%), RB1 (3/15, 20%), and BAP1 (2/15, 13%). Oncogenic activating mutations included PIK3CA (3/15, 20%), AKT1 (1/15, 7%)) and EZH2 (1/15, 7%). In conclusion, CK20-negative Merkel cell carcinoma display overlapping

  16. Peptide bond-forming reagents HOAt and HATU are not mutagenic in the bacterial reverse mutation test.

    Science.gov (United States)

    Nicolette, John; Neft, Robin E; Vanosdol, Jessica; Murray, Joel

    2016-04-01

    The peptide bond-forming reagents 1-hydroxy-7-azabenzotriazole (HOAt, CAS 39968-33-7) and O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, CAS 148893-10-1) either have structural alerts, unclassified features or are considered out of domain when evaluated for potential mutagenicity with in silico programs DEREK and CaseUltra. Since they are commonly used reagents in pharmaceutical drug syntheses, they may become drug substance or drug product impurities and would need to be either controlled to appropriately safe levels or tested for mutagenicity. Both reagents were tested in the bacterial reverse mutation (Ames) test at Covance, under GLP conditions, following the OECD test guideline and ICH S2(R1) recommendations and found to be negative. Our data show that HOAt and HATU-common pharmaceutical synthesis reagents-are not mutagenic, and can be treated as ordinary drug impurities. © 2016 Wiley Periodicals, Inc.

  17. Molecular Epidemiology of EGFR Mutations in Asian Patients with Advanced Non-Small-Cell Lung Cancer of Adenocarcinoma Histology – Mainland China Subset Analysis of the PIONEER study

    Science.gov (United States)

    Shi, Yuankai; Li, Junling; Zhang, Shucai; Wang, Mengzhao; Yang, Shujun; Li, Ning; Wu, Gang; Liu, Wei; Liao, Guoqing; Cai, Kaican; Chen, Liang’an; Zheng, Meizhen; Yu, Ping; Wang, Xiuwen; Liu, Yunpeng; Guo, Qisen; Nie, Ligong; Liu, Jiwei; Han, Xiaohong

    2015-01-01

    Epidermal growth factor receptor (EGFR) mutations are the strongest response predictors to EGFR tyrosine kinase inhibitors (TKI) therapy, but knowledge of the EGFR mutation frequency on lung adenocarcinoma is still limited to retrospective studies. The PIONEER study (NCT01185314) is a prospective molecular epidemiology study in Asian patients with newly diagnosed advanced lung adenocarcinoma, aiming to prospectively analyze EGFR mutation status in IIIB/IV treatment-naïve lung adenocarcinomas in Asia. We report the mainland China subset results. Eligible patients (≥20 yrs old, IIIB/IV adenocarcinoma and treatment-naïve) were registered in 17 hospitals in mainland China. EGFR was tested for mutations by amplification refractory mutation system using biopsy samples. Demographic and clinical characteristics were collected for subgroup analyses. A total of 747 patients were registered. Successful EGFR mutation analysis was performed in 741, with an overall mutation rate of 50.2%. The EGFR active mutation rate is 48.0% (with 1.3% of combined active and resistance mutations). Tobacco use (>30 pack-year vs. 0–10 pack-year, OR 0.27, 95%CI: 0.17–0.42) and regional lymph nodes involvement (N3 vs. N0, OR 0.47, 95%CI: 0.29–0.76) were independent predictors of EGFR mutation in multivariate analysis. However, even in regular smokers, the EGFR mutation frequency was 35.3%. The EGFR mutation frequency was similar between diverse biopsy sites and techniques. The overall EGFR mutation frequency of the mainland China subset was 50.2%, independently associated with the intensity of tobacco use and regional lymph nodes involvement. The relatively high frequency of EGFR mutations in the mainland China subset suggest that any effort to obtain tissue sample for EGFR mutation testing should be encouraged. PMID:26599344

  18. Advanced cell-based modeling of the royal disease: characterization of the mutated F9 mRNA.

    Science.gov (United States)

    Martorell, L; Luce, E; Vazquez, J L; Richaud-Patin, Y; Jimenez-Delgado, S; Corrales, I; Borras, N; Casacuberta-Serra, S; Weber, A; Parra, R; Altisent, C; Follenzi, A; Dubart-Kupperschmitt, A; Raya, A; Vidal, F; Barquinero, J

