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Sample records for automated dna mutation

  1. Automated DNA mutation detection using universal conditions direct sequencing: application to ten muscular dystrophy genes

    Directory of Open Access Journals (Sweden)

    Wu Bai-Lin

    2009-10-01

    Full Text Available Abstract Background One of the most common and efficient methods for detecting mutations in genes is PCR amplification followed by direct sequencing. Until recently, the process of designing PCR assays has been to focus on individual assay parameters rather than concentrating on matching conditions for a set of assays. Primers for each individual assay were selected based on location and sequence concerns. The two primer sequences were then iteratively adjusted to make the individual assays work properly. This generally resulted in groups of assays with different annealing temperatures that required the use of multiple thermal cyclers or multiple passes in a single thermal cycler making diagnostic testing time-consuming, laborious and expensive. These factors have severely hampered diagnostic testing services, leaving many families without an answer for the exact cause of a familial genetic disease. A search of GeneTests for sequencing analysis of the entire coding sequence for genes that are known to cause muscular dystrophies returns only a small list of laboratories that perform comprehensive gene panels. The hypothesis for the study was that a complete set of universal assays can be designed to amplify and sequence any gene or family of genes using computer aided design tools. If true, this would allow automation and optimization of the mutation detection process resulting in reduced cost and increased throughput. Results An automated process has been developed for the detection of deletions, duplications/insertions and point mutations in any gene or family of genes and has been applied to ten genes known to bear mutations that cause muscular dystrophy: DMD; CAV3; CAPN3; FKRP; TRIM32; LMNA; SGCA; SGCB; SGCG; SGCD. Using this process, mutations have been found in five DMD patients and four LGMD patients (one in the FKRP gene, one in the CAV3 gene, and two likely causative heterozygous pairs of variations in the CAPN3 gene of two other

  2. Identification of mutations leading to the Lesch-Nyhan syndrome by automated direct DNA sequencing of in vitro amplified cDNA

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    Gibbs, R.A. (Baylor College of Medicine, Houston, TX (USA)); Nguyen, Phinga (Howard Hughes Medical Institute, Houston, TX (USA)); McBride, L.J.; Koepf, S.M. (Applied Biosystems, Foster City, CA (USA)); Caskey, C.T. (Baylor College of Medicine, Houston, TX (USA) Howard Hughes Medical Institute, Houston, TX (USA))

    1989-03-01

    The Lesch-Nyhan (LN) syndrome is a severe X chromosome-linked disease that results from a deficiency of the purine salvage enzyme hypoxanthine phosphoribosyltransferase (HPRT). The mutations leading to the disease are heterogeneous and frequently arise as de novo events. The authors have identified nucleotide alterations in 15 independently arising HPRT-deficiency cases by direct DNA sequencing of in vitro amplified HPRT cDNA. They also demonstrate that the direct DNA sequence analysis can be automated, further simplifying the detection of new mutations at this locus. The mutations include DNA base substitutions, small DNA deletions, a single DNA base insertion, and errors in RNA splicing. The application of these procedures allows DNA diagnosis and carrier identification by the direct detection of the mutant alleles within individual families affected by LN.

  3. BRAF Mutation Testing in Cell-Free DNA from the Plasma of Patients with Advanced Cancers Using a Rapid, Automated Molecular Diagnostics System.

    Science.gov (United States)

    Janku, Filip; Huang, Helen J; Claes, Bart; Falchook, Gerald S; Fu, Siqing; Hong, David; Ramzanali, Nishma M; Nitti, Giovanni; Cabrilo, Goran; Tsimberidou, Apostolia M; Naing, Aung; Piha-Paul, Sarina A; Wheler, Jennifer J; Karp, Daniel D; Holley, Veronica R; Zinner, Ralph G; Subbiah, Vivek; Luthra, Rajyalakshmi; Kopetz, Scott; Overman, Michael J; Kee, Bryan K; Patel, Sapna; Devogelaere, Benoit; Sablon, Erwin; Maertens, Geert; Mills, Gordon B; Kurzrock, Razelle; Meric-Bernstam, Funda

    2016-06-01

    Cell-free (cf) DNA from plasma offers an easily obtainable material for BRAF mutation analysis for diagnostics and response monitoring. In this study, plasma-derived cfDNA samples from patients with progressing advanced cancers or malignant histiocytosis with known BRAF(V600) status from formalin-fixed paraffin-embedded (FFPE) tumors were tested using a prototype version of the Idylla BRAF Mutation Test, a fully integrated real-time PCR-based test with turnaround time about 90 minutes. Of 160 patients, BRAF(V600) mutations were detected in 62 (39%) archival FFPE tumor samples and 47 (29%) plasma cfDNA samples. The two methods had overall agreement in 141 patients [88%; κ, 0.74; SE, 0.06; 95% confidence interval (CI), 0.63-0.85]. Idylla had a sensitivity of 73% (95% CI, 0.60-0.83) and specificity of 98% (95% CI, 0.93-1.00). A higher percentage, but not concentration, of BRAF(V600) cfDNA in the wild-type background (>2% vs. ≤ 2%) was associated with shorter overall survival (OS; P = 0.005) and in patients with BRAF mutations in the tissue, who were receiving BRAF/MEK inhibitors, shorter time to treatment failure (TTF; P = 0.001). Longitudinal monitoring demonstrated that decreasing levels of BRAF(V600) cfDNA were associated with longer TTF (P = 0.045). In conclusion, testing for BRAF(V600) mutations in plasma cfDNA using the Idylla BRAF Mutation Test has acceptable concordance with standard testing of tumor tissue. A higher percentage of mutant BRAF(V600) in cfDNA corresponded with shorter OS and in patients receiving BRAF/MEK inhibitors also with shorter TTF. Mol Cancer Ther; 15(6); 1397-404. ©2016 AACR.

  4. BRAF Mutation Testing in Cell-Free DNA from the Plasma of Patients with Advanced Cancers Using a Rapid, Automated Molecular Diagnostics System.

    Science.gov (United States)

    Janku, Filip; Huang, Helen J; Claes, Bart; Falchook, Gerald S; Fu, Siqing; Hong, David; Ramzanali, Nishma M; Nitti, Giovanni; Cabrilo, Goran; Tsimberidou, Apostolia M; Naing, Aung; Piha-Paul, Sarina A; Wheler, Jennifer J; Karp, Daniel D; Holley, Veronica R; Zinner, Ralph G; Subbiah, Vivek; Luthra, Rajyalakshmi; Kopetz, Scott; Overman, Michael J; Kee, Bryan K; Patel, Sapna; Devogelaere, Benoit; Sablon, Erwin; Maertens, Geert; Mills, Gordon B; Kurzrock, Razelle; Meric-Bernstam, Funda

    2016-06-01

    Cell-free (cf) DNA from plasma offers an easily obtainable material for BRAF mutation analysis for diagnostics and response monitoring. In this study, plasma-derived cfDNA samples from patients with progressing advanced cancers or malignant histiocytosis with known BRAF(V600) status from formalin-fixed paraffin-embedded (FFPE) tumors were tested using a prototype version of the Idylla BRAF Mutation Test, a fully integrated real-time PCR-based test with turnaround time about 90 minutes. Of 160 patients, BRAF(V600) mutations were detected in 62 (39%) archival FFPE tumor samples and 47 (29%) plasma cfDNA samples. The two methods had overall agreement in 141 patients [88%; κ, 0.74; SE, 0.06; 95% confidence interval (CI), 0.63-0.85]. Idylla had a sensitivity of 73% (95% CI, 0.60-0.83) and specificity of 98% (95% CI, 0.93-1.00). A higher percentage, but not concentration, of BRAF(V600) cfDNA in the wild-type background (>2% vs. ≤ 2%) was associated with shorter overall survival (OS; P = 0.005) and in patients with BRAF mutations in the tissue, who were receiving BRAF/MEK inhibitors, shorter time to treatment failure (TTF; P = 0.001). Longitudinal monitoring demonstrated that decreasing levels of BRAF(V600) cfDNA were associated with longer TTF (P = 0.045). In conclusion, testing for BRAF(V600) mutations in plasma cfDNA using the Idylla BRAF Mutation Test has acceptable concordance with standard testing of tumor tissue. A higher percentage of mutant BRAF(V600) in cfDNA corresponded with shorter OS and in patients receiving BRAF/MEK inhibitors also with shorter TTF. Mol Cancer Ther; 15(6); 1397-404. ©2016 AACR. PMID:27207774

  5. Minisequencing mitochondrial DNA pathogenic mutations

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    Carracedo Ángel

    2008-04-01

    Full Text Available Abstract Background There are a number of well-known mutations responsible of common mitochondrial DNA (mtDNA diseases. In order to overcome technical problems related to the analysis of complete mtDNA genomes, a variety of different techniques have been proposed that allow the screening of coding region pathogenic mutations. Methods We here propose a minisequencing assay for the analysis of mtDNA mutations. In a single reaction, we interrogate a total of 25 pathogenic mutations distributed all around the whole mtDNA genome in a sample of patients suspected for mtDNA disease. Results We have detected 11 causal homoplasmic mutations in patients suspected for Leber disease, which were further confirmed by standard automatic sequencing. Mutations m.11778G>A and m.14484T>C occur at higher frequency than expected by change in the Galician (northwest Spain patients carrying haplogroup J lineages (Fisher's Exact test, P-value Conclusion We here developed a minisequencing genotyping method for the screening of the most common pathogenic mtDNA mutations which is simple, fast, and low-cost. The technique is robust and reproducible and can easily be implemented in standard clinical laboratories.

  6. Automated Extraction of DNA from clothing

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Hjort, Benjamin Benn; Nøhr Hansen, Thomas;

    2011-01-01

    Presence of PCR inhibitors in extracted DNA may interfere with the subsequent quantification and short tandem repeat (STR) reactions used in forensic genetic DNA typing. We have compared three automated DNA extraction methods based on magnetic beads with a manual method with the aim of reducing...... the amount of PCR inhibitors in the DNA extracts and increasing the proportion of reportable DNA profiles....

  7. Automated Extraction of DNA from clothing

    OpenAIRE

    Stangegaard, Michael; Hjort, Benjamin Benn; Nøhr Hansen, Thomas; Hansen, Anders Johannes; Morling, Niels

    2011-01-01

    Presence of PCR inhibitors in extracted DNA may interfere with the subsequent quantification and short tandem repeat (STR) reactions used in forensic genetic DNA typing. We have compared three automated DNA extraction methods based on magnetic beads with a manual method with the aim of reducing the amount of PCR inhibitors in the DNA extracts and increasing the proportion of reportable DNA profiles.

  8. Mutator phenotypes due to DNA replication infidelity

    OpenAIRE

    Arana, Mercedes E.; Kunkel, Thomas A.

    2010-01-01

    This article considers the fidelity of DNA replication performed by eukaryotic DNA polymerases involved in replicating the nuclear genome. DNA replication fidelity can vary widely depending on the DNA polymerase, the composition of the error, the flanking sequence, the presence of DNA damage and the ability to correct errors. As a consequence, defects in processes that determine DNA replication fidelity can confer strong mutator phenotypes whose specificity can help determine the molecular na...

  9. Natural radioactivity and human mitochondrial DNA mutations

    OpenAIRE

    Forster, Lucy; Forster, Peter; Lutz-Bonengel, Sabine; Willkomm, Horst; Brinkmann, Bernd

    2002-01-01

    Radioactivity is known to induce tumors, chromosome lesions, and minisatellite length mutations, but its effects on the DNA sequence have not previously been studied. A coastal peninsula in Kerala (India) contains the world's highest level of natural radioactivity in a densely populated area, offering an opportunity to characterize radiation-associated DNA mutations. We sampled 248 pedigrees (988 individuals) in the high-radiation peninsula and in nearby low-radiation islands as a control pop...

  10. Automated Template Quantification for DNA Sequencing Facilities

    Science.gov (United States)

    Ivanetich, Kathryn M.; Yan, Wilson; Wunderlich, Kathleen M.; Weston, Jennifer; Walkup, Ward G.; Simeon, Christian

    2005-01-01

    The quantification of plasmid DNA by the PicoGreen dye binding assay has been automated, and the effect of quantification of user-submitted templates on DNA sequence quality in a core laboratory has been assessed. The protocol pipets, mixes and reads standards, blanks and up to 88 unknowns, generates a standard curve, and calculates template concentrations. For pUC19 replicates at five concentrations, coefficients of variance were 0.1, and percent errors were from 1% to 7% (n = 198). Standard curves with pUC19 DNA were nonlinear over the 1 to 1733 ng/μL concentration range required to assay the majority (98.7%) of user-submitted templates. Over 35,000 templates have been quantified using the protocol. For 1350 user-submitted plasmids, 87% deviated by ≥ 20% from the requested concentration (500 ng/μL). Based on data from 418 sequencing reactions, quantification of user-submitted templates was shown to significantly improve DNA sequence quality. The protocol is applicable to all types of double-stranded DNA, is unaffected by primer (1 pmol/μL), and is user modifiable. The protocol takes 30 min, saves 1 h of technical time, and costs approximately $0.20 per unknown. PMID:16461949

  11. Mitochondrial DNA Mutations Associated with Aminoglycoside Ototoxicity

    Institute of Scientific and Technical Information of China (English)

    GUAN Min-Xin

    2006-01-01

    The mitochondrial 12S rRNA has been shown to be the hot spot for mutations associated with both aminoglycoside-induced and non-syndromic hearing loss. Of all the mutations, the homoplasmic A1555G and C1494T mutations at a highly conserved decoding region in the 12S rRNA have been associated with aminoglycoside-induced and non-syndromic hearing loss in many families worldwide. The A1555G or C1494T mutation is expected to form novel 1494C-G1555 or 1494U-A1555 base-pair at the highly conserved A-site of 12S rRNA. These transitions make the secondary structure of this RNA more closely resemble the corresponding region of bacterial 16S rRNA. Thus, the new U - A or G-C pair in 12S rRNA created by the C1494T or A1555G transition facilitates the binding of aminoglycosides, thereby accounting for the fact that the exposure to aminoglycosides can induce or worsen hearing loss in individuals carrying these mutations. Furthermore, the growth defect and impairment of mitochondrial translation were observed in cell lines carrying the A1555G or C1494T mutation in the presence of high concentration of aminoglycosides. In addition, nuclear modifier genes and mitochondrial haplotypes modulate the phenotypic manifestation of the A1555G and C1494T mutations. These observations provide the direct genetic and biochemical evidences that the A1555G or C1494T mutation is a pathogenic mtDNA mutation associated with aminoglycoside-induced and nonsyndromic hearing loss. Therefore, these data have been providing valuable information and technology to predict which individuals are at risk for ototoxicity, to improve the safety of aminoglycoside antibiotic therapy, and eventually to decrease the incidence of deafness.

  12. A novel fully automated molecular diagnostic system (AMDS for colorectal cancer mutation detection.

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    Shiro Kitano

    Full Text Available BACKGROUND: KRAS, BRAF and PIK3CA mutations are frequently observed in colorectal cancer (CRC. In particular, KRAS mutations are strong predictors for clinical outcomes of EGFR-targeted treatments such as cetuximab and panitumumab in metastatic colorectal cancer (mCRC. For mutation analysis, the current methods are time-consuming, and not readily available to all oncologists and pathologists. We have developed a novel, simple, sensitive and fully automated molecular diagnostic system (AMDS for point of care testing (POCT. Here we report the results of a comparison study between AMDS and direct sequencing (DS in the detection of KRAS, BRAF and PI3KCA somatic mutations. METHODOLOGY/PRINCIPAL FINDING: DNA was extracted from a slice of either frozen (n = 89 or formalin-fixed and paraffin-embedded (FFPE CRC tissue (n = 70, and then used for mutation analysis by AMDS and DS. All mutations (n = 41 among frozen and 27 among FFPE samples detected by DS were also successfully (100% detected by the AMDS. However, 8 frozen and 6 FFPE samples detected as wild-type in the DS analysis were shown as mutants in the AMDS analysis. By cloning-sequencing assays, these discordant samples were confirmed as true mutants. One sample had simultaneous "hot spot" mutations of KRAS and PIK3CA, and cloning assay comfirmed that E542K and E545K were not on the same allele. Genotyping call rates for DS were 100.0% (89/89 and 74.3% (52/70 in frozen and FFPE samples, respectively, for the first attempt; whereas that of AMDS was 100.0% for both sample sets. For automated DNA extraction and mutation detection by AMDS, frozen tissues (n = 41 were successfully detected all mutations within 70 minutes. CONCLUSIONS/SIGNIFICANCE: AMDS has superior sensitivity and accuracy over DS, and is much easier to execute than conventional labor intensive manual mutation analysis. AMDS has great potential for POCT equipment for mutation analysis.

  13. Markov chain for estimating human mitochondrial DNA mutation pattern

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    Vantika, Sandy; Pasaribu, Udjianna S.

    2015-12-01

    The Markov chain was proposed to estimate the human mitochondrial DNA mutation pattern. One DNA sequence was taken randomly from 100 sequences in Genbank. The nucleotide transition matrix and mutation transition matrix were estimated from this sequence. We determined whether the states (mutation/normal) are recurrent or transient. The results showed that both of them are recurrent.

  14. Mitochondrial DNA mutations in single human blood cells.

    Science.gov (United States)

    Yao, Yong-Gang; Kajigaya, Sachiko; Young, Neal S

    2015-09-01

    Determination mitochondrial DNA (mtDNA) sequences from extremely small amounts of DNA extracted from tissue of limited amounts and/or degraded samples is frequently employed in medical, forensic, and anthropologic studies. Polymerase chain reaction (PCR) amplification followed by DNA cloning is a routine method, especially to examine heteroplasmy of mtDNA mutations. In this review, we compare the mtDNA mutation patterns detected by three different sequencing strategies. Cloning and sequencing methods that are based on PCR amplification of DNA extracted from either single cells or pooled cells yield a high frequency of mutations, partly due to the artifacts introduced by PCR and/or the DNA cloning process. Direct sequencing of PCR product which has been amplified from DNA in individual cells is able to detect the low levels of mtDNA mutations present within a cell. We further summarize the findings in our recent studies that utilized this single cell method to assay mtDNA mutation patterns in different human blood cells. Our data show that many somatic mutations observed in the end-stage differentiated cells are found in hematopoietic stem cells (HSCs) and progenitors within the CD34(+) cell compartment. Accumulation of mtDNA variations in the individual CD34+ cells is affected by both aging and family genetic background. Granulocytes harbor higher numbers of mutations compared with the other cells, such as CD34(+) cells and lymphocytes. Serial assessment of mtDNA mutations in a population of single CD34(+) cells obtained from the same donor over time suggests stability of some somatic mutations. CD34(+) cell clones from a donor marked by specific mtDNA somatic mutations can be found in the recipient after transplantation. The significance of these findings is discussed in terms of the lineage tracing of HSCs, aging effect on accumulation of mtDNA mutations and the usage of mtDNA sequence in forensic identification. PMID:26149767

  15. Implications of mitochondrial DNA mutations and mitochondrial dysfunction in tumorigenesis

    Institute of Scientific and Technical Information of China (English)

    Jianxin Lu; Lokendra Kumar Sharma; Yidong Bai

    2009-01-01

    Alterations in oxidative phosphorylation resulting from mitochondrial dysfunction have long been hypothesized to be involved in tumorigenesis. Mitochondria have recently been shown to play an important role in regulating both programmed cell death and cell proliferation. Furthermore, mitochondrial DNA (mtDNA) mutations have been found in various cancer cells. However, the role of these mtDNA mutations in tumorigenesis remains largely unknown. This review focuses on basic mitochondrial genetics, mtDNA mutations and consequential mitochondrial dysfunction associated with cancer. The potential molecular mechanisms, mediating the pathogenesis from mtDNA mutations and mitochondrial dysfunction to tumorigenesis are also discussed.

  16. Mitochondrial DNA mutation in essential hypertension

    Institute of Scientific and Technical Information of China (English)

    Yuqi Liu; Shiwen Wang

    2008-01-01

    Essential hypertension (EH) is an escalating problem for developed and developing countries.It is currently seen as a 'complex' genetic trait caused by multiple susceptibility genes which are modulated by gene-environment and gene-gene interactions.Over the past 10 years,mitochondrial defects have been implicated in a wide variety of degenerative diseases,aging,and cancer.Recently several studies showed that human essential hypertension has excess maternal transmission which suggests a possible mitochondrial involvement.However,the exact pathophysiology of mitochondrial DNA mutation (mtDNA) in essential hypertension still remains perplexing.With the application of a variety of imaging approaches and successive mouse model of mitochonddal diseases we convince that these problems will be resolved in the near future.(J Geriatr Cardiol 2008;5(1):60-64)

  17. Novel dnaG mutation in a dnaP mutant of Escherichia coli.

    OpenAIRE

    Murakami, Y.; Nagata, T; Schwarz, W.; Wada, C.; Yura, T

    1985-01-01

    Reexamination of the dnaP18 mutant strain of Escherichia coli revealed that the mutation responsible for the arrest of DNA replication and cell growth at high temperatures resides in the dnaG gene rather than in the dnaP locus as previously thought; this mutation has been designated dnaG2903.

  18. Automation of a single-DNA molecule stretching device

    DEFF Research Database (Denmark)

    Sørensen, Kristian Tølbøl; Lopacinska, Joanna M.; Tommerup, Niels;

    2015-01-01

    We automate the manipulation of genomic-length DNA in a nanofluidic device based on real-time analysis of fluorescence images. In our protocol, individual molecules are picked from a microchannel and stretched with pN forces using pressure driven flows. The millimeter-long DNA fragments free...

  19. Capacity of DNA Data Embedding Under Substitution Mutations

    CERN Document Server

    Balado, Félix

    2011-01-01

    A number of methods have been proposed over the last decade for encoding information using deoxyribonucleic acid (DNA), giving rise to the emerging area of DNA data embedding. Since a DNA sequence is conceptually equivalent to a sequence of quaternary symbols (bases), DNA data embedding (diversely called DNA watermarking or DNA steganography) can be seen as a digital communications problem where channel errors are tantamount to mutations of DNA bases. Depending on the use of coding or noncoding DNA hosts, which, respectively, denote DNA segments that can or cannot be translated into proteins, DNA data embedding is essentially a problem of communications with or without side information at the encoder. In this paper the Shannon capacity of DNA data embedding is obtained for the case in which DNA sequences are subject to substitution mutations modelled using the Kimura model from molecular evolution studies. Inferences are also drawn with respect to the biological implications of some of the results presented.

  20. RADIA: RNA and DNA integrated analysis for somatic mutation detection.

    Directory of Open Access Journals (Sweden)

    Amie J Radenbaugh

    Full Text Available The detection of somatic single nucleotide variants is a crucial component to the characterization of the cancer genome. Mutation calling algorithms thus far have focused on comparing the normal and tumor genomes from the same individual. In recent years, it has become routine for projects like The Cancer Genome Atlas (TCGA to also sequence the tumor RNA. Here we present RADIA (RNA and DNA Integrated Analysis, a novel computational method combining the patient-matched normal and tumor DNA with the tumor RNA to detect somatic mutations. The inclusion of the RNA increases the power to detect somatic mutations, especially at low DNA allelic frequencies. By integrating an individual's DNA and RNA, we are able to detect mutations that would otherwise be missed by traditional algorithms that examine only the DNA. We demonstrate high sensitivity (84% and very high precision (98% and 99% for RADIA in patient data from endometrial carcinoma and lung adenocarcinoma from TCGA. Mutations with both high DNA and RNA read support have the highest validation rate of over 99%. We also introduce a simulation package that spikes in artificial mutations to patient data, rather than simulating sequencing data from a reference genome. We evaluate sensitivity on the simulation data and demonstrate our ability to rescue back mutations at low DNA allelic frequencies by including the RNA. Finally, we highlight mutations in important cancer genes that were rescued due to the incorporation of the RNA.

  1. RADIA: RNA and DNA integrated analysis for somatic mutation detection.

    Science.gov (United States)

    Radenbaugh, Amie J; Ma, Singer; Ewing, Adam; Stuart, Joshua M; Collisson, Eric A; Zhu, Jingchun; Haussler, David

    2014-01-01

    The detection of somatic single nucleotide variants is a crucial component to the characterization of the cancer genome. Mutation calling algorithms thus far have focused on comparing the normal and tumor genomes from the same individual. In recent years, it has become routine for projects like The Cancer Genome Atlas (TCGA) to also sequence the tumor RNA. Here we present RADIA (RNA and DNA Integrated Analysis), a novel computational method combining the patient-matched normal and tumor DNA with the tumor RNA to detect somatic mutations. The inclusion of the RNA increases the power to detect somatic mutations, especially at low DNA allelic frequencies. By integrating an individual's DNA and RNA, we are able to detect mutations that would otherwise be missed by traditional algorithms that examine only the DNA. We demonstrate high sensitivity (84%) and very high precision (98% and 99%) for RADIA in patient data from endometrial carcinoma and lung adenocarcinoma from TCGA. Mutations with both high DNA and RNA read support have the highest validation rate of over 99%. We also introduce a simulation package that spikes in artificial mutations to patient data, rather than simulating sequencing data from a reference genome. We evaluate sensitivity on the simulation data and demonstrate our ability to rescue back mutations at low DNA allelic frequencies by including the RNA. Finally, we highlight mutations in important cancer genes that were rescued due to the incorporation of the RNA. PMID:25405470

  2. Mitochondrial DNA Mutations Regulate Metastasis of Human Breast Cancer Cells

    OpenAIRE

    Hirotake Imanishi; Keisuke Hattori; Reiko Wada; Kaori Ishikawa; Sayaka Fukuda; Keizo Takenaga; Kazuto Nakada; Jun-ichi Hayashi

    2011-01-01

    Mutations in mitochondrial DNA (mtDNA) might contribute to expression of the tumor phenotypes, such as metastatic potential, as well as to aging phenotypes and to clinical phenotypes of mitochondrial diseases by induction of mitochondrial respiration defects and the resultant overproduction of reactive oxygen species (ROS). To test whether mtDNA mutations mediate metastatic pathways in highly metastatic human tumor cells, we used human breast carcinoma MDA-MB-231 cells, which simultaneously e...

  3. Mitochondrial DNA: Radically free of free-radical driven mutations.

    Science.gov (United States)

    Kauppila, Johanna H K; Stewart, James B

    2015-11-01

    Mitochondrial DNA has long been posited as a likely target of oxidative damage induced mutation during the ageing process. Research over the past decades has uncovered the accumulation of mitochondrial DNA mutations in association with a mosaic pattern of cells displaying mitochondrial dysfunction in ageing individuals. Unfortunately, the underlying mechanisms are far less straightforward than originally anticipated. Recent research on mitochondria reveals that these genomes are far less helpless than originally envisioned. Additionally, new technologies have allowed us to analyze the mutational signatures of many more somatic mitochondrial DNA mutations, revealing surprising patterns that are inconsistent with a DNA-oxidative damage based hypothesis. In this review, we will discuss these recent observations and new insights into the eccentricities of mitochondrial genetics, and their impact on our understanding of mitochondrial mutations and their role in the ageing process. This article is part of a Special Issue entitled: Mitochondrial Dysfunction in Aging.

  4. A Fast Determination of DNA Mutation Induced by Ultraviolet Radiation

    Institute of Scientific and Technical Information of China (English)

    LuFeng; LiuLili; ZhangXiaofang; WuYutian

    2001-01-01

    Electrophoresis, chromatography, immunoassay, sequencing and other time consuming ap-proaches have been developed to determine DNA base mismatching, oxidative lesion or strand breaks. Sometimes,however, only qualitative information is enough to decide whether mutation has happened to DNA and its extent.Convolution spectrometry (CS), a new technique to discover ultrafme difference on ultraviolet (UV) absorption ofdifferent substances, is originally employed to find out any subtle mutation of DNA induced by UV radiation. Muta-tive DNA is compared with ego criteria based on the spectra of the former DNA, any difference is quantitatively ex-pressed by dispersion (5). Visible changes cannot be observed on second -derivative spectra until the mutation gets 5up to 11.48%. Dimethyl sulfoxide is an intensifier of UV 254 nm induced DNA mutation and protector at 365 nm,which is simply confirmed by increasing and decreasing 5. Every convolution procedure takes less than 1 min. Convolution spectrometry provides a fast, simple, sensitive and inexpensive alternative to determine DNA mutation, andto screen anti-mutational medicines.

  5. Exercise-induced mitochondrial p53 repairs mtDNA mutations in mutator mice

    OpenAIRE

    Safdar, Adeel; Khrapko, Konstantin; Flynn, James M.; Saleem, Ayesha; De Lisio, Michael; Johnston, Adam P. W.; Kratysberg, Yevgenya; Samjoo, Imtiaz A.; Kitaoka, Yu; Ogborn, Daniel I.; Little, Jonathan P.; Raha, Sandeep; Parise, Gianni; Akhtar, Mahmood; Bart P Hettinga

    2016-01-01

    Background Human genetic disorders and transgenic mouse models have shown that mitochondrial DNA (mtDNA) mutations and telomere dysfunction instigate the aging process. Epidemiologically, exercise is associated with greater life expectancy and reduced risk of chronic diseases. While the beneficial effects of exercise are well established, the molecular mechanisms instigating these observations remain unclear. Results Endurance exercise reduces mtDNA mutation burden, alleviates multisystem pat...

  6. Mitochondrial DNA mutations in oxyphilic and chief cell parathyroid adenomas

    Directory of Open Access Journals (Sweden)

    Roth Sanford I

    2007-10-01

    Full Text Available Abstract Background The potential pathogenetic significance of mitochondrial DNA (mtDNA mutations in tumorigenesis is controversial. We hypothesized that benign tumorigenesis of a slowly replicating tissue like the human parathyroid might constitute an especially fertile ground on which a selective advantage conferred by mtDNA mutation could be manifested and might contribute to the oxyphilic phenotype observed in a subset of parathyroid tumors. Methods We sought acquired mitochondrial DNA mutations by sequencing the entire 16.6 kb mitochondrial genome of each of thirty sporadic parathyroid adenomas (18 chief cell and 12 oxyphil cell, eight independent, polyclonal, parathyroid primary chief cell hyperplasias plus corresponding normal control samples, five normal parathyroid glands, and one normal thyroid gland. Results Twenty-seven somatic mutations were identified in 15 of 30 (9 of 12 oxyphil adenomas, 6 of 18 chief cell parathyroid adenomas studied. No somatic mutations were observed in the hyperplastic parathyroid glands. Conclusion Features of the somatic mutations suggest that they may confer a selective advantage and contribute to the molecular pathogenesis of parathyroid adenomas. Importantly, the statistically significant differences in mutation prevalence in oxyphil vs. chief cell adenomas also suggest that mtDNA mutations may contribute to the oxyphil phenotype.

  7. DNA Sequence Evolution with Neighbor-Dependent Mutation

    CERN Document Server

    Arndt, P F; Hwa, T; Arndt, Peter F.; Burge, Christopher B.; Hwa, Terence

    2001-01-01

    We introduce a model of DNA sequence evolution which can account for biases in mutation rates that depend on the identity of the neighboring bases. An analytic solution for this class of non-equilibrium models is developed by adopting well-known methods of nonlinear dynamics. Results are presented for the CpG-methylation-deamination process which dominates point substitutions in vertebrates. The dinucleotide frequencies generated by the model (using empirically obtained mutation rates) match the overall pattern observed in non-coding DNA. A web-based tool has been constructed to compute single- and dinucleotide frequencies for arbitrary neighbor-dependent mutation rates. Alsoprovided is the backward procedure to infer the mutation rates using maximum likelihood analysis given the observed single- and dinucleotide frequencies. Reasonable estimates of the mutation rates can be obtained very efficiently, using generic non-coding DNA sequences as input, after masking outlong homonucleotide subsequences. Our metho...

  8. Automated Image Processing for the Analysis of DNA Repair Dynamics

    CERN Document Server

    Riess, Thorsten; Tomas, Martin; Ferrando-May, Elisa; Merhof, Dorit

    2011-01-01

    The efficient repair of cellular DNA is essential for the maintenance and inheritance of genomic information. In order to cope with the high frequency of spontaneous and induced DNA damage, a multitude of repair mechanisms have evolved. These are enabled by a wide range of protein factors specifically recognizing different types of lesions and finally restoring the normal DNA sequence. This work focuses on the repair factor XPC (xeroderma pigmentosum complementation group C), which identifies bulky DNA lesions and initiates their removal via the nucleotide excision repair pathway. The binding of XPC to damaged DNA can be visualized in living cells by following the accumulation of a fluorescent XPC fusion at lesions induced by laser microirradiation in a fluorescence microscope. In this work, an automated image processing pipeline is presented which allows to identify and quantify the accumulation reaction without any user interaction. The image processing pipeline comprises a preprocessing stage where the ima...

  9. DNA-directed mutations. Leading and lagging strand specificity

    Science.gov (United States)

    Sinden, R. R.; Hashem, V. I.; Rosche, W. A.

    1999-01-01

    The fidelity of replication has evolved to reproduce B-form DNA accurately, while allowing a low frequency of mutation. The fidelity of replication can be compromised, however, by defined order sequence DNA (dosDNA) that can adopt unusual or non B-DNA conformations. These alternative DNA conformations, including hairpins, cruciforms, triplex DNAs, and slipped-strand structures, may affect enzyme-template interactions that potentially lead to mutations. To analyze the effect of dosDNA elements on spontaneous mutagenesis, various mutational inserts containing inverted repeats or direct repeats were cloned in a plasmid containing a unidirectional origin of replication and a selectable marker for the mutation. This system allows for analysis of mutational events that are specific for the leading or lagging strands during DNA replication in Escherichia coli. Deletions between direct repeats, involving misalignment stabilized by DNA secondary structure, occurred preferentially on the lagging strand. Intermolecular strand switch events, correcting quasipalindromes to perfect inverted repeats, occurred preferentially during replication of the leading strand.

  10. Exploring the common molecular basis for the universal DNA mutation bias: Revival of Loewdin mutation model

    International Nuclear Information System (INIS)

    Highlights: → There exists a universal G:C → A:T mutation bias in three domains of life. → This universal mutation bias has not been sufficiently explained. → A DNA mutation model proposed by Loewdin 40 years ago offers a common explanation. -- Abstract: Recently, numerous genome analyses revealed the existence of a universal G:C → A:T mutation bias in bacteria, fungi, plants and animals. To explore the molecular basis for this mutation bias, we examined the three well-known DNA mutation models, i.e., oxidative damage model, UV-radiation damage model and CpG hypermutation model. It was revealed that these models cannot provide a sufficient explanation to the universal mutation bias. Therefore, we resorted to a DNA mutation model proposed by Loewdin 40 years ago, which was based on inter-base double proton transfers (DPT). Since DPT is a fundamental and spontaneous chemical process and occurs much more frequently within GC pairs than AT pairs, Loewdin model offers a common explanation for the observed universal mutation bias and thus has broad biological implications.

  11. Automated DNA extraction of single dog hairs without roots for mitochondrial DNA analysis.

    Science.gov (United States)

    Bekaert, Bram; Larmuseau, Maarten H D; Vanhove, Maarten P M; Opdekamp, Anouschka; Decorte, Ronny

    2012-03-01

    Dogs are intensely integrated in human social life and their shed hairs can play a major role in forensic investigations. The overall aim of this study was to validate a semi-automated extraction method for mitochondrial DNA analysis of telogenic dog hairs. Extracted DNA was amplified with a 95% success rate from 43 samples using two new experimental designs in which the mitochondrial control region was amplified as a single large (± 1260 bp) amplicon or as two individual amplicons (HV1 and HV2; ± 650 and 350 bp) with tailed-primers. The results prove that the extraction of dog hair mitochondrial DNA can easily be automated to provide sufficient DNA yield for the amplification of a forensically useful long mitochondrial DNA fragment or alternatively two short fragments with minimal loss of sequence in case of degraded samples.

  12. Flexible automated platform for blood group genotyping on DNA microarrays.

    Science.gov (United States)

    Paris, Sandra; Rigal, Dominique; Barlet, Valérie; Verdier, Martine; Coudurier, Nicole; Bailly, Pascal; Brès, Jean-Charles

    2014-05-01

    The poor suitability of standard hemagglutination-based assay techniques for large-scale automated screening of red blood cell antigens severely limits the ability of blood banks to supply extensively phenotype-matched blood. With better understanding of the molecular basis of blood antigens, it is now possible to predict blood group phenotype by identifying single-nucleotide polymorphisms in genomic DNA. Development of DNA-typing assays for antigen screening in blood donation qualification laboratories promises to enable blood banks to provide optimally matched donations. We have designed an automated genotyping system using 96-well DNA microarrays for blood donation screening and a first panel of eight single-nucleotide polymorphisms to identify 16 alleles in four blood group systems (KEL, KIDD, DUFFY, and MNS). Our aim was to evaluate this system on 960 blood donor samples with known phenotype. Study data revealed a high concordance rate (99.92%; 95% CI, 99.77%-99.97%) between predicted and serologic phenotypes. These findings demonstrate that our assay using a simple protocol allows accurate, relatively low-cost phenotype prediction at the DNA level. This system could easily be configured with other blood group markers for identification of donors with rare blood types or blood units for IH panels or antigens from other systems. PMID:24726279

  13. POINT MUTATIONS ON MITOCHONDRIAL DNA IN IRANIAN PATIENTS WITH FRIEDREICH’S ATAXIA

    Directory of Open Access Journals (Sweden)

    S. Etemad Ahari

    2008-11-01

    Full Text Available ObjectiveMitochondrial DNA (mtDNA is considered a candidate modifier factor for neuro-degenerative disorders. The most common type of ataxia is Friedreich's ataxia (FA. The aim of this study was to investigate different parts of mtDNA in 20 Iranian FA patients and 80 age-matched controls by polymerase chain reaction (PCR and automated DNA sequencing methods to find any probable point mutations involved in the pathogenesis of FA.Materials and MethodsWe identified 13 nucleotide substitutions including A3505G, T3335C, G3421A, G8251A, A8563G, A8563G, G8584A, T8614C, T8598C, C8684T, A8701G, G8994A and A9024G.ResultsTwelve of 13 nucleotide substitutions had already been reported as polymorphism. One of the nucleotide substitutions (A9024G had not been reported before. The A9024G nucleotide substitution does not change its amino acid. The controls were also investigated for this polymorphism which was found in two of them (2.5%.ConclusionNone of the mutations found in this study can affect the clinical manifestations of FA. This survey also provides evidence that the mtDNA A9024G allele is a new nonpathogenic polymorphism. We suggest follow-up studies for this polymorphism in different populations.Keywords:Mitochondrial DNA, Friedreich's Ataxia

  14. Arduino-based automation of a DNA extraction system.

    Science.gov (United States)

    Kim, Kyung-Won; Lee, Mi-So; Ryu, Mun-Ho; Kim, Jong-Won

    2015-01-01

    There have been many studies to detect infectious diseases with the molecular genetic method. This study presents an automation process for a DNA extraction system based on microfluidics and magnetic bead, which is part of a portable molecular genetic test system. This DNA extraction system consists of a cartridge with chambers, syringes, four linear stepper actuators, and a rotary stepper actuator. The actuators provide a sequence of steps in the DNA extraction process, such as transporting, mixing, and washing for the gene specimen, magnetic bead, and reagent solutions. The proposed automation system consists of a PC-based host application and an Arduino-based controller. The host application compiles a G code sequence file and interfaces with the controller to execute the compiled sequence. The controller executes stepper motor axis motion, time delay, and input-output manipulation. It drives the stepper motor with an open library, which provides a smooth linear acceleration profile. The controller also provides a homing sequence to establish the motor's reference position, and hard limit checking to prevent any over-travelling. The proposed system was implemented and its functionality was investigated, especially regarding positioning accuracy and velocity profile. PMID:26409535

  15. Arduino-based automation of a DNA extraction system.

    Science.gov (United States)

    Kim, Kyung-Won; Lee, Mi-So; Ryu, Mun-Ho; Kim, Jong-Won

    2015-01-01

    There have been many studies to detect infectious diseases with the molecular genetic method. This study presents an automation process for a DNA extraction system based on microfluidics and magnetic bead, which is part of a portable molecular genetic test system. This DNA extraction system consists of a cartridge with chambers, syringes, four linear stepper actuators, and a rotary stepper actuator. The actuators provide a sequence of steps in the DNA extraction process, such as transporting, mixing, and washing for the gene specimen, magnetic bead, and reagent solutions. The proposed automation system consists of a PC-based host application and an Arduino-based controller. The host application compiles a G code sequence file and interfaces with the controller to execute the compiled sequence. The controller executes stepper motor axis motion, time delay, and input-output manipulation. It drives the stepper motor with an open library, which provides a smooth linear acceleration profile. The controller also provides a homing sequence to establish the motor's reference position, and hard limit checking to prevent any over-travelling. The proposed system was implemented and its functionality was investigated, especially regarding positioning accuracy and velocity profile.

  16. Marcus model of spontaneous point mutation in DNA

    Science.gov (United States)

    Turaeva, N.; Brown-Kennerly, V.

    2015-11-01

    The theoretical model of Löwdin's mechanism of spontaneous mutation based on 2D Marcus theory of DPT has been proposed in this work. The equation for the kinetics of DPT during DNA replication has been established, and the expression for the probability of spontaneous mutation has been received. The probability of spontaneous mutation formation has been estimated for tautomeric G∗-C∗ complexes, which is in the range of experimental results. The probability of spontaneous mutation as a function of temperature, replication rate, and solvent effect has been discussed. It increases with temperature and decreases with replication rate. The solvent and pH effects on the probability of spontaneous mutation can also be discussed within the framework of the model.

  17. Prospects for DNA methods to measure human heritable mutation rates

    Energy Technology Data Exchange (ETDEWEB)

    Mendelsohn, M.L.

    1985-06-14

    A workshop cosponsored by ICPEMC and the US Department of Energy was held in Alta, Utah, December 9-13, 1984 to examine the extent to which DNA-oriented methods might provide new approaches to the important but intractable problem of measuring mutation rates in control and exposed human populations. The workshop identified and analyzed six DNA methods for detection of human heritable mutation, including several created at the meeting, and concluded that none of the methods combine sufficient feasibility and efficiency to be recommended for general application. 8 refs.

  18. mtDNA/nDNA ratio in 14484 LHON mitochondrial mutation carriers.

    Science.gov (United States)

    Nishioka, Tomoki; Soemantri, Augustinus; Ishida, Takafumi

    2004-01-01

    Leber's hereditary optic neuropathy (LHON) is a maternally inherited disease caused by mitochondrial DNA (mtDNA) mutations. In this study, the mtDNA/nuclear DNA ratio was evaluated in 11 LHON patients with the 14484 mutation, 13 asymptomatic carriers and 18 non-carrier relatives as controls, to reveal possible relationships between the disease and mtDNA content. DNAs from peripheral blood lymphocytes were subjected to quantitative PCR. Gender differences and age-dependent changes in the mtDNA content were not observed. Significant increase in the mtDNA content was observed only in the asymptomatic carriers (PLHON development, whereas those whose levels had not, had developed LHON. Since the asymptomatic carriers are the stock of the future LHON patients, monitoring the mtDNA content in patients and their relatives may help to predict the prognosis of the disease.

  19. RADIA: RNA and DNA Integrated Analysis for Somatic Mutation Detection

    OpenAIRE

    Radenbaugh, Amie J.; Ma, Singer; Ewing, Adam; Stuart, Joshua M.; Collisson, Eric A.; Zhu, Jingchun; Haussler, David

    2014-01-01

    The detection of somatic single nucleotide variants is a crucial component to the characterization of the cancer genome. Mutation calling algorithms thus far have focused on comparing the normal and tumor genomes from the same individual. In recent years, it has become routine for projects like The Cancer Genome Atlas (TCGA) to also sequence the tumor RNA. Here we present RADIA (RNA and DNA Integrated Analysis), a method that combines the patient-matched normal and tumor DNA with the tumor RN...

  20. Oxidative stress is not a major contributor to somatic mitochondrial DNA mutations.

    Directory of Open Access Journals (Sweden)

    Leslie S Itsara

    2014-02-01

    Full Text Available The accumulation of somatic mitochondrial DNA (mtDNA mutations is implicated in aging and common diseases of the elderly, including cancer and neurodegenerative disease. However, the mechanisms that influence the frequency of somatic mtDNA mutations are poorly understood. To develop a simple invertebrate model system to address this matter, we used the Random Mutation Capture (RMC assay to characterize the age-dependent frequency and distribution of mtDNA mutations in the fruit fly Drosophila melanogaster. Because oxidative stress is a major suspect in the age-dependent accumulation of somatic mtDNA mutations, we also used the RMC assay to explore the influence of oxidative stress on the somatic mtDNA mutation frequency. We found that many of the features associated with mtDNA mutations in vertebrates are conserved in Drosophila, including a comparable somatic mtDNA mutation frequency (∼10(-5, an increased frequency of mtDNA mutations with age, and a prevalence of transition mutations. Only a small fraction of the mtDNA mutations detected in young or old animals were G∶C to T∶A transversions, a signature of oxidative damage, and loss-of-function mutations in the mitochondrial superoxide dismutase, Sod2, had no detectable influence on the somatic mtDNA mutation frequency. Moreover, a loss-of-function mutation in Ogg1, which encodes a DNA repair enzyme that removes oxidatively damaged deoxyguanosine residues (8-hydroxy-2'-deoxyguanosine, did not significantly influence the somatic mtDNA mutation frequency of Sod2 mutants. Together, these findings indicate that oxidative stress is not a major cause of somatic mtDNA mutations. Our data instead suggests that somatic mtDNA mutations arise primarily from errors that occur during mtDNA replication. Further studies using Drosophila should aid in the identification of factors that influence the frequency of somatic mtDNA mutations.

  1. POINT MUTATIONS ON MITOCHONDRIAL DNA IN IRANIAN PATIENTS WITH FRIEDREICH’S ATAXIA

    Directory of Open Access Journals (Sweden)

    S. Etemad Ahari

    2007-11-01

    Full Text Available ObjectiveMitochondrial DNA (mtDNA is considered a candidate modifier factor for neuro-degenerative disorders. The most common type of ataxia is Friedreich’s ataxia (FA. The aim of this study was to investigate different parts of mtDNA in 20 Iranian FA patients and 80 age-matched controls by polymerase chain reaction (PCR and automated DNA sequencing methods to find any probable point mutations involved in the pathogenesis of FA. Materials and MethodsWe identified 13 nucleotide substitutions including A3505G, T3335C, G3421A, G8251A, A8563G, A8563G, G8584A, T8614C, T8598C, C8684T, A8701G, G8994A and A9024G. ResultsTwelve of 13 nucleotide substitutions had already been reported as polymorphism. One of the nucleotide substitutions (A9024G had not been reported before. The A9024G nucleotide substitution does not change its amino acid. The controls were also investigated for this polymorphism which was found in two of them (2.5%. ConclusionNone of the mutations found in this study can affect the clinical manifestations of FA. This survey also provides evidence that the mtDNA A9024G allele is a new nonpathogenic polymorphism. We suggest follow-up studies for this polymorphism in different populations.

  2. Holes influence the mutation spectrum of human mitochondrial DNA

    Science.gov (United States)

    Villagran, Martha; Miller, John

    Mutations drive evolution and disease, showing highly non-random patterns of variant frequency vs. nucleotide position. We use computational DNA hole spectroscopy [M.Y. Suarez-Villagran & J.H. Miller, Sci. Rep. 5, 13571 (2015)] to reveal sites of enhanced hole probability in selected regions of human mitochondrial DNA. A hole is a mobile site of positive charge created when an electron is removed, for example by radiation or contact with a mutagenic agent. The hole spectra are quantum mechanically computed using a two-stranded tight binding model of DNA. We observe significant correlation between spectra of hole probabilities and of genetic variation frequencies from the MITOMAP database. These results suggest that hole-enhanced mutation mechanisms exert a substantial, perhaps dominant, influence on mutation patterns in DNA. One example is where a trapped hole induces a hydrogen bond shift, known as tautomerization, which then triggers a base-pair mismatch during replication. Our results deepen overall understanding of sequence specific mutation rates, encompassing both hotspots and cold spots, which drive molecular evolution.

  3. Evaluation of Four Automated Protocols for Extraction of DNA from FTA Cards

    OpenAIRE

    Stangegaard, Michael; Børsting, Claus; Ferrero-Miliani, Laura; Frank-Hansen, Rune; Poulsen, Lena; Hansen, Anders J.; Morling, Niels

    2013-01-01

    Extraction of DNA using magnetic bead-based techniques on automated DNA extraction instruments provides a fast, reliable, and reproducible method for DNA extraction from various matrices. Here, we have compared the yield and quality of DNA extracted from FTA cards using four automated extraction protocols on three different instruments. The extraction processes were repeated up to six times with the same pieces of FTA cards. The sample material on the FTA cards was either blood or buccal cell...

  4. 21 CFR 864.7280 - Factor V Leiden DNA mutation detection systems.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Factor V Leiden DNA mutation detection systems....7280 Factor V Leiden DNA mutation detection systems. (a) Identification. Factor V Leiden deoxyribonucleic acid (DNA) mutation detection systems are devices that consist of different reagents...

  5. Development of an Automated DNA Detection System Using an Electrochemical DNA Chip Technology

    Science.gov (United States)

    Hongo, Sadato; Okada, Jun; Hashimoto, Koji; Tsuji, Koichi; Nikaido, Masaru; Gemma, Nobuhiro

    A new compact automated DNA detection system Genelyzer™ has been developed. After injecting a sample solution into a cassette with a built-in electrochemical DNA chip, processes from hybridization reaction to detection and analysis are all operated fully automatically. In order to detect a sample DNA, electrical currents from electrodes due to an oxidization reaction of electrochemically active intercalator molecules bound to hybridized DNAs are detected. The intercalator is supplied as a reagent solution by a fluid supply unit of the system. The feasibility test proved that the simultaneous typing of six single nucleotide polymorphisms (SNPs) associated with a rheumatoid arthritis (RA) was carried out within two hours and that all the results were consistent with those by conventional typing methods. It is expected that this system opens a new way to a DNA testing such as a test for infectious diseases, a personalized medicine, a food inspection, a forensic application and any other applications.

  6. Automated extraction of DNA and PCR setup using a Tecan Freedom EVO® liquid handler

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Frøslev, Tobias G.; Frank-Hansen, Rune;

    2009-01-01

    We have implemented and validated automated methods for DNA extraction and PCR setup developed for a Tecan Freedom EVO« liquid handler mounted with a Te-MagS(TM) magnetic separation device. The DNA was extracted using the Qiagen MagAttract« DNA Mini M48 kit. The DNA was amplified using Amp...

  7. DNA damage by reactive species: Mechanisms, mutation and repair

    Indian Academy of Sciences (India)

    N R Jena

    2012-07-01

    DNA is continuously attacked by reactive species that can affect its structure and function severely. Structural modifications to DNA mainly arise from modifications in its bases that primarily occur due to their exposure to different reactive species. Apart from this, DNA strand break, inter- and intra-strand crosslinks and DNA–protein crosslinks can also affect the structure of DNA significantly. These structural modifications are involved in mutation, cancer and many other diseases. As it has the least oxidation potential among all the DNA bases, guanine is frequently attacked by reactive species, producing a plethora of lethal lesions. Fortunately, living cells are evolved with intelligent enzymes that continuously protect DNA from such damages. This review provides an overview of different guanine lesions formed due to reactions of guanine with different reactive species. Involvement of these lesions in inter- and intra-strand crosslinks, DNA–protein crosslinks and mutagenesis are discussed. How certain enzymes recognize and repair different guanine lesions in DNA are also presented.

  8. Common Mitochondrial DNA Mutations Generated through DNA-Mediated Charge Transport

    OpenAIRE

    Merino, Edward J.; Davis, Molly L.; Barton, Jacqueline K.

    2009-01-01

    Mutation sites that arise in human mitochondrial DNA as a result of oxidation by a rhodium photooxidant have been identified. HeLa cells were incubated with [Rh(phi)2bpy]Cl3 (phi is 9,10-phenanthrenequinone diimine), an intercalating photooxidant, to allow the complex to enter the cell and bind mitochondrial DNA. Photoexcitation of DNA-bound [Rh(phi)2bpy]3+ can promote the oxidation of guanine from a distance through DNA-mediated charge transport. After two rounds of photolysis and growth of ...

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

    Energy Technology Data Exchange (ETDEWEB)

    Lushaj, Entela B., E-mail: lushaj@surgery.wisc.edu [Division of Cardiothoracic Surgery, Department of Surgery, School of Medicine and Public Health, University of Wisconsin, Madison, WI 53792 (United States); Lozonschi, Lucian; Barnes, Maria; Anstadt, Emily; Kohmoto, Takushi [Division of Cardiothoracic Surgery, Department of Surgery, School of Medicine and Public Health, University of Wisconsin, Madison, WI 53792 (United States)

    2012-06-01

    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.

  10. [UV-induced DNA mutation of peach aphid].

    Science.gov (United States)

    Du, Erxia; Guo, Jianwen; Zhao, Huiyan

    2006-07-01

    By using PCR technique and microsatellite marks, this paper studied the DNA polymorphism of peach aphid (Myzus persicae) under UV-radiation. The fragments of three primers were amplified, and the gene diversity and the rate of loci polymorphisms of their genomic DNA, which could reflect the damage degree of DNA after UV-radiation, were measured. The results revealed that after treated with different radiation intensity (15, 30, 45 W) and duration (2, 4, 6 h) , the UV-induced DNA mutations were genetic and could be delivered to F2 generation. The mutations depended on the interaction of radiation intensity and duration. Variance analysis on the gene diversity and the rate of loci polymorphisms showed that there existed a significant difference between UV-treated and control groups, except the rate of loci polymorphisms under 2 h radiation. The average value of the control was higher than that of 2 h radiation treatment. According to the cluster analysis of the genetic distance, the aphids were divided into three groups, i. e., control group, 2 h (15, 30 W) treatment group, and the other, which was consistent with the result of variance analysis.

  11. Evaluation of automated and manual DNA purification methods for detecting Ricinus communis DNA during ricin investigations.

    Science.gov (United States)

    Hutchins, Anne S; Astwood, Michael J; Saah, J Royden; Michel, Pierre A; Newton, Bruce R; Dauphin, Leslie A

    2014-03-01

    In April of 2013, letters addressed to the President of United States and other government officials were intercepted and found to be contaminated with ricin, heightening awareness about the need to evaluate laboratory methods for detecting ricin. This study evaluated commercial DNA purification methods for isolating Ricinus communis DNA as measured by real-time polymerase chain reaction (PCR). Four commercially available DNA purification methods (two automated, MagNA Pure compact and MagNA Pure LC, and two manual, MasterPure complete DNA and RNA purification kit and QIAamp DNA blood mini kit) were evaluated. We compared their ability to purify detectable levels of R. communis DNA from four different sample types, including crude preparations of ricin that could be used for biological crimes or acts of bioterrorism. Castor beans, spiked swabs, and spiked powders were included to simulate sample types typically tested during criminal and public health investigations. Real-time PCR analysis indicated that the QIAamp kit resulted in the greatest sensitivity for ricin preparations; the MasterPure kit performed best with spiked powders. The four methods detected equivalent levels by real-time PCR when castor beans and spiked swabs were used. All four methods yielded DNA free of PCR inhibitors as determined by the use of a PCR inhibition control assay. This study demonstrated that DNA purification methods differ in their ability to purify R. communis DNA; therefore, the purification method used for a given sample type can influence the sensitivity of real-time PCR assays for R. communis.

  12. Evaluation of Automated and Manual Commercial DNA Extraction Methods for Recovery of Brucella DNA from Suspensions and Spiked Swabs ▿

    OpenAIRE

    Dauphin, Leslie A.; Hutchins, Rebecca J.; Bost, Liberty A.; Bowen, Michael D.

    2009-01-01

    This study evaluated automated and manual commercial DNA extraction methods for their ability to recover DNA from Brucella species in phosphate-buffered saline (PBS) suspension and from spiked swab specimens. Six extraction methods, representing several of the methodologies which are commercially available for DNA extraction, as well as representing various throughput capacities, were evaluated: the MagNA Pure Compact and the MagNA Pure LC instruments, the IT 1-2-3 DNA sample purification kit...

  13. A patient with two mitochondrial DNA mutations causing PEO and LHON.

    Science.gov (United States)

    Melberg, Atle; Moslemi, Ali-Reza; Palm, Oscar; Raininko, Raili; Stålberg, Erik; Oldfors, Anders

    2009-01-01

    We report a 22-year-old man with PEO and optic atrophy. PEO developed before the onset of optic atrophy. The patient showed mitochondrial myopathy with cytochrome c oxidase deficient fibers. In skeletal muscle the patient was homoplasmic for the mtDNA G11778A Leber hereditary optic neuropathy (LHON) mutation and heteroplasmic for the mtDNA 5 kb "common" deletion mutation. In blood only the homoplasmic LHON mutation was identified. The occurrence of two pathogenic mtDNA mutations is exceedingly rare. The clinical findings in this patient indicate that the combination of the two mtDNA mutations resulted in the expected combined phenotype since the mtDNA deletion mutation accounted for the PEO and the mtDNA G11778A point mutation for the optic atrophy.

  14. Rapid sensitive analysis of IDH1 mutation in lower-grade gliomas by automated genetic typing involving a quenching probe.

    Science.gov (United States)

    Kurimoto, Michihiro; Suzuki, Hiromichi; Aoki, Kosuke; Ohka, Fumiharu; Kondo, Goro; Motomura, Kazuya; Iijima, Kentaro; Yamamichi, Akane; Ranjit, Melissa; Wakabayashi, Toshihiko; Kimura, Shinya; Natsume, Atsushi

    2016-01-01

    The authors recently found that 80% of lower-grade gliomas (LGGs) harbored a mutation in IDH1. Intraoperative detection of the mutated IDH1 helps not only differentiate LGGs from other type of brain tumors, but determine the resection border. In the current study, the authors have applied an automated genetic typing involving a quenching probe to detect the mutated IDH1. If tumor cells with the mutated IDH1 contained 10% or more in the mixture of normal and tumor cells, the device could detect it sensitively. The intraoperative assessment of IDH1 mutation is useful in brain tumor surgeries.

  15. Mutations in the D-loop region of mitochondrial DNA in gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Yibing Zhao; Hongyu Yang; Guoyu Chen

    2005-01-01

    Objective: To investigate the mutations in the D-loop region of mitochondrial DNA (mtDNA) in gastric cancer.Methods: The mtDNA of D-loop region was amplified by PCR and sequenced in 20 samples from gastric cancer tissue and adjacent normal membrane. Results: There were 7/20(35% ) mutations in the mtDNA of D-loop region in gastric cancer patients. There were four microsatellite instabilities among the 18 mutations. Nine new polymorphisms were identified in 20 patients. Conclusion: The mtDNA of Dloop region might be highly polymorphoric and the mutation rate is high in patients with gastric cancer.

  16. Evaluation of Four Automated Protocols for Extraction of DNA from FTA Cards

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Børsting, Claus; Ferrero-Miliani, Laura;

    2013-01-01

    Extraction of DNA using magnetic bead-based techniques on automated DNA extraction instruments provides a fast, reliable, and reproducible method for DNA extraction from various matrices. Here, we have compared the yield and quality of DNA extracted from FTA cards using four automated extraction...... protocols on three different instruments. The extraction processes were repeated up to six times with the same pieces of FTA cards. The sample material on the FTA cards was either blood or buccal cells. With the QIAamp DNA Investigator and QIAsymphony DNA Investigator kits, it was possible to extract DNA...... from the FTA cards in all six rounds of extractions in sufficient amount and quality to obtain complete short tandem repeat (STR) profiles on a QIAcube and a QIAsymphony SP. With the PrepFiler Express kit, almost all the extractable DNA was extracted in the first two rounds of extractions. Furthermore...

  17. Studies of human mutation rates: Progress report

    International Nuclear Information System (INIS)

    Progress was recorded between January 1 and July 1, 1987 on a project entitled ''Studies of Human Mutation Rates''. Studies underway include methodology for studying mutation at the DNA level, algorithms for automated analyses of two-dimensional polyacrylamide DNA gels, theoretical and applied population genetics, and studies of mutation frequency in A-bomb survivors

  18. Automated extraction of DNA from biological stains on fabric from crime cases. A comparison of a manual and three automated methods

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Hjort, Benjamin B; Hansen, Thomas N;

    2013-01-01

    that may be co-extracted with the DNA. Using 120 forensic trace evidence samples consisting of various types of fabric, we compared three automated DNA extraction methods based on magnetic beads (PrepFiler Express Forensic DNA Extraction Kit on an AutoMate Express, QIAsyphony DNA Investigator kit either......The presence of PCR inhibitors in extracted DNA may interfere with the subsequent quantification and short tandem repeat (STR) reactions used in forensic genetic DNA typing. DNA extraction from fabric for forensic genetic purposes may be challenging due to the occasional presence of PCR inhibitors...

  19. Review: Clinical aspects of hereditary DNA Mismatch repair gene mutations.

    Science.gov (United States)

    Sijmons, Rolf H; Hofstra, Robert M W

    2016-02-01

    Inherited mutations of the DNA Mismatch repair genes MLH1, MSH2, MSH6 and PMS2 can result in two hereditary tumor syndromes: the adult-onset autosomal dominant Lynch syndrome, previously referred to as Hereditary Non-Polyposis Colorectal Cancer (HNPCC) and the childhood-onset autosomal recessive Constitutional Mismatch Repair Deficiency syndrome. Both conditions are important to recognize clinically as their identification has direct consequences for clinical management and allows targeted preventive actions in mutation carriers. Lynch syndrome is one of the more common adult-onset hereditary tumor syndromes, with thousands of patients reported to date. Its tumor spectrum is well established and includes colorectal cancer, endometrial cancer and a range of other cancer types. However, surveillance for cancers other than colorectal cancer is still of uncertain value. Prophylactic surgery, especially for the uterus and its adnexa is an option in female mutation carriers. Chemoprevention of colorectal cancer with aspirin is actively being investigated in this syndrome and shows promising results. In contrast, the Constitutional Mismatch Repair Deficiency syndrome is rare, features a wide spectrum of childhood onset cancers, many of which are brain tumors with high mortality rates. Future studies are very much needed to improve the care for patients with this severe disorder. PMID:26746812

  20. Exonuclease mutations in DNA polymerase epsilon reveal replication strand specific mutation patterns and human origins of replication.

    Science.gov (United States)

    Shinbrot, Eve; Henninger, Erin E; Weinhold, Nils; Covington, Kyle R; Göksenin, A Yasemin; Schultz, Nikolaus; Chao, Hsu; Doddapaneni, HarshaVardhan; Muzny, Donna M; Gibbs, Richard A; Sander, Chris; Pursell, Zachary F; Wheeler, David A

    2014-11-01

    Tumors with somatic mutations in the proofreading exonuclease domain of DNA polymerase epsilon (POLE-exo*) exhibit a novel mutator phenotype, with markedly elevated TCT→TAT and TCG→TTG mutations and overall mutation frequencies often exceeding 100 mutations/Mb. Here, we identify POLE-exo* tumors in numerous cancers and classify them into two groups, A and B, according to their mutational properties. Group A mutants are found only in POLE, whereas Group B mutants are found in POLE and POLD1 and appear to be nonfunctional. In Group A, cell-free polymerase assays confirm that mutations in the exonuclease domain result in high mutation frequencies with a preference for C→A mutation. We describe the patterns of amino acid substitutions caused by POLE-exo* and compare them to other tumor types. The nucleotide preference of POLE-exo* leads to increased frequencies of recurrent nonsense mutations in key tumor suppressors such as TP53, ATM, and PIK3R1. We further demonstrate that strand-specific mutation patterns arise from some of these POLE-exo* mutants during genome duplication. This is the first direct proof of leading strand-specific replication by human POLE, which has only been demonstrated in yeast so far. Taken together, the extremely high mutation frequency and strand specificity of mutations provide a unique identifier of eukaryotic origins of replication.

  1. Exonuclease mutations in DNA polymerase epsilon reveal replication strand specific mutation patterns and human origins of replication.

    Science.gov (United States)

    Shinbrot, Eve; Henninger, Erin E; Weinhold, Nils; Covington, Kyle R; Göksenin, A Yasemin; Schultz, Nikolaus; Chao, Hsu; Doddapaneni, HarshaVardhan; Muzny, Donna M; Gibbs, Richard A; Sander, Chris; Pursell, Zachary F; Wheeler, David A

    2014-11-01

    Tumors with somatic mutations in the proofreading exonuclease domain of DNA polymerase epsilon (POLE-exo*) exhibit a novel mutator phenotype, with markedly elevated TCT→TAT and TCG→TTG mutations and overall mutation frequencies often exceeding 100 mutations/Mb. Here, we identify POLE-exo* tumors in numerous cancers and classify them into two groups, A and B, according to their mutational properties. Group A mutants are found only in POLE, whereas Group B mutants are found in POLE and POLD1 and appear to be nonfunctional. In Group A, cell-free polymerase assays confirm that mutations in the exonuclease domain result in high mutation frequencies with a preference for C→A mutation. We describe the patterns of amino acid substitutions caused by POLE-exo* and compare them to other tumor types. The nucleotide preference of POLE-exo* leads to increased frequencies of recurrent nonsense mutations in key tumor suppressors such as TP53, ATM, and PIK3R1. We further demonstrate that strand-specific mutation patterns arise from some of these POLE-exo* mutants during genome duplication. This is the first direct proof of leading strand-specific replication by human POLE, which has only been demonstrated in yeast so far. Taken together, the extremely high mutation frequency and strand specificity of mutations provide a unique identifier of eukaryotic origins of replication. PMID:25228659

  2. Random point mutations with major effects on protein-coding genes are the driving force behind premature aging in mtDNA mutator mice.

    NARCIS (Netherlands)

    Edgar, D.; Shabalina, I.; Camara, Y.; Wredenberg, A.; Calvaruso, M.A.; Nijtmans, L.G.J.; Nedergaard, J.; Cannon, B.; Larsson, N.G.; Trifunovic, A.

    2009-01-01

    The mtDNA mutator mice have high levels of point mutations and linear deletions of mtDNA causing a progressive respiratory chain dysfunction and a premature aging phenotype. We have now performed molecular analyses to determine the mechanism whereby these mtDNA mutations impair respiratory chain fun

  3. Streamlining DNA barcoding protocols: automated DNA extraction and a new cox1 primer in arachnid systematics.

    Directory of Open Access Journals (Sweden)

    Nina Vidergar

    Full Text Available BACKGROUND: DNA barcoding is a popular tool in taxonomic and phylogenetic studies, but for most animal lineages protocols for obtaining the barcoding sequences--mitochondrial cytochrome C oxidase subunit I (cox1 AKA CO1--are not standardized. Our aim was to explore an optimal strategy for arachnids, focusing on the species-richest lineage, spiders by (1 improving an automated DNA extraction protocol, (2 testing the performance of commonly used primer combinations, and (3 developing a new cox1 primer suitable for more efficient alignment and phylogenetic analyses. METHODOLOGY: We used exemplars of 15 species from all major spider clades, processed a range of spider tissues of varying size and quality, optimized genomic DNA extraction using the MagMAX Express magnetic particle processor-an automated high throughput DNA extraction system-and tested cox1 amplification protocols emphasizing the standard barcoding region using ten routinely employed primer pairs. RESULTS: The best results were obtained with the commonly used Folmer primers (LCO1490/HCO2198 that capture the standard barcode region, and with the C1-J-2183/C1-N-2776 primer pair that amplifies its extension. However, C1-J-2183 is designed too close to HCO2198 for well-interpreted, continuous sequence data, and in practice the resulting sequences from the two primer pairs rarely overlap. We therefore designed a new forward primer C1-J-2123 60 base pairs upstream of the C1-J-2183 binding site. The success rate of this new primer (93% matched that of C1-J-2183. CONCLUSIONS: The use of C1-J-2123 allows full, indel-free overlap of sequences obtained with the standard Folmer primers and with C1-J-2123 primer pair. Our preliminary tests suggest that in addition to spiders, C1-J-2123 will also perform in other arachnids and several other invertebrates. We provide optimal PCR protocols for these primer sets, and recommend using them for systematic efforts beyond DNA barcoding.

  4. Scar-less multi-part DNA assembly design automation

    Energy Technology Data Exchange (ETDEWEB)

    Hillson, Nathan J.

    2016-06-07

    The present invention provides a method of a method of designing an implementation of a DNA assembly. In an exemplary embodiment, the method includes (1) receiving a list of DNA sequence fragments to be assembled together and an order in which to assemble the DNA sequence fragments, (2) designing DNA oligonucleotides (oligos) for each of the DNA sequence fragments, and (3) creating a plan for adding flanking homology sequences to each of the DNA oligos. In an exemplary embodiment, the method includes (1) receiving a list of DNA sequence fragments to be assembled together and an order in which to assemble the DNA sequence fragments, (2) designing DNA oligonucleotides (oligos) for each of the DNA sequence fragments, and (3) creating a plan for adding optimized overhang sequences to each of the DNA oligos.

  5. The m.13051G>A mitochondrial DNA mutation results in variable neurology and activated mitophagy

    OpenAIRE

    Dombi, E.; Diot, A.; Morten, K.; Carver, J; Lodge, T.; Fratter, C.; Ng, Y.S.; Liao, C.; Muir, R; Blakely, E.L.; Hargreaves, I; Al-Dosary, M.; Sarkar, G; Hickman, S. J.; Downes, S M

    2016-01-01

    Maternally inherited mitochondrial DNA (mtDNA) mutations cause symptoms of Leber hereditary optic neuropathy (LHON) in -1 in 30,000 individuals. Most of the affected individuals lack respiratory chain defects1 and there is no proven prophylactic treatment.

  6. Relationship between mutations of mitochondrial DNA ND1 gene and type 2 diabetes

    Institute of Scientific and Technical Information of China (English)

    于珮; 于德民; 刘德敏; 王琨; 汤新之

    2004-01-01

    Background Recent studies have indicated that many mutations in mitochondrial (mt)DNA NDI gene region are related to diabetes mellitus. In this study we explored the relationship between various mtDNA ND1 gene mutations and type 2 diabetes mellitus (DM) among Chinese. Methods Using PCR restriction fragment length polymorphism (PCR-RFLP) analysis and gene sequencing, 4 spots of mtDNA (nt3243, nt3316, nt3394, nt3426) were screened in 478 diabetics and 430 non-diabetic subjects.Results In diabetic group, there were 13 carriers (2.72%)of 3316 G→A mutation,12 (2.51%) of 3394 T→C mutation and 2 (0.42%) of 3426A→G mutation. In controls, only 3394 T→C mutation was observed in 2 subjects (0.47%). There was significant difference in the frequency of 3316 and 3394 mutation between two groups (P<0.05, respectively). More subjects with mitochondrial DNA ND1 gene mutations had DM family history and greater tendency of maternal inheritance when compared to those patients without mutation in diabetic group(P<0.01). A 3426 mutation diabetic pedigree was studied, and we found 12 maternal members in the family had the same mutation. Conclusion mtDNA ND1 gene mutations at nt3316 (G→A), nt3394 (T→C) and 3426 (A→G) might contribute to the pathogenesis of DM with other genetic factors and environment factors.

  7. Genotoxicity of formaldehyde: Molecular basis of DNA damage and mutation

    Directory of Open Access Journals (Sweden)

    Masanobu eKawanishi

    2014-09-01

    Full Text Available Formaldehyde is commonly used in the chemical industry and is present in the environment, such as vehicle emissions, some building materials, food and tobacco smoke. It also occurs as a natural product in most organisms, the sources of which include a number of metabolic processes. It causes various acute and chronic adverse effects in humans if they inhale its fumes. Among the chronic effects on human health, we summarize data on genotoxicity and carcinogenicity in this review, and we particularly focus on the molecular mechanisms involved in the formaldehyde mutagenesis. Formaldehyde mainly induces N-hydroxymethyl mono-adducts on guanine, adenine and cytosine, and N-methylene crosslinks between adjacent purines in DNA. These crosslinks are types of DNA damage potentially fatal for cell survival if they are not removed by the nucleotide excision repair pathway. In the previous studies, we showed evidence that formaldehyde causes intra-strand crosslinks between purines in DNA using a unique method (Matsuda et al. Nucleic Acids Res. 26, 1769-1774,1998. Using shuttle vector plasmids, we also showed that formaldehyde as well as acetaldehyde induces tandem base substitutions, mainly at 5’-GG and 5’-GA sequences, which would arise from the intra-strand crosslinks. These mutation features are different from those of other aldehydes such as crotonaldehyde, acrolein, glyoxal and methylglyoxal. These findings provide molecular clues to improve our understanding of the genotoxicity and carcinogenicity of formaldehyde.

  8. An automated annotation tool for genomic DNA sequences using GeneScan and BLAST

    Indian Academy of Sciences (India)

    Andrew M. Lynn; Chakresh Kumar Jain; K. Kosalai; Pranjan Barman; Nupur Thakur; Harish Batra; Alok Bhattacharya

    2001-04-01

    Genomic sequence data are often available well before the annotated sequence is published. We present a method for analysis of genomic DNA to identify coding sequences using the GeneScan algorithm and characterize these resultant sequences by BLAST. The routines are used to develop a system for automated annotation of genome DNA sequences.

  9. Multi-center evaluation of the novel fully-automated PCR-based Idylla™ BRAF Mutation Test on formalin-fixed paraffin-embedded tissue of malignant melanoma.

    Science.gov (United States)

    Melchior, Linea; Grauslund, Morten; Bellosillo, Beatriz; Montagut, Clara; Torres, Erica; Moragón, Ester; Micalessi, Isabel; Frans, Johan; Noten, Veerle; Bourgain, Claire; Vriesema, Renske; van der Geize, Robert; Cokelaere, Kristof; Vercooren, Nancy; Crul, Katrien; Rüdiger, Thomas; Buchmüller, Diana; Reijans, Martin; Jans, Caroline

    2015-12-01

    The advent of BRAF-targeted therapies led to increased survival in patients with metastatic melanomas harboring a BRAF V600 mutation (implicated in 46-48% of malignant melanomas). The Idylla(™) System (Idylla(™)), i.e., the real-time-PCR-based Idylla(™) BRAF Mutation Test performed on the fully-automated Idylla(™) platform, enables detection of the most frequent BRAF V600 mutations (V600E/E2/D, V600K/R/M) in tumor material within approximately 90 min and with 1% detection limit. Idylla(™) performance was determined in a multi-center study by analyzing BRAF mutational status of 148 archival formalin-fixed paraffin-embedded (FFPE) tumor samples from malignant melanoma patients, and comparing Idylla(™) results with assessments made by commercial or in-house routine diagnostic methods. Of the 148 samples analyzed, Idylla(™) initially recorded 7 insufficient DNA input calls and 15 results discordant with routine method results. Further analysis learned that the quality of 8 samples was insufficient for Idylla(™) testing, 1 sample had an invalid routine test result, and Idylla(™) results were confirmed in 10 samples. Hence, Idylla(™) identified all mutations present, including 7 not identified by routine methods. Idylla(™) enables fully automated BRAF V600 testing directly on FFPE tumor tissue with increased sensitivity, ease-of-use, and much shorter turnaround time compared to existing diagnostic tests, making it a tool for rapid, simple and highly reliable analysis of therapeutically relevant BRAF mutations, in particular for diagnostic units without molecular expertise and infrastructure.

  10. Deoxyribonucleic acid initiation mutation dnaB252 is suppressed by elevated dnaC+ gene dosage.

    OpenAIRE

    Sclafani, R A; Wechsler, J A

    1981-01-01

    The Escherichia coli dnaB252 allele is the only dnaB mutation which confers a deoxyribonucleic acid initiation-defective phenotype on the cell. The presence of a multicopy hybrid plasmid containing the dnaC+ gene in a dnaB252 strain completely suppressed the temperature-sensitive phenotype. It is suggested that at high temperature the dnaB252 protein has a lowered affinity for dnaC protein, and that the formation of a dnaB-dnaC complex is mandatory for initiation.

  11. A Study on the D-loop Region of Mitochondrial DNA (mtDNA) Mutation in Cervical Carcinomas

    Institute of Scientific and Technical Information of China (English)

    XUE Wen-qun; CHEN Dao-zhen

    2009-01-01

    Objective Background-study on genesis and development of tumor is mainly concentrated on gene mutation in nucleus. In recent years, however, the role of mitochondrial DNA (mtDNA) mutation in tumor genesis has been given more and more attention, which is the only extra-nucleus DNA in cells of higher animals. Carcinoma of the uterine cervix is a common tumor in gynecology, but there are few reports of mtDNA mutation in this area. The focus of this study was to investigate the mtDNA mutation in tumor tissues of cervical carcinomas and their relationship to tumorigenesis and tumor development. Methods The D-loop region of 24 cervical carcinomas together with the adjacent normal tissues were amplified by PCR and sequenced. Results Among the 24 cervical carcinomas, 30 mutations in 9 patients′ specimen were identified with the mutations rate of 37.5%(9/24). There were 8 microsatellite instabilities among the mutations and 13 new polymorphisms which were not reported previously in the Genbank. Conclusions The D-loop region of mitochondrial DNA is a highly polymorphoric and mutable region and the mutation rate is relatively high in patients with cervical carcinomas.

  12. Significance of somatic mutations and content alteration of mitochondrial DNA in esophageal cancer

    Directory of Open Access Journals (Sweden)

    Wang Yu-Fen

    2006-04-01

    Full Text Available Abstract Background The roles of mitochondria in energy metabolism, the generation of ROS, aging, and the initiation of apoptosis have implicated their importance in tumorigenesis. In this study we aim to establish the mutation spectrum and to understand the role of somatic mtDNA mutations in esophageal cancer. Methods The entire mitochondrial genome was screened for somatic mutations in 20 pairs (18 esophageal squamous cell carcinomas, one adenosquamous carcinoma and one adenocarcinoma of tumor/surrounding normal tissue of esophageal cancers, using temporal temperature gradient gel electrophoresis (TTGE, followed by direct DNA sequencing to identify the mutations. Results Fourteen somatic mtDNA mutations were identified in 55% (11/20 of tumors analyzed, including 2 novel missense mutations and a frameshift mutation in ND4L, ATP6 subunit, and ND4 genes respectively. Nine mutations (64% were in the D-loop region. Numerous germline variations were found, at least 10 of them were novel and five were missense mutations, some of them occurred in evolutionarily conserved domains. Using real-time quantitative PCR analysis, the mtDNA content was found to increase in some tumors and decrease in others. Analysis of molecular and other clinicopathological findings does not reveal significant correlation between somatic mtDNA mutations and mtDNA content, or between mtDNA content and metastatic status. Conclusion Our results demonstrate that somatic mtDNA mutations in esophageal cancers are frequent. Some missense and frameshift mutations may play an important role in the tumorigenesis of esophageal carcinoma. More extensive biochemical and molecular studies will be necessary to determine the pathological significance of these somatic mutations.

  13. Rapid Detection of Mutation in RRDR of rpo B Gene for Rifampicin Resistance in MDR-Pulmonary Tuberculosis by DNA Sequencing

    OpenAIRE

    Patra, Surajeet Kumar; Jain, Anju; B L Sherwal; Khanna, Ashwani

    2010-01-01

    To detect the site of mutation in RRDR of rpo B gene for rifampicin resistance in MDR-TB by DNA sequencing. 50 MDR-TB patients were enrolled in our study after informed written consent. Mycobacterial DNA was extracted from sputum samples by Universal Sample Processing (USP) method and RRDR of rpo B gene was amplified by PCR using primers RP4T and RP8T and then sequenced by automated DNA sequencing. The nucleotide sequences of RRDR of rpo B gene were compared with the reference sequence. We ob...

  14. Spontaneous event of mitochondrial DNA mutation, A3243G, found in a family of identical twins.

    Science.gov (United States)

    Harihara, Shinji; Nakamura, Kennichi; Takubo, Kaiyo; Takeuchi, Fujio

    2013-04-01

    A mutation in mitochondrial DNA (mtDNA) A3243G is an important cause of some serious mitochondrial diseases, and maternal inheritance of the mutation has been reported. In order to investigate the heredity of the mutation, we measured the ratio of the mutated mtDNA molecule among 32 families of identical twins. Both twins from one family showed 20.16% and 18.49% mutated molecules, and the level is significantly high in comparison with members of other families and control subjects (0.23-0.86%). Their parents, however, showed normal level of mutated molecules (0.70% and 0.66%). The high-level mutation of the twins may be due to a spontaneous event, which occurred during development of germ line of their mother, or oogenesis of their mother, or during early stage of their development.

  15. Direct estimation of the mitochondrial DNA mutation rate in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Cathy Haag-Liautard

    2008-08-01

    Full Text Available Mitochondrial DNA (mtDNA variants are widely used in evolutionary genetics as markers for population history and to estimate divergence times among taxa. Inferences of species history are generally based on phylogenetic comparisons, which assume that molecular evolution is clock-like. Between-species comparisons have also been used to estimate the mutation rate, using sites that are thought to evolve neutrally. We directly estimated the mtDNA mutation rate by scanning the mitochondrial genome of Drosophila melanogaster lines that had undergone approximately 200 generations of spontaneous mutation accumulation (MA. We detected a total of 28 point mutations and eight insertion-deletion (indel mutations, yielding an estimate for the single-nucleotide mutation rate of 6.2 x 10(-8 per site per fly generation. Most mutations were heteroplasmic within a line, and their frequency distribution suggests that the effective number of mitochondrial genomes transmitted per female per generation is about 30. We observed repeated occurrences of some indel mutations, suggesting that indel mutational hotspots are common. Among the point mutations, there is a large excess of G-->A mutations on the major strand (the sense strand for the majority of mitochondrial genes. These mutations tend to occur at nonsynonymous sites of protein-coding genes, and they are expected to be deleterious, so do not become fixed between species. The overall mtDNA mutation rate per base pair per fly generation in Drosophila is estimated to be about 10x higher than the nuclear mutation rate, but the mitochondrial major strand G-->A mutation rate is about 70x higher than the nuclear rate. Silent sites are substantially more strongly biased towards A and T than nonsynonymous sites, consistent with the extreme mutation bias towards A+T. Strand-asymmetric mutation bias, coupled with selection to maintain specific nonsynonymous bases, therefore provides an explanation for the extreme base

  16. [Development of a DNA biochip for detection of known mtDNA mutations associated with MELAS and MERRF syndromes.].

    Science.gov (United States)

    Chen, Gang; Li, Wei; DU, Wei-Dong; Cao, Hui-Min; Tang, Hua-Yang; Tang, Xian-Fa; Sun, Zhong-Wu; Zhao, Hui; Jin, Qing-Hui; Zhao, Jian-Long; Zhang, Xue-Jun

    2008-10-01

    We developed an oligonucleotide biochip for synchronous multiplex detection of 31 known mitochondrial DNA mutations associated with MELAS (Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes) and MERRF (Myoclonic epilepsy with ragged red fibers). Allele-specific oligonucleotide probes were covalently immobilized on aldehyde modified glass slides, and then hybridized with Cy5-labled DNA fragments amplified from sample DNAs by a multiplex asymmetric PCR (MAP) method. Five patients with MELAS, 5 patients with MERRF and 20 healthy controls were investigated using the oligonucleotide biochip. The results showed that all the cases with MELAS had an A3243G mutation in the MT-TL1 gene. In the MERRF group, 4 cases were found to be an A8344G mutation and 1 case was a T8356C mutation, and both mutations were in the MT-TK gene. In the healthy controls, none of the 31 related mutations was found. The results of the DNA biochip were consistent with those by DNA sequencing. Clearly, the DNA biochip combined with MAP method would become a valuable tool in multiplex detecting of the point mutations in mtDNA leading to MELAS and/or MERRF syndrome. Moreover, this biochip format could be modified to extend to the screening scope of SNPs for any other human mitochondrial diseases.

  17. MtDNA mutation pattern in tumors and human evolution are shaped by similar selective constraints.

    Science.gov (United States)

    Zhidkov, Ilia; Livneh, Erez A; Rubin, Eitan; Mishmar, Dan

    2009-04-01

    Multiple human mutational landscapes of normal and cancer conditions are currently available. However, while the unique mutational patterns of tumors have been extensively studied, little attention has been paid to similarities between malignant and normal conditions. Here we compared the pattern of mutations in the mitochondrial genomes (mtDNAs) of cancer (98 sequences) and natural populations (2400 sequences). De novo mtDNA mutations in cancer preferentially colocalized with ancient variants in human phylogeny. A significant portion of the cancer mutations was organized in recurrent combinations (COMs), reaching a length of seven mutations, which also colocalized with ancient variants. Thus, by analyzing similarities rather than differences in patterns of mtDNA mutations in tumor and human evolution, we discovered evidence for similar selective constraints, suggesting a functional potential for these mutations.

  18. Study in mutation of alfalfa genome DNA due to low energy N+ implantation using RAPD

    International Nuclear Information System (INIS)

    After implanted by various dosage N+ beams, germination rate of alfalfa seeds appears to be saddle line with dosage increasing. The authors have studied in mutation of genome DNA due to low energy N+ implantation, and concluded that 30 differential DNA fragments have been amplified by 8 primers (S41, S42, S45, S46, S50, S52, S56, S58) in 100 primers, moreover, number of differential DNA fragments between CK and treatments increases with dosage. Consequently, low energy ion implantation can cause mutation of alfalfa genome DNA. The more dosage it is, the more mutation alfalfa will be

  19. Novel mtDNA mutations and oxidative phosphorylation dysfunction in Russian LHON families.

    Science.gov (United States)

    Brown, M D; Zhadanov, S; Allen, J C; Hosseini, S; Newman, N J; Atamonov, V V; Mikhailovskaya, I E; Sukernik, R I; Wallace, D C

    2001-07-01

    Leber's hereditary optic neuropathy (LHON) is characterized by maternally transmitted, bilateral, central vision loss in young adults. It is caused by mutations in the mitochondrial DNA (mtDNA) encoded genes that contribute polypeptides to NADH dehydrogenase or complex I. Four mtDNA variants, the nucleotide pair (np) 3460A, 11778A, 14484C, and 14459A mutations, are known as "primary" LHON mutations and are found in most, but not all, of the LHON families reported to date. Here, we report the extensive genetic and biochemical analysis of five Russian families from the Novosibirsk region of Siberia manifesting maternally transmitted optic atrophy consistent with LHON. Three of the five families harbor known LHON primary mutations. Complete sequence analysis of proband mtDNA in the other two families has revealed novel complex I mutations at nps 3635A and 4640C, respectively. These mutations are homoplasmic and have not been reported in the literature. Biochemical analysis of complex I in patient lymphoblasts and transmitochondrial cybrids demonstrated a respiration defect with complex-I-linked substrates, although the specific activity of complex I was not reduced. Overall, our data suggests that the spectrum of mtDNA mutations associated with LHON in Russia is similar to that in Europe and North America and that the np 3635A and 4640C mutations may be additional mtDNA complex I mutations contributing to LHON expression.

  20. Applications of capillary electrophoresis in DNA mutation analysis of genetic disorders.

    OpenAIRE

    Le, H; Fung, D.; Trent, R.J.

    1997-01-01

    AIM: To facilitate DNA mutation analysis by use of capillary electrophoresis. METHODS: The usefulness and applications of capillary electrophoresis in DNA fragment sizing and sequencing were evaluated. RESULTS: DNA mutation testing in disorders such as cystic fibrosis, Huntington disease, alpha thalassaemia, and hereditary fructose intolerance were undertaken effectively. However, sizing the (CAG)n repeat in the case of Huntington disease was a potential problem when using capillary electroph...

  1. Detecting the somatic mutations spectrum of Chinese lung cancer by analyzing the whole mitochondrial DNA genomes.

    Science.gov (United States)

    Fang, Yu; Huang, Jie; Zhang, Jing; Wang, Jun; Qiao, Fei; Chen, Hua-Mei; Hong, Zhi-Peng

    2015-02-01

    To detect the somatic mutations and character its spectrum in Chinese lung cancer patients. In this study, we sequenced the whole mitochondrial DNA (mtDNA) genomes for 10 lung cancer patients including the primary cancerous, matched paracancerous normal and distant normal tissues. By analyzing the 30 whole mtDNA genomes, eight somatic mutations were identified from five patients investigated, which were confirmed with the cloning and sequencing of the somatic mutations. Five of the somatic mutations were detected among control region and the rests were found at the coding region. Heterogeneity was the main character of the somatic mutations in Chinese lung cancer patients. Further potential disease-related screening showed that, except the C deletion at position 309 showed AD-weakly associated, most of them were not disease-related. Although the role of aforementioned somatic mutations was unknown, however, considering the relative higher frequency of somatic mutations among the whole mtDNA genomes, it hints that detecting the somatic mutation(s) from the whole mtDNA genomes can serve as a useful tool for the Chinese lung cancer diagnostic to some extent.

  2. Uracil-DNA glycosylase-deficient yeast exhibit a mitochondrial mutator phenotype

    OpenAIRE

    Chatterjee, Aditi; Keshav K Singh

    2001-01-01

    Mutations in mitochondrial DNA (mtDNA) have been reported in cancer and are involved in the pathogenesis of many mitochondrial diseases. Uracil-DNA glycosylase, encoded by the UNG1 gene in Saccharomyces cerevisiae, repairs uracil in DNA formed due to deamination of cytosine. Our study demonstrates that inactivation of the UNG1 gene leads to at least a 3-fold increased frequency of mutations in mtDNA compared with the wild-type. Using a Ung1p–green fluorescent protein (GFP) fusion construct, w...

  3. Mutations in the SPG7 gene cause chronic progressive external ophthalmoplegia through disordered mitochondrial DNA maintenance.

    Science.gov (United States)

    Pfeffer, Gerald; Gorman, Gráinne S; Griffin, Helen; Kurzawa-Akanbi, Marzena; Blakely, Emma L; Wilson, Ian; Sitarz, Kamil; Moore, David; Murphy, Julie L; Alston, Charlotte L; Pyle, Angela; Coxhead, Jon; Payne, Brendan; Gorrie, George H; Longman, Cheryl; Hadjivassiliou, Marios; McConville, John; Dick, David; Imam, Ibrahim; Hilton, David; Norwood, Fiona; Baker, Mark R; Jaiser, Stephan R; Yu-Wai-Man, Patrick; Farrell, Michael; McCarthy, Allan; Lynch, Timothy; McFarland, Robert; Schaefer, Andrew M; Turnbull, Douglass M; Horvath, Rita; Taylor, Robert W; Chinnery, Patrick F

    2014-05-01

    Despite being a canonical presenting feature of mitochondrial disease, the genetic basis of progressive external ophthalmoplegia remains unknown in a large proportion of patients. Here we show that mutations in SPG7 are a novel cause of progressive external ophthalmoplegia associated with multiple mitochondrial DNA deletions. After excluding known causes, whole exome sequencing, targeted Sanger sequencing and multiplex ligation-dependent probe amplification analysis were used to study 68 adult patients with progressive external ophthalmoplegia either with or without multiple mitochondrial DNA deletions in skeletal muscle. Nine patients (eight probands) were found to carry compound heterozygous SPG7 mutations, including three novel mutations: two missense mutations c.2221G>A; p.(Glu741Lys), c.2224G>A; p.(Asp742Asn), a truncating mutation c.861dupT; p.Asn288*, and seven previously reported mutations. We identified a further six patients with single heterozygous mutations in SPG7, including two further novel mutations: c.184-3C>T (predicted to remove a splice site before exon 2) and c.1067C>T; p.(Thr356Met). The clinical phenotype typically developed in mid-adult life with either progressive external ophthalmoplegia/ptosis and spastic ataxia, or a progressive ataxic disorder. Dysphagia and proximal myopathy were common, but urinary symptoms were rare, despite the spasticity. Functional studies included transcript analysis, proteomics, mitochondrial network analysis, single fibre mitochondrial DNA analysis and deep re-sequencing of mitochondrial DNA. SPG7 mutations caused increased mitochondrial biogenesis in patient muscle, and mitochondrial fusion in patient fibroblasts associated with the clonal expansion of mitochondrial DNA mutations. In conclusion, the SPG7 gene should be screened in patients in whom a disorder of mitochondrial DNA maintenance is suspected when spastic ataxia is prominent. The complex neurological phenotype is likely a result of the clonal

  4. Sensitive and specific KRAS somatic mutation analysis on whole-genome amplified DNA from archival tissues.

    Science.gov (United States)

    van Eijk, Ronald; van Puijenbroek, Marjo; Chhatta, Amiet R; Gupta, Nisha; Vossen, Rolf H A M; Lips, Esther H; Cleton-Jansen, Anne-Marie; Morreau, Hans; van Wezel, Tom

    2010-01-01

    Kirsten RAS (KRAS) is a small GTPase that plays a key role in Ras/mitogen-activated protein kinase signaling; somatic mutations in KRAS are frequently found in many cancers. The most common KRAS mutations result in a constitutively active protein. Accurate detection of KRAS mutations is pivotal to the molecular diagnosis of cancer and may guide proper treatment selection. Here, we describe a two-step KRAS mutation screening protocol that combines whole-genome amplification (WGA), high-resolution melting analysis (HRM) as a prescreen method for mutation carrying samples, and direct Sanger sequencing of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue, from which limited amounts of DNA are available. We developed target-specific primers, thereby avoiding amplification of homologous KRAS sequences. The addition of herring sperm DNA facilitated WGA in DNA samples isolated from as few as 100 cells. KRAS mutation screening using high-resolution melting analysis on wgaDNA from formalin-fixed, paraffin-embedded tissue is highly sensitive and specific; additionally, this method is feasible for screening of clinical specimens, as illustrated by our analysis of pancreatic cancers. Furthermore, PCR on wgaDNA does not introduce genotypic changes, as opposed to unamplified genomic DNA. This method can, after validation, be applied to virtually any potentially mutated region in the genome.

  5. Experimental evidence that mutated-self peptides derived from mitochondrial DNA somatic mutations have the potential to trigger autoimmunity.

    Science.gov (United States)

    Chen, Lina; Duvvuri, Bhargavi; Grigull, Jörg; Jamnik, Roni; Wither, Joan E; Wu, Gillian E

    2014-08-01

    Autoimmune disease is a critical health concern, whose etiology remains enigmatic. We hypothesized that immune responses to somatically mutated self proteins could have a role in the development of autoimmune disease. IFN-γ secretion by T cells stimulated with mitochondrial peptides encoded by published mitochondrial DNA was monitored to test the hypothesis. Human peripheral blood mononuclear cells (PBMCs) of healthy controls and autoimmune patients were assessed for their responses to the self peptides and mutated-self peptides differing from self by one amino acid. None of the self peptides but some of the mutated-self peptides elicited an immune response in healthy controls. In some autoimmune patients, PBMCs responded not only to some of the mutated-self peptides, but also to some of the self peptides, suggesting that there is a breach of self-tolerance in these patients. Although PBMCs from healthy controls failed to respond to self peptides when stimulated with self, the mutated-self peptide could elicit a response to the self peptide upon re-stimulation in vitro, suggesting that priming with mutated-self peptides elicits a cross-reactive response with self. The data raise the possibility that DNA somatic mutations are one of the events that trigger and/or sustain T cell responses in autoimmune diseases.

  6. PIK3CA mutation detection in metastatic biliary cancer using cell-free DNA

    OpenAIRE

    Kim, Seung Tae; Lira, Maruja; Deng, Shibing; Lee, Sujin; Park, Young Suk; Lim, Ho Yeong; Kang, Won Ki; Mao, Mao; Heo, Jin Seok; Kwon, Wooil; Jang, Kee-Taek; Lee, Jeeyun; Park, Joon Oh

    2015-01-01

    PIK3CA mutation is considered a good candidate for targeted therapies in cancers, especially biliary tract cancer (BTC). We evaluated the utility of cell free DNA (cfDNA) from serum by using droplet digital PCR (ddPCR) as an alternative source for PIK3CA mutation analysis. To identify matching archival tumour specimens from serum samples of advanced BTC patients, mutation detection using ddPCR with Bio-Rad's PrimePCR mutation and wild type assays were performed for PIK3CA p.E542K, p.E545K, an...

  7. Random mtDNA mutations modulate proliferation capacity in mouse embryonic fibroblasts

    International Nuclear Information System (INIS)

    Highlights: → Increased mtDNA mutations in MEFs lead to high level of spontaneous immortalization. → This process is independent of endogenous ROS production. → Aerobic glycolysis significantly contributes to spontaneous immortalization of MEFs. -- Abstract: An increase in mtDNA mutation load leads to a loss of critical cells in different tissues thereby contributing to the physiological process of organismal ageing. Additionally, the accumulation of senescent cells that display changes in metabolic function might act in an active way to further disrupt the normal tissue function. We believe that this could be the important link missing in our understanding of the molecular mechanisms of premature ageing in the mtDNA mutator mice. We tested proliferation capacity of mtDNA mutator cells in vitro. When cultured in physiological levels of oxygen (3%) their proliferation capacity is somewhat lower than wild-type cells. Surprisingly, in conditions of increased oxidative stress (20% O2) mtDNA mutator mouse embryonic fibroblasts exhibit continuous proliferation due to spontaneous immortalization, whereas the same conditions promote senescence in wild-type cells. We believe that an increase in aerobic glycolysis observed in mtDNA mutator mice is a major mechanism behind this process. We propose that glycolysis promotes proliferation and allows a fast turnover of metabolites, but also leads to energy crisis due to lower ATP production rate. This could lead to compromised replication and/or repair and therefore, in rare cases, might lead to mutations in tumor suppressor genes and spontaneous immortalization.

  8. Random mtDNA mutations modulate proliferation capacity in mouse embryonic fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Kukat, Alexandra [Division of Metabolic Diseases, Department of Laboratory Medicine, Karolinska Institute, S-17171 Stockholm (Sweden); Cologne Excellence Cluster on Cellular Stress Responses in Ageing-Associated Diseases (CECAD), Cologne University Clinic, D-50674 Cologne (Germany); Edgar, Daniel [Division of Metabolic Diseases, Department of Laboratory Medicine, Karolinska Institute, S-17171 Stockholm (Sweden); Bratic, Ivana [Division of Metabolic Diseases, Department of Laboratory Medicine, Karolinska Institute, S-17171 Stockholm (Sweden); Cologne Excellence Cluster on Cellular Stress Responses in Ageing-Associated Diseases (CECAD), Cologne University Clinic, D-50674 Cologne (Germany); Maiti, Priyanka [Cologne Excellence Cluster on Cellular Stress Responses in Ageing-Associated Diseases (CECAD), Cologne University Clinic, D-50674 Cologne (Germany); Trifunovic, Aleksandra, E-mail: aleksandra.trifunovic@ki.se [Division of Metabolic Diseases, Department of Laboratory Medicine, Karolinska Institute, S-17171 Stockholm (Sweden); Cologne Excellence Cluster on Cellular Stress Responses in Ageing-Associated Diseases (CECAD), Cologne University Clinic, D-50674 Cologne (Germany)

    2011-06-10

    Highlights: {yields} Increased mtDNA mutations in MEFs lead to high level of spontaneous immortalization. {yields} This process is independent of endogenous ROS production. {yields} Aerobic glycolysis significantly contributes to spontaneous immortalization of MEFs. -- Abstract: An increase in mtDNA mutation load leads to a loss of critical cells in different tissues thereby contributing to the physiological process of organismal ageing. Additionally, the accumulation of senescent cells that display changes in metabolic function might act in an active way to further disrupt the normal tissue function. We believe that this could be the important link missing in our understanding of the molecular mechanisms of premature ageing in the mtDNA mutator mice. We tested proliferation capacity of mtDNA mutator cells in vitro. When cultured in physiological levels of oxygen (3%) their proliferation capacity is somewhat lower than wild-type cells. Surprisingly, in conditions of increased oxidative stress (20% O{sub 2}) mtDNA mutator mouse embryonic fibroblasts exhibit continuous proliferation due to spontaneous immortalization, whereas the same conditions promote senescence in wild-type cells. We believe that an increase in aerobic glycolysis observed in mtDNA mutator mice is a major mechanism behind this process. We propose that glycolysis promotes proliferation and allows a fast turnover of metabolites, but also leads to energy crisis due to lower ATP production rate. This could lead to compromised replication and/or repair and therefore, in rare cases, might lead to mutations in tumor suppressor genes and spontaneous immortalization.

  9. DNA sequence analysis of X-ray induced Adh null mutations in Drosophila melanogaster

    Energy Technology Data Exchange (ETDEWEB)

    Mahmoud, J.; Fossett, N.G.; Arbour-Reily, P.; McDaniel, M.; Tucker, A.; Chang, S.H.; Lee, W.R. (Louisiana State Univ., Baton Rouge (United States))

    1991-01-01

    The mutational spectrum for 28 X-ray induced mutations and 2 spontaneous mutations, previously determined by genetic and cytogenetic methods, consisted of 20 multilocus deficiencies (19 induced and 1 spontaneous) and 10 intragenic mutations (9 induced and 1 spontaneous). One of the X-ray induced intragenic mutations was lost, and another was determined to be a recombinant with the allele used in the recovery scheme. The DNA sequence of two X-ray induced intragenic mutations has been published. This paper reports the results of DNA sequence analysis of the remaining intragenic mutations and a summary of the X-ray induced mutational spectrum. The combination of DNA sequence analysis with genetic complementation analysis shows a continuous distribution in size of deletions rather than two different types of mutations consisting of deletions and point mutations'. Sequencing is shown to be essential for detecting intragenic deletions. Of particular importance for future studies is the observation that all of the intragenic deletions consist of a direct repeat adjacent to the breakpoint with one of the repeats deleted.

  10. SERS-melting: a new method for discriminating mutations in dna sequences

    OpenAIRE

    Mahajan, Sumeet; Richardson, James; Brown, Tom; Bartlett, Philip N

    2008-01-01

    The reliable discrimination of mutations, single nucleotide polymorphisms (SNPs), and other differences in genomic sequence is an essential part of DNA diagnostics and forensics. It is commonly achieved using fluorescently labeled DNA probes and thermal gradients to distinguish between the matched and mismatched DNA. Here, we describe a novel method that uses surface enhanced (resonance) Raman spectroscopy (SER(R)S) to follow denaturation of dsDNA attached to a structured gold surface. T...