    2017-11-01

    Essentials The Royal disease (RD) is a form of hemophilia B predicted to be caused by a splicing mutation. We generated an iPSC-based model of the disease allowing mechanistic studies at the RNA level. F9 mRNA analysis in iPSC-derived hepatocyte-like cells showed the predicted abnormal splicing. Mutated F9 mRNA level was very low but we also found traces of wild type transcripts. Background The royal disease is a form of hemophilia B (HB) that affected many descendants of Queen Victoria in the 19th and 20th centuries. It was found to be caused by the mutation F9 c.278-3A>G. Objective To generate a physiological cell model of the disease and to study F9 expression at the RNA level. Methods Using fibroblasts from skin biopsies of a previously identified hemophilic patient bearing the F9 c.278-3A>G mutation and his mother, we generated induced pluripotent stem cells (iPSCs). Both the patient's and mother's iPSCs were differentiated into hepatocyte-like cells (HLCs) and their F9 mRNA was analyzed using next-generation sequencing (NGS). Results and Conclusion We demonstrated the previously predicted aberrant splicing of the F9 transcript as a result of an intronic nucleotide substitution leading to a frameshift and the generation of a premature termination codon (PTC). The F9 mRNA level in the patient's HLCs was significantly reduced compared with that of his mother, suggesting that mutated transcripts undergo nonsense-mediated decay (NMD), a cellular mechanism that degrades PTC-containing mRNAs. We also detected small proportions of correctly spliced transcripts in the patient's HLCs, which, combined with genetic variability in splicing and NMD machineries, could partially explain some clinical variability among affected members of the European royal families who had lifespans above the average. This work allowed the demonstration of the pathologic consequences of an intronic mutation in the F9 gene and represents the first bona fide cellular model of HB allowing the

  19. Cell culture-adaptive mutations of NS5A affect replication of hepatitis C virus differentially depending on the viral genotypes.

    Science.gov (United States)

    Chung, Aeri; Jin, Bora; Han, Kwang-Hyub; Ahn, Sang Hoon; Kim, Seungtaek

    2017-01-01

    Most of HCV RNAs require cell culture-adaptive mutations for efficient replication in cell culture and a number of such mutations have been described including a well-known S2204I substitution mutation in NS5A protein. In contrast, the replication of genotype 2a JFH1 RNA in cell culture does not require any cell culture-adaptive mutation. Rather, the presence of S2204I mutation impaired the JFH1 RNA replication. In this study, we examined the effect of reversions and substitutions of NS5A cell culture-adaptive mutations on virus replication in different genotypic backgrounds after either placing genotype 1a NS5A in the genotype 2a JFH1 or vice versa. The results from this investigation suggest that the S2204I mutation affects HCV RNA replication differentially depending on the viral genotypes but that the effect was not simply explained by the genotypic background. Perhaps, the effect of the S2204I mutation on HCV replication reflects both intra- and intergenic interactions of NS5A protein. J. Med. Virol. 89:146-152, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  20. Human Papillomavirus 16 Infection and TP53 Mutation: Two Distinct Pathogeneses for Oropharyngeal Squamous Cell Carcinoma in an Eastern Chinese Population.

    Science.gov (United States)

    Wang, Zhen; Xia, Rong-Hui; Ye, Dong-Xia; Li, Jiang

    2016-01-01

    To investigate the clinicopathological characteristics, human papillomavirus (HPV) infection, p53 expression, and TP53 mutations in oropharyngeal squamous cell carcinoma (OPSCC) and determine their utility as prognostic predictors in a primarily eastern Chinese population. The HPV infection status was tested via p16INK4A immunohistochemistry and validated using PCR, reverse blot hybridization and in situ hybridization (ISH) in 188 OPSCC samples. p53 expression levels and TP53 gene mutations were assessed through immunohistochemistry and sequencing, respectively. Clinicopathological characteristics and follow-up information were collected. Overall survival was estimated using the Log-rank test. Overall, 22 of the 188 OPSCC samples were associated with HPV infection. HPV16 was identified in all 22 samples, whereas no samples were positive for HPV18. All 22 HPV-associated OPSCC samples were p53 negative and lacked TP53 mutations. HPV16 positivity, female patients, non-smokers, and patients with histological grade I and stage N0 diseases showed better overall survival (p = 0.009, 0.003, 0.048, 0.009, and 0.004, respectively). No significant differences in overall survival between smoking and non-smoking patients were observed in the HPV-associated OPSCC group. Patients without mutations in TP53 exons 5-8 had better prognoses (p = 0.031) among the 43 sequenced specimens. Multivariate analysis indicated that HPV16 infection status (p = 0.011), histological grade (p = 0.017), and N stage (p = 0.019) were independent prognostic factors for patients with OPSCC. Distinct from the situation in Europe and America, for the patients with OPSCC in this study, HPV16 infection was relatively low, although it was still the most important independent prognostic predictor for the disease. In addition to the high smoking and drinking rate in this population, HPV16 infection and TP53 dysfunction appear to be two distinct pathogens for OPSCC patients in the eastern Chinese population.