  11. Mitochondria DNA mutations cause sex-dependent development of hypertension and alterations in cardiovascular function

    OpenAIRE

    Golob, Mark J.; Tian, Lian; Wang, Zhijie; Zimmerman, Todd A.; Caneba, Christine A.; Hacker, Timothy A.; Song, Guoqing; Chesler, Naomi C.

    2014-01-01

    Aging is associated with conduit artery stiffening that is a risk factor for and can precede hypertension and ventricular dysfunction. Increases in mitochondria DNA (mtDNA) frequency have been correlated with aging. Mice with a mutation in the encoding domain (D257A) of a proof-reading deficient version of mtDNA polymerase-γ (POLG) have musculoskeletal features of premature aging and a shortened lifespan. However, few studies using these mice have investigated the effects of mtDNA mutations o...

  12. Mitochondrial DNA Complex I and III Mutations Associated with Leber's Hereditary Optic Neuropathy

    OpenAIRE

    Brown, M D; Voljavec, A. S.; Lott, M T; Torroni, A.; Yang, C. C.; Wallace, D C

    1992-01-01

    Four new missense mutations have been identified through restriction analysis and sequencing of the mitochondrial DNAs (mtDNA) from Leber's hereditary optic neuropathy (LHON) patients who lacked the previously identified 11778 mutation. Each altered a conserved amino acid and correlated with the LHON phenotype in population and phylogenetic analyses. The nucleotide pair (np) 13708 mutation (G to A, ND5 gene) changed an alanine to a threonine and was found in 6/25 (24%) of non-11778 LHON pedig...

  13. 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

    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...

  14. Simultaneous DNA and RNA mapping of somatic mitochondrial mutations across diverse human cancers

    DEFF Research Database (Denmark)

    Stewart, James B.; Alaei-Mahabadi, Babak; Radhakrishnan, Sabarinathan;

    2015-01-01

    -coupled errors as the major source of mutations. Interestingly, while allelic ratios in general were consistent in RNA compared to DNA, some mutations in tRNAs displayed strong allelic imbalances caused by accumulation of unprocessed tRNA precursors. The effect was explained by altered secondary structure...... mitochondrial tRNA biogenesis that are difficult to address in controlled experimental systems....

  15. Nitrative and oxidative DNA damage caused by K-ras mutation in mice

    Energy Technology Data Exchange (ETDEWEB)

    Ohnishi, Shiho [Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science (Japan); Saito, Hiromitsu; Suzuki, Noboru [Department of Animal Genomics, Mie University Life Science Research Center (Japan); Ma, Ning [Faculty of Health Science, Suzuka University of Medical Science (Japan); Hiraku, Yusuke; Murata, Mariko [Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine (Japan); Kawanishi, Shosuke, E-mail: kawanisi@suzuka-u.ac.jp [Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science (Japan)

    2011-09-23

    Highlights: {yields} Mutated K-ras in transgenic mice caused nitrative DNA damage, 8-nitroguanine. {yields} The mutagenic 8-nitroguanine seemed to be generated by iNOS via Ras-MAPK signal. {yields} Mutated K-ras produces additional mutagenic lesions, as a new oncogenic role. -- Abstract: Ras mutation is important for carcinogenesis. Carcinogenesis consists of multi-step process with mutations in several genes. We investigated the role of DNA damage in carcinogenesis initiated by K-ras mutation, using conditional transgenic mice. Immunohistochemical analysis revealed that mutagenic 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) were apparently formed in adenocarcinoma caused by mutated K-ras. 8-Nitroguanine was co-localized with iNOS, eNOS, NF-{kappa}B, IKK, MAPK, MEK, and mutated K-ras, suggesting that oncogenic K-ras causes additional DNA damage via signaling pathway involving these molecules. It is noteworthy that K-ras mutation mediates not only cell over-proliferation but also the accumulation of mutagenic DNA lesions, leading to carcinogenesis.

  16. Simultaneous DNA and RNA Mapping of Somatic Mitochondrial Mutations across Diverse Human Cancers.

    Directory of Open Access Journals (Sweden)

    James B Stewart

    2015-06-01

    Full Text Available Somatic mutations in the nuclear genome are required for tumor formation, but the functional consequences of somatic mitochondrial DNA (mtDNA mutations are less understood. Here we identify somatic mtDNA mutations across 527 tumors and 14 cancer types, using an approach that takes advantage of evidence from both genomic and transcriptomic sequencing. We find that there is selective pressure against deleterious coding mutations, supporting that functional mitochondria are required in tumor cells, and also observe a strong mutational strand bias, compatible with endogenous replication-coupled errors as the major source of mutations. Interestingly, while allelic ratios in general were consistent in RNA compared to DNA, some mutations in tRNAs displayed strong allelic imbalances caused by accumulation of unprocessed tRNA precursors. The effect was explained by altered secondary structure, demonstrating that correct tRNA folding is a major determinant for processing of polycistronic mitochondrial transcripts. Additionally, the data suggest that tRNA clusters are preferably processed in the 3' to 5' direction. Our study gives insights into mtDNA function in cancer and answers questions regarding mitochondrial tRNA biogenesis that are difficult to address in controlled experimental systems.

  17. Allele-specific polymerase chain reaction for detection of a mutation in the relax circular DNA and the covalently closed circular DNA of hepatitis B virus.

    Science.gov (United States)

    Pan, Wan-Long; Hu, Jie-Li; Fang, Yan; Luo, Qiang; Xu, Ge; Xu, Lei; Jing, Zhou-Hong; Shan, Xue-Feng; Zhu, Yan-Ling; Huang, Ai-Long

    2013-12-01

    The relax circle DNA (rcDNA) sequence and the covalently closed circle DNA (cccDNA) sequence in hepatitis B virus (HBV) are crucial regions for HBV infections. To analyze mutations in rcDNA and cccDNA, DNA sequencing is often used, although it is time-consuming and expensive. Herein, we report a simple, economic, albeit accurate allele-specific polymerase chain reaction (AS-PCR) to detect mutations in these regions of HBV. This method can be extensively used to screen for mutations at specific positions of HBV genome.

  18. DNA microarray-based mutation discovery and genotyping.

    Science.gov (United States)

    Gresham, David

    2011-01-01

    DNA microarrays provide an efficient means of identifying single-nucleotide polymorphisms (SNPs) in DNA samples and characterizing their frequencies in individual and mixed samples. We have studied the parameters that determine the sensitivity of DNA probes to SNPs and found that the melting temperature (T (m)) of the probe is the primary determinant of probe sensitivity. An isothermal-melting temperature DNA microarray design, in which the T (m) of all probes is tightly distributed, can be implemented by varying the length of DNA probes within a single DNA microarray. I describe guidelines for designing isothermal-melting temperature DNA microarrays and protocols for labeling and hybridizing DNA samples to DNA microarrays for SNP discovery, genotyping, and quantitative determination of allele frequencies in mixed samples.

  19. Automated centrifugal-microfluidic platform for DNA purification using laser burst valve and coriolis effect.

    Science.gov (United States)

    Choi, Min-Seong; Yoo, Jae-Chern

    2015-04-01

    We report a fully automated DNA purification platform with a micropored membrane in the channel utilizing centrifugal microfluidics on a lab-on-a-disc (LOD). The microfluidic flow in the LOD, into which the reagents are injected for DNA purification, is controlled by a single motor and laser burst valve. The sample and reagents pass successively through the micropored membrane in the channel when each laser burst valve is opened. The Coriolis effect is used by rotating the LOD bi-directionally to increase the purity of the DNA, thereby preventing the mixing of the waste and elution solutions. The total process from the lysed sample injection into the LOD to obtaining the purified DNA was finished within 7 min with only one manual step. The experimental result for Salmonella shows that the proposed microfluidic platform is comparable to the existing devices in terms of the purity and yield of DNA.

  20. Stability of capillary gels for automated sequencing of DNA.

    Science.gov (United States)

    Swerdlow, H; Dew-Jager, K E; Brady, K; Grey, R; Dovichi, N J; Gesteland, R

    1992-08-01

    Recent interest in capillary gel electrophoresis has been fueled by the Human Genome Project and other large-scale sequencing projects. Advances in gel polymerization techniques and detector design have enabled sequencing of DNA directly in capillaries. Efforts to exploit this technology have been hampered by problems with the reproducibility and stability of gels. Gel instability manifests itself during electrophoresis as a decrease in the current passing through the capillary under a constant voltage. Upon subsequent microscopic examination, bubbles are often visible at or near the injection (cathodic) end of the capillary gel. Gels have been prepared with the polyacrylamide matrix covalently attached to the silica walls of the capillary. These gels, although more stable, still suffer from problems with bubbles. The use of actual DNA sequencing samples also adversely affects gel stability. We examined the mechanisms underlying these disruptive processes by employing polyacrylamide gel-filled capillaries in which the gel was not attached to the capillary wall. Three sources of gel instability were identified. Bubbles occurring in the absence of sample introduction were attributed to electroosmotic force; replacing the denaturant urea with formamide was shown to reduce the frequency of these bubbles. The slow, steady decline in current through capillary sequencing gels interferes with the ability to detect other gel problems. This phenomenon was shown to be a result of ionic depletion at the gel-liquid interface. The decline was ameliorated by adding denaturant and acrylamide monomers to the buffer reservoirs. Sample-induced problems were shown to be due to the presence of template DNA; elimination of the template allowed sample loading to occur without complications.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. GenePublisher: automated analysis of DNA microarray data

    DEFF Research Database (Denmark)

    Knudsen, Steen; Workman, Christopher; Sicheritz-Ponten, T.;

    2003-01-01

    GenePublisher, a system for automatic analysis of data from DNA microarray experiments, has been implemented with a web interface at http://www.cbs.dtu.dk/services/GenePublisher. Raw data are uploaded to the server together with aspecification of the data. The server performs normalization......, statistical analysis and visualization of the data. The results are run against databases of signal transduction pathways, metabolic pathways and promoter sequences in order to extract more information. The results of the entire analysis are summarized in report form and returned to the user....

  2. DNA ploidy and c-Kit mutation in gastrointestinal stromal tumors

    Institute of Scientific and Technical Information of China (English)

    Ju Han Lee; Xianglan Zhang; Woon Yong Jung; Yang Seok Chae; Jong-Jae Park; Insun Kim

    2004-01-01

    AIM: To investigate the prognostic significance of c-Kit gene mutation and DNA ploidy in gastointestinal stromal tumors (GISTs).METHODS: A total of 55 cases of GISTs were studied for the expression of c-Kit by immunohistochemistry, and the c-Kit gene mutations in exons 9, 11, 13, and 17 were detected by polymerase chain reaction-single strand confirmation polymarphism (PCR-SSCP) and denaturing high performance liquid chromatography (D-HPLC)techniques. DNA ploidy was determined by flow cytometry.RESULTS: Of the 55 cases of GISTs, 53 cases (96.4%)expressed c-Kit protein. The c-Kit gene mutations of exons 11 and 9 were found in 30 (54.5%) and 7 cases (12.7%),respectively. No mutations were found in exons 13 and 17.DNA aneuploidy was seen in 10 cases (18.2%). The c-Kit mutation positive GISTs were larger in size than the negative GISTs. The aneuploidy tumors were statistically associated with large size, high mitotic counts, high risk groups, high cellularity and severe nuclear atypia, and epithelioid type.There was a tendency that c-Kit mutations were more frequently found in aneuploidy GISTs.CONCLUSION: DNA aneuploidy and c-Kit mutations can be considered as prognostic factors in GISTs.

  3. Prevalence of migraine in persons with the 3243A>G mutation in mitochondrial DNA

    DEFF Research Database (Denmark)

    Guo, S.; Esserlind, A-L; Andersson, Z;

    2016-01-01

    BACKGROUND AND PURPOSE: Over the last three decades mitochondrial dysfunction has been postulated to be a potential mechanism in migraine pathogenesis. The lifetime prevalence of migraine in persons carrying the 3243A>G mutation in mitochondrial DNA was investigated. METHODS: In this cross...... of mitochondrial dysfunction and susceptibility to migraine. Mitochondrial DNA aberrations may contribute to the pathogenesis of migraine....

  4. The Emergent Landscape of Detecting EGFR Mutations Using Circulating Tumor DNA in Lung Cancer

    Directory of Open Access Journals (Sweden)

    Wei-Lun Huang

    2015-01-01

    Full Text Available The advances in targeted therapies for lung cancer are based on the evaluation of specific gene mutations especially the epidermal growth factor receptor (EGFR. The assays largely depend on the acquisition of tumor tissue via biopsy before the initiation of therapy or after the onset of acquired resistance. However, the limitations of tissue biopsy including tumor heterogeneity and insufficient tissues for molecular testing are impotent clinical obstacles for mutation analysis and lung cancer treatment. Due to the invasive procedure of tissue biopsy and the progressive development of drug-resistant EGFR mutations, the effective initial detection and continuous monitoring of EGFR mutations are still unmet requirements. Circulating tumor DNA (ctDNA detection is a promising biomarker for noninvasive assessment of cancer burden. Recent advancement of sensitive techniques in detecting EGFR mutations using ctDNA enables a broad range of clinical applications, including early detection of disease, prediction of treatment responses, and disease progression. This review not only introduces the biology and clinical implementations of ctDNA but also includes the updating information of recent advancement of techniques for detecting EGFR mutation using ctDNA in lung cancer.

  5. Absence of mtDNA mutations in leukocytes of CADASIL patients

    Directory of Open Access Journals (Sweden)

    Hellani Ali

    2008-05-01

    Full Text Available Abstract Background Ultrastructural and biochemical abnormalities of mitochondria have been reported in skeletal muscle biopsies of CADASIL patients with mutations in the NOTCH3 nuclear gene. Additionally, it was proposed that NOTCH3 gene mutations may predispose the mitochondrial DNA (mtDNA to mutations. Methods We sequenced the entire mitochondrial genome in five Arab patients affected by CADASIL. Results The mean number of mtDNA sequence variants (synonymous and nonsynonymous in CADASIL patients was not statistically significantly different from that in controls (p = 0.378. After excluding haplogroup specific single nucleotide polymorphisms (SNPs and proved silent polymorphisms, no known or novel pathologic mtDNA mutation(s could be detected in any patient. Additionally, there was no difference in the prevalence of different mitochondrial haplogroups between patients and controls. Conclusion Our study group is too small for any valid conclusion to be made. However, if our observation is confirmed in larger study group, then mtDNA mutations or mitochondrial haplogroups may not be important in the pathogenesis of CADASIL.

  6. Clinical Analysis of Leber's Hereditary Optic Neuropathy Harboring mtDNA Mutation at nt11778

    Institute of Scientific and Technical Information of China (English)

    Xinyu Zhang; Qiang Yu; Qingjiong Zhang; Changxian Yi

    2001-01-01

    Purpose: To improve our diagnostic technique through the analysis of clinical features ofLeber's heredita'y optic neuropathy (LHON) harboring mtDNA point mutation at nt11778. Methods: Detection of nt11778 mutation was performed on 38 patients clinically diagnosed as LHON in our ophthalmic center from year 1998 to 2000. Circumstances of onset and family history were obtained and ophthalmoscopy, fundus fluorescein angiography, visual field and visual evoked potential were performed on all 38 patients. Result: 30 In 38 patients (78.95 % ) harbor nt11778 mutation, including 28 male (93.33%) and 2 female (6.67%). The ratio of affected male to female is 14: 1. Patients harboring nt11778 mutation display typical clinical nanifestations. Ccnclusion: Identification of one of the three LHON specifically associated ntDNA mutations is essential to confirm the diagnosis. Eye Science 2001: 17:31 ~ 34.

  7. RAPD analysis of alfalfa DNA mutation via N+ implantation

    Institute of Scientific and Technical Information of China (English)

    LI Yu-Feng; HUANG Qun-Ce; LIANG Yun-Zhang; YU Zeng-Liang

    2003-01-01

    Germination capacity of alfalfa seeds under low energy N+ implantation manifests oscillations goingdown with dose strength. From analyzing alfalfa genome DNA under low energy N+ implantation by RAPD (RandomAmplified Polymorphous DNA), it is recommended that 30 polymorphic DNA fragments be amplified with 8 primersin total 100 primers, and fluorescence intensity of the identical DNA fragments amplified by RAPD is different be-tween CK and treatments. Number of different polymorphic DNA fragments between treatment and CK via N+ im-plantation manifests going up with dose strength.

  8. Hair Dye–DNA Interaction: Plausible Cause of Mutation

    Directory of Open Access Journals (Sweden)

    Swati Maiti

    2015-09-01

    Full Text Available Hair dye is one of the most popular cosmetic products which are used more widely and frequently to improve an individual’s appearance. Although the genotoxic effects of dye ingredients are widely reported, hair dye in its usable form is not reported extensively. In this contribution, we report the possible mode of interaction of hair dye with DNA which leads to genotoxicity. The effect of dye DNA interaction was studied on the most popular and globally used hair dye with Calf Thymus DNA and plasmid DNA. This interaction of dye DNA was studied by spectroscopic analyses and gel electrophoresis. The result had shown positive interaction of dye with DNA. Gel electrophoresis study confirms the binding of dye with DNA which results in linearization and fragmentation of the plasmid DNA. Dye–DNA interaction causes fragmentation and oxidation of DNA in absence of any catalyst, implies high toxicity of commercial hair dyes. Thus, it can be deduced from the present studies that hair dye in its usable form may lead to its penetration through skin affecting genomic DNA possesses genotoxic property and can be treated as one of the most common mutagen.

  9. PIK3CA mutation detection in metastatic biliary cancer using cell-free DNA

    Science.gov (United States)

    Deng, Shibing; Lee, Sujin; Park, Young Suk; Lim, Ho Yeong; Kang, Won Ki; Mao, Mao; Heo, Jin Seok; Kwon, Wooil; Jang, Kee-Taek; Lee, Jeeyun; Park, Joon Oh

    2015-01-01

    PIK3CA mutation is considered a good candidate for targeted therapies in cancers, especially biliary tract cancer (BTC). We evaluated the utility of cell free DNA (cfDNA) from serum by using droplet digital PCR (ddPCR) as an alternative source for PIK3CA mutation analysis. To identify matching archival tumour specimens from serum samples of advanced BTC patients, mutation detection using ddPCR with Bio-Rad's PrimePCR mutation and wild type assays were performed for PIK3CA p.E542K, p.E545K, and p.H1047R. Thirty-eight patients with metastatic BTC were enrolled. Only one (BTC 29T) sample (n = 38) was positive for PIK3CA p.E542K and another (BTC 27T) for p.H1047R mutation; none was positive for PIK3CA p.E545K. Matched serum sample (BTC 29P) was positive for PIK3CA p.E542K with 28 mutant copies detected, corresponding to 48 copies/ml of serum and an allelic prevalence of 0.3%. Another matched serum sample (BTC 27P) was positive for PIK3CA p.H1047R with 10 mutant copies detected, i.e. 18 copies/ml and an allelic frequency of 0.2%. High correlation was noted in the PIK3CA mutation status between tumour gDNA and serum cfDNA. Low-level PIK3CA mutations were detectable in the serum indicating the utility of cfDNA as a DNA source to detect cancer-derived mutations in metastatic biliary cancers. PMID:26498688

  10. Initial analysis of non-typical Leber hereditary optic neuropathy (LHON) at onset and late developing demyelinating disease in Italian patients by SSCP and automated DNA sequence analysis

    Energy Technology Data Exchange (ETDEWEB)

    Sartore, M.; Semeraro, A.; Fortina, P. [Univ. of Pennsylvania School of Medicine, Philadelphia, PA (United States)] [and others

    1994-09-01

    LHON is a mitochondrial genetic disease characterized by maternal inheritance and late onset of blindness caused by bilateral retinal degeneration. A number of molecular defects are known affecting expression of seven mitochondrial genes encoding subunits of respiratory chain complex I, III and IV. We screened genomic DNA from Italian patients for seven of the known point mutations in the ND-1, ND-4 and ND-6 subunits of complex I by PCR followed by SSCP and restriction enzyme digestion. Most of the patients had nonfamilial bilateral visual loss with partial or no recovery and normal neurological examination. Fundoscopic examination revealed that none of the patients had features typical of LHON. Nine of 21 patients (43%) showed multifocal CNS demyelination on MRI. Our results show aberrant SSCP patterns for a PCR product from the ND-4 subunit in one affected child and his mother. Sfa NI and Mae III digestions suggested the absence of a previously defined LHON mutation, and automated DNA sequence analysis revealed two A to G neutral sequence polymorphisms in the third position of codons 351 and 353. In addition, PCR products from the same two samples and an unrelated one showed abnormal SSCP patterns for the ND-1 subunit region of complex I due to the presence of a T to C change at nt 4,216 which was demonstrated after Nla III digestion of PCR products and further confirmed by DNA sequence analysis. Our results indicate that additional defects are present in the Italian population, and identification of abnormal SSCP patterns followed by targeted automated DNA sequence analysis is a reasonable strategy for delineation of new LHON mutations.

  11. Blocking DNA Repair in Advanced BRCA-Mutated Cancer

    Science.gov (United States)

    In this trial, patients with relapsed or refractory advanced cancer and confirmed BRCA mutations who have not previously been treated with a PARP inhibitor will be given BMN 673 by mouth once a day in 28-day cycles.

  12. The Mitochondrial DNA Mutation at Position 11778 in Chinese Families with Leber's Hereditary Optic Neuropathy

    Institute of Scientific and Technical Information of China (English)

    1994-01-01

    We amplified the 340 bp of mitochondrial DMA (mtDNA) by PCR including the recognized sequence of restriction enzyme of SfaN I . After amplification and digestion of SfaN I , two bands of 190 bp and 150 bp appeared in the mtDNA of four normal individuals but only one band of 340 bp appeared in the mtDNA with the mutation of G to A at the site of the nucleotide 11778 because such mutation destroyed the recognized sequence of SfaN I . We studied the mtDNAs of the patients with Leber's hereditary optic neur...

  13. Genetic suppression of a dnaG mutation in Escherichia coli.

    OpenAIRE

    Katayama, T; Murakami, Y.; Wada, C.; Ohmori, H; Yura, T; Nagata, T

    1989-01-01

    Escherichia coli strains with a temperature-sensitive mutation, dnaG2903, in the primase-encoding gene spontaneously reverted to the temperature-insensitive phenotype at a high frequency. Many of the reversions were caused by extragenic sdg suppressors. About 100 independently isolated sdg suppressors were analyzed. They fall into two classes. The sdgA mutations were genetically mapped very close to and upstream of the dnaG gene and were found to be cis dominant. DNA sequencing of two of them...

  14. A Preliminary Study on DNA Mutation Induction of Maize Pollen Implanted by Low Energy N+ Beam

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The maize pollens were implanted with seven different doses of 30 keV N+ beam re spectively, The genomic DNA polymorphism from treated pollens were analyzed with 104 primersby using RAPD respectively. The results showed that N+ beam-induced mutation of maize pollenscan result in the change of their DNA bases. The mutation is not properly random and its frequency increases with a rise in 30 keV N+ beam doses. It is conformed with A-G transformation,which is one of the most important factors in DNA bases induced by N+ beam.

  15. Automated microfluidic DNA/RNA extraction with both disposable and reusable components

    International Nuclear Information System (INIS)

    An automated microfluidic nucleic extraction system was fabricated with a multilayer polydimethylsiloxane (PDMS) structure that consists of sample wells, microvalves, a micropump and a disposable microfluidic silica cartridge. Both the microvalves and micropump structures were fabricated in a single layer and are operated pneumatically using a 100 µm PDMS membrane. To fabricate the disposable microfluidic silica cartridge, two-cavity structures were made in a PDMS replica to fit the stacked silica membranes. A handheld controller for the microvalves and pumps was developed to enable system automation. With purified ribonucleic acid (RNA), whole blood and E. coli samples, the automated microfluidic nucleic acid extraction system was validated with a guanidine-based solid phase extraction procedure. An extraction efficiency of ∼90% for deoxyribonucleic acid (DNA) and ∼54% for RNA was obtained in 12 min from whole blood and E. coli samples, respectively. In addition, the same quantity and quality of extracted DNA was confirmed by polymerase chain reaction (PCR) amplification. The PCR also presented the appropriate amplification and melting profiles. Automated, programmable fluid control and physical separation of the reusable components and the disposable components significantly decrease the assay time and manufacturing cost and increase the flexibility and compatibility of the system with downstream components

  16. Identification of pathways controlling DNA damage induced mutation in Saccharomyces cerevisiae.

    Science.gov (United States)

    Lis, Ewa T; O'Neill, Bryan M; Gil-Lamaignere, Cristina; Chin, Jodie K; Romesberg, Floyd E

    2008-05-01

    Mutation in response to most types of DNA damage is thought to be mediated by the error-prone sub-branch of post-replication repair and the associated translesion synthesis polymerases. To further understand the mutagenic response to DNA damage, we screened a collection of 4848 haploid gene deletion strains of Saccharomyces cerevisiae for decreased damage-induced mutation of the CAN1 gene. Through extensive quantitative validation of the strains identified by the screen, we identified ten genes, which included error-prone post-replication repair genes known to be involved in induced mutation, as well as two additional genes, FYV6 and RNR4. We demonstrate that FYV6 and RNR4 are epistatic with respect to induced mutation, and that they function, at least partially, independently of post-replication repair. This pathway of induced mutation appears to be mediated by an increase in dNTP levels that facilitates lesion bypass by the replicative polymerase Pol delta, and it is as important as error-prone post-replication repair in the case of UV- and MMS-induced mutation, but solely responsible for EMS-induced mutation. We show that Rnr4/Pol delta-induced mutation is efficiently inhibited by hydroxyurea, a small molecule inhibitor of ribonucleotide reductase, suggesting that if similar pathways exist in human cells, intervention in some forms of mutation may be possible.

  17. DNA transposon activity is associated with increased mutation rates in genes of rice and other grasses.

    Science.gov (United States)

    Wicker, Thomas; Yu, Yeisoo; Haberer, Georg; Mayer, Klaus F X; Marri, Pradeep Reddy; Rounsley, Steve; Chen, Mingsheng; Zuccolo, Andrea; Panaud, Olivier; Wing, Rod A; Roffler, Stefan

    2016-01-01

    DNA (class 2) transposons are mobile genetic elements which move within their 'host' genome through excising and re-inserting elsewhere. Although the rice genome contains tens of thousands of such elements, their actual role in evolution is still unclear. Analysing over 650 transposon polymorphisms in the rice species Oryza sativa and Oryza glaberrima, we find that DNA repair following transposon excisions is associated with an increased number of mutations in the sequences neighbouring the transposon. Indeed, the 3,000 bp flanking the excised transposons can contain over 10 times more mutations than the genome-wide average. Since DNA transposons preferably insert near genes, this is correlated with increases in mutation rates in coding sequences and regulatory regions. Most importantly, we find this phenomenon also in maize, wheat and barley. Thus, these findings suggest that DNA transposon activity is a major evolutionary force in grasses which provide the basis of most food consumed by humankind. PMID:27599761

  18. Mutation induction in Haemophilus influenzae by ICR-191 II. Role of DNA replication and repair

    Energy Technology Data Exchange (ETDEWEB)

    Kimball, R.F.; Perdue, S.W.

    1981-01-01

    Evidence is presented to show that presumptive frameshift mutations induced in Haemophilus influenzae by ICR-191 are fixed very repidly, essentially at the time of treatment. DNA synthesis during treatment is essential for fixation, but DNA synthesis after treatment has no effect. The conclusion is drawn that the mutagen acts at the replication fork, possibly to stabilize misannealings arising in association with the discontinuities in the newly synthesized DNA. (JMT)

  19. Aptaligner: Automated Software for Aligning Pseudorandom DNA X-Aptamers from Next-Generation Sequencing Data

    OpenAIRE

    Lu, Emily; Elizondo-Riojas, Miguel-Angel; Chang, Jeffrey T.; Volk, David E.

    2014-01-01

    Next-generation sequencing results from bead-based aptamer libraries have demonstrated that traditional DNA/RNA alignment software is insufficient. This is particularly true for X-aptamers containing specialty bases (W, X, Y, Z, ...) that are identified by special encoding. Thus, we sought an automated program that uses the inherent design scheme of bead-based X-aptamers to create a hypothetical reference library and Markov modeling techniques to provide improved alignments. Aptaligner provid...

  20. Somatic mutations in stilbene estrogen-induced Syrian hamster kidney tumors identified by DNA fingerprinting

    Directory of Open Access Journals (Sweden)

    Roy Deodutta

    2004-01-01

    Full Text Available Abstract Kidney tumors from stilbene estrogen (diethylstilbestrol-treated Syrian hamsters were screened for somatic genetic alterations by Random Amplified Polymorphic DNA-polymerase chain-reaction (RAPD-PCR fingerprinting. Fingerprints from tumor tissue were generated by single arbitrary primers and compared with fingerprints for normal tissue from the same animal, as well as normal and tumor tissues from different animals. Sixty one of the arbitrary primers amplified 365 loci that contain approximately 476 kbp of the hamster genome. Among these amplified DNA fragments, 44 loci exhibited either qualitative or quantitative differences between the tumor tissues and normal kidney tissues. RAPD-PCR loci showing decreased and increased intensities in tumor tissue DNA relative to control DNA indicate that loci have undergone allelic losses and gains, respectively, in the stilbene estrogen-induced tumor cell genome. The presence or absence of the amplified DNA fragments indicate homozygous insertions or deletions in the kidney tumor DNA compared to the age-matched normal kidney tissue DNA. Seven of 44 mutated loci also were present in the kidney tissues adjacent to tumors (free of macroscopic tumors. The presence of mutated loci in uninvolved (non-tumor surrounding tissue adjacent to tumors from stilbene estrogen-treated hamsters suggests that these mutations occurred in the early stages of carcinogenesis. The cloning and sequencing of RAPD amplified loci revealed that one mutated locus had significant sequence similarity with the hamster Cyp1A1 gene. The results show the ability of RAPD-PCR to detect and isolate, in a single step, DNA sequences representing genetic alterations in stilbene estrogen-induced cancer cells, including losses of heterozygosity, and homozygous deletion and insertion mutations. RAPD-PCR provides an alternative molecular approach for studying cancer cytogenetics in stilbene estrogen-induced tumors in humans and experimental

  1. Automated screening for small organic ligands using DNA-encoded chemical libraries.

    Science.gov (United States)

    Decurtins, Willy; Wichert, Moreno; Franzini, Raphael M; Buller, Fabian; Stravs, Michael A; Zhang, Yixin; Neri, Dario; Scheuermann, Jörg

    2016-04-01

    DNA-encoded chemical libraries (DECLs) are collections of organic compounds that are individually linked to different oligonucleotides, serving as amplifiable identification barcodes. As all compounds in the library can be identified by their DNA tags, they can be mixed and used in affinity-capture experiments on target proteins of interest. In this protocol, we describe the screening process that allows the identification of the few binding molecules within the multiplicity of library members. First, the automated affinity selection process physically isolates binding library members. Second, the DNA codes of the isolated binders are PCR-amplified and subjected to high-throughput DNA sequencing. Third, the obtained sequencing data are evaluated using a C++ program and the results are displayed using MATLAB software. The resulting selection fingerprints facilitate the discrimination of binding from nonbinding library members. The described procedures allow the identification of small organic ligands to biological targets from a DECL within 10 d. PMID:26985574

  2. Direct DNA isolation from solid biological sources without pretreatments with proteinase-K and/or homogenization through automated DNA extraction.

    Science.gov (United States)

    Ki, Jang-Seu; Chang, Ki Byum; Roh, Hee June; Lee, Bong Youb; Yoon, Joon Yong; Jang, Gi Young

    2007-03-01

    Genomic DNA from solid biomaterials was directly isolated with an automated DNA extractor, which was based on magnetic bead technology with a bore-mediated grinding (BMG) system. The movement of the bore broke down the solid biomaterials, mixed crude lysates thoroughly with reagents to isolate the DNA, and carried the beads to the next step. The BMG system was suitable for the mechanical homogenization of the solid biomaterials and valid as an automated system for purifying the DNA from the solid biomaterials without the need for pretreatment or disruption procedures prior to the application of the solid biomaterials.

  3. Increased transmission of mutations by low-condition females: evidence for condition-dependent DNA repair.

    Directory of Open Access Journals (Sweden)

    Aneil F Agrawal

    2008-02-01

    Full Text Available Evidence is mounting that mutation rates are sufficiently high for deleterious alleles to be a major evolutionary force affecting the evolution of sex, the maintenance of genetic variation, and many other evolutionary phenomena. Though point estimates of mutation rates are improving, we remain largely ignorant of the biological factors affecting these rates at the individual level. Of special importance is the possibility that mutation rates are condition-dependent with low-condition individuals experiencing more mutation. Theory predicts that such condition dependence would dramatically increase the rate at which populations adapt to new environments and the extent to which populations suffer from mutation load. Despite its importance, there has been little study of this phenomenon in multicellular organisms. Here, we examine whether DNA repair processes are condition-dependent in Drosophila melanogaster. In this species, damaged DNA in sperm can be repaired by maternal repair processes after fertilization. We exposed high- and low-condition females to sperm containing damaged DNA and then assessed the frequency of lethal mutations on paternally derived X chromosomes transmitted by these females. The rate of lethal mutations transmitted by low-condition females was 30% greater than that of high-condition females, indicating reduced repair capacity of low-condition females. A separate experiment provided no support for an alternative hypothesis based on sperm selection.

  4. Random mutagenesis strategies for construction of large and diverse clone libraries of mutated DNA fragments.

    Science.gov (United States)

    Chusacultanachai, Sudsanguan; Yuthavong, Yongyuth

    2004-01-01

    The first important step toward a successful preparation of large and diverse DNA libraries with desired complexity is to select a suitable mutagenesis strategy. This chapter describes three different methods for random mutagenesis, the use of XL1-red cells, error-prone polymerase chain reaction (PCR), and degenerate oligonucleotides-Pfu (DOP). These mutagenesis strategies possess different benefits and pitfalls; thus, they are differentially useful for production of DNA libraries with different density and complexity. The use of XL1-red, an engineered Escherichia coli with DNA repair deficiency, is one of the simplest mutagenesis and requires no subcloning step. After plasmid encoding DNA of inter-est is transformed into the cells, the mutations are simply generated during each round of DNA replication. The mutation frequency of this method is reported to be 1 base change per 2000 nucleotides; however, it can be slightly increased by extending the culture period to allow the accumulation of more mutations. This strategy is suitable for generation of random mutations with low frequency in a large target DNA. Error-prone PCR is one of the most widely used random mutagenesis. During DNA amplification, misincorporation of nucleotides can be promoted by altering the nucleotide ratio and the concentration of divalent cations in the reaction. We discovered that, by adjusting template concentration, frequency of mutation could be controlled easily and a library with desired mutation rate could be obtained. Additionally, efficiency of subsequent cloning steps to insert the PCR product into plasmid DNA is also a key factor determining size and complexity of the libraries. DOP mutagenesis is a rapid and effective method for random mutagenesis of small DNA and peptides. This strategy uses two chemically synthesized degenerate oligonucleotides as primers. By controlling the positions and ratios of degenerate nucleotides used during oligonucleotide synthesis, it is possible to

  5. Origins and functional consequences of somatic mitochondrial DNA mutations in human cancer.

    Science.gov (United States)

    Ju, Young Seok; Alexandrov, Ludmil B; Gerstung, Moritz; Martincorena, Inigo; Nik-Zainal, Serena; Ramakrishna, Manasa; Davies, Helen R; Papaemmanuil, Elli; Gundem, Gunes; Shlien, Adam; Bolli, Niccolo; Behjati, Sam; Tarpey, Patrick S; Nangalia, Jyoti; Massie, Charles E; Butler, Adam P; Teague, Jon W; Vassiliou, George S; Green, Anthony R; Du, Ming-Qing; Unnikrishnan, Ashwin; Pimanda, John E; Teh, Bin Tean; Munshi, Nikhil; Greaves, Mel; Vyas, Paresh; El-Naggar, Adel K; Santarius, Tom; Collins, V Peter; Grundy, Richard; Taylor, Jack A; Hayes, D Neil; Malkin, David; Foster, Christopher S; Warren, Anne Y; Whitaker, Hayley C; Brewer, Daniel; Eeles, Rosalind; Cooper, Colin; Neal, David; Visakorpi, Tapio; Isaacs, William B; Bova, G Steven; Flanagan, Adrienne M; Futreal, P Andrew; Lynch, Andy G; Chinnery, Patrick F; McDermott, Ultan; Stratton, Michael R; Campbell, Peter J

    2014-10-01

    Recent sequencing studies have extensively explored the somatic alterations present in the nuclear genomes of cancers. Although mitochondria control energy metabolism and apoptosis, the origins and impact of cancer-associated mutations in mtDNA are unclear. In this study, we analyzed somatic alterations in mtDNA from 1675 tumors. We identified 1907 somatic substitutions, which exhibited dramatic replicative strand bias, predominantly C > T and A > G on the mitochondrial heavy strand. This strand-asymmetric signature differs from those found in nuclear cancer genomes but matches the inferred germline process shaping primate mtDNA sequence content. A number of mtDNA mutations showed considerable heterogeneity across tumor types. Missense mutations were selectively neutral and often gradually drifted towards homoplasmy over time. In contrast, mutations resulting in protein truncation undergo negative selection and were almost exclusively heteroplasmic. Our findings indicate that the endogenous mutational mechanism has far greater impact than any other external mutagens in mitochondria and is fundamentally linked to mtDNA replication.

  6. X-ray induced mutations, DNA and target theory

    International Nuclear Information System (INIS)

    It is stated that X-ray induced, specific locus, germ-line mutation rates vary significantly in eukaryotes. A consideration of the radiobiology of the individual gene by the author leads to three major conclusions. (1) Mutation rates tend to be much lower than radiation theory predicts. (2) Selection and/or repair are major factors that determine the rates. (3) The mouse 7-locus test, which provides a principal data base for the standards of human radiation hygiene, may not provide adequate overall representation of the mutability of the mammalian genome, so more research is needed. (U.K.)

  7. DNA polymerase ι functions in the generation of tandem mutations during somatic hypermutation of antibody genes.

    Science.gov (United States)

    Maul, Robert W; MacCarthy, Thomas; Frank, Ekaterina G; Donigan, Katherine A; McLenigan, Mary P; Yang, William; Saribasak, Huseyin; Huston, Donald E; Lange, Sabine S; Woodgate, Roger; Gearhart, Patricia J

    2016-08-22

    DNA polymerase ι (Pol ι) is an attractive candidate for somatic hypermutation in antibody genes because of its low fidelity. To identify a role for Pol ι, we analyzed mutations in two strains of mice with deficiencies in the enzyme: 129 mice with negligible expression of truncated Pol ι, and knock-in mice that express full-length Pol ι that is catalytically inactive. Both strains had normal frequencies and spectra of mutations in the variable region, indicating that loss of Pol ι did not change overall mutagenesis. We next examined if Pol ι affected tandem mutations generated by another error-prone polymerase, Pol ζ. The frequency of contiguous mutations was analyzed using a novel computational model to determine if they occur during a single DNA transaction or during two independent events. Analyses of 2,000 mutations from both strains indicated that Pol ι-compromised mice lost the tandem signature, whereas C57BL/6 mice accumulated significant amounts of double mutations. The results support a model where Pol ι occasionally accesses the replication fork to generate a first mutation, and Pol ζ extends the mismatch with a second mutation. PMID:27455952

  8. Selenium supplementation reduced oxidative DNA damage in adnexectomized BRCA1 mutations carriers.

    Science.gov (United States)

    Dziaman, Tomasz; Huzarski, Tomasz; Gackowski, Daniel; Rozalski, Rafal; Siomek, Agnieszka; Szpila, Anna; Guz, Jolanta; Lubinski, Jan; Wasowicz, Wojciech; Roszkowski, Krzysztof; Olinski, Ryszard

    2009-11-01

    Some experimental evidence suggests that BRCA1 plays a role in repair of oxidative DNA damage. Selenium has anticancer properties that are linked with protection against oxidative stress. To assess whether supplementation of BRCA1 mutation carriers with selenium have a beneficial effect concerning oxidative stress/DNA damage in the present double-blinded placebo control study, we determined 8-oxodG level in cellular DNA and urinary excretion of 8-oxodG and 8-oxoGua in the mutation carriers. We found that 8-oxodG level in leukocytes DNA is significantly higher in BRCA1 mutation carriers. In the distinct subpopulation of BRCA1 mutation carriers without symptoms of cancer who underwent adnexectomy and were supplemented with selenium, the level of 8-oxodG in DNA decreased significantly in comparison with the subgroup without supplementation. Simultaneously in the same group, an increase of urinary 8-oxoGua, the product of base excision repair (hOGG1 glycosylase), was observed. Therefore, it is likely that the selenium supplementation of the patients is responsible for the increase of BER enzymes activities, which in turn may result in reduction of oxidative DNA damage. Importantly, in a double-blinded placebo control prospective study, it was shown that in the same patient groups, reduction in cancer incidents was observed. Altogether, these results suggest that BRCA1 deficiency contributes to 8-oxodG accumulation in cellular DNA, which in turn may be a factor responsible for cancer development in women with mutations, and that the risk to developed breast cancer in BRCA1 mutation carriers may be reduced in selenium-supplemented patients who underwent adnexectomy. PMID:19843683

  9. DNA methylation patterns of candidate genes regulated by thymine DNA glycosylase in patients with TP53 germline mutations

    Energy Technology Data Exchange (ETDEWEB)

    Fortes, F.P. [CIPE, Laboratrio de Oncogentica Molecular, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Kuasne, H. [CIPE, Laboratrio NeoGene, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Departamento de Urologia, Faculdade de Medicina, Universidade Estadual Paulista, Botucatu, SP (Brazil); Marchi, F.A. [CIPE, Laboratrio NeoGene, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Programa Inter-Institucional em Bioinformtica, Instituto de Matemtica e Estatstica, Universidade So Paulo, So Paulo, SP (Brazil); Miranda, P.M. [CIPE, Laboratrio NeoGene, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Rogatto, S.R. [CIPE, Laboratrio NeoGene, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Departamento de Urologia, Faculdade de Medicina, Universidade Estadual Paulista, Botucatu, SP (Brazil); Achatz, M.I. [CIPE, Laboratrio de Oncogentica Molecular, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Departamento de Oncogentica, A.C. Camargo Cancer Center, So Paulo, SP (Brazil)

    2015-04-28

    Li-Fraumeni syndrome (LFS) is a rare, autosomal dominant, hereditary cancer predisposition disorder. In Brazil, the p.R337H TP53 founder mutation causes the variant form of LFS, Li-Fraumeni-like syndrome. The occurrence of cancer and age of disease onset are known to vary, even in patients carrying the same mutation, and several mechanisms such as genetic and epigenetic alterations may be involved in this variability. However, the extent of involvement of such events has not been clarified. It is well established that p53 regulates several pathways, including the thymine DNA glycosylase (TDG) pathway, which regulates the DNA methylation of several genes. This study aimed to identify the DNA methylation pattern of genes potentially related to the TDG pathway (CDKN2A, FOXA1, HOXD8, OCT4, SOX2, and SOX17) in 30 patients with germline TP53mutations, 10 patients with wild-type TP53, and 10 healthy individuals. We also evaluated TDG expression in patients with adrenocortical tumors (ADR) with and without the p.R337H TP53 mutation. Gene methylation patterns of peripheral blood DNA samples assessed by pyrosequencing revealed no significant differences between the three groups. However, increased TDG expression was observed by quantitative reverse transcription PCR in p.R337H carriers with ADR. Considering the rarity of this phenotype and the relevance of these findings, further studies using a larger sample set are necessary to confirm our results.

  10. DNA methylation patterns of candidate genes regulated by thymine DNA glycosylase in patients with TP53 germline mutations

    International Nuclear Information System (INIS)

    Li-Fraumeni syndrome (LFS) is a rare, autosomal dominant, hereditary cancer predisposition disorder. In Brazil, the p.R337H TP53 founder mutation causes the variant form of LFS, Li-Fraumeni-like syndrome. The occurrence of cancer and age of disease onset are known to vary, even in patients carrying the same mutation, and several mechanisms such as genetic and epigenetic alterations may be involved in this variability. However, the extent of involvement of such events has not been clarified. It is well established that p53 regulates several pathways, including the thymine DNA glycosylase (TDG) pathway, which regulates the DNA methylation of several genes. This study aimed to identify the DNA methylation pattern of genes potentially related to the TDG pathway (CDKN2A, FOXA1, HOXD8, OCT4, SOX2, and SOX17) in 30 patients with germline TP53mutations, 10 patients with wild-type TP53, and 10 healthy individuals. We also evaluated TDG expression in patients with adrenocortical tumors (ADR) with and without the p.R337H TP53 mutation. Gene methylation patterns of peripheral blood DNA samples assessed by pyrosequencing revealed no significant differences between the three groups. However, increased TDG expression was observed by quantitative reverse transcription PCR in p.R337H carriers with ADR. Considering the rarity of this phenotype and the relevance of these findings, further studies using a larger sample set are necessary to confirm our results

  11. Establishing a novel automated magnetic bead-based method for the extraction of DNA from a variety of forensic samples.

    Science.gov (United States)

    Witt, Sebastian; Neumann, Jan; Zierdt, Holger; Gébel, Gabriella; Röscheisen, Christiane

    2012-09-01

    Automated systems have been increasingly utilized for DNA extraction by many forensic laboratories to handle growing numbers of forensic casework samples while minimizing the risk of human errors and assuring high reproducibility. The step towards automation however is not easy: The automated extraction method has to be very versatile to reliably prepare high yields of pure genomic DNA from a broad variety of sample types on different carrier materials. To prevent possible cross-contamination of samples or the loss of DNA, the components of the kit have to be designed in a way that allows for the automated handling of the samples with no manual intervention necessary. DNA extraction using paramagnetic particles coated with a DNA-binding surface is predestined for an automated approach. For this study, we tested different DNA extraction kits using DNA-binding paramagnetic particles with regard to DNA yield and handling by a Freedom EVO(®)150 extraction robot (Tecan) equipped with a Te-MagS magnetic separator. Among others, the extraction kits tested were the ChargeSwitch(®)Forensic DNA Purification Kit (Invitrogen), the PrepFiler™Automated Forensic DNA Extraction Kit (Applied Biosystems) and NucleoMag™96 Trace (Macherey-Nagel). After an extensive test phase, we established a novel magnetic bead extraction method based upon the NucleoMag™ extraction kit (Macherey-Nagel). The new method is readily automatable and produces high yields of DNA from different sample types (blood, saliva, sperm, contact stains) on various substrates (filter paper, swabs, cigarette butts) with no evidence of a loss of magnetic beads or sample cross-contamination.

  12. Mitochondrial DNA mutations affect calcium handling in differentiated neurons.

    NARCIS (Netherlands)

    Trevelyan, A.J.; Kirby, D.M.; Smulders-Srinivasan, T.K.; Nooteboom, M.; Acin-Perez, R.; Enriquez, J.A.; Whittington, M.A.; Lightowlers, R.N.; Turnbull, D.M.

    2010-01-01

    Mutations in the mitochondrial genome are associated with a wide range of neurological symptoms, but many aspects of the basic neuronal pathology are not understood. One candidate mechanism, given the well-established role of mitochondria in calcium buffering, is a deficit in neuronal calcium homoeo

  13. The impact of SF3B1 mutations in CLL on the DNA-damage response

    DEFF Research Database (Denmark)

    Te Raa, G D; Derks, I A M; Navrkalova, V;

    2015-01-01

    Mutations or deletions in TP53 or ATM are well-known determinants of poor prognosis in chronic lymphocytic leukemia (CLL), but only account for approximately 40% of chemo-resistant patients. Genome-wide sequencing has uncovered novel mutations in the splicing factor sf3b1, that were in part...... associated with ATM aberrations, suggesting functional synergy. We first performed detailed genetic analyses in a CLL cohort (n=110) containing ATM, SF3B1 and TP53 gene defects. Next, we applied a newly developed multiplex assay for p53/ATM target gene induction and measured apoptotic responses to DNA damage....... Interestingly, SF3B1 mutated samples without concurrent ATM and TP53 aberrations (sole SF3B1) displayed partially defective ATM/p53 transcriptional and apoptotic responses to various DNA-damaging regimens. In contrast, NOTCH1 or K/N-RAS mutated CLL displayed normal responses in p53/ATM target gene induction...

  14. Hair Dye–DNA Interaction: Plausible Cause of Mutation

    OpenAIRE

    Swati Maiti; Sudarson Sekhar Sinha; Mukesh Singh

    2015-01-01

    Hair dye is one of the most popular cosmetic products which are used more widely and frequently to improve an individual’s appearance. Although the genotoxic effects of dye ingredients are widely reported, hair dye in its usable form is not reported extensively. In this contribution, we report the possible mode of interaction of hair dye with DNA which leads to genotoxicity. The effect of dye DNA interaction was studied on the most popular and globally used hair dye with Calf Thymus DNA and p...

  15. Response to: DNA identification by pedigree likelihood ratio accommodating population substructure and mutations

    Directory of Open Access Journals (Sweden)

    Egeland Thore

    2011-03-01

    Full Text Available Abstract Mutation models are important in many areas of genetics including forensics. This letter criticizes the model of the paper 'DNA identification by pedigree likelihood ratio accommodating population substructure and mutations' by Ge et al. (2010. Furthermore, we argue that the paper in some cases misrepresents previously published papers. Please see related letter: http://www.investigativegenetics.com/content/2/1/8.

  16. New mutation detection system of repackaged λ gt11 DNA containing LacZ gene

    Institute of Scientific and Technical Information of China (English)

    LIU Yong; CAO Jia; WU Tao; YANG Lu-jun; SUN Hua-ming; YANG Ming-jie; QIAN Ping

    2002-01-01

    Objective: To establish a reformative detection system which has sound ability of providing information on molecular mutagenesis spectrum and the specificity of detection system of repackaged λ phage.Methods: LacZ gene, as mutational target gene and reporter gene, was applied into the detection system.The λ gt11 DNA treated with ENU (1-ethyl-1-nitrosourea) and 9-AA (9-aminoacridine) was repackaged in vitro. The packaged λ phage was then grown in E. coli Y1090 on a selective plate containing X-gel and IPTG. The survival and mutation frequencies were determined by counting the clear-plaque and blue-plaque,and the molecular mutation mechanism was studied by extracting and sequencing the LacZ gene of mutants.Results: The survival of repackaged λ phages treated with 9-AA and ENU apparently decreased in consistent dose-dependence. The mutation frequency of clear-plaque mutants showed a linear dose-related increase. The predominant mutations induced by 9-AA were ±1 frameshift mutation, and 9-AA induced -1 frameshift was much more effective than induced + 1 frameshift. 9-AA also induced substitutions with transversions more common. ENU-induced mutations were chiefly occurred at G: C sites. Substitutions induced by ENU were mainly G: C→A: T, G: C→C: G and A: T→T: A transversion. Conclusion: Mutation detection system of λgt11 DNA containing LacZ gene is proven better than that of λDNA without LacZ gene. The combination of survival, mutant frequency and sequence spectrum can not only increase the sensitivity and specificity of the new method, but also provide a better understanding of the molecular mechanism of mutation for ultimate extrapolation to risk assessment.

  17. Limited clinical relevance of mitochondrial DNA mutation and gene expression analyses in ovarian cancer

    Directory of Open Access Journals (Sweden)

    Rachinger Andrea

    2008-10-01

    Full Text Available Abstract Background In recent years, numerous studies have investigated somatic mutations in mitochondrial DNA in various tumours. The observed high mutation rates might reflect mitochondrial deregulation; consequently, mutation analyses could be clinically relevant. The purpose of this study was to determine if mutations in the mitochondrial D-loop region and/or the level of mitochondrial gene expression could influence the clinical course of human ovarian carcinomas. Methods We sequenced a 1320-base-pair DNA fragment of the mitochondrial genome (position 16,000-750 in 54 cancer samples and in 44 corresponding germline control samples. In addition, six transcripts (MT-ATP6, MT-CO1, MT-CYB, MT-ND1, MT-ND6, and MT-RNR1 were quantified in 62 cancer tissues by real-time RT-PCR. Results Somatic mutations in the D-loop sequence were found in 57% of ovarian cancers. Univariate analysis showed no association between mitochondrial DNA mutation status or mitochondrial gene expression and any of the examined clinicopathologic parameters. A multivariate logistic regression model revealed that the expression of the mitochondrial gene RNR1 might be used as a predictor of tumour sensitivity to chemotherapy. Conclusion In contrast to many previously published papers, our study indicates rather limited clinical relevance of mitochondrial molecular analyses in ovarian carcinomas. These discrepancies in the clinical utility of mitochondrial molecular tests in ovarian cancer require additional large, well-designed validation studies.

  18. A new mtDNA mutation showing accumulation with time and restriction to skeletal muscle

    Energy Technology Data Exchange (ETDEWEB)

    Weber, K.; Wilson, J.N.; Taylor, L. [Middlesbrough General Hospital (United Kingdom)] [and others

    1997-02-01

    We have identified a new mutation in mtDNA, involving tRNA{sup Leu(CUN)} in a patient manifesting an isolated skeletal myopathy. This heteroplasmic A{r_arrow}G transition at position 12320 affects the T{Psi}C loop at a conserved site and was not found in 120 controls. Analysis of cultured fibroblasts, white blood cells/platelets, and skeletal muscle showed that only skeletal muscle contained the mutation and that only this tissue demonstrated a biochemical defect of respiratory-chain activity. In a series of four muscle-biopsy specimens taken over a 12-year period, there was a gradual increase, from 70% to 90%, in the overall level of mutation, as well as a marked clinical deterioration. Single-fiber PCR confirmed that the proportion of mutant mtDNA was highest in cytochrome c oxidase-negative fibers. This study, which reports a mutation involving tRNA{sup Leu(CUN)}, demonstrates clearly that mtDNA point mutations can accumulate over time and may be restricted in their tissue distribution. Furthermore, clinical deterioration seemed to follow the increase in the level of mutation, although, interestingly, the appearance of fibers deficient in respiratory-chain activity showed a lag period. 32 refs., 4 figs., 1 tab.

  19. Chemical cleavage reactions of DNA on solid support: application in mutation detection

    Directory of Open Access Journals (Sweden)

    Cotton Richard GH

    2003-05-01

    Full Text Available Abstract Background The conventional solution-phase Chemical Cleavage of Mismatch (CCM method is time-consuming, as the protocol requires purification of DNA after each reaction step. This paper describes a new version of CCM to overcome this problem by immobilizing DNA on silica solid supports. Results DNA test samples were loaded on to silica beads and the DNA bound to the solid supports underwent chemical modification reactions with KMnO4 (potassium permanganate and hydroxylamine in 3M TEAC (tetraethylammonium chloride solution. The resulting modified DNA was then simultaneously cleaved by piperidine and removed from the solid supports to afford DNA fragments without the requirement of DNA purification between reaction steps. Conclusions The new solid-phase version of CCM is a fast, cost-effective and sensitive method for detection of mismatches and mutations.

  20. Frequent mtDNA mutations and its role in gastric carcino-genesis

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    In order to disclose the relationship between mutations of mitochondrial DNA (mtDNA) and gastric carcinogenesis, we screened the entire mtDNA sequence in 30 cases of human gastric cancer and matched normal tissues by using denaturing high-performance liquid chromatography (DHPLC) and DNA sequencing. Our data showed that high frequency (66.7%, 20/30) of mitochondrial genome mutation occur red in gastric cancer. Among these variants, 17 cases (56.7%, 17/30) were identified to be somatic mutation. High level mutant frequency was found in ND4, ND5 c oding genes and D-loop control region, which was 36.7%, 26.7% and 30% respective ly. Comparing with complexes Ⅲ, Ⅳ and Ⅴof the electron transport chain, we found that variants appeared to be more frequent in the subunit genes of complexⅠ . Most of mutations were base substitutions (85.4%, 41/48). Our results suggested that mutations of subunit genes encoding complex Ⅰ, especially ND3, ND4 and N D5 genes, might contribute to human gastric carcinogenesis.

  1. Identification of DNA polymerase molecules repairing DNA irradiated damage and molecular biological study on modified factors of mutation rate

    International Nuclear Information System (INIS)

    To explain the development mechanism of mutation by radiation, DNA polymerase molecules repairing DNA should be identified. In this study, plasmid was constructed in order to express anti sense DNA of DNA polymerase in the cell and it was introduced into the cell by the calcium phosphate method. Polyclonal antibody of DNA polymerase δ and ε were produced so as to prove no existence of specific polymerase molecules in the cell. When center part of polymerase ε was immunized, antiserum with high antibody titer was obtained. Near terminal C of polymerase δ was immunized, then antiserum was obtained. We discovered very interesting fact that base sequence of polymerase ε published by Syvaoja was not correct. (S.Y.)

  2. Evolutionary constraints and the neutral theory. [mutation-caused nucleotide substitutions in DNA

    Science.gov (United States)

    Jukes, T. H.; Kimura, M.

    1984-01-01

    The neutral theory of molecular evolution postulates that nucleotide substitutions inherently take place in DNA as a result of point mutations followed by random genetic drift. In the absence of selective constraints, the substitution rate reaches the maximum value set by the mutation rate. The rate in globin pseudogenes is about 5 x 10 to the -9th substitutions per site per year in mammals. Rates slower than this indicate the presence of constraints imposed by negative (natural) selection, which rejects and discards deleterious mutations.

  3. Clustering of Caucasian Leber hereditary optic neuropathy patients containing the 11778 or 14484 mutations on an mtDNA lineage

    Energy Technology Data Exchange (ETDEWEB)

    Brown, M.D.; Sun, F.; Wallace, D.C. [Emory Univ. School of Medicine, Atlanta, GA (United States)

    1997-02-01

    Leber hereditary optic neuropathy (LHON) is a type of blindness caused by mtDNA mutations. Three LHON mtDNA mutations at nucleotide positions 3460, 11778, and 14484 are specific for LHON and account for 90% of worldwide cases and are thus designated as {open_quotes}primary{close_quotes} LHON mutations. Fifteen other {open_quotes}secondary{close_quotes} LHON mtDNA mutations have been identified, but their pathogenicity is unclear. mtDNA haplotype and phylogenetic analysis of the primary LHON mutations in North American Caucasian patients and controls has shown that, unlike the 3460 and 11778 mutations, which are distributed throughout the European-derived (Caucasian) mtDNA phylogeny, patients containing the 14484 mutation tended to be associated with European mtDNA haplotype J. To investigate this apparent clustering, we performed {chi}{sup 2}-based statistical analyses to compare the distribution of LHON patients on the Caucasian phylogenetic tree. Our results indicate that, unlike the 3460 and 11778 mutations, the 14484 mutation was not distributed on the phylogeny in proportion to the frequencies of the major Caucasian mtDNA haplogroups found in North America. The 14484 mutation was next shown to occur on the haplogroup J background more frequently that expected, consistent with the observation that {approximately}75% of worldwide 14484-positive LHON patients occur in association with haplogroup J. The 11778 mutation also exhibited a moderate clustering on haplogroup J. These observations were supported by statistical analysis using all available mutation frequencies reported in the literature. This paper thus illustrates the potential importance of genetic background in certain mtDNA-based diseases, speculates on a pathogenic role for a subset of LHON secondary mutations and their interaction with primary mutations, and provides support for a polygenic model for LHON expression in some cases. 18 refs., 3 tabs.

  4. Deficiency of the DNA repair protein nibrin increases the basal but not the radiation induced mutation frequency in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Wessendorf, Petra [Institute of Medical and Human Genetics, Charité – Universitätsmedizin Berlin, Augustenburger Platz 1, D-13353 Berlin (Germany); Vijg, Jan [Albert Einstein College of Medicine, Michael F. Price Center, 1301 Morris Park Avenue, Bronx, NY 10461 (United States); Nussenzweig, André [Laboratory of Genome Integrity, National Cancer Institute, National Institute of Health, 37 Convent Drive, Room 1106, Bethesda, MD 20892 (United States); Digweed, Martin, E-mail: martin.digweed@charite.de [Institute of Medical and Human Genetics, Charité – Universitätsmedizin Berlin, Augustenburger Platz 1, D-13353 Berlin (Germany)

    2014-11-15

    Highlights: • lacZ mutant frequencies measured in vivo in mouse models of radiosensitive Nijmegen Breakage Syndrome. • Spontaneous mutation frequencies are increased in lymphatic tissue due to Nbn mutation. • Single base transitions, not deletions, dominate the mutation spectrum. • Radiation induced mutation frequencies are not increased due to Nbn mutation. - Abstract: Nibrin (NBN) is a member of a DNA repair complex together with MRE11 and RAD50. The complex is associated particularly with the repair of DNA double strand breaks and with the regulation of cell cycle check points. Hypomorphic mutation of components of the complex leads to human disorders characterised by radiosensitivity and increased tumour occurrence, particularly of the lymphatic system. We have examined here the relationship between DNA damage, mutation frequency and mutation spectrum in vitro and in vivo in mouse models carrying NBN mutations and a lacZ reporter plasmid. We find that NBN mutation leads to increased spontaneous DNA damage in fibroblasts in vitro and high basal mutation rates in lymphatic tissue of mice in vivo. The characteristic mutation spectrum is dominated by single base transitions rather than the deletions and complex rearrangements expected after abortive repair of DNA double strand breaks. We conclude that in the absence of wild type nibrin, the repair of spontaneous errors, presumably arising during DNA replication, makes a major contribution to the basal mutation rate. This applies also to cells heterozygous for an NBN null mutation. Mutation frequencies after irradiation in vivo were not increased in mice with nibrin mutations as might have been expected considering the radiosensitivity of NBS patient cells in vitro. Evidently apoptosis is efficient, even in the absence of wild type nibrin.

  5. Deficiency of the DNA repair protein nibrin increases the basal but not the radiation induced mutation frequency in vivo

    International Nuclear Information System (INIS)

    Highlights: • lacZ mutant frequencies measured in vivo in mouse models of radiosensitive Nijmegen Breakage Syndrome. • Spontaneous mutation frequencies are increased in lymphatic tissue due to Nbn mutation. • Single base transitions, not deletions, dominate the mutation spectrum. • Radiation induced mutation frequencies are not increased due to Nbn mutation. - Abstract: Nibrin (NBN) is a member of a DNA repair complex together with MRE11 and RAD50. The complex is associated particularly with the repair of DNA double strand breaks and with the regulation of cell cycle check points. Hypomorphic mutation of components of the complex leads to human disorders characterised by radiosensitivity and increased tumour occurrence, particularly of the lymphatic system. We have examined here the relationship between DNA damage, mutation frequency and mutation spectrum in vitro and in vivo in mouse models carrying NBN mutations and a lacZ reporter plasmid. We find that NBN mutation leads to increased spontaneous DNA damage in fibroblasts in vitro and high basal mutation rates in lymphatic tissue of mice in vivo. The characteristic mutation spectrum is dominated by single base transitions rather than the deletions and complex rearrangements expected after abortive repair of DNA double strand breaks. We conclude that in the absence of wild type nibrin, the repair of spontaneous errors, presumably arising during DNA replication, makes a major contribution to the basal mutation rate. This applies also to cells heterozygous for an NBN null mutation. Mutation frequencies after irradiation in vivo were not increased in mice with nibrin mutations as might have been expected considering the radiosensitivity of NBS patient cells in vitro. Evidently apoptosis is efficient, even in the absence of wild type nibrin

  6. Discovery of rare mutations in extensively pooled DNA samples using multiple target enrichment.

    Science.gov (United States)

    Chi, Xu; Zhang, Yingchun; Xue, Zheyong; Feng, Laibao; Liu, Huaqing; Wang, Feng; Qi, Xiaoquan

    2014-08-01

    Chemical mutagenesis is routinely used to create large numbers of rare mutations in plant and animal populations, which can be subsequently subjected to selection for beneficial traits and phenotypes that enable the characterization of gene functions. Several next-generation sequencing (NGS)-based target enrichment methods have been developed for the detection of mutations in target DNA regions. However, most of these methods aim to sequence a large number of target regions from a small number of individuals. Here, we demonstrate an effective and affordable strategy for the discovery of rare mutations in a large sodium azide-induced mutant rice population (F2 ). The integration of multiplex, semi-nested PCR combined with NGS library construction allowed for the amplification of multiple target DNA fragments for sequencing. The 8 × 8 × 8 tridimensional DNA sample pooling strategy enabled us to obtain DNA sequences of 512 individuals while only sequencing 24 samples. A stepwise filtering procedure was then elaborated to eliminate most of the false positives expected to arise through sequencing error, and the application of a simple Student's t-test against position-prone error allowed for the discovery of 16 mutations from 36 enriched targeted DNA fragments of 1024 mutagenized rice plants, all without any false calls.

  7. Development of an Automated Microfluidic System for DNA Collection, Amplification, and Detection of Pathogens

    Energy Technology Data Exchange (ETDEWEB)

    Hagan, Bethany S.; Bruckner-Lea, Cynthia J.

    2002-12-01

    This project was focused on developing and testing automated routines for a microfluidic Pathogen Detection System. The basic pathogen detection routine has three primary components; cell concentration, DNA amplification, and detection. In cell concentration, magnetic beads are held in a flow cell by an electromagnet. Sample liquid is passed through the flow cell and bacterial cells attach to the beads. These beads are then released into a small volume of fluid and delivered to the peltier device for cell lysis and DNA amplification. The cells are lysed during initial heating in the peltier device, and the released DNA is amplified using polymerase chain reaction (PCR) or strand displacement amplification (SDA). Once amplified, the DNA is then delivered to a laser induced fluorescence detection unit in which the sample is detected. These three components create a flexible platform that can be used for pathogen detection in liquid and sediment samples. Future developments of the system will include on-line DNA detection during DNA amplification and improved capture and release methods for the magnetic beads during cell concentration.

  8. Automated Line Tracking of lambda-DNA for Single-Molecule Imaging

    CERN Document Server

    Guan, Juan; Granick, Steve

    2011-01-01

    We describe a straightforward, automated line tracking method to visualize within optical resolution the contour of linear macromolecules as they rearrange shape as a function of time by Brownian diffusion and under external fields such as electrophoresis. Three sequential stages of analysis underpin this method: first, "feature finding" to discriminate signal from noise; second, "line tracking" to approximate those shapes as lines; third, "temporal consistency check" to discriminate reasonable from unreasonable fitted conformations in the time domain. The automated nature of this data analysis makes it straightforward to accumulate vast quantities of data while excluding the unreliable parts of it. We implement the analysis on fluorescence images of lambda-DNA molecules in agarose gel to demonstrate its capability to produce large datasets for subsequent statistical analysis.

  9. Rapid DNA analysis for automated processing and interpretation of low DNA content samples

    OpenAIRE

    Turingan, Rosemary S.; Vasantgadkar, Sameer; Palombo, Luke; Hogan, Catherine; Jiang, Hua; Tan, Eugene; Selden, Richard F.

    2016-01-01

    Background Short tandem repeat (STR) analysis of casework samples with low DNA content include those resulting from the transfer of epithelial cells from the skin to an object (e.g., cells on a water bottle, or brim of a cap), blood spatter stains, and small bone and tissue fragments. Low DNA content (LDC) samples are important in a wide range of settings, including disaster response teams to assist in victim identification and family reunification, military operations to identify friend or f...

  10. Branch migration displacement assay with automated heuristic analysis for discrete DNA length measurement using DNA microarrays

    OpenAIRE

    Pourmand, Nader; Caramuta, Stefano; Villablanca, Andrea; Mori, Silvia; Karhanek, Miloslav; Wang, Shan X.; Ronald W Davis

    2007-01-01

    The analysis of short tandem repeats (STRs) plays an important role in forensic science, human identification, genetic mapping, and disease diagnostics. Traditional STR analysis utilizes gel- or column-based approaches to analyze DNA repeats. Individual STR alleles are separated and distinguished according to fragment length; thus the assay is generally hampered by its low multiplex capacity. However, use of DNA microarray would employ a simple hybridization and detection for field forensics ...

  11. Primer effect in the detection of mitochondrial DNA point heteroplasmy by automated sequencing.

    Science.gov (United States)

    Calatayud, Marta; Ramos, Amanda; Santos, Cristina; Aluja, Maria Pilar

    2013-06-01

    The correct detection of mitochondrial DNA (mtDNA) heteroplasmy by automated sequencing presents methodological constraints. The main goals of this study are to investigate the effect of sense and distance of primers in heteroplasmy detection and to test if there are differences in the accurate determination of heteroplasmy involving transitions or transversions. A gradient of the heteroplasmy levels was generated for mtDNA positions 9477 (transition G/A) and 15,452 (transversion C/A). Amplification and subsequent sequencing with forward and reverse primers, situated at 550 and 150 bp from the heteroplasmic positions, were performed. Our data provide evidence that there is a significant difference between the use of forward and reverse primers. The forward primer is the primer that seems to give a better approximation to the real proportion of the variants. No significant differences were found concerning the distance at which the sequencing primers were placed neither between the analysis of transitions and transversions. The data collected in this study are a starting point that allows to glimpse the importance of the sequencing primers in the accurate detection of point heteroplasmy, providing additional insight into the overall automated sequencing strategy.

  12. Detection of HIV cDNA Point Mutations with Rolling-Circle Amplification Arrays

    OpenAIRE

    Zhongwei Wu; Zuhong Lu; Quanjun Liu; Lingwei Wu

    2010-01-01

    In this paper we describe an isothermal rolling-circle amplification (RCA) protocol to detect gene point mutations on chips. The method is based on an allele-specific oligonucleotide circularization mediated by a special DNA ligase. The probe is circularized when perfect complementary sequences between the probe oligonucleotide and HIV cDNA gene. Mismatches around the ligation site can prevent probe circularization. The circularized probe (C-probe) can be amplified by rolling circle amplifica...

  13. DNA transposons and the role of recombination in mutation accumulation in Daphnia pulex

    OpenAIRE

    Schaack, Sarah; Choi, Eunjin; Lynch, Michael; Pritham, Ellen J

    2010-01-01

    Background We identify DNA transposons from the completed draft genome sequence of Daphnia pulex, a cyclically parthenogenetic, aquatic microcrustacean of the class Branchiopoda. In addition, we experimentally quantify the abundance of six DNA transposon families in mutation-accumulation lines in which sex is either promoted or prohibited in order to better understand the role of recombination in transposon proliferation. Results We identified 55 families belonging to 10 of the known superfam...

  14. Both microsatellite length and sequence context determine frameshift mutation rates in defective DNA mismatch repair.

    Science.gov (United States)

    Chung, Heekyung; Lopez, Claudia G; Holmstrom, Joy; Young, Dennis J; Lai, Jenny F; Ream-Robinson, Deena; Carethers, John M

    2010-07-01

    It is generally accepted that longer microsatellites mutate more frequently in defective DNA mismatch repair (MMR) than shorter microsatellites. Indeed, we have previously observed that the A10 microsatellite of transforming growth factor beta type II receptor (TGFBR2) frameshifts -1 bp at a faster rate than the A8 microsatellite of activin type II receptor (ACVR2), although both genes become frameshift-mutated in >80% of MMR-defective colorectal cancers. To experimentally determine the effect of microsatellite length upon frameshift mutation in gene-specific sequence contexts, we altered the microsatellite length within TGFBR2 exon 3 and ACVR2 exon 10, generating A7, A10 and A13 constructs. These constructs were cloned 1 bp out of frame of EGFP, allowing a -1 bp frameshift to drive EGFP expression, and stably transfected into MMR-deficient cells. Subsequent non-fluorescent cells were sorted, cultured for 7-35 days and harvested for EGFP analysis and DNA sequencing. Longer microsatellites within TGFBR2 and ACVR2 showed significantly higher mutation rates than shorter ones, with TGFBR2 A13, A10 and A7 frameshifts measured at 22.38x10(-4), 2.17x10(-4) and 0.13x10(-4), respectively. Surprisingly, shorter ACVR2 constructs showed three times higher mutation rates at A7 and A10 lengths than identical length TGFBR2 constructs but comparably lower at the A13 length, suggesting influences from both microsatellite length as well as the sequence context. Furthermore, the TGFBR2 A13 construct mutated into 33% A11 sequences (-2 bp) in addition to expected A12 (-1 bp), indicating that this construct undergoes continual subsequent frameshift mutation. These data demonstrate experimentally that both the length of a mononucleotide microsatellite and its sequence context influence mutation rate in defective DNA MMR.

  15. Mutation in D-loop region of mitochondrial DNA in gastric cancer and its significance

    Institute of Scientific and Technical Information of China (English)

    Yi-Bing Zhao; Hong-Yu Yang; Xi-Wei Zhang; Guo-Yu Chen

    2005-01-01

    AIM: lo investigate the mutation in D-loop region of mitochondrial DNA in gastric cancer and its influence on the changes of reactive oxygen species (ROS) and cell cycle. METHODS: The D-loop region was amplified by PCR and sequenced. Reactive oxygen species and cell cycle were detected by flow cytometry in 20 specimens from gastriccancer and adjacent normal tissues. According to the sequence results, gastric cancer tissue was divided into mutation group and control group. Reactive oxygen species, apoptosis and proliferation in the two groups were compared.RESULTS: Among the 20 gastric cancer specimens, 18 mutations were identified in 7 patients, the mutation rate being 35%. There were four microsatellite instabilities in the mutations. No mutation was found in the adjacent tissues. Reactive oxygen species, apoptosis, and proliferation in the mutation group were all significantly higher than those in control group.CONCLUSION: Mutation in D-loop region plays a role in the genesis and development of gastric cancer.

  16. A novel class of mutations that affect DNA replication in E. coli

    DEFF Research Database (Denmark)

    Nordman, Jared; Skovgaard, Ole; Wright, Andrew

    2007-01-01

    Over-initiation of DNA replication in cells containing the cold-sensitive dnaA(cos) allele has been shown to lead to extensive DNA damage, potentially due to head-to-tail replication fork collisions that ultimately lead to replication fork collapse, growth stasis and/or cell death. Based on the...... assumption that suppressors of the cold-sensitive phenotype of the cos mutant should include mutations that affect the efficiency and/or regulation of DNA replication, we subjected a dnaA(cos) mutant strain to transposon mutagenesis and selected mutant derivatives that could form colonies at 30°C. Four...... suppressors of the dnaA(cos)-mediated cold sensitivity were identified and further characterized. Based on origin to terminus ratios, chromosome content per cell, measured by flow cytometry, and sensitivity to the replication fork inhibitor hydroxyurea, the suppressors fell into two distinct categories: those...

  17. Effect of cold atmospheric pressure He-plasma jet on DNA change and mutation

    Science.gov (United States)

    Yaopromsiri, C.; Yu, L. D.; Sarapirom, S.; Thopan, P.; Boonyawan, D.

    2015-12-01

    Cold atmospheric pressure plasma jet (CAPPJ) effect on DNA change was studied for assessment of its safety. The experiment utilized a home-developed CAPPJ using 100% helium to directly treat naked DNA plasmid pGFP (plasmid green fluorescent protein). A traversal electric field was applied to separate the plasma components and both dry and wet sample conditions were adopted to investigate various factor roles in changing DNA. Plasma species were measured by using optical emission spectroscopy. DNA topological form change was analyzed by gel electrophoresis. The plasma jet treated DNA was transferred into bacterial Escherichia coli cells for observing mutation. The results show that the He-CAPPJ could break DNA strands due to actions from charge, radicals and neutrals and potentially cause genetic modification of living cells.

  18. Dyes as bifunctional markers of DNA hybridization on surfaces and mutation detection.

    Science.gov (United States)

    García-Mendiola, Tania; Cerro, María Ramos; López-Moreno, José María; Pariente, Félix; Lorenzo, Encarnación

    2016-10-01

    The interaction of small molecules with DNA has found diagnostic and therapeutic applications. In this work, we propose the use of two different dyes, in particular Azure A and Safranine, as bifunctional markers of on-surface DNA hybridization and potent tools for screening of specific gene mutations directly in real DNA PCR amplicons extracted from blood cells. By combining spectroscopic and electrochemical methods we demonstrate that both dyes can interact with single and double stranded DNA to a different extent, allowing reliable hybridization detection. From these data, we have also elucidated the nature of the interaction. We conclude that the binding mode is fundamentally intercalative with an electrostatic component. The dye fluorescence allows their use as nucleic acid stains for the detection of on-surfaces DNA hybridization. Its redox activity is exploited in the development of selective electrochemical DNA biosensors. PMID:27317997

  19. Aptaligner: automated software for aligning pseudorandom DNA X-aptamers from next-generation sequencing data.

    Science.gov (United States)

    Lu, Emily; Elizondo-Riojas, Miguel-Angel; Chang, Jeffrey T; Volk, David E

    2014-06-10

    Next-generation sequencing results from bead-based aptamer libraries have demonstrated that traditional DNA/RNA alignment software is insufficient. This is particularly true for X-aptamers containing specialty bases (W, X, Y, Z, ...) that are identified by special encoding. Thus, we sought an automated program that uses the inherent design scheme of bead-based X-aptamers to create a hypothetical reference library and Markov modeling techniques to provide improved alignments. Aptaligner provides this feature as well as length error and noise level cutoff features, is parallelized to run on multiple central processing units (cores), and sorts sequences from a single chip into projects and subprojects.

  20. Screening for mtDNA diabetes mutations in Pima Indians with NIDDM

    Energy Technology Data Exchange (ETDEWEB)

    Sepehrnia, B.; Prezant, T.R.; Rotter, J.I. [UCLA School of Medicine, Los Angeles, CA (United States)] [and others

    1995-03-27

    More than half of the Pima Indians over age 35 years have non-insulin-dependent (type II) diabetes mellitus (NIDDM). Extensive data indicate the importance of maternal diabetes in determining their risk for diabetes. Generally, the risk of having NIDDM is higher in patients with affected mothers than affected fathers. This has been attributed to intrauterine factors, but recently mitochondrial inheritance has been raised as an alternative hypothesis. In other populations, several families and individuals with diabetes due to a mitochondrial DNA point mutation at nucleotide 3243 in the tRNA{sup leu(UUR)} gene have been described, as has one family with a 10.4 kb mitochondrial DNA duplication/deletion. We tested whether these specific mitochondrial gene mutations could explain a portion of the excess maternal transmission seen in the Pima Indians. Mitochondrial DNA obtained from blood lymphocytes of 148 Pima Indians with NIDDM was screened both for the point mutation at nt 3243, and the 10.4 kb duplication/deletion. Neither of these mutations was detected, and although a small proportion of the excess maternal transmission in Pima Indians could still be due to yet undescribed mitochondrial mutations or imprinted nuclear genes, our data support the role of the intrauterine environment in this population. 32 refs, 21 figs.

  1. Soluble normal and mutated DNA sequences from single-copy genes in human blood.

    Science.gov (United States)

    Sorenson, G D; Pribish, D M; Valone, F H; Memoli, V A; Bzik, D J; Yao, S L

    1994-01-01

    Healthy individuals have soluble (extracellular) DNA in their blood, and increased amounts are present in cancer patients. Here we report the detection of specific sequences of the cystic fibrosis and K-ras genes in plasma DNA from normal donors by amplification with the polymerase chain reaction. In addition, mutated K-ras sequences are identified by polymerase chain reaction utilizing allele-specific primers in the plasma or serum from three patients with pancreatic carcinoma that contain mutated K-ras genes. The mutations are confirmed by direct sequencing. These results indicate that sequences of single-copy genes can be identified in normal plasma and that the sequences of mutated oncogenes can be detected and identified with allele-specific amplification by polymerase chain reaction in plasma or serum from patients with malignant tumors containing identical mutated genes. Mutated oncogenes in plasma and serum may represent tumor markers that could be useful for diagnosis, determining response to treatment, and predicting prognosis. PMID:8118388

  2. Detection of somatic mutations by high-resolution DNA melting (HRM analysis in multiple cancers.

    Directory of Open Access Journals (Sweden)

    Jesus Gonzalez-Bosquet

    Full Text Available Identification of somatic mutations in cancer is a major goal for understanding and monitoring the events related to cancer initiation and progression. High resolution melting (HRM curve analysis represents a fast, post-PCR high-throughput method for scanning somatic sequence alterations in target genes. The aim of this study was to assess the sensitivity and specificity of HRM analysis for tumor mutation screening in a range of tumor samples, which included 216 frozen pediatric small rounded blue-cell tumors as well as 180 paraffin-embedded tumors from breast, endometrial and ovarian cancers (60 of each. HRM analysis was performed in exons of the following candidate genes known to harbor established commonly observed mutations: PIK3CA, ERBB2, KRAS, TP53, EGFR, BRAF, GATA3, and FGFR3. Bi-directional sequencing analysis was used to determine the accuracy of the HRM analysis. For the 39 mutations observed in frozen samples, the sensitivity and specificity of HRM analysis were 97% and 87%, respectively. There were 67 mutation/variants in the paraffin-embedded samples, and the sensitivity and specificity for the HRM analysis were 88% and 80%, respectively. Paraffin-embedded samples require higher quantity of purified DNA for high performance. In summary, HRM analysis is a promising moderate-throughput screening test for mutations among known candidate genomic regions. Although the overall accuracy appears to be better in frozen specimens, somatic alterations were detected in DNA extracted from paraffin-embedded samples.

  3. Somatic mtDNA mutation spectra in the aging human putamen.

    Directory of Open Access Journals (Sweden)

    Siôn L Williams

    Full Text Available The accumulation of heteroplasmic mitochondrial DNA (mtDNA deletions and single nucleotide variants (SNVs is a well-accepted facet of the biology of aging, yet comprehensive mutation spectra have not been described. To address this, we have used next generation sequencing of mtDNA-enriched libraries (Mito-Seq to investigate mtDNA mutation spectra of putamen from young and aged donors. Frequencies of the "common" deletion and other "major arc" deletions were significantly increased in the aged cohort with the fold increase in the frequency of the common deletion exceeding that of major arc deletions. SNVs also increased with age with the highest rate of accumulation in the non-coding control region which contains elements necessary for translation and replication. Examination of predicted amino acid changes revealed a skew towards pathogenic SNVs in the coding region driven by mutation bias. Levels of the pathogenic m.3243A>G tRNA mutation were also found to increase with age. Novel multimeric tandem duplications that resemble murine control region multimers and yeast ρ(- mtDNAs, were identified in both young and aged specimens. Clonal ∼50 bp deletions in the control region were found at high frequencies in aged specimens. Our results reveal the complex manner in which the mitochondrial genome alters with age and provides a foundation for studies of other tissues and disease states.

  4. A New Approach To the Diagnosis of Point Mutations in Native DNA Using Graphene Oxide

    Science.gov (United States)

    Kuznetsov, A.A.; Maksimova, N.R.; Kaimonov, V.S.; Alexandrov, G.N.; Smagulova, S.A.

    2016-01-01

    Development of new methods for the diagnosis of point mutations is a pressing issue. We have developed a new approach to the design of graphene oxide-based test systems for the diagnosis of point mutations in native DNA. This new approach is based on the use of graphene oxide for the adsorption and quenching of fluorescently labeled primers in a post-amplification PCR mixture followed by detection of fluorescently labeled PCR products. It is possible to detect fluorescently labelled amplicons in the presence of an excess of primers in a PCR product solution due to the different affinities of single-stranded and double-stranded DNA molecules to graphene oxide, as well as the ability of graphene oxide to act as a quencher of the fluorophores adsorbed on its surface. The new approach was tested by designing a graphene oxide-based test system for the DNA diagnosis of the point mutation associated with the development of the 3M syndrome in Yakuts. The developed approach enables one to design graphene oxide-based test systems suitable for the diagnosis of any point mutations in native DNA. PMID:27437142

  5. A New Approach To the Diagnosis of Point Mutations in Native DNA Using Graphene Oxide.

    Science.gov (United States)

    Kuznetsov, A A; Maksimova, N R; Kaimonov, V S; Alexandrov, G N; Smagulova, S A

    2016-01-01

    Development of new methods for the diagnosis of point mutations is a pressing issue. We have developed a new approach to the design of graphene oxide-based test systems for the diagnosis of point mutations in native DNA. This new approach is based on the use of graphene oxide for the adsorption and quenching of fluorescently labeled primers in a post-amplification PCR mixture followed by detection of fluorescently labeled PCR products. It is possible to detect fluorescently labelled amplicons in the presence of an excess of primers in a PCR product solution due to the different affinities of single-stranded and double-stranded DNA molecules to graphene oxide, as well as the ability of graphene oxide to act as a quencher of the fluorophores adsorbed on its surface. The new approach was tested by designing a graphene oxide-based test system for the DNA diagnosis of the point mutation associated with the development of the 3M syndrome in Yakuts. The developed approach enables one to design graphene oxide-based test systems suitable for the diagnosis of any point mutations in native DNA. PMID:27437142

  6. CSF studies facilitate DNA diagnosis in familial Alzheimer's disease due to a presenilin-1 mutation

    NARCIS (Netherlands)

    de Bot, Susanne T; Kremer, H P H; Dooijes, Dennis; Verbeek, Marcel M

    2009-01-01

    In sporadic Alzheimer's disease (AD), cerebrospinal fluid (CSF) analysis is becoming increasingly relevant to establish an early diagnosis. We present a case of familial AD due to a presenilin-1 mutation in which CSF studies suggested appropriate DNA diagnostics. A 38 year old Dutch man presented wi

  7. 4-Aminobiphenyl-DNA adducts and p53 mutations in bladder cancer.

    Science.gov (United States)

    Martone, T; Airoldi, L; Magagnotti, C; Coda, R; Randone, D; Malaveille, C; Avanzi, G; Merletti, F; Hautefeuille, A; Vineis, P

    1998-02-01

    Epidemiologic studies have suggested that smokers of air-cured tobacco (rich in arylamines) are at higher risk of bladder cancer than smokers of flue-cured tobacco. The risk has been shown to be modulated by the N-acetyltransferase genotype. We analyzed the biopsies of 45 patients with bladder cancer. p53 mutations were sought by direct sequencing, and 4-aminobiphenyl-DNA adducts were measured by negative ion gas chromatography-mass spectrometry. 4-Aminobiphenyl-DNA adducts were higher in smokers of air-cured tobacco and in current smokers, but no relationship with the number of cigarettes smoked was found. Adducts were higher in more advanced histologic grades of tumors. No pattern was evident for p53 mutations. Seven of 9 mutations occurred in grade 3 tumors. No association was found between 4-ABP adducts and GSTM1 or NAT2 genetic polymorphisms. PMID:9466649

  8. Mutations in BALB mitochondrial DNA induce CCL20 up-regulation promoting tumorigenic phenotypes

    Energy Technology Data Exchange (ETDEWEB)

    Sligh, James [Department of Medicine—Dermatology Division, University of Arizona, Tucson, AZ 857 24 (United States); University of Arizona Cancer Center, Tucson, AZ 85724 (United States); Janda, Jaroslav [University of Arizona Cancer Center, Tucson, AZ 85724 (United States); Jandova, Jana, E-mail: jjandova@email.arizona.edu [Department of Medicine—Dermatology Division, University of Arizona, Tucson, AZ 857 24 (United States); University of Arizona Cancer Center, Tucson, AZ 85724 (United States)

    2014-11-15

    Highlights: • Alterations in mitochondrial DNA are commonly found in various human cancers. • Mutations in BALB mitochondrial DNA induce up-regulation of chemokine CCL20. • Increased growth and motility of mtBALB cells is associated with CCL20 levels. • mtDNA changes in BALB induce in vivo tumor growth through CCL20 up-regulation. • Mutations in mitochondrial DNA play important roles in keratinocyte neoplasia. - Abstract: mtDNA mutations are common in human cancers and are thought to contribute to the process of neoplasia. We examined the role of mtDNA mutations in skin cancer by generating fibroblast cybrids harboring a mutation in the gene encoding the mitochondrial tRNA for arginine. This somatic mutation (9821insA) was previously reported in UV-induced hyperkeratotic skin tumors in hairless mice and confers specific tumorigenic phenotypes to mutant cybrids. Microarray analysis revealed and RT-PCR along with Western blot analysis confirmed the up-regulation of CCL20 and its receptor CCR6 in mtBALB haplotype containing the mt-Tr 9821insA allele compared to wild type mtB6 haplotype. Based on reported role of CCL20 in cancer progression we examined whether the hyper-proliferation and enhanced motility of mtBALB haplotype would be associated with CCL20 levels. Treatment of both genotypes with recombinant CCL20 (rmCCL20) resulted in enhanced growth and motility of mtB6 cybrids. Furthermore, the acquired somatic alteration increased the in vivo tumor growth of mtBALB cybrids through the up-regulation of CCL20 since neutralizing antibody significantly decreased in vivo tumor growth of these cells; and tumors from anti-CCL20 treated mice injected with mtBALB cybrids showed significantly decreased CCL20 levels. When rmCCL20 or mtBALB cybrids were used as chemotactic stimuli, mtB6 cybrids showed increased motility while anti-CCL20 antibody decreased the migration and in vivo tumor growth of mtBALB cybrids. Moreover, the inhibitors of MAPK signaling and NF

  9. A fluoroquinolone resistance associated mutation in gyrA affects DNA supercoiling in Campylobacter jejuni

    Directory of Open Access Journals (Sweden)

    Jing eHan

    2012-03-01

    Full Text Available The prevalence of fluoroquinolone (FQ-resistant Campylobacter has become a concern for public health. To facilitate the control of FQ-resistant Campylobacter, it is necessary to understand the impact of FQ resistance on the fitness of Campylobacter in its natural hosts as understanding fitness will help to determine and predict the persistence of FQ-resistant Campylobacter. Previously it was shown that acquisition of resistance to FQ antimicrobials enhanced the in vivo fitness of FQ-resistant Campylobacter. In this study, we confirmed the role of the Thr-86-Ile mutation in GyrA in modulating Campylobacter fitness by reverting the mutation to the wild-type allele, which resulted in the loss of the fitness advantage. Additionally, we determined if the resistance-conferring GyrA mutations alter the enzymatic function of the DNA gyrase. Recombinant wild-type gyrase and mutant gyrases with three different types of mutations (Thr-86-Ile, Thr-86-Lys, and Asp-90-Asn, which are associated with FQ resistance in Campylobacter, were generated in E. coli and compared for their supercoiling activities using an in vitro assay. The mutant gyrase with the Thr-86-Ile change showed a greatly reduced supercoiling activity compared with the wild-type gyrase, while other mutant gyrases did not show an altered supercoiling. Furthermore, we measured DNA supercoiling within Campylobacter cells using a reporter plasmid. Consistent with the results from the in vitro supercoiling assay, the FQ-resistant mutant carrying the Thr-86-Ile change in GyrA showed much less DNA supercoiling than the wild-type strain and the mutant strains carrying other mutations. Together, these results indicate that the Thr-86-Ile mutation, which is predominant in clinical FQ-resistant Campylobacter, modulates DNA supercoiling homeostasis in FQ-resistant Campylobacter.

  10. A mitochondrial DNA (mtDNA) mutation associated with maternally inherited Parkinson`s disease (PD) and deafness

    Energy Technology Data Exchange (ETDEWEB)

    Shoffner, J.M.; Brown, M.; Huoponen, K. [Emory Univ., Atlanta, GA (United States)] [and others

    1994-09-01

    A pedigree was characterized in which PD and deafness is expressed along the maternal lineage. The proband is 74 years old and has PD. Her mother and 3 of 7 siblings have PD and a maternal lineage cousin may have early signs of PD. The proband`s mother, a sibling, and all four of her daughters have premature deafness. Since manifestations of PD begin after 50 years of age, the 30-40 year old daughters have not reached an age where extrapyramidal symptoms are likely to appear. Although all 4 daughters have premature deafness, one daughter experienced a rapid reduction of her hearing after receiving a short course during childhood of the aminoglycoside streptomycin. Muscle biopsies from the proband who has PD and 3 daughters with deafness revealed normal histology. Oxidative phosphorylation biochemistry showed Complex I and IV defects in the proband and 2 daughters and a Complex I defect in the other daughter. The proband`s mtDNA was sequenced. Of the nucleotide variants observed, the only significant nucleotide change was a homoplasmic A-to-G point mutation in the 12S rRNA gene at position 1555 of the mtDNA. This site is homologous to the E. coli aminoglycoside binding site and has been found in a large Arab-Israeli pedigree with spontaneously occurring deafness and three Chinese pedigrees with aminoglycoside-induced deafness. Hence, this family shows a direct link between PD, deafness, Complex I and IV defects, and a mutation in a gene that functions in mitochondrial protein synthesis. Furthermore, the interaction between aminoglycosides and the mtDNA in a manner that augments the pathogenic effects of this mutation provides an excellent example of how environmental toxins and mtDNA mutations can interact to give a spectrum of clinical presentations.

  11. A high sensitivity, high throughput, automated single-cell gel electrophoresis ('Comet') DNA damage assay

    International Nuclear Information System (INIS)

    A fully automated microscopy machine vision image capture and analysis system for the collection of data from slides of 'comets' has been developed. The novel image processing algorithms employed in delineating the 'comet head' from the 'comet tail' allow us to determine accurately very low levels of damage. In conjunction with calibrated and automated image capture methods, we are able to eliminate operator subjectivity and analyse large numbers of cells (>2500) in a short time (<1 hour). The image processing algorithm is designed to handle particularly difficult nuclei containing a high degree of structure, due to DNA clumping. We also present techniques used to extend the assay's dynamic range by removing interfering background fluorescence and to define a region of interest. If subtle biological variations are to be quantified (e.g. cell cycle dependant damage), then the use of large cell populations is dictated. Under those circumstances, the use of a fully automated system is particularly advantageous providing that the manner in which data is extracted does not introduce any inadvertent bias. In practice, it is essential that the image processing steps are geared towards the correct recognition of an acceptable cell nucleus, i.e. comet 'head'. We acknowledge the financial support of CRUK, Programme Grant C133/A1812 - SP 2195-01/02 and the US Department of Energy Low Dose Radiation Research Program grant DE-FG07-99ER62878

  12. Rapid photometric detection of thymine residues partially flipped out of double helix as a method for direct scanning of point mutations and apurinic DNA sites.

    Science.gov (United States)

    Logvina, N A; Yakubovskaya, M G; Dolinnaya, N G

    2011-02-01

    A spectroscopic assay for detection of extrahelical thymine residues in DNA heteroduplexes under their modification by potassium permanganate has been developed. The assay is based on increase in absorbance at 420 nm due to accumulation of thymidine oxidation intermediates and soluble manganese dioxide. The analysis was carried out using a set of 19-bp DNA duplexes containing unpaired thymidines opposite tetrahydrofuranyl derivatives mimicking a widespread DNA damage (apurinic (AP) sites) and a library of 50-bp DNA duplexes containing all types of base mismatches in different surroundings. The relation between the selectivity of unpaired T oxidation and the thermal stability of DNA double helix was investigated. The method described here was shown to discriminate between DNA duplexes with one or two AP sites and to reveal thymine-containing mismatches and all noncanonical base pairs in AT-surroundings. Comparative results of CCM analysis and the rapid photometric assay for mismatch detection are demonstrated for the first time in the same model system. The chemical reactivity of target thymines was shown to correlate with local disturbance of double helix at the mismatch site. As the spectroscopic assay does not require the DNA cleavage reaction and gel electrophoresis, it can be easily automated and used for primary screening of somatic mutations. PMID:21568858

  13. Detection of HIV cDNA Point Mutations with Rolling-Circle Amplification Arrays

    Directory of Open Access Journals (Sweden)

    Zhongwei Wu

    2010-01-01

    Full Text Available In this paper we describe an isothermal rolling-circle amplification (RCA protocol to detect gene point mutations on chips. The method is based on an allele-specific oligonucleotide circularization mediated by a special DNA ligase. The probe is circularized when perfect complementary sequences between the probe oligonucleotide and HIV cDNA gene. Mismatches around the ligation site can prevent probe circularization. The circularized probe (C-probe can be amplified by rolling circle amplification to generate multimeric singlestranded DNA (ssDNA under isothermal conditions. There are four sequence regions to bind respectively with fluorescent probe, RCA primer, solid probe and HIV cDNA template in the C-probe which we designed. These ssDNA products are hybridized with fluorescent probes and solid probes which are immobilized on a glass slide composing a regular microarray pattern. The fluorescence signals can be monitored by a scanner in the presence of HIV cDNA templates, whereas the probes cannot be circularized and signal of fluorescence cannot be found. The RCA array has capability of high-throughput detection of the point mutation and the single-nucleotide polymorphism (SNP.The development of C-probe-based technologies offers a promising prospect for situ detection, microarray, molecular diagnosis, single nucleotide polymorphism, and whole genome amplification.

  14. Relationship Between Mitochondrial DNA Mutations and Aging. Estimation of Age-at-death.

    Science.gov (United States)

    Zapico, Sara C; Ubelaker, Douglas H

    2016-04-01

    Some studies have pointed to the relationship between mitochondrial DNA (mtDNA) mutations and age in different tissues, which are potentially interesting in aging research and in forensic identification because they could help to improve the estimation of age-at-death. The present study aims to evaluate the mutations in mtDNA from dentin and pulp and their relation with age. Healthy erupted third molars were extracted from individuals from two Spanish populations, aged 20-70. When analyzing the amplification of hypervariable region 2 of the mtDNA by real-time polymerase chain reaction, a negative strong linear correlation was found between the mtDNA amplification and age in dentin from both populations. In contrast, a significant correlation between mtDNA amplification and age in pulp was not discovered, probably due to the majority of the mitochondria are placed in dentin. A difference in mtDNA damage between these two populations was also detected, indicating the role of ancestry as a component. The findings from this research enrich the current studies related to aging and mitochondrial damage and provide a new quantitative tool for estimating the age-at-death that, in combination with traditional age markers, could improve identification accuracy in forensic cases. PMID:26286606

  15. Mutation Processes in 293-Based Clones Overexpressing the DNA Cytosine Deaminase APOBEC3B.

    Directory of Open Access Journals (Sweden)

    Monica K Akre

    Full Text Available Molecular, cellular, and clinical studies have combined to demonstrate a contribution from the DNA cytosine deaminase APOBEC3B (A3B to the overall mutation load in breast, head/neck, lung, bladder, cervical, ovarian, and other cancer types. However, the complete landscape of mutations attributable to this enzyme has yet to be determined in a controlled human cell system. We report a conditional and isogenic system for A3B induction, genomic DNA deamination, and mutagenesis. Human 293-derived cells were engineered to express doxycycline-inducible A3B-eGFP or eGFP constructs. Cells were subjected to 10 rounds of A3B-eGFP exposure that each caused 80-90% cell death. Control pools were subjected to parallel rounds of non-toxic eGFP exposure, and dilutions were done each round to mimic A3B-eGFP induced population fluctuations. Targeted sequencing of portions of TP53 and MYC demonstrated greater mutation accumulation in the A3B-eGFP exposed pools. Clones were generated and microarray analyses were used to identify those with the greatest number of SNP alterations for whole genome sequencing. A3B-eGFP exposed clones showed global increases in C-to-T transition mutations, enrichments for cytosine mutations within A3B-preferred trinucleotide motifs, and more copy number aberrations. Surprisingly, both control and A3B-eGFP clones also elicited strong mutator phenotypes characteristic of defective mismatch repair. Despite this additional mutational process, the 293-based system characterized here still yielded a genome-wide view of A3B-catalyzed mutagenesis in human cells and a system for additional studies on the compounded effects of simultaneous mutation mechanisms in cancer cells.

  16. Structural Insight into Processive Human Mitochondrial DNA Synthesis and Disease-Related Polymerase Mutations

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Young-Sam; Kennedy, W. Dexter; Yin, Y. Whitney; (Texas)

    2010-09-07

    Human mitochondrial DNA polymerase (Pol {gamma}) is the sole replicase in mitochondria. Pol {gamma} is vulnerable to nonselective antiretroviral drugs and is increasingly associated with mutations found in patients with mitochondriopathies. We determined crystal structures of the human heterotrimeric Pol {gamma} holoenzyme and, separately, a variant of its processivity factor, Pol {gamma}B. The holoenzyme structure reveals an unexpected assembly of the mitochondrial DNA replicase where the catalytic subunit Pol {gamma}A interacts with its processivity factor primarily via a domain that is absent in all other DNA polymerases. This domain provides a structural module for supporting both the intrinsic processivity of the catalytic subunit alone and the enhanced processivity of holoenzyme. The Pol {gamma} structure also provides a context for interpreting the phenotypes of disease-related mutations in the polymerase and establishes a foundation for understanding the molecular basis of toxicity of anti-retroviral drugs targeting HIV reverse transcriptase.

  17. Automated extraction of DNA and PCR setup using a Tecan Freedom EVO® liquid handler

    DEFF Research Database (Denmark)

    Frøslev, Tobias Guldberg; Hansen, Anders Johannes; Stangegaard, Michael;

    We have implemented and validated automated protocols for DNA extraction and PCR setup using a Tecan Freedom EVO® liquid handler mounted with the TeMagS magnetic separation device. The methods were validated for accredited, forensic genetic work according to ISO 17025 using the Qiagen Mag......Attract® DNA Mini M48 kit from fresh, whole blood and blood from deceased. The methods were simplified by returning the DNA extracts to the original tubes reducing the risk of misplacing samples. The original tubes that had contained the samples were washed with 700 µl Milli-Q water prior to the return...... of the DNA extracts. The PCR setup protocols were designed for 96 well microtiter plates. The methods were validated for the kits: AmpFlSTR® Identifiler® and Y-filer® (Applied Biosystems), GenePrint® FFFL and PowerPlex® Y (Promega). Within 3.5 hours, 96 samples were extracted and PCR master mix was added...

  18. Rapid screening mitochondrial DNA mutation by using denaturing high-performance liquid chromatography

    Institute of Scientific and Technical Information of China (English)

    Man-Ran Liu; Kai-Feng Pan; Zhen-Fu Li; Yi Wang; Da-Jun Deng; Lian Zhang; You-Yong Lu

    2002-01-01

    AIM: To optimize conditions of DHPLC and analyze theeffectiveness of various DNA polymerases on DHPLCresolution, and evaluate the sensitivity of DHPLC in themutation screening of mitochondrial DNA (mtDNA).METHODS: Two fragments of 16s gene of mitochondrial DNA(one of them F2 is a mutant fragment) and an A3243Gmutated fragment were used to analyze the UV detectionlimit and determine the minimum percentage of mutant PCRproducts for DHPLC and evaluate effects of DNApolymerases on resolution of DHPLC. Under the optimalconditions, we analyzed the mtDNA mutations from muscletissues of mitochondrial encephalomyopathy with lacticacidosis and stroke-like episodes (NELAS) and screenedblindly for variances in D-loop region of mtDNA from humangastric tumor specimen.RESULTS: Ten A3243G variants were detected in 12 cases ofMELAS, no alterations were detected in controls and theseresults were consistent with the results obtained by analysisof RFLP with Apel. We also identified 26 D-loop variances in46 cases of human gastric cancer tissues and 38 alterationsin 13 gastric cancer cell lines. The mutation of mtDNA at 80 ngPCFI products containing a minimum of 5 % mutant sequencescould be detected by using DHPLC with UV detector.Moreover, Ampli-Taq Gold polymerase was equally as good asthe proofreading DNA polymerase (e. g., Pfu) in eliminatingthe false positive produced by Taq DNA polymerases.CONCLUSION: DHPLC is a powerful, rapid and sensitivemutation screening method for mtDNA. Proofreading DNApolymerase is more suitable for DHPLC analysis than Taqpolymerase.

  19. Transcription Restores DNA Repair to Heterochromatin, Determining Regional Mutation Rates in Cancer Genomes

    Directory of Open Access Journals (Sweden)

    Christina L. Zheng

    2014-11-01

    Full Text Available Somatic mutations in cancer are more frequent in heterochromatic and late-replicating regions of the genome. We report that regional disparities in mutation density are virtually abolished within transcriptionally silent genomic regions of cutaneous squamous cell carcinomas (cSCCs arising in an XPC−/− background. XPC−/− cells lack global genome nucleotide excision repair (GG-NER, thus establishing differential access of DNA repair machinery within chromatin-rich regions of the genome as the primary cause for the regional disparity. Strikingly, we find that increasing levels of transcription reduce mutation prevalence on both strands of gene bodies embedded within H3K9me3-dense regions, and only to those levels observed in H3K9me3-sparse regions, also in an XPC-dependent manner. Therefore, transcription appears to reduce mutation prevalence specifically by relieving the constraints imposed by chromatin structure on DNA repair. We model this relationship among transcription, chromatin state, and DNA repair, revealing a new, personalized determinant of cancer risk.

  20. [Clinical features and DGUOK mutations of an infant with mitochondrial DNA depletion syndrome].

    Science.gov (United States)

    Deng, Mei; Lin, Wei-Xia; Guo, Li; Zhang, Zhan-Hui; Song, Yuan-Zong

    2016-06-01

    The aim of this study was to investigate the clinical features and DGUOK gene mutations of an infant with mitochondrial DNA depletion syndrome (MDS). The patient (more than 7 months old) manifested as hepatosplenomegaly, abnormal liver function, nystagmus and psychomotor retardation. Genetic DNA was extracted from peripheral blood samples of the patient and her parents. Targeted Exome Sequencing was performed to explore the genetic causes. Sanger sequencing was carried out to confirm the detected mutations. The sequencing results showed that the patient was a compound heterozygote for c.679G>A and c.817delT in the DGUOK gene. The former was a reportedly pathogenic missense mutation of maternal origin, while the latter, a frameshift mutation from the father, has not been described yet. The findings in this study expand the mutation spectrum of DGUOK gene, and provide molecular evidence for the etiologic diagnosis of the patient as well as for the genetic counseling and prenatal diagnosis in the family. PMID:27324545

  1. Comparison of mitochondrial mutation spectra in ageing human colonic epithelium and disease: absence of evidence for purifying selection in somatic mitochondrial DNA point mutations

    NARCIS (Netherlands)

    Greaves, L.C.; Elson, J.L.; Nooteboom, M.; Grady, J.P.; Taylor, G.A.; Taylor, R.W.; Mathers, J.C.; Kirkwood, T.B.; Turnbull, D.M.

    2012-01-01

    Human ageing has been predicted to be caused by the accumulation of molecular damage in cells and tissues. Somatic mitochondrial DNA (mtDNA) mutations have been documented in a number of ageing tissues and have been shown to be associated with cellular mitochondrial dysfunction. It is unknown whethe

  2. Complete Spectrum of CRISPR/Cas9-induced Mutations on HBV cccDNA

    Science.gov (United States)

    Seeger, Christoph; Sohn, Ji A

    2016-01-01

    Hepatitis B virus (HBV) causes chronic infections that cannot yet be cured. The virus persists in infected hepatocytes, because covalently closed circular DNA (cccDNA), the template for the transcription of viral RNAs, is stable in nondividing cells. Antiviral therapies with nucleoside analogues inhibit HBV DNA synthesis in capsids in the cytoplasm of infected hepatocytes, but do not destroy nuclear cccDNA. Because over 200 million people are still infected, a cure for chronic hepatitis B (CHB) has become one of the major challenges in antiviral therapy. As a first step toward the development of curative therapies, we previously demonstrated that the CRISPR/Cas9 system can be used to functionally inactivate cccDNA derived from infectious HBV. Moreover, some evidence suggests that certain cytokines might induce an APOBEC-mediated cascade leading to the destruction of cccDNA. In this report we investigated whether a combination of the two mechanisms could act synergistically to inactivate cccDNA. Using next generation sequencing (NGS), we determined the complete spectrum of mutations in cccDNA following Cas9 cleavage and repair by nonhomologous end joining (NHEJ). We found that over 90% of HBV DNA was cleaved by Cas9. In addition our results showed that editing of HBV DNA after Cas9 cleavage is at least 15,000 times more efficient that APOBEC-mediated cytosine deamination following treatment of infected cells with interferon alpha (IFNα). We also found that a previously used method to detect cytosine deaminated DNA, termed 3D-PCR, overestimates the amount and frequency of edited HBV DNA. Taken together, our results demonstrated that the CRISPR/Cas9 system is so far the best method to functionally inactivate HBV cccDNA and provide a cure for CHB. PMID:27203444

  3. Complete Spectrum of CRISPR/Cas9-induced Mutations on HBV cccDNA.

    Science.gov (United States)

    Seeger, Christoph; Sohn, Ji A

    2016-08-01

    Hepatitis B virus (HBV) causes chronic infections that cannot yet be cured. The virus persists in infected hepatocytes, because covalently closed circular DNA (cccDNA), the template for the transcription of viral RNAs, is stable in nondividing cells. Antiviral therapies with nucleoside analogues inhibit HBV DNA synthesis in capsids in the cytoplasm of infected hepatocytes, but do not destroy nuclear cccDNA. Because over 200 million people are still infected, a cure for chronic hepatitis B (CHB) has become one of the major challenges in antiviral therapy. As a first step toward the development of curative therapies, we previously demonstrated that the CRISPR/Cas9 system can be used to functionally inactivate cccDNA derived from infectious HBV. Moreover, some evidence suggests that certain cytokines might induce an APOBEC-mediated cascade leading to the destruction of cccDNA. In this report we investigated whether a combination of the two mechanisms could act synergistically to inactivate cccDNA. Using next generation sequencing (NGS), we determined the complete spectrum of mutations in cccDNA following Cas9 cleavage and repair by nonhomologous end joining (NHEJ). We found that over 90% of HBV DNA was cleaved by Cas9. In addition our results showed that editing of HBV DNA after Cas9 cleavage is at least 15,000 times more efficient that APOBEC-mediated cytosine deamination following treatment of infected cells with interferon alpha (IFNα). We also found that a previously used method to detect cytosine deaminated DNA, termed 3D-PCR, overestimates the amount and frequency of edited HBV DNA. Taken together, our results demonstrated that the CRISPR/Cas9 system is so far the best method to functionally inactivate HBV cccDNA and provide a cure for CHB. PMID:27203444

  4. Human aging and somatic point mutations in mtDNA: a comparative study of generational differences (grandparents and grandchildren

    Directory of Open Access Journals (Sweden)

    Anderson Nonato do Rosário Marinho

    2011-01-01

    Full Text Available The accumulation of somatic mutations in mtDNA is correlated with aging. In this work, we sought to identify somatic mutations in the HVS-1 region (D-loop of mtDNA that might be associated with aging. For this, we compared 31 grandmothers (mean age: 63 ± 2.3 years and their 62 grandchildren (mean age: 15 ± 4.1 years, the offspring of their daughters. Direct DNA sequencing showed that mutations absent in the grandchildren were detected in a presumably homoplasmic state in three grandmothers and in a heteroplasmic state in an additional 13 grandmothers; no mutations were detected in the remaining 15 grandmothers. However, cloning followed by DNA sequencing in 12 grandmothers confirmed homoplasia in only one of the three mutations previously considered to be homoplasmic and did not confirm heteroplasmy in three out of nine grandmothers found to be heteroplasmic by direct sequencing. Thus, of 12 grandmothers in whom mtDNA was analyzed by cloning, eight were heteroplasmic for mutations not detected in their grandchildren. In this study, the use of genetically related subjects allowed us to demonstrate the occurrence of age-related (> 60 years old mutations (homoplasia and heteroplasmy. It is possible that both of these situations (homoplasia and heteroplasmy were a long-term consequence of mitochondrial oxidative phosphorylation that can lead to the accumulation of mtDNA mutations throughout life.

  5. FAN1 mutations cause karyomegalic interstitial nephritis, linking chronic kidney failure to defective DNA damage repair.

    Science.gov (United States)

    Zhou, Weibin; Otto, Edgar A; Cluckey, Andrew; Airik, Rannar; Hurd, Toby W; Chaki, Moumita; Diaz, Katrina; Lach, Francis P; Bennett, Geoffrey R; Gee, Heon Yung; Ghosh, Amiya K; Natarajan, Sivakumar; Thongthip, Supawat; Veturi, Uma; Allen, Susan J; Janssen, Sabine; Ramaswami, Gokul; Dixon, Joanne; Burkhalter, Felix; Spoendlin, Martin; Moch, Holger; Mihatsch, Michael J; Verine, Jerome; Reade, Richard; Soliman, Hany; Godin, Michel; Kiss, Denes; Monga, Guido; Mazzucco, Gianna; Amann, Kerstin; Artunc, Ferruh; Newland, Ronald C; Wiech, Thorsten; Zschiedrich, Stefan; Huber, Tobias B; Friedl, Andreas; Slaats, Gisela G; Joles, Jaap A; Goldschmeding, Roel; Washburn, Joseph; Giles, Rachel H; Levy, Shawn; Smogorzewska, Agata; Hildebrandt, Friedhelm

    2012-08-01

    Chronic kidney disease (CKD) represents a major health burden. Its central feature of renal fibrosis is not well understood. By exome sequencing, we identified mutations in FAN1 as a cause of karyomegalic interstitial nephritis (KIN), a disorder that serves as a model for renal fibrosis. Renal histology in KIN is indistinguishable from that of nephronophthisis, except for the presence of karyomegaly. The FAN1 protein has nuclease activity and acts in DNA interstrand cross-link (ICL) repair within the Fanconi anemia DNA damage response (DDR) pathway. We show that cells from individuals with FAN1 mutations have sensitivity to the ICL-inducing agent mitomycin C but do not exhibit chromosome breakage or cell cycle arrest after diepoxybutane treatment, unlike cells from individuals with Fanconi anemia. We complemented ICL sensitivity with wild-type FAN1 but not with cDNA having mutations found in individuals with KIN. Depletion of fan1 in zebrafish caused increased DDR, apoptosis and kidney cysts. Our findings implicate susceptibility to environmental genotoxins and inadequate DNA repair as novel mechanisms contributing to renal fibrosis and CKD.

  6. Mutations in the ND5 subunit of complex I of the mitochondrial DNA are a frequent cause of oxidative phosphorylation disease.

    NARCIS (Netherlands)

    Blok, M.J.; Spruijt, L.; Coo, I.F.M. de; Schoonderwoerd, K.C.; Hendrickx, A.; Smeets, H.J.M.

    2007-01-01

    BACKGROUND: Detection of mutations in the mitochondrial DNA (mtDNA) is usually limited to common mutations and the transfer RNA genes. However, mutations in other mtDNA regions can be an important cause of oxidative phosphorylation (OXPHOS) disease as well. OBJECTIVE: To investigate whether regions

  7. Evaluation of Sample Stability and Automated DNA Extraction for Fetal Sex Determination Using Cell-Free Fetal DNA in Maternal Plasma

    Directory of Open Access Journals (Sweden)

    Elena Ordoñez

    2013-01-01

    Full Text Available Objective. The detection of paternally inherited sequences in maternal plasma, such as the SRY gene for fetal sexing or RHD for fetal blood group genotyping, is becoming part of daily routine in diagnostic laboratories. Due to the low percentage of fetal DNA, it is crucial to ensure sample stability and the efficiency of DNA extraction. We evaluated blood stability at 4°C for at least 24 hours and automated DNA extraction, for fetal sex determination in maternal plasma. Methods. A total of 158 blood samples were collected, using EDTA-K tubes, from women in their 1st trimester of pregnancy. Samples were kept at 4°C for at least 24 hours before processing. An automated DNA extraction was evaluated, and its efficiency was compared with a standard manual procedure. The SRY marker was used to quantify cfDNA by real-time PCR. Results. Although lower cfDNA amounts were obtained by automated DNA extraction (mean 107,35 GE/mL versus 259,43 GE/mL, the SRY sequence was successfully detected in all 108 samples from pregnancies with male fetuses. Conclusion. We successfully evaluated the suitability of standard blood tubes for the collection of maternal blood and assessed samples to be suitable for analysis at least 24 hours later. This would allow shipping to a central reference laboratory almost from anywhere in Europe.

  8. Heterogeneity of six children and their mothers with mitochondrial DNA 3243 A>G mutation.

    Science.gov (United States)

    Ma, Yan-Yan; Wu, Tong-Fei; Liu, Yu-Peng; Wang, Qiao; Li, Xi-Yuan; Song, Jin-Qing; Shi, Xiu-Yu; Zhang, Wei-Na; Zhao, Meng; Hu, Ling-Yan; Yang, Yan-Ling; Zou, Li-Ping

    2013-06-01

    To study the clinical, biochemical, and genetic heterogeneity of six Chinese patients and their mothers with the 3243 A>G mutation, six patients (ranging from 5 to 11 years) were hospitalized. All the mothers were healthy. Mitochondrial respiratory chain enzyme activities were determined by spectrophotometry. Mitochondrial gene was analyzed in all patients. Six core pedigrees were investigated. Two patients had mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes syndrome and one had Leigh syndrome. The common initial symptoms were headache, vomiting, blurred vision, and epilepsy. m.3243A>G mutation was detected in all patients and their mothers. The mutation loads ranged from 43.6% to 58% and those of their mothers ranged from 14.1% to 28.6%. Varied respiratory chain deficiencies were observed in all patients and two mothers. m.3243A>G mutation can result in a wide spectrum of respiratory chain complex deficiencies. Mitochondrial DNA mutation detected in blood may be likely to transmit to offspring, and the mutation load may increase.

  9. Two Mechanisms Produce Mutation Hotspots at DNA Breaks in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Chandan Shee

    2012-10-01

    Full Text Available Mutation hotspots and showers occur across phylogeny and profoundly influence genome evolution, yet the mechanisms that produce hotspots remain obscure. We report that DNA double-strand breaks (DSBs provoke mutation hotspots via stress-induced mutation in Escherichia coli. With tet reporters placed 2 kb to 2 Mb (half the genome away from an I-SceI site, RpoS/DinB-dependent mutations occur maximally within the first 2 kb and decrease logarithmically to ∼60 kb. A weak mutation tail extends to 1 Mb. Hotspotting occurs independently of I-site/tet-reporter-pair position in the genome, upstream and downstream in the replication path. RecD, which allows RecBCD DSB-exonuclease activity, is required for strong local but not long-distance hotspotting, indicating that double-strand resection and gap-filling synthesis underlie local hotspotting, and newly illuminating DSB resection in vivo. Hotspotting near DSBs opens the possibility that specific genomic regions could be targeted for mutagenesis, and could also promote concerted evolution (coincident mutations within genes/gene clusters, an important issue in the evolution of protein functions.

  10. Suppression of thermosensitive initiation of DNA replication in a dnaR mutant of Escherichia coli by a rifampin resistance mutation in the rpoB gene.

    OpenAIRE

    Sakakibara, Y

    1995-01-01

    The thermosensitivity of the Escherichia coli dnaR130 mutant in initiation of DNA replication was suppressed by a spontaneous rifampin resistance mutation in rpoB, the gene for the beta subunit of RNA polymerase. Among the dnaR-suppressing rpoB alleles obtained was rpoB22, which was able to suppress the thermosensitivity of the dnaA46 or dnaA167 mutant, but not that of the dnaA5 mutant, in initiation of replication. Some dnaA-suppressing rpoB alleles obtained from rifampin-resistant derivativ...

  11. KRAS G12V Mutation Detection by Droplet Digital PCR in Circulating Cell-Free DNA of Colorectal Cancer Patients

    OpenAIRE

    Olmedillas López, Susana; García-Olmo, Dolores C; García-Arranz, Mariano; Guadalajara, Héctor; Pastor, Carlos; García-Olmo, Damián

    2016-01-01

    KRAS mutations are responsible for resistance to anti-epidermal growth factor receptor (EGFR) therapy in colorectal cancer patients. These mutations sometimes appear once treatment has started. Detection of KRAS mutations in circulating cell-free DNA in plasma (“liquid biopsy”) by droplet digital PCR (ddPCR) has emerged as a very sensitive and promising alternative to serial biopsies for disease monitoring. In this study, KRAS G12V mutation was analyzed by ddPCR in plasma DNA from 10 colorect...

  12. Human aging and somatic point mutations in mtDNA: A comparative study of generational differences (grandparents and grandchildren)

    OpenAIRE

    Anderson Nonato do Rosário Marinho; Milene Raiol de Moraes; Sidney Santos; Ândrea Ribeiro-dos- Santos

    2011-01-01

    The accumulation of somatic mutations in mtDNA is correlated with aging. In this work, we sought to identify somatic mutations in the HVS-1 region (D-loop) of mtDNA that might be associated with aging. For this, we compared 31 grandmothers (mean age: 63 ± 2.3 years) and their 62 grandchildren (mean age: 15 ± 4.1 years), the offspring of their daughters. Direct DNA sequencing showed that mutations absent in the grandchildren were detected in a presumably homoplasmic state in three grandmothers...

  13. Recent mitochondrial DNA mutations increase the risk of developing common late-onset human diseases.

    Directory of Open Access Journals (Sweden)

    Gavin Hudson

    2014-05-01

    Full Text Available Mitochondrial DNA (mtDNA is highly polymorphic at the population level, and specific mtDNA variants affect mitochondrial function. With emerging evidence that mitochondrial mechanisms are central to common human diseases, it is plausible that mtDNA variants contribute to the "missing heritability" of several complex traits. Given the central role of mtDNA genes in oxidative phosphorylation, the same genetic variants would be expected to alter the risk of developing several different disorders, but this has not been shown to date. Here we studied 38,638 individuals with 11 major diseases, and 17,483 healthy controls. Imputing missing variants from 7,729 complete mitochondrial genomes, we captured 40.41% of European mtDNA variation. We show that mtDNA variants modifying the risk of developing one disease also modify the risk of developing other diseases, thus providing independent replication of a disease association in different case and control cohorts. High-risk alleles were more common than protective alleles, indicating that mtDNA is not at equilibrium in the human population, and that recent mutations interact with nuclear loci to modify the risk of developing multiple common diseases.

  14. DNA repair in Haemophilus influenzae: isolation and characterization of an ultraviolet sensitive mutator mutant

    Energy Technology Data Exchange (ETDEWEB)

    Walter, R.B.

    1985-01-01

    DNA repair in Haemophilus influenzae appears to be quite different from that seen in Escherichia coli in that H. influenzae shows neither SOS nor adaptation phenomena. Repair of DNA lesions in H. influenzae has been seen to occur via recombinational, excision, and mismatch repair pathways acting independently of one another. The author has isolated an ultraviolet (UV)-sensitive mutator mutant (mutB1) of H. influenzae Rd which shows deficiencies in both recombinational and mismatch repair pathways. This mutant is sensitive to a variety of DNA damaging agents as well as being hypermutable by alkylating agents and base analogues. MutB1 cells do not show post-UV DNA breakdown but do begin excision after UV irradiation. Genetic transformation with UV-irradiated DNA on mut B1 recipients shows that high (HE) and low (LE) efficiency markers are transformed at a ratio of 1.0 as in the mismatch repair deficient hex 1 mutant; however, kinetics of UV-inactivation experiments indicate that HE markers are sensitized and act as LE markers do on wild type recipients. Thus, the mutB gene product appears to play a role in both DNA repair and genetic transformation. A model is outlined which presents a role for a DNA helicase in both DNA repair and genetic transformation of H. influenzae.

  15. Automation and integration of multiplexed on-line sample preparation with capillary electrophoresis for DNA sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Tan, H.

    1999-03-31

    The purpose of this research is to develop a multiplexed sample processing system in conjunction with multiplexed capillary electrophoresis for high-throughput DNA sequencing. The concept from DNA template to called bases was first demonstrated with a manually operated single capillary system. Later, an automated microfluidic system with 8 channels based on the same principle was successfully constructed. The instrument automatically processes 8 templates through reaction, purification, denaturation, pre-concentration, injection, separation and detection in a parallel fashion. A multiplexed freeze/thaw switching principle and a distribution network were implemented to manage flow direction and sample transportation. Dye-labeled terminator cycle-sequencing reactions are performed in an 8-capillary array in a hot air thermal cycler. Subsequently, the sequencing ladders are directly loaded into a corresponding size-exclusion chromatographic column operated at {approximately} 60 C for purification. On-line denaturation and stacking injection for capillary electrophoresis is simultaneously accomplished at a cross assembly set at {approximately} 70 C. Not only the separation capillary array but also the reaction capillary array and purification columns can be regenerated after every run. DNA sequencing data from this system allow base calling up to 460 bases with accuracy of 98%.

  16. Specific mutation screening of TP53 gene by low-density DNA microarray

    Directory of Open Access Journals (Sweden)

    Angélica Rangel-López

    2009-01-01

    Full Text Available Angélica Rangel-López1–3, Alfonso Méndez-Tenorio3, Kenneth L Beattie4, Rogelio Maldonado3, Patricia Mendoza1, Guelaguetza Vázquez1, Carlos Pérez-Plasencia5, Martha Sánchez2, Guillermo Navarro6, Mauricio Salcedo11Laboratorio de Oncología Genómica, Unidad de Investigación Médica en Enfermedades Oncológicas, Hospital de Oncología, CMN Siglo XXI-IMSS, Mexico City, Mexico; 2Unidad de Investigación Médica en Enfermedades Nefrológicas, Hospital de Especialidades, CMN Siglo XXI-IMSS, Mexico City; Mexico; 3Laboratorio de Biotecnología y Bioinformática Genómica, Escuela Nacional de Ciencias Biológicas, IPN Mexico City, Mexico; 4Amerigenics, Inc., Crossville, TN, USA; 5Unidad de Investigación Biomédica en Cáncer, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México UNAM, Instituto Nacional de Cancerología INCAN, Mexico City, Mexico; 6Laboratorio de Organometálicos UNAM, Mexico City, MexicoAbstract: TP53 is the most commonly mutated gene in human cancers. Approximately 90% of mutations in this gene are localized between domains encoding exons 5 to 8. The aim of this investigation was to examine the ability of the low density DNA microarray with the assistance of double tandem hybridization platform to characterize TP53 mutational hotspots in exons 5, 7, and 8 of the TP53. Nineteen capture probes specific to each potential mutation site were designed to hybridize to specific site. Virtual hybridization was used to predict the stability of hybridization of each capture probe with the target. Thirty-three DNA samples from different sources were analyzed for mutants in these exons. A total of 32 codon substitutions were found by DNA sequencing. 24 of them a showed a perfect correlation with the hybridization pattern system and DNA sequencing analysis of the regions scanned. Although in this work we directed our attention to some of the most representative mutations of the TP503 gene, the results suggest

  17. Mitochondrial DNA (mt DNA) A3243G mutation associated with an annular perimacular retinal atrophy

    OpenAIRE

    Adjadj E.

    2010-01-01

    Résumé:Background:La mutation 3243 de 1'ADN mitochondrial est associee avec le syndrome l\\/HDD (surdite, diabète transmis par la mère) et le syndrome MELAS (Myopathie, Encéphalopathie, acidose Lactique et attaques cérébrales). Elle est aussi associe à des troubles cardiaques, digestifs, endo- et exocrines. Nous rapportons deux cas de maculopathie associée à cette mutation.Histoire et symptomes: pCas l: il s'agit d'une femme de 60 ans soufrant d'un diabète et d'une surdité sans plainte visuell...

  18. A novel mtDNA ND6 gene mutation associated with LHON in a Caucasian family.

    Science.gov (United States)

    Zhadanov, Sergey I; Atamanov, Vasily V; Zhadanov, Nikolay I; Oleinikov, Oleg V; Osipova, Ludmila P; Schurr, Theodore G

    2005-07-15

    Leber's hereditary optic neuropathy (LHON) is a frequent cause of inherited blindness. A routine screening for common mtDNA mutations constitutes an important first in its diagnosis. However, a substantial number of LHON patients do not harbor known variants, both pointing to the genetic heterogeneity of LHON and bringing into question its genetic diagnosis. We report a familial case that exhibited typical features of LHON but lacked any of the common mutations. Genetic analysis revealed a novel pathogenic defect in the ND6 gene at 14279A that was not detected in any haplogroup-matched controls screened for it, nor has it been previously reported. This mutation causes a substantial conformational change in the secondary structure of the polypeptide matrix coil and may explain the LHON expression. Thus, it expands the spectrum of deleterious changes affecting ND6-encoding subunit and further highlights the functional significance of this gene, providing additional clues to the disease pathogenesis.

  19. Oxidative stress at low levels can induce clustered DNA lesions leading to NHEJ mediated mutations.

    Science.gov (United States)

    Sharma, Vyom; Collins, Leonard B; Chen, Ting-Huei; Herr, Natalie; Takeda, Shunichi; Sun, Wei; Swenberg, James A; Nakamura, Jun

    2016-05-01

    DNA damage and mutations induced by oxidative stress are associated with various different human pathologies including cancer. The facts that most human tumors are characterized by large genome rearrangements and glutathione depletion in mice results in deletions in DNA suggest that reactive oxygen species (ROS) may cause gene and chromosome mutations through DNA double strand breaks (DSBs). However, the generation of DSBs at low levels of ROS is still controversial. In the present study, we show that H2O2 at biologically-relevant levels causes a marked increase in oxidative clustered DNA lesions (OCDLs) with a significant elevation of replication-independent DSBs. Although it is frequently reported that OCDLs are fingerprint of high-energy IR, our results indicate for the first time that H2O2, even at low levels, can also cause OCDLs leading to DSBs specifically in G1 cells. Furthermore, a reverse genetic approach revealed a significant contribution of the non-homologous end joining (NHEJ) pathway in H2O2-induced DNA repair & mutagenesis. This genomic instability induced by low levels of ROS may be involved in spontaneous mutagenesis and the etiology of a wide variety of human diseases like chronic inflammation-related disorders, carcinogenesis, neuro-degeneration and aging. PMID:27015367

  20. Enzyme-Free Detection of Mutations in Cancer DNA Using Synthetic Oligonucleotide Probes and Fluorescence Microscopy

    DEFF Research Database (Denmark)

    Miotke, Laura; Maity, Arindam; Ji, Hanlee;

    2015-01-01

    BACKGROUND: Rapid reliable diagnostics of DNA mutations are highly desirable in research and clinical assays. Current development in this field goes simultaneously in two directions: 1) high-throughput methods, and 2) portable assays. Non-enzymatic approaches are attractive for both types...... microscopy and nucleic acid analogues have been proposed so far. METHODS AND RESULTS: Here we report a novel enzyme-free approach to efficiently detect cancer mutations. This assay includes gene-specific target enrichment followed by annealing to oligonucleotides containing locked nucleic acids (LNAs...... 1000-fold above the potential detection limit. CONCLUSION: Overall, the novel assay we describe could become a new approach to rapid, reliable and enzyme-free diagnostics of cancer or other associated DNA targets. Importantly, stoichiometry of wild type and mutant targets is conserved in our assay...

  1. Alternative mechanisms of telomere lengthening: Permissive mutations, DNA repair proteins and tumorigenic progression

    Energy Technology Data Exchange (ETDEWEB)

    Gocha, April Renee Sandy; Harris, Julia [Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States); Groden, Joanna, E-mail: joanna.groden@osumc.edu [Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States)

    2013-03-15

    Highlights: ► Neoplastic cells maintain telomeres by telomerase or ALT. ► Genetic mutations in p53, ATRX, DAXX or H3F3A may activate ALT. ► Many DNA repair proteins are involved in ALT. ► Tumor progression is favored by telomerase expression. - Abstract: Telomeres protect chromosome termini to maintain genomic stability and regulate cellular lifespan. Maintenance of telomere length is required for neoplastic cells after the acquisition of mutations that deregulate cell cycle control and increase cellular proliferation, and can occur through expression of the enzyme telomerase or in a telomerase-independent manner termed alternative lengthening of telomeres (ALT). The precise mechanisms that govern the activation of ALT or telomerase in tumor cells are unknown, although cellular origin may favor one or the other mechanisms. ALT pathways are incompletely understood to date; however, recent publications have increasingly broadened our understanding of how ALT is activated, how it proceeds, and how it influences tumor growth. Specific mutational events influence ALT activation, as mutations in genes that suppress recombination and/or alterations in the regulation of telomerase expression are associated with ALT. Once engaged, ALT uses DNA repair proteins to maintain telomeres in the absence of telomerase; experiments that manipulate the expression of specific proteins in cells using ALT are illuminating some of its mechanisms. Furthermore, ALT may influence tumor growth, as experimental and clinical data suggest that telomerase expression may favor tumor progression. This review summarizes recent findings in mammalian cells and models, as well as clinical data, that identify the genetic mutations permissive to ALT, the DNA repair proteins involved in ALT mechanisms and the importance of telomere maintenance mechanisms for tumor progression. A comprehensive understanding of the mechanisms that permit tumor cell immortalization will be important for identifying

  2. Mutations in the DNA methyltransferase gene DNMT3A cause an overgrowth syndrome with intellectual disability

    DEFF Research Database (Denmark)

    Tatton-Brown, Katrina; Seal, Sheila; Ruark, Elise;

    2014-01-01

    Overgrowth disorders are a heterogeneous group of conditions characterized by increased growth parameters and other variable clinical features such as intellectual disability and facial dysmorphism. To identify new causes of human overgrowth, we performed exome sequencing in ten proband...... and histone binding. Similar mutations were not present in 1,000 UK population controls (13/152 cases versus 0/1,000 controls; P intellectual disability and greater height. DNMT3A encodes a DNA methyltransferase essential for establishing...

  3. Mutation patterns of mitochondrial H- and L-strand DNA in closely related Cyprinid fishes.

    OpenAIRE

    Bielawski, Joseph P.; John R. Gold

    2002-01-01

    Mitochondrial genome replication is asymmetric. Replication starts from the origin of heavy (H)-strand replication, displacing the parental H-strand as it proceeds along the molecule. The H-strand remains single stranded until light (L)-strand replication is initiated from a second origin of replication. It has been suggested that single-stranded H-strand DNA is more sensitive to mutational damage, giving rise to substitutional rate differences between the two strands and among genes in mamma...

  4. MtDNA analysis reveals enriched pathogenic mutations in Tibetan highlanders

    Science.gov (United States)

    Kang, Longli; Zheng, Hong-Xiang; Zhang, Menghan; Yan, Shi; Li, Lei; Liu, Lijun; Liu, Kai; Hu, Kang; Chen, Feng; Ma, Lifeng; Qin, Zhendong; Wang, Yi; Wang, Xiaofeng; Jin, Li

    2016-01-01

    Tibetan highlanders, including Tibetans, Monpas, Lhobas, Dengs and Sherpas, are considered highly adaptive to severe hypoxic environments. Mitochondrial DNA (mtDNA) might be important in hypoxia adaptation given its role in coding core subunits of oxidative phosphorylation. In this study, we employed 549 complete highlander mtDNA sequences (including 432 random samples) to obtain a comprehensive view of highlander mtDNA profile. In the phylogeny of a total of 36,914 sequences, we identified 21 major haplogroups representing founding events of highlanders, most of which were coalesced in 10 kya. Through founder analysis, we proposed a three-phase model of colonizing the plateau, i.e., pre-LGM Time (30 kya, 4.68%), post-LGM Paleolithic Time (16.8 kya, 29.31%) and Neolithic Time (after 8 kya, 66.01% in total). We observed that pathogenic mutations occurred far more frequently in 22 highlander-specific lineages (five lineages carrying two pathogenic mutations and six carrying one) than in the 6,857 haplogroups of all the 36,914 sequences (P = 4.87 × 10−8). Furthermore, the number of possible pathogenic mutations carried by highlanders (in average 3.18 ± 1.27) were significantly higher than that in controls (2.82 ± 1.40) (P = 1.89 × 10−4). Considering that function-altering and pathogenic mutations are enriched in highlanders, we therefore hypothesize that they may have played a role in hypoxia adaptation. PMID:27498855

  5. Obtaining insurance after DNA diagnostics: a survey among hypertrophic cardiomyopathy mutation carriers

    OpenAIRE

    Christiaans, Imke; Kok, Tjitske M; van Langen, Irene M.; Birnie, Erwin; Bonsel, Gouke J.; Wilde, Arthur A. M.; Smets, Ellen M. A.

    2009-01-01

    Hypertrophic cardiomyopathy (HCM) is a common hereditary heart disease associated with increased mortality. Disclosure of DNA test results may have social implications such as low access to insurance. In the Netherlands, insurance companies are restricted in the use of genetic information of their clients by the Medical Examination Act. A cross-sectional survey was used to assess the frequency and type of problems encountered by HCM mutation carriers applying for insurance, and associations w...

  6. DNA crosslinking, sister-chromatid exchange and specific-locus mutations.

    Science.gov (United States)

    Carrano, A V; Thompson, L H; Stetka, D G; Minkler, J L; Mazrimas, J A; Fong, S

    1979-11-01

    Chinese hamster ovary cells were treated with the DNA-crosslinking chemicals, mitomycin C (MMC) and porfiromycin (POR), and their monofunctional derivative decarbamoyl mitomycin C (DCMMC). After exposure, the cells were studied for the induction of sister-chromatid exchanges (SCEs) and mutations at the hypoxanthine phosphoribosyltransferase and adenine phosphoribosyltransferase loci. The frequency of SCEs varied significantly in successive sampling intervals, requiring the weighting of each interval by the percentage of second-division mitosis in that interval to obtain the mean SCE frequency for each dose. All 3 compounds were potent inducers of SCEs but weakly mutagenic. All 3 chemicals by concentration were approximately equally effective in inducing SCEs or mutations. When the induced SCEs and mutations were compared at equal levels of survival, DCMMC was slightly more effective than MMC or POR in inducing SCEs and somewhat less mutagenic. These results indicate that the DNA interstrand crosslink is not the major lesion responsible for the induction of SCE or mutation by these compounds. PMID:522865

  7. DNA crosslinking, sister-chromatid exchange and specific-locus mutations

    Energy Technology Data Exchange (ETDEWEB)

    Carrano, A.V.; Thompson, L.H.; Stetka, D.G.; Minkler, J.L.; Mazrimas, J.A.; Fong, S.

    1979-01-01

    Chinese hamster ovary cells were treated with the DNA-crosslinking chemicals, mitomycin C (MMC) and porfiromycin (POR), and their monofunctional derivative decarbamoyl mitomycin C (DCMMC). After exposure, the cells were studied for the induction of sister-chromatid exchanges (SCEs) and mutations at the hypoxanthine phosphoribosyltransferase and adenine phosphoribosyltransferase loci. The frequency of SCEs varied significantly in successive sampling intervals, requiring the weighting of each interval by the percentage of second-division mitosis in that interval to obtain the mean SCE frequency for each dose. All 3 compounds were potent inducers of SCEs but weakly mutagenic. All 3 chemicals by concentration were approximately equally effective in inducing SCEs or mutations. When the induced SCEs and mutations were compared at equal levels of survival, DCMMC was slightly more effective than MMC or POR in inducing SCEs and somewhat less mutagenic. These results indicate that the DNA interstrand crosslink is not the major lesion responsible for the induction of SCE or mutation by these compounds.

  8. Increasing Nucleosome Occupancy Is Correlated with an Increasing Mutation Rate so Long as DNA Repair Machinery Is Intact.

    Science.gov (United States)

    Yazdi, Puya G; Pedersen, Brian A; Taylor, Jared F; Khattab, Omar S; Chen, Yu-Han; Chen, Yumay; Jacobsen, Steven E; Wang, Ping H

    2015-01-01

    Deciphering the multitude of epigenomic and genomic factors that influence the mutation rate is an area of great interest in modern biology. Recently, chromatin has been shown to play a part in this process. To elucidate this relationship further, we integrated our own ultra-deep sequenced human nucleosomal DNA data set with a host of published human genomic and cancer genomic data sets. Our results revealed, that differences in nucleosome occupancy are associated with changes in base-specific mutation rates. Increasing nucleosome occupancy is associated with an increasing transition to transversion ratio and an increased germline mutation rate within the human genome. Additionally, cancer single nucleotide variants and microindels are enriched within nucleosomes and both the coding and non-coding cancer mutation rate increases with increasing nucleosome occupancy. There is an enrichment of cancer indels at the theoretical start (74 bp) and end (115 bp) of linker DNA between two nucleosomes. We then hypothesized that increasing nucleosome occupancy decreases access to DNA by DNA repair machinery and could account for the increasing mutation rate. Such a relationship should not exist in DNA repair knockouts, and we thus repeated our analysis in DNA repair machinery knockouts to test our hypothesis. Indeed, our results revealed no correlation between increasing nucleosome occupancy and increasing mutation rate in DNA repair knockouts. Our findings emphasize the linkage of the genome and epigenome through the nucleosome whose properties can affect genome evolution and genetic aberrations such as cancer.

  9. Segregation pattern and biochemical effect of the G3460A mtDNA mutation in 27 members of LHON family.

    Science.gov (United States)

    Kaplanová, Vilma; Zeman, Jirí; Hansíková, Hana; Cerná, Leona; Houst'ková, Hana; Misovicová, Nadezda; Houstek, Josef

    2004-08-30

    Inheritance and expression of mitochondrial DNA (mtDNA) mutations are crucial for the pathogenesis of Leber hereditary optic neuropathy (LHON). We have investigated the segregation and functional consequences of G3460A mtDNA mutation in 27 members of a three-generation family with LHON syndrome. Specific activity of respiratory chain complex I in platelets was reduced in average to 56%, but no direct correlation between the mutation load and its biochemical expression was found. Heteroplasmy in blood, platelets and hair follicles varied from 7% to 100%. Segregation pattern exhibited tissue specificity and influence of different nuclear backgrounds in four branches of the pedigree. Longitudinal analysis revealed a significant (p=0.02) decrease in blood mutation load. Although enzyme assay showed reduction of complex I activity, our results give additional support to the hypothesis that expression of LHON mutation depends on complex nuclear-mitochondrial interaction.

  10. Triplet repeat DNA structures and human genetic disease: dynamic mutations from dynamic DNA

    Indian Academy of Sciences (India)

    Richard R Sinden; Vladimir N Potaman; Elena A Oussatcheva; Christopher E Pearson; Yuri L Lyubchenko; Luda S Shlyakhtenko

    2002-02-01

    Fourteen genetic neurodegenerative diseases and three fragile sites have been associated with the expansion of (CTG)n•(CAG)n, (CGG)n•(CCG)n, or (GAA)n•(TTC)n repeat tracts. Different models have been proposed for the expansion of triplet repeats, most of which presume the formation of alternative DNA structures in repeat tracts. One of the most likely structures, slipped strand DNA, may stably and reproducibly form within triplet repeat sequences. The propensity to form slipped strand DNA is proportional to the length and homogeneity of the repeat tract. The remarkable stability of slipped strand DNA may, in part, be due to loop-loop interactions facilitated by the sequence complementarity of the loops and the dynamic structure of three-way junctions formed at the loop-outs.

  11. Mitochondrial DNA mutations provoke dominant inhibition of mitochondrial inner membrane fusion.

    Directory of Open Access Journals (Sweden)

    Cécile Sauvanet

    Full Text Available Mitochondria are highly dynamic organelles that continuously move, fuse and divide. Mitochondrial dynamics modulate overall mitochondrial morphology and are essential for the proper function, maintenance and transmission of mitochondria and mitochondrial DNA (mtDNA. We have investigated mitochondrial fusion in yeast cells with severe defects in oxidative phosphorylation (OXPHOS due to removal or various specific mutations of mtDNA. We find that, under fermentative conditions, OXPHOS deficient cells maintain normal levels of cellular ATP and ADP but display a reduced mitochondrial inner membrane potential. We demonstrate that, despite metabolic compensation by glycolysis, OXPHOS defects are associated to a selective inhibition of inner but not outer membrane fusion. Fusion inhibition was dominant and hampered the fusion of mutant mitochondria with wild-type mitochondria. Inhibition of inner membrane fusion was not systematically associated to changes of mitochondrial distribution and morphology, nor to changes in the isoform pattern of Mgm1, the major fusion factor of the inner membrane. However, inhibition of inner membrane fusion correlated with specific alterations of mitochondrial ultrastructure, notably with the presence of aligned and unfused inner membranes that are connected to two mitochondrial boundaries. The fusion inhibition observed upon deletion of OXPHOS related genes or upon removal of the entire mtDNA was similar to that observed upon introduction of point mutations in the mitochondrial ATP6 gene that are associated to neurogenic ataxia and retinitis pigmentosa (NARP or to maternally inherited Leigh Syndrome (MILS in humans. Our findings indicate that the consequences of mtDNA mutations may not be limited to OXPHOS defects but may also include alterations in mitochondrial fusion. Our results further imply that, in healthy cells, the dominant inhibition of fusion could mediate the exclusion of OXPHOS-deficient mitochondria from

  12. BRAF mutation analysis in circulating free tumor DNA of melanoma patients treated with BRAF inhibitors.

    Science.gov (United States)

    Gonzalez-Cao, Maria; Mayo-de-Las-Casas, Clara; Molina-Vila, Miguel A; De Mattos-Arruda, Leticia; Muñoz-Couselo, Eva; Manzano, Jose L; Cortes, Javier; Berros, Jose P; Drozdowskyj, Ana; Sanmamed, Miguel; Gonzalez, Alvaro; Alvarez, Carlos; Viteri, Santiago; Karachaliou, Niki; Martin Algarra, Salvador; Bertran-Alamillo, Jordi; Jordana-Ariza, Nuria; Rosell, Rafael

    2015-12-01

    BRAFV600E is a unique molecular marker for metastatic melanoma, being the most frequent somatic point mutation in this malignancy. Detection of BRAFV600E in blood could have prognostic and predictive value and could be useful for monitoring response to BRAF-targeted therapy. We developed a rapid, sensitive method for the detection and quantification of BRAFV600E in circulating free DNA (cfDNA) isolated from plasma and serum on the basis of a quantitative 5'-nuclease PCR (Taqman) in the presence of a peptide-nucleic acid. We validated the assay in 92 lung, colon, and melanoma archival serum and plasma samples with paired tumor tissue (40 wild-type and 52 BRAFV600E). The correlation of cfDNA BRAFV600E with clinical parameters was further explored in 22 metastatic melanoma patients treated with BRAF inhibitors. Our assay could detect and quantify BRAFV600E in mixed samples with as little as 0.005% mutant DNA (copy number ratio 1 : 20 000), with a specificity of 100% and a sensitivity of 57.7% in archival serum and plasma samples. In 22 melanoma patients treated with BRAF inhibitors, the median progression-free survival was 3.6 months for those showing BRAFV600E in pretreatment cfDNA compared with 13.4 months for those in whom the mutation was not detected (P=0.021). Moreover, the median overall survival for positive versus negative BRAFV600E tests in pretreatment cfDNA differed significantly (7 vs. 21.8 months, P=0.017). This finding indicates that the sensitive detection and accurate quantification of low-abundance BRAFV600E alleles in cfDNA using our assay can be useful for predicting treatment outcome.

  13. Rare primary mitochondrial DNA mutations and probable synergistic variants in Leber's hereditary optic neuropathy.

    Directory of Open Access Journals (Sweden)

    Alessandro Achilli

    Full Text Available BACKGROUND: Leber's hereditary optic neuropathy (LHON is a maternally inherited blinding disorder, which in over 90% of cases is due to one of three primary mitochondrial DNA (mtDNA point mutations (m.11778G>A, m.3460G>A and m.14484T>C, respectively in MT-ND4, MT-ND1 and MT-ND6 genes. However, the spectrum of mtDNA mutations causing the remaining 10% of cases is only partially and often poorly defined. METHODOLOGY/PRINCIPAL FINDINGS: In order to improve such a list of pathological variants, we completely sequenced the mitochondrial genomes of suspected LHON patients from Italy, France and Germany, lacking the three primary common mutations. Phylogenetic and conservation analyses were performed. Sixteen mitochondrial genomes were found to harbor at least one of the following nine rare LHON pathogenic mutations in genes MT-ND1 (m.3700G>A/p.A132T, m.3733G>A-C/p.E143K-Q, m.4171C>A/p.L289M, MT-ND4L (m.10663T>C/p.V65A and MT-ND6 (m.14459G>A/p.A72V, m.14495A>G/p.M64I, m.14482C>A/p.L60S, and m.14568C>T/p.G36S. Phylogenetic analyses revealed that these substitutions were due to independent events on different haplogroups, whereas interspecies comparisons showed that they affected conserved amino acid residues or domains in the ND subunit genes of complex I. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that these nine substitutions are all primary LHON mutations. Therefore, despite their relative low frequency, they should be routinely tested for in all LHON patients lacking the three common mutations. Moreover, our sequence analysis confirms the major role of haplogroups J1c and J2b (over 35% in our probands versus 6% in the general population of Western Europe and other putative synergistic mtDNA variants in LHON expression.

  14. Actionable mutations in plasma cell-free DNA in patients with advanced cancers referred for experimental targeted therapies

    OpenAIRE

    Janku, Filip; Angenendt, Philipp; Tsimberidou, Apostolia M.; Fu, Siqing; Naing, Aung; Falchook, Gerald S.; David S Hong; Holley, Veronica R.; Cabrilo, Goran; Jennifer J Wheler; Piha-Paul, Sarina A.; Zinner, Ralph G.; Bedikian, Agop Y.; Overman, Michael J.; Kee, Bryan K.

    2015-01-01

    Cell-free (cf) DNA in the plasma of cancer patients offers an easily obtainable source of biologic material for mutation analysis. Plasma samples from 157 patients with advanced cancers who progressed on systemic therapy were tested for 21 mutations in BRAF, EGFR, KRAS, and PIK3CA using the BEAMing method and results were compared to mutation analysis of archival tumor tissue from a CLIA-certified laboratory obtained as standard of care from diagnostic or therapeutic procedures. Results were ...

  15. Exome Sequencing of Cell-Free DNA from Metastatic Cancer Patients Identifies Clinically Actionable Mutations Distinct from Primary Disease.

    Directory of Open Access Journals (Sweden)

    Timothy M Butler

    Full Text Available The identification of the molecular drivers of cancer by sequencing is the backbone of precision medicine and the basis of personalized therapy; however, biopsies of primary tumors provide only a snapshot of the evolution of the disease and may miss potential therapeutic targets, especially in the metastatic setting. A liquid biopsy, in the form of cell-free DNA (cfDNA sequencing, has the potential to capture the inter- and intra-tumoral heterogeneity present in metastatic disease, and, through serial blood draws, track the evolution of the tumor genome. In order to determine the clinical utility of cfDNA sequencing we performed whole-exome sequencing on cfDNA and tumor DNA from two patients with metastatic disease; only minor modifications to our sequencing and analysis pipelines were required for sequencing and mutation calling of cfDNA. The first patient had metastatic sarcoma and 47 of 48 mutations present in the primary tumor were also found in the cell-free DNA. The second patient had metastatic breast cancer and sequencing identified an ESR1 mutation in the cfDNA and metastatic site, but not in the primary tumor. This likely explains tumor progression on Anastrozole. Significant heterogeneity between the primary and metastatic tumors, with cfDNA reflecting the metastases, suggested separation from the primary lesion early in tumor evolution. This is best illustrated by an activating PIK3CA mutation (H1047R which was clonal in the primary tumor, but completely absent from either the metastasis or cfDNA. Here we show that cfDNA sequencing supplies clinically actionable information with minimal risks compared to metastatic biopsies. This study demonstrates the utility of whole-exome sequencing of cell-free DNA from patients with metastatic disease. cfDNA sequencing identified an ESR1 mutation, potentially explaining a patient's resistance to aromatase inhibition, and gave insight into how metastatic lesions differ from the primary tumor.

  16. Exome Sequencing of Cell-Free DNA from Metastatic Cancer Patients Identifies Clinically Actionable Mutations Distinct from Primary Disease

    Science.gov (United States)

    Butler, Timothy M.; Johnson-Camacho, Katherine; Peto, Myron; Wang, Nicholas J.; Macey, Tara A.; Korkola, James E.; Koppie, Theresa M.; Corless, Christopher L.; Gray, Joe W.; Spellman, Paul T.

    2015-01-01

    The identification of the molecular drivers of cancer by sequencing is the backbone of precision medicine and the basis of personalized therapy; however, biopsies of primary tumors provide only a snapshot of the evolution of the disease and may miss potential therapeutic targets, especially in the metastatic setting. A liquid biopsy, in the form of cell-free DNA (cfDNA) sequencing, has the potential to capture the inter- and intra-tumoral heterogeneity present in metastatic disease, and, through serial blood draws, track the evolution of the tumor genome. In order to determine the clinical utility of cfDNA sequencing we performed whole-exome sequencing on cfDNA and tumor DNA from two patients with metastatic disease; only minor modifications to our sequencing and analysis pipelines were required for sequencing and mutation calling of cfDNA. The first patient had metastatic sarcoma and 47 of 48 mutations present in the primary tumor were also found in the cell-free DNA. The second patient had metastatic breast cancer and sequencing identified an ESR1 mutation in the cfDNA and metastatic site, but not in the primary tumor. This likely explains tumor progression on Anastrozole. Significant heterogeneity between the primary and metastatic tumors, with cfDNA reflecting the metastases, suggested separation from the primary lesion early in tumor evolution. This is best illustrated by an activating PIK3CA mutation (H1047R) which was clonal in the primary tumor, but completely absent from either the metastasis or cfDNA. Here we show that cfDNA sequencing supplies clinically actionable information with minimal risks compared to metastatic biopsies. This study demonstrates the utility of whole-exome sequencing of cell-free DNA from patients with metastatic disease. cfDNA sequencing identified an ESR1 mutation, potentially explaining a patient’s resistance to aromatase inhibition, and gave insight into how metastatic lesions differ from the primary tumor. PMID:26317216

  17. A novel missense mutation in SUCLG1 associated with mitochondrial DNA depletion, encephalomyopathic form, with methylmalonic aciduria

    DEFF Research Database (Denmark)

    Østergaard, Elsebet; Schwartz, Marianne; Batbayli, Mustafa;

    2010-01-01

    Mitochondrial DNA depletion, encephalomyopathic form, with methylmalonic aciduria is associated with mutations in SUCLA2, the gene encoding a beta subunit of succinate-CoA ligase, where 17 patients have been reported. Mutations in SUCLG1, encoding the alpha subunit of the enzyme, have been reported...

  18. Actionable mutations in plasma cell-free DNA in patients with advanced cancers referred for experimental targeted therapies

    Science.gov (United States)

    Janku, Filip; Angenendt, Philipp; Tsimberidou, Apostolia M.; Fu, Siqing; Naing, Aung; Falchook, Gerald S.; Hong, David S.; Holley, Veronica R.; Cabrilo, Goran; Wheler, Jennifer J.; Piha-Paul, Sarina A.; Zinner, Ralph G.; Bedikian, Agop Y.; Overman, Michael J.; Kee, Bryan K.; Kim, Kevin B.; Kopetz, E. Scott; Luthra, Rajyalakshmi; Diehl, Frank; Meric-Bernstam, Funda; Kurzrock, Razelle

    2015-01-01

    Cell-free (cf) DNA in the plasma of cancer patients offers an easily obtainable source of biologic material for mutation analysis. Plasma samples from 157 patients with advanced cancers who progressed on systemic therapy were tested for 21 mutations in BRAF, EGFR, KRAS, and PIK3CA using the BEAMing method and results were compared to mutation analysis of archival tumor tissue from a CLIA-certified laboratory obtained as standard of care from diagnostic or therapeutic procedures. Results were concordant for archival tissue and plasma cfDNA in 91% cases for BRAF mutations (kappa = 0.75, 95% confidence interval [CI] 0.63 – 0.88), in 99% cases for EGFR mutations (kappa = 0.90, 95% CI 0.71– 1.00), in 83% cases for KRAS mutations (kappa = 0.67, 95% CI 0.54 – 0.80) and in 91% cases for PIK3CA mutations (kappa = 0.65, 95% CI 0.46 – 0.85). Patients (n = 41) with > 1% of KRAS mutant cfDNA had a shorter median survival compared to 20 patients with 1% of mutant cfDNA (BRAF, EGFR, KRAS, or PIK3CA) had a shorter median survival compared to 33 patients with DNA (5.5 vs. 9.8 months, p = 0.001), which was confirmed in multivariable analysis. PMID:25980577

  19. A mutation in the XPB/ERCC3 DNA repair transcription gene, associated with trichothiodystrophy

    Energy Technology Data Exchange (ETDEWEB)

    Weeda, G.; Donker, I.; Vermeulen, W. [Erasmus Univ., Rotterdam (Netherlands)] [and others

    1997-02-01

    Trichothiodystrophy (TTD) is a rare, autosomal recessive disorder characterized by sulfur-deficient brittle hair and nails, mental retardation, impaired sexual development, and ichthyosis. Photosensitivity has been reported in {approximately}50% of the cases, but no skin cancer is associated with TTD. Virtually all photosensitive TTD patients have a deficiency in the nucleotide excision repair (NER) of UV-induced DNA damage that is indistinguishable from that of xeroderma pigmentosum (XP) complementation group D (XP-D) patients. DNA repair defects in XP-D are associated with two additional, quite different diseases; XP, a sun-sensitive and cancer-prone repair disorder, and Cockayne syndrome (CS), a photosensitive condition characterized by physical and mental retardation and wizened facial appearance. One photosensitive TTD case constitutes a new repair-deficient complementation group, TTD-A. Remarkably, both TTD-A and XP-D defects are associated with subunits of TFIIH, a basal transcription factor with a second function in DNA repair. Thus, mutations in TFIIH components may, on top of a repair defect, also cause transcriptional insufficiency, which may explain part of the non-XP clinical features of TTD. To date, three patients with the remarkable conjunction of XP and CS but not TM have been assigned to XP complementation group B (XP-B). Here we present the characterization of the NER defect in two mild TTD patients (TTD6VI and TTD4VI) and confirm the assignment to X-PB. The causative mutation was found to be a single base substitution resulting in a missense mutation (T119P) in a region of the XPB protein. These findings define a third TTD complementation group, extend the clinical heterogeneity associated with XP-B, stress the exclusive relationship between TTD and mutations in subunits of repair/transcription factor TFIIH, and strongly support the concept of {open_quotes}transcription syndromes.{close_quotes} 46 refs., 6 figs., 2 tabs.

  20. ALS-associated mutation FUS-R521C causes DNA damage and RNA splicing defects.

    Science.gov (United States)

    Qiu, Haiyan; Lee, Sebum; Shang, Yulei; Wang, Wen-Yuan; Au, Kin Fai; Kamiya, Sherry; Barmada, Sami J; Finkbeiner, Steven; Lui, Hansen; Carlton, Caitlin E; Tang, Amy A; Oldham, Michael C; Wang, Hejia; Shorter, James; Filiano, Anthony J; Roberson, Erik D; Tourtellotte, Warren G; Chen, Bin; Tsai, Li-Huei; Huang, Eric J

    2014-03-01

    Autosomal dominant mutations of the RNA/DNA binding protein FUS are linked to familial amyotrophic lateral sclerosis (FALS); however, it is not clear how FUS mutations cause neurodegeneration. Using transgenic mice expressing a common FALS-associated FUS mutation (FUS-R521C mice), we found that mutant FUS proteins formed a stable complex with WT FUS proteins and interfered with the normal interactions between FUS and histone deacetylase 1 (HDAC1). Consequently, FUS-R521C mice exhibited evidence of DNA damage as well as profound dendritic and synaptic phenotypes in brain and spinal cord. To provide insights into these defects, we screened neural genes for nucleotide oxidation and identified brain-derived neurotrophic factor (Bdnf) as a target of FUS-R521C-associated DNA damage and RNA splicing defects in mice. Compared with WT FUS, mutant FUS-R521C proteins formed a more stable complex with Bdnf RNA in electrophoretic mobility shift assays. Stabilization of the FUS/Bdnf RNA complex contributed to Bdnf splicing defects and impaired BDNF signaling through receptor TrkB. Exogenous BDNF only partially restored dendrite phenotype in FUS-R521C neurons, suggesting that BDNF-independent mechanisms may contribute to the defects in these neurons. Indeed, RNA-seq analyses of FUS-R521C spinal cords revealed additional transcription and splicing defects in genes that regulate dendritic growth and synaptic functions. Together, our results provide insight into how gain-of-function FUS mutations affect critical neuronal functions.

  1. Evidence that in xeroderma pigmentosum variant cells, which lack DNA polymerase eta, DNA polymerase iota causes the very high frequency and unique spectrum of UV-induced mutations.

    Science.gov (United States)

    Wang, Yun; Woodgate, Roger; McManus, Terrence P; Mead, Samantha; McCormick, J Justin; Maher, Veronica M

    2007-04-01

    Xeroderma pigmentosum variant (XPV) patients have normal DNA excision repair, yet are predisposed to develop sunlight-induced cancer. They exhibit a 25-fold higher than normal frequency of UV-induced mutations and very unusual kinds (spectrum), mainly transversions. The primary defect in XPV cells is the lack of functional DNA polymerase (Pol) eta, the translesion synthesis DNA polymerase that readily inserts adenine nucleotides opposite photoproducts involving thymine. The high frequency and striking difference in kinds of UV-induced mutations in XPV cells strongly suggest that, in the absence of Pol eta, an abnormally error-prone polymerase substitutes. In vitro replication studies of Pol iota show that it replicates past 5'T-T3' and 5'T-U3' cyclobutane pyrimidine dimers, incorporating G or T nucleotides opposite the 3' nucleotide. To test the hypothesis that Pol iota causes the high frequency and abnormal spectrum of UV-induced mutations in XPV cells, we identified an unlimited lifespan XPV cell line expressing two forms of Pol iota, whose frequency of UV-induced mutations is twice that of XPV cells expressing one form. We eliminated expression of one form and compared the parental cells and derivatives for the frequency and kinds of UV-induced mutations. All exhibited similar sensitivity to the cytotoxicity of UV((254 nm)), and the kinds of mutations induced were identical, but the frequency of mutations induced in the derivatives was reduced to UV-induced mutations, and ultimately their malignant transformation.

  2. Platelet hexosaminidase a enzyme assay effectively detects carriers missed by targeted DNA mutation analysis.

    Science.gov (United States)

    Nakagawa, Sachiko; Zhan, Jie; Sun, Wei; Ferreira, Jose Carlos; Keiles, Steven; Hambuch, Tina; Kammesheidt, Anja; Mark, Brian L; Schneider, Adele; Gross, Susan; Schreiber-Agus, Nicole

    2012-01-01

    Biochemical testing of hexosaminidase A (HexA) enzyme activity has been available for decades and has the ability to detect almost all Tay-Sachs disease (TSD) carriers, irrespective of ethnic background. This is increasingly important, as the gene pool of those who identify as Ashkenazi Jewish is diversifying. Here we describe the analysis of a cohort of 4,325 individuals arising from large carrier screening programs and tested by the serum and/or platelet HexA enzyme assays and by targeted DNA mutation analysis. Our results continue to support the platelet assay as a highly effective method for TSD carrier screening, with a low inconclusive rate and the ability to detect possible disease-causing mutation carriers that would have been missed by targeted DNA mutation analysis. Sequence analysis performed on one such platelet assay carrier, who had one non-Ashkenazi Jewish parent, identified the amino acid change Thr259Ala (A775G). Based on crystallographic modeling, this change is predicted to be deleterious, as threonine 259 is positioned proximal to the HexA alpha subunit active site and helps to stabilize key residues therein. Accordingly, if individuals are screened for TSD in broad-based programs by targeted molecular testing alone, they must be made aware that there is a more sensitive and inexpensive test available that can identify additional carriers. Alternatively, the enzyme assays can be offered as a first tier test, especially when screening individuals of mixed or non-Jewish ancestry. PMID:23430931

  3. A BRCA1-mutation associated DNA methylation signature in blood cells predicts sporadic breast cancer incidence and survival.

    OpenAIRE

    Anjum, S; Fourkala, E O; Zikan, M.; Wong, A.; Gentry-Maharaj, A.; Jones, A.; HARDY, R.; Cibula, D.; Kuh, D.; Jacobs, I. J.; Teschendorff, A.E.; Menon, U; Widschwendter, M

    2014-01-01

    Background BRCA1 mutation carriers have an 85% risk of developing breast cancer but the risk of developing non-hereditary breast cancer is difficult to assess. Our objective is to test whether a DNA methylation (DNAme) signature derived from BRCA1 mutation carriers is able to predict non-hereditary breast cancer. Methods In a case/control setting (72 BRCA1 mutation carriers and 72 BRCA1/2 wild type controls) blood cell DNA samples were profiled on the Illumina 27 k methylation array. Using th...

  4. Prognostic role of PIK3CA mutations of cell‐free DNA in early‐stage triple negative breast cancer

    OpenAIRE

    Takeshita, Takashi; Yamamoto, Yutaka; Yamamoto‐Ibusuki, Mutsuko; Inao, Toko; Sueta, Aiko; Fujiwara, Saori; Omoto, Yoko; Iwase, Hirotaka

    2015-01-01

    PIK3CA is an oncogene that encodes the p110α component of phosphatidylinositol 3‐kinase (PI3K); it is the second most frequently mutated gene following the TP53 gene. In the clinical setting, PIK3CA mutations may have favorable prognostic value for hormone receptor‐positive breast cancer patients and, during the past few years, PIK3CA mutations of cell‐free DNA (cfDNA) have attracted attention as a potential noninvasive biomarker of cancer. However, there are few reports on the clinical impli...

  5. DNA mutations mediate microevolution between host-adapted forms of the pathogenic fungus Cryptococcus neoformans.

    Directory of Open Access Journals (Sweden)

    Denise A Magditch

    Full Text Available The disease cryptococcosis, caused by the fungus Cryptococcus neoformans, is acquired directly from environmental exposure rather than transmitted person-to-person. One explanation for the pathogenicity of this species is that interactions with environmental predators select for virulence. However, co-incubation of C. neoformans with amoeba can cause a "switch" from the normal yeast morphology to a pseudohyphal form, enabling fungi to survive exposure to amoeba, yet conversely reducing virulence in mammalian models of cryptococcosis. Like other human pathogenic fungi, C. neoformans is capable of microevolutionary changes that influence the biology of the organism and outcome of the host-pathogen interaction. A yeast-pseudohyphal phenotypic switch also happens under in vitro conditions. Here, we demonstrate that this morphological switch, rather than being under epigenetic control, is controlled by DNA mutation since all pseudohyphal strains bear mutations within genes encoding components of the RAM pathway. High rates of isolation of pseudohyphal strains can be explained by the physical size of RAM pathway genes and a hypermutator phenotype of the strain used in phenotypic switching studies. Reversion to wild type yeast morphology in vitro or within a mammalian host can occur through different mechanisms, with one being counter-acting mutations. Infection of mice with RAM mutants reveals several outcomes: clearance of the infection, asymptomatic maintenance of the strains, or reversion to wild type forms and progression of disease. These findings demonstrate a key role of mutation events in microevolution to modulate the ability of a fungal pathogen to cause disease.

  6. Mutation rates of TGFBR2 and ACVR2 coding microsatellites in human cells with defective DNA mismatch repair.

    Directory of Open Access Journals (Sweden)

    Heekyung Chung

    Full Text Available Microsatellite instability promotes colonic tumorigenesis through generating frameshift mutations at coding microsatellites of tumor suppressor genes, such as TGFBR2 and ACVR2. As a consequence, signaling through these TGFbeta family receptors is abrogated in DNA Mismatch repair (MMR-deficient tumors. How these mutations occur in real time and mutational rates of these human coding sequences have not previously been studied. We utilized cell lines with different MMR deficiencies (hMLH1-/-, hMSH6-/-, hMSH3-/-, and MMR-proficient to determine mutation rates. Plasmids were constructed in which exon 3 of TGFBR2 and exon 10 of ACVR2 were cloned +1 bp out of frame, immediately after the translation initiation codon of an enhanced GFP (EGFP gene, allowing a -1 bp frameshift mutation to drive EGFP expression. Mutation-resistant plasmids were constructed by interrupting the coding microsatellite sequences, preventing frameshift mutation. Stable cell lines were established containing portions of TGFBR2 and ACVR2, and nonfluorescent cells were sorted, cultured for 7-35 days, and harvested for flow cytometric mutation detection and DNA sequencing at specific time points. DNA sequencing revealed a -1 bp frameshift mutation (A9 in TGFBR2 and A7 in ACVR2 in the fluorescent cells. Two distinct fluorescent populations, M1 (dim, representing heteroduplexes and M2 (bright, representing full mutants were identified, with the M2 fraction accumulating over time. hMLH1 deficiency revealed 11 (5.91 x 10(-4 and 15 (2.18 x 10(-4 times higher mutation rates for the TGFBR2 and ACVR2 microsatellites compared to hMSH6 deficiency, respectively. The mutation rate of the TGFBR2 microsatellite was approximately 3 times higher in both hMLH1 and hMSH6 deficiencies than the ACVR2 microsatellite. The -1 bp frameshift mutation rates of TGFBR2 and ACVR2 microsatellite sequences are dependent upon the human MMR background.

  7. SOLAR RADIATION AND INDUCTION OF DNA DAMAGE, MUTATIONS AND SKIN CANCERS.

    Energy Technology Data Exchange (ETDEWEB)

    SETLOW,R.B.

    2007-05-10

    An understanding of the effects of sunlight on human skin begins with the effects on DNA and extends to cells, animals and humans. The major DNA photoproducts arising from UVB (280-320 nm) exposures are cyclobutane pyrimidine dimers. If unrepaired, they may kill or mutate cells and result in basal and squamous cell carcinomas. Although UVA (320-400 nm) and visible wavelengths are poorly absorbed by DNA, the existing data indicate clearly that exposures to these wavelengths are responsible, in an animal model, for {approx}95 % of the incidence of cutaneous malignant melanoma (CMM). Six lines of evidence, to be discussed in detail, support the photosensitizing role of melanin in the induction of this cancer. They are: (1) Melanomas induced in backcross hybrids of small tropical fish of the genus Xiphophorus, exposed to wavelengths from 302-547 nm, indicate that {approx}95% of the cancers induced by exposure to sunlight would arise from UVA + visible wavelengths; (2) The action spectrum for inducing melanin-photosensitized oxidant production is very similar to the spectrum for inducing melanoma; (3) Albino whites and blacks, although very sensitive to sunburn and the sunlight induction of non-CMM, have very low incidences of CMM; (4) The incidence of CMM as a function of latitude is very similar to that of UVA, but not UVB; (5) Use of UVA-exposing sun-tanning parlors by the young increases the incidence rate of CMM and (6) Major mutations observed in CMM are not UVB-induced.

  8. A triple-helix forming oligonucleotide targeting genomic DNA fails to induce mutation.

    Science.gov (United States)

    Reshat, Reshat; Priestley, Catherine C; Gooderham, Nigel J

    2012-11-01

    Purine tracts in duplex DNA can bind oligonucleotide strands in a sequence specific manner to form triple-helix structures. Triple-helix forming oligonucleotides (TFOs) targeting supFG1 constructs have previously been shown to be mutagenic raising safety concerns for oligonucleotide-based pharmaceuticals. We have engineered a TFO, TFO27, to target the genomic Hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus to define the mutagenic potential of such structures at genomic DNA. We report that TFO27 was resistant to nuclease degradation and readily binds to its target motif in a cell free system. Contrary to previous studies using the supFG1 reporter construct, TFO27 failed to induce mutation within the genomic HPRT locus. We suggest that it is possible that previous reports of triplex-mediated mutation using the supFG1 reporter construct could be confounded by DNA quadruplex formation. Although the present study indicates that a TFO targeting a genomic locus lacks mutagenic activity, it is unclear if this finding can be generalised to all TFOs and their targets. For the present, we suggest that it is prudent to avoid large purine stretches in oligonucleotide pharmaceutical design to minimise concern regarding off-target genotoxicity. PMID:22914677

  9. 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 wi

  10. Clonal expansion of early to mid-life mitochondrial DNA point mutations drives mitochondrial dysfunction during human ageing.

    NARCIS (Netherlands)

    Greaves, L.C.; Nooteboom, M.; Elson, J.L.; Tuppen, H.A.; Taylor, G.A.; Commane, D.M.; Arasaradnam, R.P.; Khrapko, K.; Taylor, R.W.; Kirkwood, T.B.; Mathers, J.C.; Turnbull, D.M.

    2014-01-01

    Age-related decline in the integrity of mitochondria is an important contributor to the human ageing process. In a number of ageing stem cell populations, this decline in mitochondrial function is due to clonal expansion of individual mitochondrial DNA (mtDNA) point mutations within single cells. Ho

  11. Mutation analysis of circulating plasma DNA to determine response to EGFR tyrosine kinase inhibitor therapy of lung adenocarcinoma patients

    Science.gov (United States)

    Riediger, Anja Lisa; Dietz, Steffen; Schirmer, Uwe; Meister, Michael; Heinzmann-Groth, Ingrid; Schneider, Marc; Muley, Thomas; Thomas, Michael; Sültmann, Holger

    2016-01-01

    Long-lasting success in lung cancer therapy using tyrosine kinase inhibitors (TKIs) is rare since the tumors develop resistance due to the occurrence of molecularly altered subclones. The aim of this study was to monitor tumors over time based on the quantity of mutant plasma DNA and to identify early indications for therapy response and tumor progression. Serial plasma samples from lung adenocarcinoma patients treated with TKIs were used to quantify EGFR and KRAS mutations in circulating DNA by digital PCR. Mutant DNA levels were compared with the courses of responses to treatment with TKIs, conventional chemotherapy, radiotherapy, or combinations thereof. Variations in plasma DNA mutation levels over time were found in 15 patients. We categorize three major courses: First, signs of therapy response are associated with a fast clearing of plasma DNA mutations within a few days. Second, periods of stable disease are accompanied by either absence of mutations or fluctuation at low levels. Finally, dramatic increase of mutational load is followed by rapid tumor progression and poor patient survival. In summary, the serial assessment of EGFR mutations in the plasma of NSCLC patients allows conclusions about controlled disease and tumor progression earlier than currently available methods. PMID:27640882

  12. Microelectronic DNA assay for the detection of BRCA1 gene mutations

    Science.gov (United States)

    Chen, Hua; Han, Jie; Li, Jun; Meyyappan, Meyya

    2004-01-01

    Mutations in BRCA1 are characterized by predisposition to breast cancer, ovarian cancer and prostate cancer as well as colon cancer. Prognosis for this cancer survival depends upon the stage at which cancer is diagnosed. Reliable and rapid mutation detection is crucial for the early diagnosis and treatment. We developed an electronic assay for the detection of a representative single nucleotide polymorphism (SNP), deletion and insertion in BRCA1 gene by the microelectronics microarray instrumentation. The assay is rapid, and it takes 30 minutes for the immobilization of target DNA samples, hybridization, washing and readout. The assay is multiplexing since it is carried out at the same temperature and buffer conditions for each step. The assay is also highly specific, as the signal-to-noise ratio is much larger than recommended value (72.86 to 321.05 vs. 5) for homozygotes genotyping, and signal ratio close to the perfect value 1 for heterozygotes genotyping (1.04).

  13. Analysis of morphology, DNA and isozyme of leaf mutation in Brassica napus L

    International Nuclear Information System (INIS)

    This paper aims to study the rule of irradiating effects, provide the effective way of analyzing mutant, and discuss the production application of mutant. By irradiating the 040B of Brassica napus L with . 0Co γ- ray, an obvious leaf mutation (ML) with large leaf area was found. The ML which has been inherited stably after three generations was compared with wide-type (CK) on the morphologic, DNA and isozymic levels. Results showed that S 4 and S17 from RAPD were two molecular markers which can express good polymorphism and have close relationships with leaf mutation sites. And in the analysis of EST and POD between ML and CK, the polymorphisms also proved that many discrepancies exist between ML and CK on the protein level. In addition, the research results in question can be applied to the breeding and genetic research of Brassica napus L

  14. Mismatch oxidation assay: detection of DNA mutations using a standard UV/Vis microplate reader.

    Science.gov (United States)

    Tabone, Tania; Sallmann, Georgina; Cotton, Richard G H

    2009-01-01

    Simple, low-cost mutation detection assays that are suitable for low-throughput analysis are essential for diagnostic applications where the causative mutation may be different in every family. The mismatch oxidation assay is a simple optical absorbance assay to detect nucleotide substitutions, insertions, and deletions in heteroduplex DNA. The method relies on detecting the oxidative modification products of mismatched thymine and cytosine bases by potassium permanganate as it is reduced to manganese dioxide. This approach, unlike other methods commonly used to detect sequence variants, does not require costly labeled probes or primers, toxic chemicals, or a time-consuming electrophoretic separation step. The oxidation rate, and hence the presence of a sequence variant, is detected by measuring the formation of the potassium permanganate reduction product (hypomanganate diester), which absorbs at the 420-nm visible wavelength, using a standard UV/vis microplate reader. PMID:19768598

  15. Modulating the DNA affinity of Elk-1 with computationally selected mutations.

    Science.gov (United States)

    Park, Sheldon; Boder, Eric T; Saven, Jeffery G

    2005-04-22

    In order to regulate gene expression, transcription factors must first bind their target DNA sequences. The affinity of this binding is determined by both the network of interactions at the interface and the entropy change associated with the complex formation. To study the role of structural fluctuation in fine-tuning DNA affinity, we performed molecular dynamics simulations of two highly homologous proteins, Elk-1 and SAP-1, that exhibit different sequence specificity. Simulation studies show that several residues in Elk have significantly higher main-chain root-mean-square deviations than their counterparts in SAP. In particular, a single residue, D69, may contribute to Elk's lower DNA affinity for P(c-fos) by structurally destabilizing the carboxy terminus of the recognition helix. While D69 does not contact DNA directly, the increased mobility in the region may contribute to its weaker binding. We measured the ability of single point mutants of Elk to bind P(c-fos) in a reporter assay, in which D69 of wild-type Elk has been mutated to other residues with higher helix propensity in order to stabilize the local conformation. The gains in transcriptional activity and the free energy of binding suggested from these measurements correlate well with stability gains computed from helix propensity and charge-macrodipole interactions. The study suggests that residues that are distal to the binding interface may indirectly modulate the binding affinity by stabilizing the protein scaffold required for efficient DNA interaction.

  16. DNA analysis of an uncommon missense mutation in a Gaucher disease patient of Jewish-Polish-Russian descent

    Energy Technology Data Exchange (ETDEWEB)

    Choy, F.Y.M.; Wei, C.; Applegarth, D.A.; McGillivray, B.C. [Univ. of British Columbia, Vancouver (Canada)

    1994-06-01

    Gaucher disease is the most frequent lysosomal lipid storage disease. It results from deficient glucocerebrosidase activity and is transmitted as an autosomal recessive trait. Three clinical forms of Gaucher disease have been described: type 1, non-neuronopathic; type 2, acute neuronopathic; and type 3, subacute neuronopathic. We have sequenced the full length cDNA of the glucocerebrosidase gene and identified an uncommon mutation in nucleotide position 1604 (genoma DNA nucleotide position 6683) from a Gaucher disease patient of Jewish-Polish-Russian descent with type 1 Gaucher disease. It is a G{yields}A transition in exon 11 that results in {sup 496}Arg{yields}{sup 496}His of glucocerebrosidase. This missense mutation is present in the heterozygous form and creates a new cleavage site for the endonuclease HphI. We have developed a simple method to detect the presence of this mutation by using HphI restriction fragment length polymorphism analysis of glucocerebrosidase genomic DNA or cDNA. The mutation in the other Gaucher allele of this patient is an A{yields}G transition at cDNA nucleotide position 1226 which creates an XhoI cleavage site after PCR mismatch amplification. The presence of this mutation was also confirmed by sequence analysis. Based on previous reports that mutation 1226 is present only in type 1 Gaucher disease and the observation that there is no neurological involvement in this patient, we conclude that our patient with the 1226/1604 genotype is diagnosed as having type 1 Gaucher disease. Since it was also postulated that mutation 1226 in the homozygous form will usually result in a good prognosis, we speculate that the orthopedic complications and the unusual presence of glomerulosclerosis in this patient may be attributable to the mutation at nucleotide 1604. This speculation will require a description of more patients with this mutation for confirmation. 32 refs., 5 figs.

  17. Robust DNA Damage Response and Elevated Reactive Oxygen Species in TINF2-Mutated Dyskeratosis Congenita Cells.

    Directory of Open Access Journals (Sweden)

    Larisa Pereboeva

    Full Text Available Dyskeratosis Congenita (DC is an inherited multisystem premature aging disorder with characteristic skin and mucosal findings as well as a predisposition to cancer and bone marrow failure. DC arises due to gene mutations associated with the telomerase complex or telomere maintenance, resulting in critically shortened telomeres. The pathogenesis of DC, as well as several congenital bone marrow failure (BMF syndromes, converges on the DNA damage response (DDR pathway and subsequent elevation of reactive oxygen species (ROS. Historically, DC patients have had poor outcomes following bone marrow transplantation (BMT, perhaps as a consequence of an underlying DNA hypersensitivity to cytotoxic agents. Previously, we demonstrated an activated DDR and increased ROS, augmented by chemotherapy and radiation, in somatic cells isolated from DC patients with a mutation in the RNA component of telomerase, TERC. The current study was undertaken to determine whether previous findings related to ROS and DDR in TERC patients' cells could be extended to other DC mutations. Of particular interest was whether an antioxidant approach could counter increased ROS and decrease DC pathologies. To test this, we examined lymphocytes from DC patients from different DC mutations (TERT, TINF2, and TERC for the presence of an active DDR and increased ROS. All DC mutations led to increased steady-state p53 (2-fold to 10-fold and ROS (1.5-fold to 2-fold. Upon exposure to ionizing radiation (XRT, DC cells increased in both DDR and ROS to a significant degree. Exposing DC cells to hydrogen peroxide also revealed that DC cells maintain a significant oxidant burden compared to controls (1.5-fold to 3-fold. DC cell culture supplemented with N-acetylcysteine, or alternatively grown in low oxygen, afforded significant proliferative benefits (proliferation: maximum 2-fold increase; NAC: 5-fold p53 decrease; low oxygen: maximum 3.5-fold p53 decrease. Together, our data supports a

  18. Robust DNA Damage Response and Elevated Reactive Oxygen Species in TINF2-Mutated Dyskeratosis Congenita Cells.

    Science.gov (United States)

    Pereboeva, Larisa; Hubbard, Meredith; Goldman, Frederick D; Westin, Erik R

    2016-01-01

    Dyskeratosis Congenita (DC) is an inherited multisystem premature aging disorder with characteristic skin and mucosal findings as well as a predisposition to cancer and bone marrow failure. DC arises due to gene mutations associated with the telomerase complex or telomere maintenance, resulting in critically shortened telomeres. The pathogenesis of DC, as well as several congenital bone marrow failure (BMF) syndromes, converges on the DNA damage response (DDR) pathway and subsequent elevation of reactive oxygen species (ROS). Historically, DC patients have had poor outcomes following bone marrow transplantation (BMT), perhaps as a consequence of an underlying DNA hypersensitivity to cytotoxic agents. Previously, we demonstrated an activated DDR and increased ROS, augmented by chemotherapy and radiation, in somatic cells isolated from DC patients with a mutation in the RNA component of telomerase, TERC. The current study was undertaken to determine whether previous findings related to ROS and DDR in TERC patients' cells could be extended to other DC mutations. Of particular interest was whether an antioxidant approach could counter increased ROS and decrease DC pathologies. To test this, we examined lymphocytes from DC patients from different DC mutations (TERT, TINF2, and TERC) for the presence of an active DDR and increased ROS. All DC mutations led to increased steady-state p53 (2-fold to 10-fold) and ROS (1.5-fold to 2-fold). Upon exposure to ionizing radiation (XRT), DC cells increased in both DDR and ROS to a significant degree. Exposing DC cells to hydrogen peroxide also revealed that DC cells maintain a significant oxidant burden compared to controls (1.5-fold to 3-fold). DC cell culture supplemented with N-acetylcysteine, or alternatively grown in low oxygen, afforded significant proliferative benefits (proliferation: maximum 2-fold increase; NAC: 5-fold p53 decrease; low oxygen: maximum 3.5-fold p53 decrease). Together, our data supports a mechanism

  19. HotSpot Wizard 2.0: automated design of site-specific mutations and smart libraries in protein engineering.

    Science.gov (United States)

    Bendl, Jaroslav; Stourac, Jan; Sebestova, Eva; Vavra, Ondrej; Musil, Milos; Brezovsky, Jan; Damborsky, Jiri

    2016-07-01

    HotSpot Wizard 2.0 is a web server for automated identification of hot spots and design of smart libraries for engineering proteins' stability, catalytic activity, substrate specificity and enantioselectivity. The server integrates sequence, structural and evolutionary information obtained from 3 databases and 20 computational tools. Users are guided through the processes of selecting hot spots using four different protein engineering strategies and optimizing the resulting library's size by narrowing down a set of substitutions at individual randomized positions. The only required input is a query protein structure. The results of the calculations are mapped onto the protein's structure and visualized with a JSmol applet. HotSpot Wizard lists annotated residues suitable for mutagenesis and can automatically design appropriate codons for each implemented strategy. Overall, HotSpot Wizard provides comprehensive annotations of protein structures and assists protein engineers with the rational design of site-specific mutations and focused libraries. It is freely available at http://loschmidt.chemi.muni.cz/hotspotwizard.

  20. DNA Hydroxymethylation Profiling Reveals that WT1 Mutations Result in Loss of TET2 Function in Acute Myeloid Leukemia

    Directory of Open Access Journals (Sweden)

    Raajit Rampal

    2014-12-01

    Full Text Available Somatic mutations in IDH1/IDH2 and TET2 result in impaired TET2-mediated conversion of 5-methylcytosine (5mC to 5-hydroxymethylcytosine (5hmC. The observation that WT1 inactivating mutations anticorrelate with TET2/IDH1/IDH2 mutations in acute myeloid leukemia (AML led us to hypothesize that WT1 mutations may impact TET2 function. WT1 mutant AML patients have reduced 5hmC levels similar to TET2/IDH1/IDH2 mutant AML. These mutations are characterized by convergent, site-specific alterations in DNA hydroxymethylation, which drive differential gene expression more than alterations in DNA promoter methylation. WT1 overexpression increases global levels of 5hmC, and WT1 silencing reduced 5hmC levels. WT1 physically interacts with TET2 and TET3, and WT1 loss of function results in a similar hematopoietic differentiation phenotype as observed with TET2 deficiency. These data provide a role for WT1 in regulating DNA hydroxymethylation and suggest that TET2 IDH1/IDH2 and WT1 mutations define an AML subtype defined by dysregulated DNA hydroxymethylation.

  1. Functional Characterization of Three Concomitant MtDNA LHON Mutations Shows No Synergistic Effect on Mitochondrial Activity.

    Science.gov (United States)

    Cruz-Bermúdez, Alberto; Vicente-Blanco, Ramiro J; Hernández-Sierra, Rosana; Montero, Mayte; Alvarez, Javier; González Manrique, Mar; Blázquez, Alberto; Martín, Miguel Angel; Ayuso, Carmen; Garesse, Rafael; Fernández-Moreno, Miguel A

    2016-01-01

    The presence of more than one non-severe pathogenic mutation in the same mitochondrial DNA (mtDNA) molecule is very rare. Moreover, it is unclear whether their co-occurrence results in an additive impact on mitochondrial function relative to single mutation effects. Here we describe the first example of a mtDNA molecule harboring three Leber's hereditary optic neuropathy (LHON)-associated mutations (m.11778G>A, m.14484T>C, m.11253T>C) and the analysis of its genetic, biochemical and molecular characterization in transmitochondrial cells (cybrids). Extensive characterization of cybrid cell lines harboring either the 3 mutations or the single classic m.11778G>A and m.14484T>C mutations revealed no differences in mitochondrial function, demonstrating the absence of a synergistic effect in this model system. These molecular results are in agreement with the ophthalmological characteristics found in the triple mutant patient, which were similar to those carrying single mtDNA LHON mutations.

  2. Functional Characterization of Three Concomitant MtDNA LHON Mutations Shows No Synergistic Effect on Mitochondrial Activity.

    Directory of Open Access Journals (Sweden)

    Alberto Cruz-Bermúdez

    Full Text Available The presence of more than one non-severe pathogenic mutation in the same mitochondrial DNA (mtDNA molecule is very rare. Moreover, it is unclear whether their co-occurrence results in an additive impact on mitochondrial function relative to single mutation effects. Here we describe the first example of a mtDNA molecule harboring three Leber's hereditary optic neuropathy (LHON-associated mutations (m.11778G>A, m.14484T>C, m.11253T>C and the analysis of its genetic, biochemical and molecular characterization in transmitochondrial cells (cybrids. Extensive characterization of cybrid cell lines harboring either the 3 mutations or the single classic m.11778G>A and m.14484T>C mutations revealed no differences in mitochondrial function, demonstrating the absence of a synergistic effect in this model system. These molecular results are in agreement with the ophthalmological characteristics found in the triple mutant patient, which were similar to those carrying single mtDNA LHON mutations.

  3. Functional Characterization of Three Concomitant MtDNA LHON Mutations Shows No Synergistic Effect on Mitochondrial Activity

    Science.gov (United States)

    Cruz-Bermúdez, Alberto; Vicente-Blanco, Ramiro J.; Hernández-Sierra, Rosana; Montero, Mayte; Alvarez, Javier; González Manrique, Mar; Blázquez, Alberto; Martín, Miguel Angel; Ayuso, Carmen; Garesse, Rafael; Fernández-Moreno, Miguel A.

    2016-01-01

    The presence of more than one non-severe pathogenic mutation in the same mitochondrial DNA (mtDNA) molecule is very rare. Moreover, it is unclear whether their co-occurrence results in an additive impact on mitochondrial function relative to single mutation effects. Here we describe the first example of a mtDNA molecule harboring three Leber's hereditary optic neuropathy (LHON)-associated mutations (m.11778G>A, m.14484T>C, m.11253T>C) and the analysis of its genetic, biochemical and molecular characterization in transmitochondrial cells (cybrids). Extensive characterization of cybrid cell lines harboring either the 3 mutations or the single classic m.11778G>A and m.14484T>C mutations revealed no differences in mitochondrial function, demonstrating the absence of a synergistic effect in this model system. These molecular results are in agreement with the ophthalmological characteristics found in the triple mutant patient, which were similar to those carrying single mtDNA LHON mutations. PMID:26784702

  4. Low penetrance of the 14484 LHON mutation when it arises in a non-haplogroup J mtDNA background.

    Science.gov (United States)

    Howell, Neil; Herrnstadt, Corinna; Shults, Cliff; Mackey, David A

    2003-06-01

    The penetrance in Leber's hereditary optic neuropathy (LHON) pedigrees is determined primarily by a mutation in the mitochondrial genome (mtDNA), but secondary factors are also necessary for manifestation of the disorder. It has been proposed that mtDNA polymorphisms affect penetrance in LHON pedigrees. In particular, it has been postulated that one or more polymorphisms associated with European haplogroup J mtDNAs substantially increase the penetrance of the primary LHON mutation at nucleotide 14484. We report here a haplogroup H matrilineal pedigree (VIC14) in which the single affected member carries the 14484 LHON mutation, but who manifested a milder and atypical optic nerve disorder. In addition, during a population screen, we identified an individual who carried the 14484 mutation but who had normal vision. Finally, the 14484 mutation is under-represented among haplogroup H mtDNAs that carry a LHON mutation. These results, in conjunction with other studies that are reviewed, indicate that 14484 LHON mutations have a low penetrance when they arise in a haplogroup H mtDNA background.

  5. Diamondoid-functionalized gold nanogaps as sensors for natural, mutated, and epigenetically modified DNA nucleotides

    Science.gov (United States)

    Sivaraman, Ganesh; Amorim, Rodrigo G.; Scheicher, Ralph H.; Fyta, Maria

    2016-05-01

    Modified tiny hydrogen-terminated diamond structures, known as diamondoids, show a high efficiency in sensing DNA molecules. These diamond cages, as recently proposed, could offer functionalization possibilities for gold junction electrodes. In this investigation, we report on diamondoid-functionalized electrodes, showing that such a device would have a high potential in sensing and sequencing DNA. The smallest diamondoid including an amine modification was chosen for the functionalization. Here, we report on the quantum tunneling signals across diamondoid-functionalized Au(111) electrodes. Our work is based on quantum-transport calculations and predicts the expected signals arising from different DNA units within the break junctions. Different gating voltages are proposed in order to tune the sensitivity of the functionalized electrodes with respect to specific nucleotides. The relation of this sensitivity to the coupling or decoupling of the electrodes is discussed. Our results also shed light on the sensing capability of such a device in distinguishing the DNA nucleotides, in their natural and mutated forms.

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

    International Nuclear Information System (INIS)

    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 32P-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

  7. Target DNA sequence directly regulates the frequency of activation-induced deaminase-dependent mutations.

    Science.gov (United States)

    Chen, Zhangguo; Viboolsittiseri, Sawanee S; O'Connor, Brian P; Wang, Jing H

    2012-10-15

    Activation-induced deaminase (AID) catalyses class switch recombination (CSR) and somatic hypermutation (SHM) in B lymphocytes to enhance Ab diversity. CSR involves breaking and rejoining highly repetitive switch (S) regions in the IgH (Igh) locus. S regions appear to be preferential targets of AID. To determine whether S region sequence per se, independent of Igh cis regulatory elements, can influence AID targeting efficiency and mutation frequency, we established a knock-in mouse model by inserting a core Sγ1 region into the first intron of proto-oncogene Bcl6, which is a non-Ig target of SHM. We found that the mutation frequency of the inserted Sγ1 region was dramatically higher than that of the adjacent Bcl6 endogenous sequence. Mechanistically, S region-enhanced SHM was associated with increased recruitment of AID and RNA polymerase II, together with Spt5, albeit to a lesser extent. Our studies demonstrate that target DNA sequences influence mutation frequency via regulating AID recruitment. We propose that the nucleotide sequence preference may serve as an additional layer of AID regulation by restricting its mutagenic activity to specific sequences despite the observation that AID has the potential to access the genome widely.

  8. Detection of TP53 mutation, loss of heterozygosity and DNA content in fine-needle aspirates of breast carcinoma.

    Science.gov (United States)

    Lavarino, C.; Corletto, V.; Mezzelani, A.; Della Torre, G.; Bartoli, C.; Riva, C.; Pierotti, M. A.; Rilke, F.; Pilotti, S.

    1998-01-01

    Recent preclinical and clinical data suggest that TP53 status and TP53 mutations may be important in determining tumour aggressiveness and therapy response. In this study we investigate the feasibility of a structural and quantitative analysis of TP53 on fine-needle aspiration (FNA) material obtained from 31 consecutive female patients with breast carcinoma, enrolled in a primary chemotherapy protocol. Tumours were screened for p53 protein overexpression and TP53 mutations (exons 5-8) using immunocytochemistry, polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and DNA sequencing analyses, and finally using fluorescence in situ hybridization (FISH) analysis. Positive nuclear staining was identified in six cases whereas mutations were detected in nine. Although the immunoreactive pattern fitted fully with the characterized TP53 mutation type, the considerable number of null p53 mutations (i.e. four) coupled with the lack of information regarding the localization of TP53 mutations make immunocytochemistry an inadequate indicator of TP53 function deregulation. Combining molecular and FISH analyses, we detected three cases with TP53 deletion and one case with deletion and mutation. Finally, DNA static-image analysis performed on 29 cases showed aneuploidy in 26 cases, which included all TP53-mutated cases. The present results show that FNA may assist clinical decisions by allowing the evaluation of a variety of biological parameters relevant for prognosis and treatment planning. Images Figure 1 PMID:9459157

  9. Distinct nuclear gene expression profiles in cells with mtDNA depletion and homoplasmic A3243G mutation

    Energy Technology Data Exchange (ETDEWEB)

    Jahangir Tafrechi, Roshan S. [Department of Molecular Cell Biology, Leiden University Medical Center, P.O. Box 9503, 2300 RA Leiden (Netherlands); Svensson, Peter J. [Department of Toxicogenetics, Leiden University Medical Center, P.O. Box 9503, 2300 RA Leiden (Netherlands); Department of Oncology, Radiology and Clinical Immunology, University Hospital, 75185 Uppsala (Sweden); Janssen, George M.C. [Department of Molecular Cell Biology, Leiden University Medical Center, P.O. Box 9503, 2300 RA Leiden (Netherlands); Szuhai, Karoly [Department of Molecular Cell Biology, Leiden University Medical Center, P.O. Box 9503, 2300 RA Leiden (Netherlands); Maassen, J. Antonie [Department of Molecular Cell Biology, Leiden University Medical Center, P.O. Box 9503, 2300 RA Leiden (Netherlands); Raap, Anton K. [Department of Molecular Cell Biology, Leiden University Medical Center, P.O. Box 9503, 2300 RA Leiden (Netherlands)]. E-mail: A.K.Raap@lumc.nl

    2005-10-15

    The pathobiochemical pathways determining the wide variability in phenotypic expression of mitochondrial DNA (mtDNA) mutations are not well understood. Most pathogenic mtDNA mutations induce a general defect in mitochondrial respiration and thereby ATP synthesis. Yet phenotypic expression of the different mtDNA mutations shows large variations that are difficult to reconcile with ATP depletion as sole pathogenic factor, implying that additional mechanisms contribute to the phenotype. Here, we use DNA microarrays to identify changes in nuclear gene expression resulting from the presence of the A3243G diabetogenic mutation and from a depletion of mtDNA ({rho}{sup 0} cells). We find that cells respond mildly to these mitochondrial states with both general and specific changes in nuclear gene expression. This observation indicates that cells can sense the status of mtDNA. A number of genes show divergence in expression in {rho}{sup 0} cells compared to cells with the A3243G mutation, such as genes involved in oxidative phosphorylation. As a common response in A3243G and {rho}{sup 0} cells, mRNA levels for extracellular matrix genes are up-regulated, while the mRNA levels of genes involved in ubiquitin-mediated protein degradation and in ribosomal protein synthesis is down-regulated. This reduced expression is reflected at the level of cytosolic protein synthesis in both A3243G and {rho}{sup 0} cells. Our finding that mitochondrial dysfunction caused by different mutations affects nuclear gene expression in partially distinct ways suggests that multiple pathways link mitochondrial function to nuclear gene expression and contribute to the development of the different phenotypes in mitochondrial disease.

  10. A novel approach to detect KRAS/BRAF mutation for colon cancer: Highly sensitive simultaneous detection of mutations and simple pre-treatment without DNA extraction.

    Science.gov (United States)

    Suzuki, Shun-Ichi; Matsusaka, Satoshi; Hirai, Mitsuharu; Shibata, Harumi; Takagi, Koichi; Mizunuma, Nobuyuki; Hatake, Kiyohiko

    2015-07-01

    It has been reported that colon cancer patients with KRAS and BRAF mutations that lie downstream of epidermal growth factor receptor (EGFR) acquire resistance against therapy with anti‑EGFR antibodies, cetuximab and panitumumab. On the other hand, some reports say KRAS codon 13 mutation (p.G13D) has lower resistance against anti-EGFR antibodies, thus there is a substantial need for detection of specific KRAS mutations. We have established a state-of-the-art measurement system using QProbe (QP) method that allows simultaneous measurement of KRAS codon 12/13, p.G13D and BRAF mutation, and compared this method against Direct Sequencing (DS) using 182 specimens from colon cancer patients. In addition, 32 biopsy specimens were processed with a novel pre-treatment method without DNA purification in order to detect KRAS/BRAF. As a result of KRAS mutation measurement, concordance rate between the QP method and DS method was 81.4% (144/177) except for the 5 specimens that were undeterminable. Among them, 29 specimens became positive with QP method and negative with DS method. BRAF was measured with QP method only, and the mutation detection rate was 3.9% (6/153). KRAS measurement using a simple new pre-treatment method without DNA extraction resulted in 31 good results out of 32, all of them matching with the DS method. We have established a simple but highly sensitive simultaneous detection system for KRAS/BRAF. Moreover, introduction of the novel pre-treatment technology eliminated the inconvenient DNA extraction process. From this research achievement, we not only anticipate quick and accurate results returned in the clinical field but also contribution in improving the test quality and work efficiency.

  11. PCR-SSCP-DNA sequencing method in detecting PTEN gene mutation and its significance in human gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Chuan-Yong Guo; Xuan-Fu Xu; Jian-Ye Wu; Shu-Fang Liu

    2008-01-01

    AIM: To discuss the possible effect of PTEN gene mutations on occurrence and development of gastric cancer.METHODS: Fifty-three gastric cancer specimens were selected to probe PTEN gene mutations in genome of gastric cancer and paracancerous tissues using PCR-SSCP-DNA sequencing method based on microdissection and to observe the protein expression by immunohistochemistry technique.RESULTS: PCR-SSCP-DNA sequencing indicated that 4 kinds of mutation sites were found in 5 of 53 gastric cancer specimens.One kind of mutation was found in exons.AA-TCC mutation was located at 40bp upstream of 3' lateral exert 7 (115946 AA-TCC).Such mutations led to terminator formation in the 297th codon of the PTEN gene.The other 3 kinds of mutation were found in introns,including a G-C point mutation at 91 bp upstream of 5' lateral exon 5(90896 G-C),a T-G point mutation at 24 bp upstream of 5' lateral exon 5 (90963 T-G),and a single base A mutation at 7 bp upstream of 5' lateral exon 5 (90980 A del).The PTEN protein expression in gastric cancer and paracancerous tissues detected using immunohistochemistry technique indicated that the total positive rate of PTEN protein expression was 66% in gastric cancer tissue,which was significantly lower than that (100%) in paracancerous tissues (P<0.005).CONCLUSION: PTEN gene mutation and expression may play an important role in the occurrence and development of gastric cancer.(C)2008 The WJG Press.All rights reserved.

  12. The Use of EGFR Exon 19 and 21 Unlabeled DNA Probes to Screen for Activating Mutations in Non–Small Cell Lung Cancer

    OpenAIRE

    Willmore-Payne, Carlynn; Holden, Joseph A.; Wittwer, Carl T.; Layfield, Lester J.

    2008-01-01

    Activating mutations in epidermal growth factor receptor-1 (EGFR) are found in 10–15% of Caucasian patients with non–small cell lung carcinoma (NSCLC). Approximately 90% of the mutations are deletions of several amino acids in exon 19 or point mutations in exon 21. Some studies suggest that these mutations identify patients that might benefit from targeted EGFR inhibitor therapy. DNA melting analysis of polymerase chain reaction products can screen for these mutations to identify this patient...

  13. KRAS G12V Mutation Detection by Droplet Digital PCR in Circulating Cell-Free DNA of Colorectal Cancer Patients.

    Science.gov (United States)

    Olmedillas López, Susana; García-Olmo, Dolores C; García-Arranz, Mariano; Guadalajara, Héctor; Pastor, Carlos; García-Olmo, Damián

    2016-04-01

    KRAS mutations are responsible for resistance to anti-epidermal growth factor receptor (EGFR) therapy in colorectal cancer patients. These mutations sometimes appear once treatment has started. Detection of KRAS mutations in circulating cell-free DNA in plasma ("liquid biopsy") by droplet digital PCR (ddPCR) has emerged as a very sensitive and promising alternative to serial biopsies for disease monitoring. In this study, KRAS G12V mutation was analyzed by ddPCR in plasma DNA from 10 colorectal cancer patients and compared to six healthy donors. The percentage of KRAS G12V mutation relative to wild-type sequences in tumor-derived DNA was also determined. KRAS G12V mutation circulating in plasma was detected in 9 of 10 colorectal cancer patients whose tumors were also mutated. Colorectal cancer patients had 35.62 copies of mutated KRAS/mL plasma, whereas in healthy controls only residual copies were found (0.62 copies/mL, p = 0.0066). Interestingly, patients with metastatic disease showed a significantly higher number of mutant copies than M0 patients (126.25 versus 9.37 copies/mL, p = 0.0286). Wild-type KRAS was also significantly elevated in colorectal cancer patients compared to healthy controls (7718.8 versus 481.25 copies/mL, p = 0.0002). In conclusion, KRAS G12V mutation is detectable in plasma of colorectal cancer patients by ddPCR and could be used as a non-invasive biomarker.

  14. KRAS G12V Mutation Detection by Droplet Digital PCR in Circulating Cell-Free DNA of Colorectal Cancer Patients

    Directory of Open Access Journals (Sweden)

    Susana Olmedillas López

    2016-04-01

    Full Text Available KRAS mutations are responsible for resistance to anti-epidermal growth factor receptor (EGFR therapy in colorectal cancer patients. These mutations sometimes appear once treatment has started. Detection of KRAS mutations in circulating cell-free DNA in plasma (“liquid biopsy” by droplet digital PCR (ddPCR has emerged as a very sensitive and promising alternative to serial biopsies for disease monitoring. In this study, KRAS G12V mutation was analyzed by ddPCR in plasma DNA from 10 colorectal cancer patients and compared to six healthy donors. The percentage of KRAS G12V mutation relative to wild-type sequences in tumor-derived DNA was also determined. KRAS G12V mutation circulating in plasma was detected in 9 of 10 colorectal cancer patients whose tumors were also mutated. Colorectal cancer patients had 35.62 copies of mutated KRAS/mL plasma, whereas in healthy controls only residual copies were found (0.62 copies/mL, p = 0.0066. Interestingly, patients with metastatic disease showed a significantly higher number of mutant copies than M0 patients (126.25 versus 9.37 copies/mL, p = 0.0286. Wild-type KRAS was also significantly elevated in colorectal cancer patients compared to healthy controls (7718.8 versus 481.25 copies/mL, p = 0.0002. In conclusion, KRAS G12V mutation is detectable in plasma of colorectal cancer patients by ddPCR and could be used as a non-invasive biomarker.

  15. KRAS G12V Mutation Detection by Droplet Digital PCR in Circulating Cell-Free DNA of Colorectal Cancer Patients.

    Science.gov (United States)

    Olmedillas López, Susana; García-Olmo, Dolores C; García-Arranz, Mariano; Guadalajara, Héctor; Pastor, Carlos; García-Olmo, Damián

    2016-01-01

    KRAS mutations are responsible for resistance to anti-epidermal growth factor receptor (EGFR) therapy in colorectal cancer patients. These mutations sometimes appear once treatment has started. Detection of KRAS mutations in circulating cell-free DNA in plasma ("liquid biopsy") by droplet digital PCR (ddPCR) has emerged as a very sensitive and promising alternative to serial biopsies for disease monitoring. In this study, KRAS G12V mutation was analyzed by ddPCR in plasma DNA from 10 colorectal cancer patients and compared to six healthy donors. The percentage of KRAS G12V mutation relative to wild-type sequences in tumor-derived DNA was also determined. KRAS G12V mutation circulating in plasma was detected in 9 of 10 colorectal cancer patients whose tumors were also mutated. Colorectal cancer patients had 35.62 copies of mutated KRAS/mL plasma, whereas in healthy controls only residual copies were found (0.62 copies/mL, p = 0.0066). Interestingly, patients with metastatic disease showed a significantly higher number of mutant copies than M0 patients (126.25 versus 9.37 copies/mL, p = 0.0286). Wild-type KRAS was also significantly elevated in colorectal cancer patients compared to healthy controls (7718.8 versus 481.25 copies/mL, p = 0.0002). In conclusion, KRAS G12V mutation is detectable in plasma of colorectal cancer patients by ddPCR and could be used as a non-invasive biomarker. PMID:27043547

  16. Mutation of mtDNA ND1 Gene in 20 Type 2 Diabetes Mellitus Patients of Gorontalonese and Javanese Ethnicity

    Directory of Open Access Journals (Sweden)

    AMIEN RAMADHAN ISHAK

    2014-12-01

    Full Text Available Mitochondrial gene mutation plays a role in the development of type two diabetes mellitus (T2DM. A point mutation in the mitochondrial gene Nicotinamide adenine dinucleotide dehydrogenase 1 (mtDNA ND1 gene mainly reported as the most common mutation related to T2DM. However, several studies have identified another SNP (single-nucleotide polymorphisms in the RNA region of mtDNA from patients from specific ethnic populations in Indonesia. Building on those findings, this study aimed to use PCR and DNA sequencing technology to identify nucleotides in RNA and ND1 fragment from 20 Gorontalonese and 20 Javanese T2DM patients, that may trigger T2DM expression. The results showed successful amplification of RNA along 294 bp for all samples. From these samples, we found two types of point mutation in Javanese patients in the G3316A and T3200C points of the rRNA and ND1 gene. In samples taken from Gorontalonese patients, no mutation were found in the RNA or ND1 region. We conclude that T2DM was triggered differently in our two populations. While genetic mutation is implicated for the 20 Javanese patients, T2DM pathogenesis in the Gorontalonese patients must be traced to other genetic, environmental, or behavioral factors.

  17. Identification of an mtDNA mutation hot spot in UV-induced mouse skin tumors producing altered cellular biochemistry.

    Science.gov (United States)

    Jandova, Jana; Eshaghian, Alex; Shi, Mingjian; Li, Meiling; King, Lloyd E; Janda, Jaroslav; Sligh, James E

    2012-02-01

    There is increasing awareness of the role of mtDNA alterations in the development of cancer, as mtDNA point mutations are found at high frequency in a variety of human tumors. To determine the biological effects of mtDNA mutations in UV-induced skin tumors, hairless mice were irradiated to produce tumors, and the tumor mtDNAs were screened for single-nucleotide changes using temperature gradient capillary electrophoresis (TGCE), followed by direct sequencing. A mutation hot spot (9821insA) in the mitochondrially encoded tRNA arginine (mt-Tr) locus (tRNA(Arg)) was discovered in approximately one-third of premalignant and malignant skin tumors. To determine the functional relevance of this particular mutation in vitro, cybrid cell lines containing different mt-Tr (tRNA(Arg)) alleles were generated. The resulting cybrid cell lines contained the same nuclear genotype and differed only in their mtDNAs. The biochemical analysis of the cybrids revealed that the mutant haplotype is associated with diminished levels of complex I protein (CI), resulting in lower levels of baseline oxygen consumption and lower cellular adenosine triphosphate (ATP) production. We hypothesize that this specific mtDNA mutation alters cellular biochemistry, supporting the development of keratinocyte neoplasia.

  18. Long-term bezafibrate treatment improves skin and spleen phenotypes of the mtDNA mutator mouse.

    Directory of Open Access Journals (Sweden)

    Lloye M Dillon

    Full Text Available Pharmacological agents, such as bezafibrate, that activate peroxisome proliferator-activated receptors (PPARs and PPAR γ coactivator-1α (PGC-1α pathways have been shown to improve mitochondrial function and energy metabolism. The mitochondrial DNA (mtDNA mutator mouse is a mouse model of aging that harbors a proofreading-deficient mtDNA polymerase γ. These mice develop many features of premature aging including hair loss, anemia, osteoporosis, sarcopenia and decreased lifespan. They also have increased mtDNA mutations and marked mitochondrial dysfunction. We found that mutator mice treated with bezafibrate for 8-months had delayed hair loss and improved skin and spleen aging-like phenotypes. Although we observed an increase in markers of fatty acid oxidation in these tissues, we did not detect a generalized increase in mitochondrial markers. On the other hand, there were no improvements in muscle function or lifespan of the mutator mouse, which we attributed to the rodent-specific hepatomegaly associated with fibrate treatment. These results showed that despite its secondary effects in rodent's liver, bezafibrate was able to improve some of the aging phenotypes in the mutator mouse. Because the associated hepatomegaly is not observed in primates, long-term bezafibrate treatment in humans could have beneficial effects on tissues undergoing chronic bioenergetic-related degeneration.

  19. A nonsense mutation in the DNA repair factor Hebo causes mild bone marrow failure and microcephaly.

    Science.gov (United States)

    Zhang, Shu; Pondarre, Corinne; Pennarun, Gaelle; Labussiere-Wallet, Helene; Vera, Gabriella; France, Benoit; Chansel, Marie; Rouvet, Isabelle; Revy, Patrick; Lopez, Bernard; Soulier, Jean; Bertrand, Pascale; Callebaut, Isabelle; de Villartay, Jean-Pierre

    2016-05-30

    Inherited bone marrow failure syndromes are human conditions in which one or several cell lineages of the hemopoietic system are affected. They are present at birth or may develop progressively. They are sometimes accompanied by other developmental anomalies. Three main molecular causes have been recognized to result in bone marrow failure syndromes: (1) defects in the Fanconi anemia (FA)/BRCA DNA repair pathway, (2) defects in telomere maintenance, and (3) abnormal ribosome biogenesis. We analyzed a patient with mild bone marrow failure and microcephaly who did not present with the typical FA phenotype. Cells from this patient showed increased sensitivity to ionizing radiations and phleomycin, attesting to a probable DNA double strand break (dsb) repair defect. Linkage analysis and whole exome sequencing revealed a homozygous nonsense mutation in the ERCC6L2 gene. We identified a new ERCC6L2 alternative transcript encoding the DNA repair factor Hebo, which is critical for complementation of the patient's DNAdsb repair defect. Sequence analysis revealed three structured regions within Hebo: a TUDOR domain, an adenosine triphosphatase domain, and a new domain, HEBO, specifically present in Hebo direct orthologues. Hebo is ubiquitously expressed, localized in the nucleus, and rapidly recruited to DNAdsb's in an NBS1-dependent manner.

  20. Methods for detection of ataxia telangiectasia mutations

    Science.gov (United States)

    Gatti, Richard A.

    2005-10-04

    The present invention is directed to a method of screening large, complex, polyexonic eukaryotic genes such as the ATM gene for mutations and polymorphisms by an improved version of single strand conformation polymorphism (SSCP) electrophoresis that allows electrophoresis of two or three amplified segments in a single lane. The present invention also is directed to new mutations and polymorphisms in the ATM gene that are useful in performing more accurate screening of human DNA samples for mutations and in distinguishing mutations from polymorphisms, thereby improving the efficiency of automated screening methods.

  1. Investigating the effects of dietary folic acid on sperm count, DNA damage and mutation in Balb/c mice

    Energy Technology Data Exchange (ETDEWEB)

    Swayne, Breanne G.; Kawata, Alice [Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario, K1A 0K9 (Canada); Behan, Nathalie A. [Nutrition Research Division, Food Directorate, Health Products and Food Branch, Health Canada, Ottawa, Ontario, K1A 0K9 (Canada); Williams, Andrew; Wade, Mike G. [Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario, K1A 0K9 (Canada); MacFarlane, Amanda J. [Nutrition Research Division, Food Directorate, Health Products and Food Branch, Health Canada, Ottawa, Ontario, K1A 0K9 (Canada); Yauk, Carole L., E-mail: carole.yauk@hc-sc.ga.ca [Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario, K1A 0K9 (Canada)

    2012-09-01

    To date, fewer than 50 mutagens have been studied for their ability to cause heritable mutations. The majority of those studied are classical mutagens like radiation and anti-cancer drugs. Very little is known about the dietary variables influencing germline mutation rates. Folate is essential for DNA synthesis and methylation and can impact chromatin structure. We therefore determined the effects of folic acid-deficient (0 mg/kg), control (2 mg/kg) and supplemented (6 mg/kg) diets in early development and during lactation or post-weaning on mutation rates and chromatin quality in sperm of adult male Balb/c mice. The sperm chromatin structure assay and mutation frequencies at expanded simple tandem repeats (ESTRs) were used to evaluate germline DNA integrity. Treatment of a subset of mice fed the control diet with the mutagen ethylnitrosourea (ENU) at 8 weeks of age was included as a positive control. ENU treated mice exhibited decreased cauda sperm counts, increased DNA fragmentation and increased ESTR mutation frequencies relative to non-ENU treated mice fed the control diet. Male mice weaned to the folic acid deficient diet had decreased cauda sperm numbers, increased DNA fragmentation index, and increased ESTR mutation frequency. Folic acid deficiency in early development did not lead to changes in sperm counts or chromatin integrity in adult mice. Folic acid supplementation in early development or post-weaning did not affect germ cell measures. Therefore, adequate folic acid intake in adulthood is important for preventing chromatin damage and mutation in the male germline. Folic acid supplementation at the level achieved in this study does not improve nor is it detrimental to male germline chromatin integrity.

  2. Investigating the effects of dietary folic acid on sperm count, DNA damage and mutation in Balb/c mice

    International Nuclear Information System (INIS)

    To date, fewer than 50 mutagens have been studied for their ability to cause heritable mutations. The majority of those studied are classical mutagens like radiation and anti-cancer drugs. Very little is known about the dietary variables influencing germline mutation rates. Folate is essential for DNA synthesis and methylation and can impact chromatin structure. We therefore determined the effects of folic acid-deficient (0 mg/kg), control (2 mg/kg) and supplemented (6 mg/kg) diets in early development and during lactation or post-weaning on mutation rates and chromatin quality in sperm of adult male Balb/c mice. The sperm chromatin structure assay and mutation frequencies at expanded simple tandem repeats (ESTRs) were used to evaluate germline DNA integrity. Treatment of a subset of mice fed the control diet with the mutagen ethylnitrosourea (ENU) at 8 weeks of age was included as a positive control. ENU treated mice exhibited decreased cauda sperm counts, increased DNA fragmentation and increased ESTR mutation frequencies relative to non-ENU treated mice fed the control diet. Male mice weaned to the folic acid deficient diet had decreased cauda sperm numbers, increased DNA fragmentation index, and increased ESTR mutation frequency. Folic acid deficiency in early development did not lead to changes in sperm counts or chromatin integrity in adult mice. Folic acid supplementation in early development or post-weaning did not affect germ cell measures. Therefore, adequate folic acid intake in adulthood is important for preventing chromatin damage and mutation in the male germline. Folic acid supplementation at the level achieved in this study does not improve nor is it detrimental to male germline chromatin integrity.

  3. Mitochondrial DNA mutation m.10680G > A is associated with Leber hereditary optic neuropathy in Chinese patients

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    Zhang A-Mei

    2012-03-01

    Full Text Available Abstract Background Leber hereditary optic neuropathy (LHON is a mitochondrial disorder with gender biased and incomplete penetrance. The majority of LHON patients are caused by one of the three primary mutations (m.3460G > A, m.11778G > A and m.14484T > C. Rare pathogenic mutations have been occasionally reported in LHON patients. Methods We screened mutation m.10680G > A in the MT-ND4L gene in 774 Chinese patients with clinical features of LHON but lacked the three primary mutations by using allele specific PCR (AS-PCR. Patients with m.10680G > A were further determined entire mtDNA genome sequence. Results The optimal AS-PCR could detect as low as 10% heteroplasmy of mutation m.10680G > A. Two patients (Le1263 and Le1330 were identified to harbor m.10680G > A. Analysis of the complete mtDNA sequences of the probands suggested that they belonged to haplogroups B4a1 and D6a1. There was no other potentially pathogenic mutation, except for a few private yet reported variants in the MT-ND1 and MT-ND5 genes, in the two lineages. A search in reported mtDNA genome data set (n = 9277; excluding Chinese LHON patients identified no individual with m.10680G > A. Frequency of m.10680G > A in Chinese LHON patients analyzed in this study and our previous studies (3/784 was significantly higher than that of the general populations (0/9277 (P = 0.0005. Conclusion Taken together, we speculated that m.10680G > A may be a rare pathogenic mutation for LHON in Chinese. This mutation should be included in future clinical diagnosis.

  4. Comparing the DNA hypermethylome with gene mutations in human colorectal cancer.

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    Kornel E Schuebel

    2007-09-01

    Full Text Available We have developed a transcriptome-wide approach to identify genes affected by promoter CpG island DNA hypermethylation and transcriptional silencing in colorectal cancer. By screening cell lines and validating tumor-specific hypermethylation in a panel of primary human colorectal cancer samples, we estimate that nearly 5% or more of all known genes may be promoter methylated in an individual tumor. When directly compared to gene mutations, we find larger numbers of genes hypermethylated in individual tumors, and a higher frequency of hypermethylation within individual genes harboring either genetic or epigenetic changes. Thus, to enumerate the full spectrum of alterations in the human cancer genome, and to facilitate the most efficacious grouping of tumors to identify cancer biomarkers and tailor therapeutic approaches, both genetic and epigenetic screens should be undertaken.

  5. The role of mtDNA background in disease expression: a new primary LHON mutation associated with Western Eurasian haplogroup J.

    Science.gov (United States)

    Brown, Michael D; Starikovskaya, Elena; Derbeneva, Olga; Hosseini, Seyed; Allen, Jon C; Mikhailovskaya, Irina E; Sukernik, Rem I; Wallace, Douglas C

    2002-02-01

    Leber's hereditary optic neuropathy (LHON) is a maternally transmitted form of blindness caused by mitochondrial DNA (mtDNA) mutations. Approximately 90% of LHON cases are caused by 3460A, 11778A, or 14484C mtDNA mutations. These are designated "primary" mutations because they impart a high risk for LHON expression. Although the 11778A and 14484C mutations unequivocally predispose carriers to LHON, they are preferentially associated with mtDNA haplogroup J, one of nine Western Eurasian mtDNA lineages, suggesting a synergistic and deleterious interaction between these LHON mutations and haplogroup J polymorphism(s). We report here the characterization of a new primary LHON mutation in the mtDNA ND4L gene at nucleotide pair 10663. The homoplasmic 10663C mutation has been found in three independent LHON patients who lack a known primary mutation and all of which belong to haplogroup J. This mutation has not been found in a large number of haplotype-matched or non-haplogroup-J control mtDNAs. Phylogenetic analysis with primarily complete mtDNA sequence data demonstrates that the 10663C mutation has arisen at least three independent times in haplogroup J, indicating that it is not a rare lineage-specific polymorphism. Analysis of complex I function in patient lymphoblasts and transmitochondrial cybrids has revealed a partial complex I defect similar in magnitude to the 14484C mutation. Thus, the 10663C mutation appears to be a new primary LHON mutation that is pathogenic when co-occurring with haplogroup J. These results strongly support a role for haplogroup J in the expression of certain LHON mutations.

  6. Arsenic trioxide promotes mitochondrial DNA mutation and cell apoptosis in primary APL cells and NB4 cell lines

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    This study aimed to investigate the effects of arsenic trioxide(As2O3) on the mitochondrial DNA(mtDNA) of acute promyelocytic leukemia(APL) cells.The NB4 cell line was treated with 2.0 μmol/L As2O3 in vitro,and the primary APL cells were treated with 2.0 μmol/L As2O3 in vitro and 0.16 mg kg-1 d-1 As2O3 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 As2O3 use,but the mutation spots were remarkably increased after As2O3 treatment,which was positively correlated to the rates of cellular apoptosis,the correlation coefficient:rNB4-As2O3=0.973818,and rAPL-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 As2O3 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 As2O3 in APL treatment.

  7. Leber's hereditary optic neuroretinopathy (LHON) associated with mitochondrial DNA point mutation G11778A in two Croatian families.

    Science.gov (United States)

    Martin-Kleiner, Irena; Gabrilovac, Jelka; Bradvica, Mario; Vidović, Tomislav; Cerovski, Branimir; Fumić, Ksenija; Boranić, Milivoj

    2006-03-01

    Leber's hereditary optic neuroretinopathy (LHON) is manifested as a bilateral acute or subacute loss of central vision due to optic atrophy. It is linked to point mutations of mitochondrial DNA, which is inherited maternally. The most common mitochondrial DNA point mutations associated with LHON are G3460A, G11778A and T14484C. These mutations are linked with the defects of subunits of the complex I (NADH-dehydrogenase-ubiquinone reductase) in mitochondria. The G11778A mitochondrial DNA point mutation is manifested by a severe visual impairment. In this paper two Croatian families with the LHON G11778A mutation are presented. Three LHON patients from two families were younger males which had the visual acuity of 0.1 or below, the ophthalmoscopy revealed telangiectatic microangiopathy and papilloedema, while Goldmann kinetic perimetry showed a central scotoma. The mothers and female relatives were LHON mutants without symptoms, whereas their sons suffered from a severe visual impairment. Molecular diagnosis helps to explain the cause of LHON disease.

  8. Frequent mutations in EGFR, KRAS and TP53 genes in human lung cancer tumors detected by ion torrent DNA sequencing.

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    Xin Cai

    Full Text Available Lung cancer is the most common malignancy and the leading cause of cancer deaths worldwide. While smoking is by far the leading cause of lung cancer, other environmental and genetic factors influence the development and progression of the cancer. Since unique mutations patterns have been observed in individual cancer samples, identification and characterization of the distinctive lung cancer molecular profile is essential for developing more effective, tailored therapies. Until recently, personalized DNA sequencing to identify genetic mutations in cancer was impractical and expensive. The recent technological advancements in next-generation DNA sequencing, such as the semiconductor-based Ion Torrent sequencing platform, has made DNA sequencing cost and time effective with more reliable results. Using the Ion Torrent Ampliseq Cancer Panel, we sequenced 737 loci from 45 cancer-related genes to identify genetic mutations in 76 human lung cancer samples. The sequencing analysis revealed missense mutations in KRAS, EGFR, and TP53 genes in the breast cancer samples of various histologic types. Thus, this study demonstrates the necessity of sequencing individual human cancers in order to develop personalized drugs or combination therapies to effectively target individual, breast cancer-specific mutations.

  9. GTF2E2 Mutations Destabilize the General Transcription Factor Complex TFIIE in Individuals with DNA Repair-Proficient Trichothiodystrophy.

    Science.gov (United States)

    Kuschal, Christiane; Botta, Elena; Orioli, Donata; Digiovanna, John J; Seneca, Sara; Keymolen, Kathelijn; Tamura, Deborah; Heller, Elizabeth; Khan, Sikandar G; Caligiuri, Giuseppina; Lanzafame, Manuela; Nardo, Tiziana; Ricotti, Roberta; Peverali, Fiorenzo A; Stephens, Robert; Zhao, Yongmei; Lehmann, Alan R; Baranello, Laura; Levens, David; Kraemer, Kenneth H; Stefanini, Miria

    2016-04-01

    The general transcription factor IIE (TFIIE) is essential for transcription initiation by RNA polymerase II (RNA pol II) via direct interaction with the basal transcription/DNA repair factor IIH (TFIIH). TFIIH harbors mutations in two rare genetic disorders, the cancer-prone xeroderma pigmentosum (XP) and the cancer-free, multisystem developmental disorder trichothiodystrophy (TTD). The phenotypic complexity resulting from mutations affecting TFIIH has been attributed to the nucleotide excision repair (NER) defect as well as to impaired transcription. Here, we report two unrelated children showing clinical features typical of TTD who harbor different homozygous missense mutations in GTF2E2 (c.448G>C [p.Ala150Pro] and c.559G>T [p.Asp187Tyr]) encoding the beta subunit of transcription factor IIE (TFIIEβ). Repair of ultraviolet-induced DNA damage was normal in the GTF2E2 mutated cells, indicating that TFIIE was not involved in NER. We found decreased protein levels of the two TFIIE subunits (TFIIEα and TFIIEβ) as well as decreased phosphorylation of TFIIEα in cells from both children. Interestingly, decreased phosphorylation of TFIIEα was also seen in TTD cells with mutations in ERCC2, which encodes the XPD subunit of TFIIH, but not in XP cells with ERCC2 mutations. Our findings support the theory that TTD is caused by transcriptional impairments that are distinct from the NER disorder XP.

  10. GTF2E2 Mutations Destabilize the General Transcription Factor Complex TFIIE in Individuals with DNA Repair-Proficient Trichothiodystrophy

    Science.gov (United States)

    Kuschal, Christiane; Botta, Elena; Orioli, Donata; Digiovanna, John J.; Seneca, Sara; Keymolen, Kathelijn; Tamura, Deborah; Heller, Elizabeth; Khan, Sikandar G.; Caligiuri, Giuseppina; Lanzafame, Manuela; Nardo, Tiziana; Ricotti, Roberta; Peverali, Fiorenzo A.; Stephens, Robert; Zhao, Yongmei; Lehmann, Alan R.; Baranello, Laura; Levens, David; Kraemer, Kenneth H.; Stefanini, Miria

    2016-01-01

    The general transcription factor IIE (TFIIE) is essential for transcription initiation by RNA polymerase II (RNA pol II) via direct interaction with the basal transcription/DNA repair factor IIH (TFIIH). TFIIH harbors mutations in two rare genetic disorders, the cancer-prone xeroderma pigmentosum (XP) and the cancer-free, multisystem developmental disorder trichothiodystrophy (TTD). The phenotypic complexity resulting from mutations affecting TFIIH has been attributed to the nucleotide excision repair (NER) defect as well as to impaired transcription. Here, we report two unrelated children showing clinical features typical of TTD who harbor different homozygous missense mutations in GTF2E2 (c.448G>C [p.Ala150Pro] and c.559G>T [p.Asp187Tyr]) encoding the beta subunit of transcription factor IIE (TFIIEβ). Repair of ultraviolet-induced DNA damage was normal in the GTF2E2 mutated cells, indicating that TFIIE was not involved in NER. We found decreased protein levels of the two TFIIE subunits (TFIIEα and TFIIEβ) as well as decreased phosphorylation of TFIIEα in cells from both children. Interestingly, decreased phosphorylation of TFIIEα was also seen in TTD cells with mutations in ERCC2, which encodes the XPD subunit of TFIIH, but not in XP cells with ERCC2 mutations. Our findings support the theory that TTD is caused by transcriptional impairments that are distinct from the NER disorder XP. PMID:26996949

  11. GTF2E2 Mutations Destabilize the General Transcription Factor Complex TFIIE in Individuals with DNA Repair-Proficient Trichothiodystrophy.

    Science.gov (United States)

    Kuschal, Christiane; Botta, Elena; Orioli, Donata; Digiovanna, John J; Seneca, Sara; Keymolen, Kathelijn; Tamura, Deborah; Heller, Elizabeth; Khan, Sikandar G; Caligiuri, Giuseppina; Lanzafame, Manuela; Nardo, Tiziana; Ricotti, Roberta; Peverali, Fiorenzo A; Stephens, Robert; Zhao, Yongmei; Lehmann, Alan R; Baranello, Laura; Levens, David; Kraemer, Kenneth H; Stefanini, Miria

    2016-04-01

    The general transcription factor IIE (TFIIE) is essential for transcription initiation by RNA polymerase II (RNA pol II) via direct interaction with the basal transcription/DNA repair factor IIH (TFIIH). TFIIH harbors mutations in two rare genetic disorders, the cancer-prone xeroderma pigmentosum (XP) and the cancer-free, multisystem developmental disorder trichothiodystrophy (TTD). The phenotypic complexity resulting from mutations affecting TFIIH has been attributed to the nucleotide excision repair (NER) defect as well as to impaired transcription. Here, we report two unrelated children showing clinical features typical of TTD who harbor different homozygous missense mutations in GTF2E2 (c.448G>C [p.Ala150Pro] and c.559G>T [p.Asp187Tyr]) encoding the beta subunit of transcription factor IIE (TFIIEβ). Repair of ultraviolet-induced DNA damage was normal in the GTF2E2 mutated cells, indicating that TFIIE was not involved in NER. We found decreased protein levels of the two TFIIE subunits (TFIIEα and TFIIEβ) as well as decreased phosphorylation of TFIIEα in cells from both children. Interestingly, decreased phosphorylation of TFIIEα was also seen in TTD cells with mutations in ERCC2, which encodes the XPD subunit of TFIIH, but not in XP cells with ERCC2 mutations. Our findings support the theory that TTD is caused by transcriptional impairments that are distinct from the NER disorder XP. PMID:26996949

  12. Roles of DNA mutation in the coding region and DNA methylation in the 5' flanking region of BRCA1 in canine mammary tumors.

    Science.gov (United States)

    Qiu, Hengbin; Lin, Deigui

    2016-07-01

    The Breast cancer 1, early onset gene (BRCA1) is known to be significantly associated with human familial breast cancer and is identified to play an important role in canine mammary tumors. Here, genetic variations in the coding region and DNA methylation in the 5' flanking region of BRCA1 in canine mammary tumor samples, 15 each of benign and malignant against 10 normal canine mammary tissue samples, were analyzed using the direct sequencing method. The results indicated two point mutations each in the coding region of canine BRCA1 in one benign mammary tumor sample (4702G >T and 4765G >T) and in one malignant canine mammary tumor sample (3619A >G and 4006G >A). No mutations were detected in the normal canine mammary tissue samples. The 4702G >T mutation was found to terminate further translation. The physical effect of the 4765G >T mutation was found to be the repalacement of the glutamate residue with glutamine. The physical effect of the 3619A >G mutation was found to be the replacement of the threonine residue with alanine, and that of mutation 4006G >A was the replacement of the valine residue with isoleucine in the BRCA1 protein. Bisulfite sequencing detected methylated CpG sites in one canine malignant mammary tumor sample. In conclusion, the present study elucidated the mutational status of the BRCA1 coding region and methylation status of the 5' flanking region of BRCA1 in canine mammary tumors. PMID:26888582

  13. Novel TK2 mutations as a cause of delayed muscle maturation in mtDNA depletion syndrome.

    Science.gov (United States)

    Termglinchan, Thanes; Hisamatsu, Seito; Ohmori, Junko; Suzumura, Hiroshi; Sumitomo, Noriko; Imataka, George; Arisaka, Osamu; Murakami, Nobuyuki; Minami, Narihiro; Akihiko, Ishiyama; Sasaki, Masayuki; Goto, Yuichi; Noguchi, Satoru; Nonaka, Ikuya; Mitsuhashi, Satomi; Nishino, Ichizo

    2016-10-01

    Recessive mutations in TK2 cause a severe mitochondrial DNA depletion syndrome (MDS),(1) characterized by severe myopathy from early infancy. Recent reports have suggested a wider clinical spectrum including encephalomyopathic form.(1,2) We report a patient with infantile-onset fatal encephalomyopathy presenting with extreme muscle fiber immaturity.

  14. EGFR Mutations Detection in Non-small Cell Lung Cancer Tissues by Real-time PCR and DNA Sequencing

    Directory of Open Access Journals (Sweden)

    Qinghua ZHOU

    2009-12-01

    Full Text Available Background and objective Small molecule tyrosine kinase inhibitors (TKIs, such as gefitinib and erlotinib that target the kinase domain of epidermal growth factor receptor (EGFR are making successful progression for advanced non-small cell lung cancer patients treatment. The growing evidences revealed that EGFR exon 19 and 21 mutation status in NSCLC patients was correlated with the outcome for EGFR-TKI treatment. In this study, two methods of Real-time PCR and DNA sequencing were compared to detected EGFR exon 19 and 21 mutations. Methods EGFR exon19 mutation del-E746-A750 and exon 21mutation L858R were detected by Real-time PCR and DNA sequencing in 103 NSCLC patients. Chi-square test was used to analyze the consistance. Results There was no significant difference between the two methods. However, Real-time PCR was more convenient and sensitive compared to DNA sequencing. Conclusion Real-time PCR was more suitable for clinical testing than DNA sequencing.

  15. Leber Hereditary Optic Neuropathy: Do Folate Pathway Gene Alterations Influence the Expression of Mitochondrial DNA Mutation?

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    A Aleyasin

    2010-09-01

    Full Text Available "nBackground: Leber hereditary optic neuropathy (LHON is an inherited form of bilateral optic atrophy leading to the loss of central vision.  The primary cause of vision loss is mutation in the mitochondrial DNA (mtDNA, however, unknown secon­dary genetic and/or epigenetic risk factors are suggested to influence its neuropathology.  In this study folate gene polymor­phisms were examined as a possible LHON secondary genetic risk factor in Iranian patients."nMethods: Common polymorphisms in the MTHFR (C677T and A1298C and MTRR (A66G genes were tested in 21 LHON patients and 150 normal controls."nResults:  Strong associations were observed between the LHON syndrome and C677T (P= 0.00 and A66G (P= 0.00 polymor­phisms.  However, no significant association was found between A1298C (P =0.69 and the LHON syndrome."nConclusion: This is the first study that shows MTHFR C677T and MTRR A66G polymorphisms play a role in the etiology of the LHON syndrome.  This finding may help in the better understanding of mechanisms involved in neural degeneration and vision loss by LHON and hence the better treatment of patients.

  16. Creation of chimeric human/rabbit APOBEC1 with HIV-1 restriction and DNA mutation activities

    Science.gov (United States)

    Ikeda, Terumasa; Ong, Eugene Boon Beng; Watanabe, Nobumoto; Sakaguchi, Nobuo; Maeda, Kazuhiko; Koito, Atsushi

    2016-01-01

    APOBEC1 (A1) proteins from lagomorphs and rodents have deaminase-dependent restriction activity against HIV-1, whereas human A1 exerts a negligible effect. To investigate these differences in the restriction of HIV-1 by A1 proteins, a series of chimeric proteins combining rabbit and human A1s was constructed. Homology models of the A1s indicated that their activities derive from functional domains that likely act in tandem through a dimeric interface. The C-terminal region containing the leucine-rich motif and the dimerization domains of rabbit A1 is important for its anti-HIV-1 activity. The A1 chimeras with strong anti-HIV-1 activity were incorporated into virions more efficiently than those without anti-HIV-1 activity, and exhibited potent DNA-mutator activity. Therefore, the C-terminal region of rabbit A1 is involved in both its packaging into the HIV-1 virion and its deamination activity against both viral cDNA and genomic RNA. This study identifies the novel molecular mechanism underlying the target specificity of A1.

  17. Mutational analysis of Mycobacterium UvrD1 identifies functional groups required for ATP hydrolysis, DNA unwinding, and chemomechanical coupling

    OpenAIRE

    Sinha, Krishna Murari; Glickman, Michael S.; Shuman, Stewart

    2009-01-01

    Mycobacterial UvrD1 is a DNA-dependent ATPase and a Ku-dependent 3’ to 5’ DNA helicase. The UvrD1 motor domain resembles that of the prototypal superfamily I helicases UvrD and PcrA. Here we performed a mutational analysis of UvrD1 guided by the crystal structure of a DNA-bound E. coli UvrD-ADP-MgF3 transition state mimetic. Alanine scanning and conservative substitutions identified five amino acids essential for both ATP hydrolysis and duplex unwinding, including those implicated in phosphoh...

  18. Temporal changes of EGFR mutations and T790M levels in tumour and plasma DNA following AZD9291 treatment.

    Science.gov (United States)

    Chia, Puey Ling; Do, Hongdo; Morey, Adrienne; Mitchell, Paul; Dobrovic, Alexander; John, Thomas

    2016-08-01

    AZD9291, a T790M specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), has demonstrated impressive response rates in tumours harbouring the EGFR T790M resistance mutation. Emergence of resistance to AZD9291 has been shown to occur through several different mechanisms including the development of new mutations (e.g. C797S) in the EGFR tyrosine kinase domain. We studied two patients with paired tumour biopsies and blood samples pre- and post-progression on AZD9291 to explore possible resistance mechanisms. Pre- and Post-AZD9291 tumour biopsies as well as serial plasma samples were collected from two patients on the AURA clinical study (AZD9291 First Time in Patients Ascending Dose study). Droplet digital PCR (ddPCR) assays were used to quantify T790M, the driver EGFR mutation, and the C797S mutation in genomic DNA from paired tumour biopsies and plasma cell-free DNA. In the first patient, both EGFR T790M and L858R became undetectable in the plasma within 1 month after treatment with AZD9291. However, the T790M and the original L858R mutation re-emerged with radiologically confirmed resistance to AZD9291. In patient two, the levels of T790M were undetectable at the time of radiological resistance to AZD9291 but increasing levels of the original EGFR exon 19 deletion was detected. MET amplification was detected in a biopsy performed on progression. The EGFR C797S mutation was not detected in either patient at the time of relapse. ddPCR of cell free DNA enables real time monitoring of patients on 3rd generation TKIs. As resistance mechanisms are variable, monitoring levels of the initial activating EGFR mutation may facilitate more reliable detection of progression. PMID:27393503

  19. Molecular analysis of the human laminin alpha3a chain gene (LAMA3a): a strategy for mutation identification and DNA-based prenatal diagnosis in Herlitz junctional epidermolysis bullosa.

    Science.gov (United States)

    Pulkkinen, L; Cserhalmi-Friedman, P B; Tang, M; Ryan, M C; Uitto, J; Christiano, A M

    1998-09-01

    Mutations in the genes (LAMA3, LAMB3, and LAMC2) encoding the subunit polypeptides of the cutaneous basement membrane zone protein laminin 5 have been reported in different forms of junctional epidermolysis bullosa (JEB), an inherited blistering skin disease. In this study, we present the complete exon-intron organization of the "a" transcript of the laminin alpha3 chain gene, LAMA3a, which is expressed primarily in the skin. We have performed fine-resolution mapping of this gene on chromosome 18q11.2 using a human-hamster radiation hybrid panel. We have also developed a mutation-detection strategy based on the exon-intron structure of LAMA3a. This strategy, based on PCR amplification of genomic sequences, followed by heteroduplex scanning and automated nucleotide sequencing, was used for successful mutation screening in a family with the lethal (Herlitz) type of JEB, and two novel LAMA3 mutations were identified in the proband. The mutations consisted of a single-base pair deletion in LAMA3a exon A11 on the paternal allele, designated 1239delC, and a two-base pair deletion in LAMA3a exon A23 on the maternal allele, designated 2959delGG. This information was also used for DNA-based prenatal testing in a subsequent pregnancy in this family. Collectively, these results attest to our expanding capability to elucidate the genetic basis of various forms of epidermolysis bullosa using molecular techniques. PMID:9759651

  20. POLG1 p.R722H mutation associated with multiple mtDNA deletions and a neurological phenotype

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    Finnilä Saara

    2010-05-01

    Full Text Available Abstract Background The c.2447G>A (p.R722H mutation in the gene POLG1 of the catalytic subunit of human mitochondrial polymerase gamma has been previously found in a few occasions but its pathogenicity has remained uncertain. We set out to ascertain its contribution to neuromuscular disease. Methods Probands from two families with probable mitochondrial disease were examined clinically, muscle and buccal epithelial DNA were analyzed for mtDNA deletions, and the POLG1, POLG2, ANT1 and Twinkle genes were sequenced. Results An adult proband presented with progressive external ophthalmoplegia, sensorineural hearing impairment, diabetes mellitus, dysphagia, a limb myopathy and dementia. Brain MRI showed central and cortical atrophy, and 18F-deoxyglucose PET revealed reduced glucose uptake. Histochemical analysis of muscle disclosed ragged red fibers and cytochrome c oxidase-negative fibers. Electron microscopy showed subsarcolemmal aggregates of morphologically normal mitochondria. Multiple mtDNA deletions were found in the muscle, and sequencing of the POLG1 gene revealed a homozygous c.2447G>A (p.R722H mutation. His two siblings were also homozygous with respect to the p.R722H mutation and presented with dementia and sensorineural hearing impairment. In another family the p.R722H mutation was found as compound heterozygosity with the common p.W748S mutation in two siblings with mental retardation, ptosis, epilepsy and psychiatric symptoms. The estimated carrier frequency of the p.R722H mutation was 1:135 in the Finnish population. No mutations in POLG2, ANT1 and Twinkle genes were found. Analysis of the POLG1 sequence by homology modeling supported the notion that the p.R722H mutation is pathogenic. Conclusions The recessive c.2447G>A (p.R722H mutation in the linker region of the POLG1 gene is pathogenic for multiple mtDNA deletions in muscle and is associated with a late-onset neurological phenotype as a homozygous state. The onset of the disease

  1. Paths of Heritable Mitochondrial DNA Mutation and Heteroplasmy in Reference and gas-1 Strains of Caenorhabditis elegans

    Science.gov (United States)

    Wernick, Riana I.; Estes, Suzanne; Howe, Dana K.; Denver, Dee R.

    2016-01-01

    Heteroplasmy—the presence of more than one mitochondrial DNA (mtDNA) sequence type in a cell, tissue, or individual—impacts human mitochondrial disease and numerous aging-related syndromes. Understanding the trans-generational dynamics of mtDNA is critical to understanding the underlying mechanisms of mitochondrial disease and evolution. We investigated mtDNA mutation and heteroplasmy using a set of wild-type (N2 strain) and mitochondrial electron transport chain (ETC) mutant (gas-1) mutant Caenorhabditis elegans mutation-accumulation (MA) lines. The N2 MA lines, derived from a previous experiment, were bottlenecked for 250 generations. The gas-1 MA lines were created for this study, and bottlenecked in the laboratory for up to 50 generations. We applied Illumina-MiSeq DNA sequencing to L1 larvae from five gas-1 MA lines and five N2 MA lines to detect and characterize mtDNA mutation and heteroplasmic inheritance patterns evolving under extreme drift. mtDNA copy number increased in both sets of MA lines: three-fold on average among the gas-1 MA lines and five-fold on average among N2 MA lines. Eight heteroplasmic single base substitution polymorphisms were detected in the gas-1 MA lines; only one was observed in the N2 MA lines. Heteroplasmy frequencies ranged broadly in the gas-1 MA lines, from as low as 2.3% to complete fixation (homoplasmy). An initially low-frequency (<5%) heteroplasmy discovered in the gas-1 progenitor was observed to fix in one gas-1 MA line, achieve higher frequency (37.4%) in another, and be lost in the other three lines. A similar low-frequency heteroplasmy was detected in the N2 progenitor, but was lost in all five N2 MA lines. We identified three insertion-deletion (indel) heteroplasmies in gas-1 MA lines and six indel variants in the N2 MA lines, most occurring at homopolymeric nucleotide runs. The observed bias toward accumulation of single nucleotide polymorphisms in gas-1 MA lines is consistent with the idea that impaired

  2. Paths of heritable mitochondrial DNA mutation and heteroplasmy in reference and gas-1 strains of Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Riana eWernick

    2016-04-01

    Full Text Available Heteroplasmy—the presence of more than one mitochondrial DNA (mtDNA sequence type in a cell, tissue, or individual—impacts human mitochondrial disease and numerous aging-related syndromes. Understanding the trans-generational dynamics of mtDNA is critical to understanding the underlying mechanisms of mitochondrial disease and evolution. We investigated mtDNA mutation and heteroplasmy using a set of wild-type (N2 strain and mitochondrial electron transport chain mutant (gas-1 mutant Caenohabditis elegans mutation-accumulation (MA lines. The N2 MA lines, derived from a previous experiment, were bottlenecked for 250 generations. The gas-1 MA lines were created for this study, and bottlenecked in the laboratory for up to 50 generations. We applied Illumina-MiSeq DNA sequencing to L1 larvae from five gas-1 MA lines and five N2 MA lines to detect and characterize mtDNA mutation and heteroplasmic inheritance patterns evolving under extreme drift. mtDNA copy number increased in both sets of MA lines: 3-fold on average among the gas-1 MA lines and 5-fold on average among N2 MA lines. Eight heteroplasmic single base substitution polymorphisms were detected in the gas-1 MA lines; only one was observed in the N2 MA lines. Heteroplasmy frequencies ranged broadly in the gas-1 MA lines, from as low as 2.3% to complete fixation (homoplasmy. An initially low-frequency (<5% heteroplasmy discovered in the gas-1 progenitor was observed to fix in one gas-1 MA line, achieve higher frequency (37.4% in another, and be lost in the other three lines. A similar low-frequency heteroplasmy was detected in the N2 progenitor, but was lost in all five N2 MA lines. We identified three insertion-deletion (indel heteroplasmies in gas-1 MA lines and six indel variants in the N2 MA lines, most occurring at homopolymeric nucleotide runs. The observed bias toward accumulation of single nucleotide polymorphisms in gas-1 MA lines is consistent with the idea that impaired

  3. High quality DNA obtained with an automated DNA extraction method with 70+ year old formalin-fixed celloidin-embedded (FFCE) blocks from the indiana medical history museum.

    Science.gov (United States)

    Niland, Erin E; McGuire, Audrey; Cox, Mary H; Sandusky, George E

    2012-01-01

    DNA and RNA have been used as markers of tissue quality and integrity throughout the last few decades. In this research study, genomic quality DNA of kidney, liver, heart, lung, spleen, and brain were analyzed in tissues from post-mortem patients and surgical cancer cases spanning the past century. DNA extraction was performed on over 180 samples from: 70+ year old formalin-fixed celloidin-embedded (FFCE) tissues, formalin-fixed paraffin-embedded (FFPE) tissue samples from surgical cases and post-mortem cases from the 1970's, 1980's, 1990's, and 2000's, tissues fixed in 10% neutral buffered formalin/stored in 70% ethanol from the 1990's, 70+ year old tissues fixed in unbuffered formalin of various concentrations, and fresh tissue as a control. To extract DNA from FFCE samples and ethanol-soaked samples, a modified standard operating procedure was used in which all tissues were homogenized, digested with a proteinase K solution for a long period of time (24-48 hours), and DNA was extracted using the Autogen Flexstar automated extraction machine. To extract DNA from FFPE, all tissues were soaked in xylene to remove the paraffin from the tissue prior to digestion, and FFPE tissues were not homogenized. The results were as follows: celloidin-embedded and paraffin-embedded tissues yielded the highest DNA concentration and greatest DNA quality, while the formalin in various concentrations, and long term formalin/ethanol-stored tissue yielded both the lowest DNA concentration and quality of the tissues tested. The average DNA yield for the various fixatives was: 367.77 μg/ mL FFCE, 590.7 μg/mL FFPE, 53.74 μg/mL formalin-fixed/70% ethanol-stored and 33.2 μg/mL unbuffered formalin tissues. The average OD readings for FFCE, FFPE, formalin-fixed/70% ethanol-stored tissues, and tissues fixed in unbuffered formalin were 1.86, 1.87, 1.43, and 1.48 respectively. The results show that usable DNA can be extracted from tissue fixed in formalin and embedded in celloidin or

  4. The use of EGFR exon 19 and 21 unlabeled DNA probes to screen for activating mutations in non-small cell lung cancer.

    Science.gov (United States)

    Willmore-Payne, Carlynn; Holden, Joseph A; Wittwer, Carl T; Layfield, Lester J

    2008-07-01

    Activating mutations in epidermal growth factor receptor-1 (EGFR) are found in 10-15% of Caucasian patients with non-small cell lung carcinoma (NSCLC). Approximately 90% of the mutations are deletions of several amino acids in exon 19 or point mutations in exon 21. Some studies suggest that these mutations identify patients that might benefit from targeted EGFR inhibitor therapy. DNA melting analysis of polymerase chain reaction products can screen for these mutations to identify this patient population. However, amplicon DNA melting analysis, although easily capable of detecting heterozygous mutations by heterodimer formation, becomes more difficult if mutations are homozygous or if the mutant allele is selectively amplified over wild type. Amplification of EGFR is common in NSCLC and this could compromise mutation detection by amplicon melting analysis. To overcome this potential limitation, we developed unlabeled, single-stranded DNA probes, complimentary to EGFR exon 19 and exon 21 where the common activating mutations occur. The unlabeled probes are incorporated into a standard polymerase chain reaction during the amplification of EGFR exons 19 and 21. The probe melting peak is easily distinguished from the amplicon melting peak, and probe melting is altered if mutations are present. This allows for easy identification of activating mutations even in homozygous or amplified states and is useful in the screening of NSCLC for the common EGFR activating mutations. PMID:19137110

  5. Chronic mucocutaneous candidiasis caused by a gain-of-function mutation in the STAT1 DNA-binding domain.

    Science.gov (United States)

    Takezaki, Shunichiro; Yamada, Masafumi; Kato, Masahiko; Park, Myoung-Ja; Maruyama, Kenichi; Yamazaki, Yasuhiro; Chida, Natsuko; Ohara, Osamu; Kobayashi, Ichiro; Ariga, Tadashi

    2012-08-01

    Chronic mucocutaneous candidiasis (CMC) is a heterogeneous group of primary immunodeficiency diseases characterized by chronic and recurrent Candida infections of the skin, nails, and oropharynx. Gain-of-function mutations in STAT1 were very recently shown to be responsible for autosomal-dominant or sporadic cases of CMC. The reported mutations have been exclusively localized in the coiled-coil domain, resulting in impaired dephosphorylation of STAT1. However, recent crystallographic analysis and direct mutagenesis experiments indicate that mutations affecting the DNA-binding domain of STAT1 could also lead to persistent phosphorylation of STAT1. To our knowledge, this study shows for the first time that a DNA-binding domain mutation of c.1153C>T in exon 14 (p.T385M) is the genetic cause of sporadic CMC in two unrelated Japanese patients. The underlying mechanisms involve a gain of STAT1 function due to impaired dephosphorylation as observed in the coiled-coil domain mutations.

  6. Molecular diagnosis of mucopolysaccharidosis Type II (Hunter syndrome) by automated sequencing and computer-assisted interpretation: Toward mutation mapping of the Iduronate-2-sulfatase gene

    Energy Technology Data Exchange (ETDEWEB)

    Jonsson, J.J.; Aronovich, E.L.; Braun, S.E.; Whitley, C.B. [Univ. of Minnesota Medical School, Minneapolis, MN (United States)

    1995-03-01

    Virtually all mutations causing Hunter syndrome (mucopolysaccharidosis type II) are expected to be new mutations. Therefore, as a means of molecular diagnosis, we developed a rapid method to sequence the entire iduronate-2-sulfatase (IDS) coding region. PCR amplicons representing the IDS cDNA were sequenced with an automatic instrument, and output was analyzed by computer-assisted interpretation of tracings, using Staden programs on a Sun computer. Mutations were found in 10 of 11 patients studied. Unique missense mutations were identified in five patients: H229Y (685{r_arrow}T, severe phenotype); P358R (1073C{r_arrow}G, severe); R468W (1402C{r_arrow}T, mild); P469H (1406C{r_arrow}A, mild); and Y523C (1568A{r_arrow}G, mild). Nonsense mutations were identified in two patients: R172X (514C{r_arrow}T, severe) and Q389X (1165C{r_arrow}T, severe). Two other patients with severe disease had insertions of 1 and 14 bp, in exons 3 and 6, respectively. In another patient with severe disease, the predominant (<95%) IDS message resulted from aberrant splicing, which skipped exon 3. In this last case, consensus sequences for splice sites in exon 3 were intact, but a 395C{r_arrow}G mutation was identified 24 bp upstream from the 3` splice of exon 3. This mutation created a cryptic 5` splice site with a better consensus sequence for 5` splice sites than the natural 5` splice site of intron 3. A minor population of the IDS message was processed by using this cryptic splice site; however, no correctly spliced message was detected in leukocytes from this patient. The mutational topology of the IDS gene is presented. 46 refs., 6 figs., 2 tabs.

  7. DNA synthesis time in germinating rice and pattern of diethylsulphate induced mutations in pre-soaked seeds

    International Nuclear Information System (INIS)

    DNA synthesis pattern in germinating rice seeds, pre-soaked in water for varying periods upto 48 hr, was determined by following the pulse incorporation of 3H-thymidine into the TCA-insoluble nucleoprotein. Synthesis of DNA commenced at 24 hr, progressively increased to a first peak at about 38 hr, thereafter showed a 1/3rd drop and subsequently increased to a 2nd and still higher peak at 46 to 48 hr of pre-soaking. Treatments of diethylsulphate (dES) at a low concentration (0.2%-2hr) administered at various progressing stages of DNA synthesis resulted in decrease in seedling height and survival, and increase in mutation frequency at 45 hr. pre-soaking, maximum mutation frequencies of 20, 10 and 2% on M1 plants, M1 spikes and M2 seedling bases, respectively were observed. Higher dES concentration (0.3%-2hr) given at later periods of pre-soaking showed near lethal effects and consequently decreased mutation frequencies. Treatments of sodium fluoride given singly or in combination with dES did not show any substantially different results as compared to those of the respective controls. Mutation spectra observed after dES treatments to germinating seeds, at different pre-soaking periods, were quite dissimilar. Specific mutations of economic importance like semi-dwarf mutants were isolated from the treatment of germinating seeds pre-soaked for 37.5 hr or more when shoot apex cells were undergoing DNA synthesis. (author)

  8. Detection of Onchocerca volvulus in Latin American black flies for pool screening PCR using high-throughput automated DNA isolation for transmission surveillance.

    Science.gov (United States)

    Rodríguez-Pérez, Mario A; Gopal, Hemavathi; Adeleke, Monsuru Adebayo; De Luna-Santillana, Erick Jesús; Gurrola-Reyes, J Natividad; Guo, Xianwu

    2013-11-01

    The posttreatment entomological surveillance (ES) of onchocerciasis in Latin America requires quite large numbers of flies to be examined for parasite infection to prove that the control strategies have worked and that the infection is on the path of elimination. Here, we report a high-throughput automated DNA isolation of Onchocerca volvulus for PCR using a major Latin American black fly vector of onchocerciasis. The sensitivity and relative effectiveness of silica-coated paramagnetic beads was evaluated in comparison with phenol chloroform (PC) method which is known as the gold standard of DNA extraction for ES in Latin America. The automated method was optimized in the laboratory and validated in the field to detect parasite DNA in Simulium ochraceum sensu lato flies in comparison with PC. The optimization of the automated method showed that it is sensitive to detect O. volvulus with a pool size of 100 flies as compared with PC which utilizes 50 flies pool size. The validation of the automated method in comparison with PC in an endemic community showed that 5/67 and 3/134 heads pools were positive for the two methods, respectively. There was no statistical variation (P < 0.05) in the estimation of transmission indices generated by automated method when compared with PC method. The fact that the automated method is sensitive to pool size up to 100 confers advantage over PC method and can, therefore, be employed in large-scale ES of onchocerciasis transmission in endemic areas of Latin America. PMID:24030195

  9. Genome-wide DNA methylation analysis of transient neonatal diabetes type 1 patients with mutations in ZFP57

    DEFF Research Database (Denmark)

    Bak, Mads; Boonen, Susanne E; Dahl, Christina;

    2016-01-01

    involved in establishment and maintenance of methylation of imprinted loci. Our objective was to investigate whether additional regions are aberrantly methylated in ZFP57 mutation carriers. METHODS: Genome-wide DNA methylation analysis was performed on four individuals with homozygous or compound...... and HYMAI. A subset of patients with maternal hypomethylation at PLAGL1 have hypomethylation at additional imprinted loci throughout the genome, including GRB10, ZIM2 (PEG3), MEST (PEG1), KCNQ1OT1 and NESPAS (GNAS-AS1). About half of the TNDM1 patients carry mutations in ZFP57, a transcription factor...

  10. The mitochondrial DNA 10197 G > A mutation causes MELAS/Leigh overlap syndrome presenting with acute auditory agnosia.

    Science.gov (United States)

    Leng, Yinglin; Liu, Yuhe; Fang, Xiaojing; Li, Yao; Yu, Lei; Yuan, Yun; Wang, Zhaoxia

    2015-04-01

    Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes/Leigh (MELAS/LS) overlap syndrome is a mitochondrial disorder subtype with clinical and magnetic resonance imaging (MRI) features that are characteristic of both MELAS and Leigh syndrome (LS). Here, we report an MELAS/LS case presenting with cortical deafness and seizures. Cranial MRI revealed multiple lesions involving bilateral temporal lobes, the basal ganglia and the brainstem, which conformed to neuroimaging features of both MELAS and LS. Whole mitochondrial DNA (mtDNA) sequencing and PCR-RFLP revealed a de novo heteroplasmic m.10197 G > A mutation in the NADH dehydrogenase subunit 3 gene (ND3), which was predicted to cause an alanine to threonine substitution at amino acid 47. Although the mtDNA m.10197 G > A mutation has been reported in association with LS, Leber hereditary optic neuropathy and dystonia, it has never been linked with MELAS/LS overlap syndrome. Our patient therefore expands the phenotypic spectrum of the mtDNA m.10197 G > A mutation.

  11. Mitochondrial DNA A10398G Mutation is not Associated with Breast Cancer Risk in a Sample of Iraqi Women

    Directory of Open Access Journals (Sweden)

    Rawaa A. Zahid

    2013-05-01

    Full Text Available The aim of this study was to investigate if there is a relationship between mtDNA polymorphism (A10398G and breast cancer in a sample of 59 Iraqi women. Breast cancer is the second most common diagnosed cause of cancer death in the developed countries and accounts for 23% of the total cancers. Different studies reported that breast cancer accounts for 14% of all cancer deaths in females. It is well documented that the different factors such as genetics and environment factors are involved in tumorigenesis. Mutations in the mitochondrial DNA D-loop region and somatic mutations are emerging as early genetic markers of cancer. Identification of such markers for breast cancer would prevent late detection and increase the chance of recovery and survival rate. In breast cancer different mtDNA alterations were reported. The A10398G mutation in NADH Dehyrogenase (ND3 a subunit of complex I of the Oxidative Phosphorylation process (OXPHOS is perhaps one of the most studied mutations with conflicting reports of its association with breast cancer. Genomic DNA was extracted from 21 unrelated women with malignant tumors, 22 women with benign tumors and 16 healthy women blood donors. Subsequently, PCR amplification was performed using specific primers, PCR products were subjected to a suitable restriction enzyme. No genetic variants were identified in mtDNA among malignant tumoral group and controls while 9% of benign tumor cases exhibited the variant. Our finding indicated that A10398G polymorphism cannot be used as a biomarker for breast cancer detection in Iraqi women.

  12. Quantitative cell-free DNA, KRAS, and BRAF mutations in plasma from patients with metastatic colorectal cancer during treatment with cetuximab and irinotecan

    DEFF Research Database (Denmark)

    Spindler, Karen-Lise Garm; Pallisgaard, Niels; Vogelius, Ivan Storgaard;

    2012-01-01

    The present study investigated the levels of circulating cell-free DNA (cfDNA) in plasma from patients with metastatic colorectal cancer (mCRC) in relation to third-line treatment with cetuximab and irinotecan and the quantitative relationship of cfDNA with tumor-specific mutations in plasma....

  13. Imprinting mutations suggested by abnormal DNA methylation patterns in familial angelman and Prader-Willi syndromes

    Energy Technology Data Exchange (ETDEWEB)

    Reis, A. (Freie Universitaet, Berlin (Germany)); Dittrich, B.; Buiting, K.; Gillessen-Kaesbach, G.; Horsthemke, B. (Institut fuer Humangenetik, Essen (United Kingdom)); Greger, V.; Lalande, M. (Harvard Medical School, Boston, MA (United States)); Anvret, M. (Karolinska Hospital, Stockholm (Sweden))

    1994-05-01

    The D15S9 and D15S63 loci in the Prader-Willi/Angelman syndrome region on chromosome 15 are subject to parent-of-origin-specific DNA methylation. The authors have found two Prader-Willi syndrome families in which the patients carry a maternal methylation imprint on the paternal chromosome. In one of these families, the patients have a small deletion encompassing the gene for the small nuclear ribonucleoprotein polypeptide N, which maps 130 kb telomeric to D15S63. Furthermore, they have identified a pair of nondeletion Angelman syndrome sibs and two isolated Angelman syndrome patients who carry a paternal methylation imprint on the maternal chromosome. These Angelman and Prader-Willi syndrome patients may have a defect in the imprinting process in 15q11-13. The authors propose a model in which a cis-acting mutation prevents the resetting of the imprinting signal in the germ line and thus disturbs the expression of imprinted genes in this region. 39 refs., 4 figs., 1 tab.

  14. Automated property optimization via ab initio O(N) elongation method: Application to (hyper-)polarizability in DNA.

    Science.gov (United States)

    Orimoto, Yuuichi; Aoki, Yuriko

    2016-07-14

    An automated property optimization method was developed based on the ab initio O(N) elongation (ELG) method and applied to the optimization of nonlinear optical (NLO) properties in DNA as a first test. The ELG method mimics a polymerization reaction on a computer, and the reaction terminal of a starting cluster is attacked by monomers sequentially to elongate the electronic structure of the system by solving in each step a limited space including the terminal (localized molecular orbitals at the terminal) and monomer. The ELG-finite field (ELG-FF) method for calculating (hyper-)polarizabilities was used as the engine program of the optimization method, and it was found to show linear scaling efficiency while maintaining high computational accuracy for a random sequenced DNA model. Furthermore, the self-consistent field convergence was significantly improved by using the ELG-FF method compared with a conventional method, and it can lead to more feasible NLO property values in the FF treatment. The automated optimization method successfully chose an appropriate base pair from four base pairs (A, T, G, and C) for each elongation step according to an evaluation function. From test optimizations for the first order hyper-polarizability (β) in DNA, a substantial difference was observed depending on optimization conditions between "choose-maximum" (choose a base pair giving the maximum β for each step) and "choose-minimum" (choose a base pair giving the minimum β). In contrast, there was an ambiguous difference between these conditions for optimizing the second order hyper-polarizability (γ) because of the small absolute value of γ and the limitation of numerical differential calculations in the FF method. It can be concluded that the ab initio level property optimization method introduced here can be an effective step towards an advanced computer aided material design method as long as the numerical limitation of the FF method is taken into account. PMID:27421397

  15. Automated property optimization via ab initio O(N) elongation method: Application to (hyper-)polarizability in DNA

    Science.gov (United States)

    Orimoto, Yuuichi; Aoki, Yuriko

    2016-07-01

    An automated property optimization method was developed based on the ab initio O(N) elongation (ELG) method and applied to the optimization of nonlinear optical (NLO) properties in DNA as a first test. The ELG method mimics a polymerization reaction on a computer, and the reaction terminal of a starting cluster is attacked by monomers sequentially to elongate the electronic structure of the system by solving in each step a limited space including the terminal (localized molecular orbitals at the terminal) and monomer. The ELG-finite field (ELG-FF) method for calculating (hyper-)polarizabilities was used as the engine program of the optimization method, and it was found to show linear scaling efficiency while maintaining high computational accuracy for a random sequenced DNA model. Furthermore, the self-consistent field convergence was significantly improved by using the ELG-FF method compared with a conventional method, and it can lead to more feasible NLO property values in the FF treatment. The automated optimization method successfully chose an appropriate base pair from four base pairs (A, T, G, and C) for each elongation step according to an evaluation function. From test optimizations for the first order hyper-polarizability (β) in DNA, a substantial difference was observed depending on optimization conditions between "choose-maximum" (choose a base pair giving the maximum β for each step) and "choose-minimum" (choose a base pair giving the minimum β). In contrast, there was an ambiguous difference between these conditions for optimizing the second order hyper-polarizability (γ) because of the small absolute value of γ and the limitation of numerical differential calculations in the FF method. It can be concluded that the ab initio level property optimization method introduced here can be an effective step towards an advanced computer aided material design method as long as the numerical limitation of the FF method is taken into account.

  16. Mutations altering the interplay between GkDnaC helicase and DNA reveal an insight into helicase unwinding.

    Directory of Open Access Journals (Sweden)

    Yu-Hua Lo

    Full Text Available Replicative helicases are essential molecular machines that utilize energy derived from NTP hydrolysis to move along nucleic acids and to unwind double-stranded DNA (dsDNA. Our earlier crystal structure of the hexameric helicase from Geobacillus kaustophilus HTA426 (GkDnaC in complex with single-stranded DNA (ssDNA suggested several key residues responsible for DNA binding that likely play a role in DNA translocation during the unwinding process. Here, we demonstrated that the unwinding activities of mutants with substitutions at these key residues in GkDnaC are 2-4-fold higher than that of wild-type protein. We also observed the faster unwinding velocities in these mutants using single-molecule experiments. A partial loss in the interaction of helicase with ssDNA leads to an enhancement in helicase efficiency, while their ATPase activities remain unchanged. In strong contrast, adding accessory proteins (DnaG or DnaI to GkDnaC helicase alters the ATPase, unwinding efficiency and the unwinding velocity of the helicase. It suggests that the unwinding velocity of helicase could be modulated by two different pathways, the efficiency of ATP hydrolysis or protein-DNA interaction.

  17. Comprehensive and accurate mutation scanning of the CFTR gene by two-dimensional DNA electrophoresis

    NARCIS (Netherlands)

    Wu, Y; Hofstra, RMW; Scheffer, H; Uitterlinden, AG; Mullaart, E; Buys, CHCM; Vijg, J

    1996-01-01

    The large number of possible disease causing mutations in the 27 exons of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has severely limited direct diagnosis of cystic fibrosis (CF) patients and carriers by mutation detection. Here we show that in principle testing for mutation

  18. Prospective blinded study of somatic mutation detection in cell-free DNA utilizing a targeted 54-gene next generation sequencing panel in metastatic solid tumor patients

    Science.gov (United States)

    Lanman, Richard B.; Mortimer, Stefanie; Zill, Oliver A.; Kim, Kyoung-Mee; Jang, Kee Taek; Kim, Seok-Hyung; Park, Se Hoon; Park, Joon Oh; Park, Young Suk; Lim, Ho Yeong; Eltoukhy, Helmy; Kang, Won Ki; Lee, Woo Yong; Kim, Hee-Cheol; Park, Keunchil; Lee, Jeeyun; Talasaz, AmirAli

    2015-01-01

    Sequencing of the mutant allele fraction of circulating cell-free DNA (cfDNA) derived from tumors is increasingly utilized to detect actionable genomic alterations in cancer. We conducted a prospective blinded study of a comprehensive cfDNA sequencing panel with 54 cancer genes. To evaluate the concordance between cfDNA and tumor DNA (tDNA), sequencing results were compared between cfDNA from plasma and genomic tumor DNA (tDNA). Utilizing next generation digital sequencing technology (DST), we profiled approximately 78,000 bases encoding 512 complete exons in the targeted genes in cfDNA from plasma. Seventy-five patients were prospectively enrolled between February 2013 and March 2014, including 61 metastatic cancer patients and 14 clinical stage II CRC patients with matched plasma and tissue samples. Using the 54-gene panel, we detected at least one somatic mutation in 44 of 61 tDNA (72.1%) and 29 of 44 (65.9%) cfDNA. The overall concordance rate of cfDNA to tDNA was 85.9%, when all detected mutations were considered. We collected serial cfDNAs during cetuximab-based treatment in 2 metastatic KRAS wild-type CRC patients, one with acquired resistance and one with primary resistance. We demonstrate newly emerged KRAS mutation in cfDNA 1.5 months before radiologic progression. Another patient had a newly emerged PIK3CA H1047R mutation on cfDNA analysis at progression during cetuximab/irinotecan chemotherapy with gradual increase in allele frequency from 0.8 to 2.1%. This blinded, prospective study of a cfDNA sequencing showed high concordance to tDNA suggesting that the DST approach may be used as a non-invasive biopsy-free alternative to conventional sequencing using tumor biopsy. PMID:26452027

  19. A model for triplet mutation formation based on error-prone translesional DNA synthesis opposite UV photolesions.

    Science.gov (United States)

    Ikehata, Hironobu; Ono, Tetsuya; Tanaka, Kiyoji; Todo, Takeshi

    2007-05-01

    A triplet mutation is defined as multiple base substitutions or frameshifts within a three-nucleotide sequence which includes a dipyrimidine sequence. Triplet mutations have recently been identified as a new type of UV-specific mutation, although the mechanism of their formation is unknown. A total of 163 triplet mutations were identified through an extensive search of previously published data on UV-induced mutations, including mutations from skin, skin cancer, and cultured mammalian cells. Seven common patterns of sequence changes were found: Type I, NTC-->TTT; Type IIa, NCC-->PyTT or PyCT (Py, pyrimidine); Type IIb, TCC-->PuTT or PuCT (Pu, purine); Type III, NCC-->NAT or NTA; Type IV, NTT-->AAT; Type Va, NCT-->NTX; and Type Vb, PuCT-->XTT (N and X, independent anonymous bases). Furthermore, it is suggested that the type of UV lesion responsible for each of these triplet mutation classes are (a) pyrimidine(6-4)pyrimidone photoproducts for Types I, IIb, III, IV and Vb, (b) cyclobutane pyrimidine dimers for Type Va, and (c) Dewar valence isomers for Types IIa and IIb. These estimations are based primarily on results from previous studies using photolyases specific for each type of UV lesion. A model is proposed to explain the formation of each type of triplet mutation, based on error-prone translesional DNA synthesis opposite UV-specific photolesions. The model is largely consistent with the 'A-rule', and predicts error-prone insertions not only opposite photolesions but also opposite the undamaged template base one-nucleotide downstream from the lesions.

  20. Detection of DNA Aneuploidy in Exfoliated Airway Epithelia Cells of Sputum Specimens by the Automated Image Cytometry and Its Clinical Value in the Identification of Lung Cancer

    Institute of Scientific and Technical Information of China (English)

    杨健; 周宜开

    2004-01-01

    To evaluate the value of detecton of DNA aneuploidy in exfoliated airway epithelia cells of sputum specimens by the automated image cytometry for the identification of lung cancer, 100patients were divided into patient group (50 patients with lung cancer)and control group (30 patients with tuberculosis and 20 healthy people). Sputum was obtained for the quantitative analysis of DNA content of exfoliated airway epithelial cells with the automated image cytometry, together with the examinations of brush cytology and conventional sputum cytology. Our results showed that DNA aneuploidy (DI>2.5 or 5c) was found in 20 out of 50 sputum samples of lung cancer, 1 out of 30 sputum samples from tuberculosis patients, and none of 20 sputum samples from healthy people. The positive rates of conventional sputum cytology and brush cytology were 16 % and 32 %,which was lower than that of DNA aneuploidy detection by the automated image cytometry (P<0.01 ,P>0.05). Our study showed that automated image cytometry, which uses DNA aneuploidy as a marker for tumor, can detect the malignant cells in sputum samples of lung cancer and it is a sensitive and specific method serving as a complement for the diagnosis of lung cancer.

  1. Significance of the pathogenic mutation T372R in the Yin Yang 1 protein interaction with DNA--thermodynamic studies.

    Science.gov (United States)

    Nieborak, Anna; Górecki, Andrzej

    2016-03-01

    This work focuses on the pathogenic missense mutation in YY1 protein correlated with insulinomas. Based on in vitro studies, we demonstrate that the mutation does not affect the secondary structure of either zinc fingers or the N-terminal fragment (NTF) of the protein. Apart from a slight increase in the protein's compactness, no changes in the tertiary structure were observed. The introduced mutation significantly alters DNA-binding properties, both the affinity and enthalpy-entropy contribution of the process, which are highly dependent on the recognized sequence. Obtained results indicate concerted rather than a modular mode of sequence recognition by YY1 with the significant impact of a disordered NTF. PMID:26910132

  2. Distinctive Drug-resistant Mutation Profiles and Interpretations of HIV-1 Proviral DNA Revealed by Deep Sequencing in Reverse Transcriptase

    Institute of Scientific and Technical Information of China (English)

    YIN Qian Qian; SHAO Yi Ming; MA Li Ying; LI Zhen Peng; ZHAO Hai; PAN Dong; WANG Yan; XU Wei Si; XING Hui; FENGYi; JIANG Shi Bo

    2016-01-01

    ObjectiveTo investigate distinctive features in drug-resistant mutations(DRMs) and interpretations for reverse transcriptase inhibitors (RTIs) between proviral DNA and paired viral RNA in HIV-1-infected patients. MethodsForty-three HIV-1-infected individuals receiving first-line antiretroviral therapy were recruited to participate in a multicenter AIDS Cohort Study in Anhui and Henan Provinces in China in 2004. Drug resistance genotyping was performed by bulk sequencing and deep sequencing on the plasma and whole blood of 77 samples, respectively. Drug-resistance interpretation was compared between viral RNA and paired proviral DNA. ResultsCompared with bulk sequencing, deep sequencing could detect more DRMs and samples with DRMs in both viral RNA and proviral DNA. The mutations M184I and M230I were more prevalent in proviral DNA than in viral RNA (Fisher’s exact test,P ConclusionCompared with viral RNA, the distinctive information of DRMsand drug resistance interpretations for proviral DNA could be obtained by deep sequencing, which could provide more detailed and precise information for drug resistance monitoring and the rational design of optimal antiretroviral therapy regimens.

  3. SIMILARITY ANALYSIS OF DNA SEQUENCES BASED ON THE CHEMICAL PROPERTIES OF NUCLEOTIDE BASES, FREQUENCY AND POSITION OF GROUP MUTATIONS

    Directory of Open Access Journals (Sweden)

    Fatima KABLI

    2016-01-01

    Full Text Available The DNA sequences similarity analysis approaches have been based on the representation and the frequency of sequences components; however, the position inside sequence is important information for the sequence data. Whereas, insufficient information in sequences representations is important reason that causes poor similarity results. Based on three classifications of the DNA bases according to their chemical properties, the frequencies and average positions of group mutations have been grouped into two twelve-components vectors, the Euclidean distances among introduced vectors applied to compare the coding sequences of the first exon of beta globin gene of 11 species.

  4. A Mitochondrial DNA A8701G Mutation Associated with Maternally Inherited Hypertension and Dilated Cardiomyopathy in a Chinese Pedigree of a Consanguineous Marriage

    Institute of Scientific and Technical Information of China (English)

    Ye Zhu; Xiang Gu; Chao Xu

    2016-01-01

    Background: Cardiovascular diseases, including dilated cardiomyopathy (DCM) and hypertension, are the leading cause of death worldwide.The role of mitochondrial DNA (mtDNA) in the pathogenesis of these diseases has not been completely clarified.In this study, we evaluate whether A8701G mutation is associated with maternally inherited hypertension and DCM in a Chinese pedigree of a consanguineous marriage.Methods: Fourteen subjects in a three-generation Han Chinese family with hypertension and DCM, in which consanguineous marriage was present in the parental generation, were interviewed.We divided all the family members into case (7 maternal members) and control group (7 nonmaternal members) for comparison.Clinical evaluations and sequence analysis ofmtDNA were obtained from all participants.Frequency differences between maternal and nonmaternal members were tested to locate the disease-associated mutations.Results: The majority of the family members presented with a maternal inheritance of hypertension and DCM.Sequence analysis of mtDNA in this pedigree identified eight mtDNA mutations.Among the mutations identified, there was only one significant mutation: A8701G (P =0.005), which is a homoplasmic mitochondrial missense mutation in all the matrilineal relatives.There was no clear evidence for any synergistic effects between A8701G and other mutations.Conclusions: A8701G mutation may act as an inherited risk factor for the matrilineal transmission of hypertension and DCM in conj unction with genetic disorders caused by consanguineous marriage.

  5. DNA mutation detection with chip-based temperature gradient capillary electrophoresis using a slantwise radiative heating system.

    Science.gov (United States)

    Zhang, Hui-Dan; Zhou, Jing; Xu, Zhang-Run; Song, Jin; Dai, Jing; Fang, Jin; Fang, Zhao-Lun

    2007-09-01

    A simple and robust chip-based temperature gradient capillary electrophoresis (TGCE) system was developed for DNA mutation/single-nucleotide polymorphism (SNP) analysis using a radiative heating system. Reproducible, stable and uniform temperature gradients were established along a 3 cm length of the electrophoretic separation channel using a single thermostated aluminium heater plate. The heater was slightly slanted relative to the plane of the glass chip at 0.2-1.3 degrees by inserting thin spacers between the plate and chip at one end to produce differences in radiative heating that created the temperature gradient. On-chip TGCE analyses of 4 mutant DNA model samples amplified from plasmid templates, each containing a single base substitution, with a wide range of melting temperatures, showed that mutations were successfully detected under a wide temperature gradient of 10 degrees C and within a short gradient region of about 3 cm (3.3 degrees C cm(-1) gradient). The radiative heating system was able to establish stable spatial temperature gradients along short microfluidic separation channels using simple peripheral equipment and manipulation while ensuring good resolution for detecting a wide range of mutations. Effectiveness of the system was demonstrated by the successful detection of K-ras gene mutations in 6 colon cancer cell lines.

  6. A combinatorial role for MutY and Fpg DNA glycosylases in mutation avoidance in Mycobacterium smegmatis

    Energy Technology Data Exchange (ETDEWEB)

    Hassim, Farzanah; Papadopoulos, Andrea O.; Kana, Bavesh D.; Gordhan, Bhavna G., E-mail: bhavna.gordhan@nhls.ac.za

    2015-09-15

    Highlights: • We studied the combined role of MutY and Fpg DNA glycosylases in M. smegmatis. • Loss of MutY showed increased sensitivity to oxidative damage. • Loss of MutY together with the Fpg glycosylases showed increased mutation rates. • Our data indicate interplay between these enzymes to control mutagenesis. - Abstract: Hydroxyl radical (·OH) among reactive oxygen species cause damage to nucleobases with thymine being the most susceptible, whilst in contrast, the singlet oxygen ({sup 1}0{sub 2}) targets only guanine bases. The high GC content of mycobacterial genomes predisposes these organisms to oxidative damage of guanine. The exposure of cellular DNA to ·OH and one-electron oxidants results in the formation of two main degradation products, the pro-mutagenic 8-oxo-7,8-dihydroguanine (8-oxoGua) and the cytotoxic 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua). These lesions are repaired through the base excision repair (BER) pathway and we previously, demonstrated a combinatorial role for the mycobacterial Endonuclease III (Nth) and the Nei family of DNA glycosylases in mutagenesis. In addition, the formamidopyrimidine (Fpg/MutM) and MutY DNA glycosylases have also been implicated in mutation avoidance and BER in mycobacteria. In this study, we further investigate the combined role of MutY and the Fpg/Nei DNA glycosylases in Mycobacterium smegmatis and demonstrate that deletion of mutY resulted in enhanced sensitivity to oxidative stress, an effect which was not exacerbated in Δfpg1 Δfpg2 or Δnei1 Δnei2 double mutant backgrounds. However, combinatorial loss of the mutY, fpg1 and fpg2 genes resulted in a significant increase in mutation rates suggesting interplay between these enzymes. Consistent with this, there was a significant increase in C → A mutations with a corresponding change in cell morphology of rifampicin resistant mutants in the Δfpg1 Δfpg2 ΔmutY deletion mutant. In contrast, deletion of mutY together with the nei

  7. A combinatorial role for MutY and Fpg DNA glycosylases in mutation avoidance in Mycobacterium smegmatis

    International Nuclear Information System (INIS)

    Highlights: • We studied the combined role of MutY and Fpg DNA glycosylases in M. smegmatis. • Loss of MutY showed increased sensitivity to oxidative damage. • Loss of MutY together with the Fpg glycosylases showed increased mutation rates. • Our data indicate interplay between these enzymes to control mutagenesis. - Abstract: Hydroxyl radical (·OH) among reactive oxygen species cause damage to nucleobases with thymine being the most susceptible, whilst in contrast, the singlet oxygen (102) targets only guanine bases. The high GC content of mycobacterial genomes predisposes these organisms to oxidative damage of guanine. The exposure of cellular DNA to ·OH and one-electron oxidants results in the formation of two main degradation products, the pro-mutagenic 8-oxo-7,8-dihydroguanine (8-oxoGua) and the cytotoxic 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua). These lesions are repaired through the base excision repair (BER) pathway and we previously, demonstrated a combinatorial role for the mycobacterial Endonuclease III (Nth) and the Nei family of DNA glycosylases in mutagenesis. In addition, the formamidopyrimidine (Fpg/MutM) and MutY DNA glycosylases have also been implicated in mutation avoidance and BER in mycobacteria. In this study, we further investigate the combined role of MutY and the Fpg/Nei DNA glycosylases in Mycobacterium smegmatis and demonstrate that deletion of mutY resulted in enhanced sensitivity to oxidative stress, an effect which was not exacerbated in Δfpg1 Δfpg2 or Δnei1 Δnei2 double mutant backgrounds. However, combinatorial loss of the mutY, fpg1 and fpg2 genes resulted in a significant increase in mutation rates suggesting interplay between these enzymes. Consistent with this, there was a significant increase in C → A mutations with a corresponding change in cell morphology of rifampicin resistant mutants in the Δfpg1 Δfpg2 ΔmutY deletion mutant. In contrast, deletion of mutY together with the nei homologues did

  8. Mutational Analysis of Bacterial NAD+-dependent DNA Ligase:Role of Motif Ⅳ in Ligation Catalysis

    Institute of Scientific and Technical Information of China (English)

    Hong FENG

    2007-01-01

    The bacterial DNA ligase as a multiple domain protein is involved in DNA replication, repair and recombination. Its catalysis of ligation can be divided into three steps. To delineate the roles of amino acid residues in motif Ⅳ in ligation catalysis, site-directed mutants were constructed in a bacterial NAD+-dependent DNA ligase from Thermus sp. TAK16D. It was shown that four conserved residues (D286, G287, V289 and K291) in motif Ⅳ had significant roles on the overall ligation. Under single turnover conditions, the observed apparent rates of D286E, G287A, V289I and K291R mutants were clearly reduced compared with that of WT ligase on both match and mismatch nicked substrates. The effects of D286E mutation on overall ligation may not only be ascribed to the third step. The G287A mutation has a major effect on the second step. The effects of V289I and K291R mutation on overall ligation are not on the third step, perhaps other aspects, such as conformation change of ligase protein in ligation catalysis, are involved. Moreover, the amino acid substitutions of above four residues were more sensitive on mismatch nicked substrate, indicating an enhanced ligation fidelity.

  9. Automated seamless DNA co-transformation cloning with direct expression vectors applying positive or negative insert selection

    Directory of Open Access Journals (Sweden)

    Frey Daniel

    2010-08-01

    Full Text Available Abstract Background Molecular DNA cloning is crucial to many experiments and with the trend to higher throughput of modern approaches automated techniques are urgently required. We have established an automated, fast and flexible low-cost expression cloning approach requiring only vector and insert amplification by PCR and co-transformation of the products. Results Our vectors apply positive selection for the insert or negative selection against empty vector molecules and drive strong expression of target proteins in E.coli cells. Variable tags are available both in N-terminal or C-terminal position. A newly developed β-lactamase (ΔW290 selection cassette contains a segment inside the β-lactamase open reading frame encoding a stretch of hydrophilic amino acids that result in a T7 promoter when back-translated. This position of the promoter permits positive selection and attenuated expression of fusion proteins with C-terminal tags. We have tested eight vectors by inserting six target sequences of variable length, provenience and function. The target proteins were cloned, expressed and detected using an automated Tecan Freedom Evo II liquid handling work station. Only two colonies had to be picked to score with 85% correct inserts while 80% of those were positive in expression tests. Conclusions Our results establish co-transformation and positive/negative selection cloning in conjunction with the provided vectors and selection cassettes as an automatable alternative to commercialized high-throughput cloning systems like Gateway® or ligase-independent cloning (LIC .

  10. Mitochondrial DNA A1555G mutation screening using a testing kit method and its significance in preventing aminoglycoside-related hearing loss

    Institute of Scientific and Technical Information of China (English)

    LIU Xin; YANG Weiyan; HAN Dongyi; JIN Zhengce; GUAN Minxin; DAI Pu; HUANG Deliang; YUAN Huijun; LI Weiming; YU Fei; ZHANG Xin; KANG Dongyang; CAO Juyang

    2006-01-01

    To report a new screening method for mitochondrial DNA 1555A→G mutation and the results of genotype analysis in 19 maternal inherited deafness pedigrees. Method Five hundred and forty-six non-syndromic neuro-sensory hearing loss patients were tested for 1555A→G mutation using a new compact testing kit, which allows clear distinction between wild type and 1555 A→G mutated mtDNAs. Results Nineteen subjects among the 546 patients (3.48%) were found to carry mtDNA A1555G mutation. The results were confirmed by sequencing in an ABI 3100 Avant sequencer. Conclusions Maternal inherited deafness families are a frequently seen in outpatient group. The detection ofmtDNA 1555 A→G mutation with a low cost, ready to use detection kit is needed and suitable in China for large scale screening and preventive testing before usage of aminoglycoside antibiotics.

  11. Molecular dynamics simulations demonstrate the regulation of DNA-DNA attraction by H4 histone tail acetylations and mutations.

    Science.gov (United States)

    Korolev, Nikolay; Yu, Hang; Lyubartsev, Alexander P; Nordenskiöld, Lars

    2014-10-01

    The positively charged N-terminal histone tails play a crucial role in chromatin compaction and are important modulators of DNA transcription, recombination, and repair. The detailed mechanism of the interaction of histone tails with DNA remains elusive. To model the unspecific interaction of histone tails with DNA, all-atom molecular dynamics (MD) simulations were carried out for systems of four DNA 22-mers in the presence of 20 or 16 short fragments of the H4 histone tail (variations of the 16-23 a. a. KRHRKVLR sequence, as well as the unmodified fragment a. a.13-20, GGAKRHRK). This setup with high DNA concentration, explicit presence of DNA-DNA contacts, presence of unstructured cationic peptides (histone tails) and K(+) mimics the conditions of eukaryotic chromatin. A detailed account of the DNA interactions with the histone tail fragments, K(+) and water is presented. Furthermore, DNA structure and dynamics and its interplay with the histone tail fragments binding are analysed. The charged side chains of the lysines and arginines play major roles in the tail-mediated DNA-DNA attraction by forming bridges and by coordinating to the phosphate groups and to the electronegative sites in the minor groove. Binding of all species to DNA is dynamic. The structure of the unmodified fully-charged H4 16-23 a.a. fragment KRHRKVLR is dominated by a stretched conformation. The H4 tail a. a. fragment GGAKRHRK as well as the H4 Lys16 acetylated fragment are highly flexible. The present work allows capturing typical features of the histone tail-counterion-DNA structure, interaction and dynamics.

  12. Retrospective assessment of the most common mitochondrial DNA mutations in a large Hungarian cohort of suspect mitochondrial cases.

    Science.gov (United States)

    Remenyi, Viktoria; Inczedy-Farkas, Gabriella; Komlosi, Katalin; Horvath, Rita; Maasz, Anita; Janicsek, Ingrid; Pentelenyi, Klara; Gal, Aniko; Karcagi, Veronika; Melegh, Bela; Molnar, Maria Judit

    2015-08-01

    Prevalence estimations for mitochondrial disorders still vary widely and only few epidemiologic studies have been carried out so far. With the present work we aim to give a comprehensive overview about frequencies of the most common mitochondrial mutations in Hungarian patients. A total of 1328 patients were tested between 1999 and 2012. Among them, 882 were screened for the m.3243A > G, m.8344A > G, m.8993T > C/G mutations and deletions, 446 for LHON primary mutations. The mutation frequency in our cohort was 2.61% for the m.3243A > G, 1.47% for the m.8344A > G, 17.94% for Leber's Hereditary Optic Neuropathy (m.3460G > A, m.11778G > A, m.14484T > C) and 0.45% for the m.8993T > C/G substitutions. Single mtDNA deletions were detected in 14.97%, while multiple deletions in 6.01% of the cases. The mutation frequency in Hungarian patients suggestive of mitochondrial disease was similar to other Caucasian populations. Further retrospective studies of different populations are needed in order to accurately assess the importance of mitochondrial diseases and manage these patients.

  13. Microsatellite DNA mutations in double-crested cormorants (Phalacrocorax auritus) associated with exposure to PAH-containing industrial air pollution.

    Science.gov (United States)

    King, L E; de Solla, S R; Small, J M; Sverko, E; Quinn, J S

    2014-10-01

    Hamilton Harbour, Ontario, Canada is one of the most polluted sites on the Great Lakes, and is subject to substantial airborne pollution due to emissions from both heavy industry and intense vehicle traffic. Mutagenic Polycyclic aromatic hydrocarbons (PAHs) are present at very high concentrations in the air and sediment of Hamilton Harbour. We used five variable DNA microsatellites to screen for mutations in 97 families of Double-crested Cormorants (Phalacrocorax auritus) from three wild colonies, two in Hamilton Harbour and one in cleaner northeastern Lake Erie. Mutations were identified in all five microsatellites at low frequencies, with the majority of mutations found in chicks from the Hamilton Harbour site closest to industrial sources of PAH contamination. Microsatellite mutation rates were 6-fold higher at the Hamilton Harbour site closest to the industrial sources of PAH contamination than the other Hamilton Harbour site, and both were higher than the reference colony. A Phase I metabolite of the PAH benzo[a]pyrene identified by LC-MS/MS in bile and liver from Hamilton Harbour cormorant chicks suggests that these cormorants are exposed to and metabolizing PAHs, highlighting their potential to have caused the observed mutations.

  14. Concise Review: Heteroplasmic Mitochondrial DNA Mutations and Mitochondrial Diseases: Toward iPSC-Based Disease Modeling, Drug Discovery, and Regenerative Therapeutics.

    Science.gov (United States)

    Hatakeyama, Hideyuki; Goto, Yu-Ichi

    2016-04-01

    Mitochondria contain multiple copies of their own genome (mitochondrial DNA; mtDNA). Once mitochondria are damaged by mutant mtDNA, mitochondrial dysfunction is strongly induced, followed by symptomatic appearance of mitochondrial diseases. Major genetic causes of mitochondrial diseases are defects in mtDNA, and the others are defects of mitochondria-associating genes that are encoded in nuclear DNA (nDNA). Numerous pathogenic mutations responsible for various types of mitochondrial diseases have been identified in mtDNA; however, it remains uncertain why mitochondrial diseases present a wide variety of clinical spectrum even among patients carrying the same mtDNA mutations (e.g., variations in age of onset, in affected tissues and organs, or in disease progression and phenotypic severity). Disease-relevant induced pluripotent stem cells (iPSCs) derived from mitochondrial disease patients have therefore opened new avenues for understanding the definitive genotype-phenotype relationship of affected tissues and organs in various types of mitochondrial diseases triggered by mtDNA mutations. In this concise review, we briefly summarize several recent approaches using patient-derived iPSCs and their derivatives carrying various mtDNA mutations for applications in human mitochondrial disease modeling, drug discovery, and future regenerative therapeutics.

  15. Identification of DNA polymerase molecules repairing DNA irradiated damage and molecular biological study on modified factors of mutation rate

    Energy Technology Data Exchange (ETDEWEB)

    Yamada, Koichi; Inoue, Shuji [National Inst. of Healthand Nutrition, Tokyo (Japan)

    1999-02-01

    DNA repairing polymerase has not been identified in human culture cells because the specificities of enzyme inhibitors used in previous studies were not so high. In this study, anti-sense oligonucleotides were transfected into human fibroblast cells by electroporation and several clones selected by geneticin treatment were found to express the RNA of the incorporated DNA. However, the expression was not significant and its reproducibility was poor. Then, a study on repairing mechanism was made using XP30 RO and XP 115 LO cells which are variant cells of xeroderma pigmentosum, a human hereditary disease aiming to identify the DNA polymerase related to the disease. There were abnormalities in DNA polymerase subunit {delta} or {epsilon} which consists DNA replication complex. Thus, it was suggested that the DNA replication of these mutant cells might terminate at the site containing such abnormality. (M.N.)

  16. Identification of DNA polymerase molecules repairing DNA irradiated damage and molecular biological study on modified factors of mutation rate

    International Nuclear Information System (INIS)

    DNA repairing polymerase has not been identified in human culture cells because the specificities of enzyme inhibitors used in previous studies were not so high. In this study, anti-sense oligonucleotides were transfected into human fibroblast cells by electroporation and several clones selected by geneticin treatment were found to express the RNA of the incorporated DNA. However, the expression was not significant and its reproducibility was poor. Then, a study on repairing mechanism was made using XP30 RO and XP 115 LO cells which are variant cells of xeroderma pigmentosum, a human hereditary disease aiming to identify the DNA polymerase related to the disease. There were abnormalities in DNA polymerase subunit δ or ε which consists DNA replication complex. Thus, it was suggested that the DNA replication of these mutant cells might terminate at the site containing such abnormality. (M.N.)

  17. Identification of a nonsense mutation in the carboxyl-terminal region of DNA-dependent protein kinase catalytic subunit in the scid mouse.

    Science.gov (United States)

    Blunt, T; Gell, D; Fox, M; Taccioli, G E; Lehmann, A R; Jackson, S P; Jeggo, P A

    1996-01-01

    DNA-dependent protein kinase (DNA-PK) consists of a heterodimeric protein (Ku) and a large catalytic subunit (DNA-PKcs). The Ku protein has double-stranded DNA end-binding activity that serves to recruit the complex to DNA ends. Despite having serine/threonine protein kinase activity, DNA-PKcs falls into the phosphatidylinositol 3-kinase superfamily. DNA-PK functions in DNA double-strand break repair and V(D)J recombination, and recent evidence has shown that mouse scid cells are defective in DNA-PKcs. In this study we have cloned the cDNA for the carboxyl-terminal region of DNA-PKcs in rodent cells and identified the existence of two differently spliced products in human cells. We show that DNA-PKcs maps to the same chromosomal region as the mouse scid gene. scid cells contain approximately wild-type levels of DNA-PKcs transcripts, whereas the V-3 cell line, which is also defective in DNA-PKcs, contains very reduced transcript levels. Sequence comparison of the carboxyl-terminal region of scid and wild-type mouse cells enabled us to identify a nonsense mutation within a highly conserved region of the gene in mouse scid cells. This represents a strong candidate for the inactivating mutation in DNA-PKcs in the scid mouse. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8816792

  18. Analysis of association among clinical features and shorter leukocyte telomere length in mitochondrial diabetes with m.3243A>G mitochondrial DNA mutation

    OpenAIRE

    Zhou, Mei-Cen; Min, Rui; Ji, Jian-Jun; Zhang, Shi; Tong, An-li; Xu, Jian-Ping; Li, Zeng-Yi; Zhang, Hua-Bing; Li, Yu-xiu

    2015-01-01

    Background Mitochondrial diabetes is a kind of rare diabetes caused by monogenic mutation in mitochondia. The study aimed to summarize the clinical phenotype profiles in mitochondrial diabetes withm.3243A>G mitochondrial DNA mutation and to investigate the mechanism in this kind of diabetes by analyzing the relationship among clinical phenotypes and peripheral leukocyte DNA telomere length. Methods Fifteen patients with maternally inherited diabetes in five families were confirmed as carrying...

  19. High prevalence of impaired glucose homeostasis and myopathy in asymptomatic and oligosymptomatic 3243A>G mitochondrial DNA mutation-positive subjects

    DEFF Research Database (Denmark)

    Frederiksen, A.L.; Jeppesen, T.D.; Vissing, J.;

    2009-01-01

    INTRODUCTION: The point mutation of 3243A>G mtDNA is the most frequent cause of mitochondrial diabetes, often presenting as the syndrome maternally inherited diabetes and deafness (MIDD). The mutation may also cause myopathy, ataxia, strokes, ophthalmoplegia, epilepsy, and cardiomyopathy in vario...

  20. Homoplasmy of the G7444A mtDNA and heterozygosity of the GJB2 c.35delG mutations in a family with hearing loss

    DEFF Research Database (Denmark)

    Kokotas, Haris; Grigoriadou, Maria; Li, Yang;

    2011-01-01

    Mitochondrial mutations have been shown to be responsible for syndromic as well as non-syndromic hearing loss. The G7444A mitochondrial DNA mutation affects COI/the precursor of tRNA(Ser(UCN)), encoding the first subunit of cytochrome oxidase. Here we report on the first Greek family with the G7444...

  1. High prevalence of impaired glucose homeostasis and myopathy in asymptomatic and oligosymptomatic 3243A>G mitochondrial DNA mutation-positive subjects

    DEFF Research Database (Denmark)

    Frederiksen, Anja Lisbeth; Jeppesen, Tina Dysgaard; Vissing, John;

    2009-01-01

    INTRODUCTION: The point mutation of 3243A>G mtDNA is the most frequent cause of mitochondrial diabetes, often presenting as the syndrome maternally inherited diabetes and deafness (MIDD). The mutation may also cause myopathy, ataxia, strokes, ophthalmoplegia, epilepsy, and cardiomyopathy in various...

  2. Mutational analysis of Mycobacterium UvrD1 identifies functional groups required for ATP hydrolysis, DNA unwinding, and chemomechanical coupling.

    Science.gov (United States)

    Sinha, Krishna Murari; Glickman, Michael S; Shuman, Stewart

    2009-05-19

    Mycobacterial UvrD1 is a DNA-dependent ATPase and a Ku-dependent 3' to 5' DNA helicase. The UvrD1 motor domain resembles that of the prototypal superfamily I helicases UvrD and PcrA. Here we performed a mutational analysis of UvrD1 guided by the crystal structure of a DNA-bound Escherichia coli UvrD-ADP-MgF(3) transition state mimetic. Alanine scanning and conservative substitutions identified amino acids essential for both ATP hydrolysis and duplex unwinding, including those implicated in phosphohydrolase chemistry via transition state stabilization (Arg308, Arg648, Gln275), divalent cation coordination (Glu236), or activation of the nucleophilic water (Glu236, Gln275). Other residues important for ATPase/helicase activity include Phe280 and Phe72, which interact with the DNA 3' single strand tail. ATP hydrolysis was uncoupled from duplex unwinding by mutations at Glu609 (in helicase motif V), which contacts the ATP ribose sugar. Introducing alanine in lieu of the adenine-binding "Q motif" glutamine (Gln24) relaxed the substrate specificity in NTP hydrolysis, e.g., eliciting a gain of function as a UTPase/TTPase, although the Q24A mutant still relied on ATP/dATP for duplex unwinding. Our studies highlight the role of the Q motif as a substrate filter and the contributions of adenosine-binding residues as couplers of NTP hydrolysis to motor activity. The Ku-binding function of UvrD1 lies within its C-terminal 270 amino acid segment. Here we found that deleting the 90 amino acid C-terminal domain, which is structurally uncharacterized, diminished DNA unwinding, without affecting ATP hydrolysis or binding to the DNA helicase substrate, apparently by affecting the strength of the UvrD1-Ku interaction. PMID:19317511

  3. Analysis of UV-induced mutation spectra in Escherichia coli by DNA polymerase η from Arabidopsis thaliana

    International Nuclear Information System (INIS)

    DNA polymerase η belongs to the Y-family of DNA polymerases, enzymes that are able to synthesize past template lesions that block replication fork progression. This polymerase accurately bypasses UV-associated cis-syn cyclobutane thymine dimers in vitro and therefore may contributes to resistance against sunlight in vivo, both ameliorating survival and decreasing the level of mutagenesis. We cloned and sequenced a cDNA from Arabidopsis thaliana which encodes a protein containing several sequence motifs characteristics of Polη homologues, including a highly conserved sequence reported to be present in the active site of the Y-family DNA polymerases. The gene, named AtPOLH, contains 14 exons and 13 introns and is expressed in different plant tissues. A strain from Saccharomyces cerevisiae, deficient in Polη activity, was transformed with a yeast expression plasmid containing the AtPOLH cDNA. The rate of survival to UV irradiation in the transformed mutant increased to similar values of the wild type yeast strain, showing that AtPOLH encodes a functional protein. In addition, when AtPOLH is expressed in Escherichia coli, a change in the mutational spectra is detected when bacteria are irradiated with UV light. This observation might indicate that AtPOLH could compete with DNA polymerase V and then bypass cyclobutane pyrimidine dimers incorporating two adenylates

  4. Analysis of UV-induced mutation spectra in Escherichia coli by DNA polymerase eta from Arabidopsis thaliana.

    Science.gov (United States)

    Santiago, María Jesús; Alejandre-Durán, Encarna; Ruiz-Rubio, Manuel

    2006-10-10

    DNA polymerase eta belongs to the Y-family of DNA polymerases, enzymes that are able to synthesize past template lesions that block replication fork progression. This polymerase accurately bypasses UV-associated cis-syn cyclobutane thymine dimers in vitro and therefore may contributes to resistance against sunlight in vivo, both ameliorating survival and decreasing the level of mutagenesis. We cloned and sequenced a cDNA from Arabidopsis thaliana which encodes a protein containing several sequence motifs characteristics of Pol eta homologues, including a highly conserved sequence reported to be present in the active site of the Y-family DNA polymerases. The gene, named AtPOLH, contains 14 exons and 13 introns and is expressed in different plant tissues. A strain from Saccharomyces cerevisiae, deficient in Pol eta activity, was transformed with a yeast expression plasmid containing the AtPOLH cDNA. The rate of survival to UV irradiation in the transformed mutant increased to similar values of the wild type yeast strain, showing that AtPOLH encodes a functional protein. In addition, when AtPOLH is expressed in Escherichia coli, a change in the mutational spectra is detected when bacteria are irradiated with UV light. This observation might indicate that AtPOLH could compete with DNA polymerase V and then bypass cyclobutane pyrimidine dimers incorporating two adenylates.

  5. SNPase-ARMS qPCR: Ultrasensitive Mutation-Based Detection of Cell-Free Tumor DNA in Melanoma Patients

    Science.gov (United States)

    Stadler, Julia; Eder, Johanna; Pratscher, Barbara; Brandt, Sabine; Schneller, Doris; Müllegger, Robert; Vogl, Claus; Trautinger, Franz; Brem, Gottfried; Burgstaller, Joerg P.

    2015-01-01

    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. PMID:26562020

  6. 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.

  7. Effect of increased intake of dietary animal fat and fat energy on oxidative damage, mutation frequency, DNA adduct level and DNA repair in rat colon and liver

    DEFF Research Database (Denmark)

    Vogel, Ulla Birgitte; Danesvar, B.; Autrup, H.;

    2003-01-01

    The effect of high dietary intake of animal fat and an increased fat energy intake on colon and liver genotoxicity and on markers of oxidative damage and antioxidative defence in colon, liver and plasma was investigated in Big Blue rats. The rats were fed ad libitum with semi-synthetic feed...... supplemented with 0, 3, 10 or 30% w/w lard. After 3 weeks, the mutation frequency, DNA repair gene expression, DNA damage and oxidative markers were determined in liver, colon and plasma. The mutation frequency of the lambda gene cII did not increase with increased fat or energy intake in colon or liver....... The DNA-adduct level measured by P-32-postlabelling decreased in both liver and colon with increased fat intake. In liver, this was accompanied by a 2-fold increase of the mRNA level of nucleotide excision repair (NER) gene ERCC1. In colon, a non-statistically significant increase in the ERCC1 mRNA levels...

  8. Prevalence of Germline Mutations in Genes Engaged in DNA Damage Repair by Homologous Recombination in Patients with Triple-Negative and Hereditary Non-Triple-Negative Breast Cancers.

    Directory of Open Access Journals (Sweden)

    Pawel Domagala

    Full Text Available This study sought to assess the prevalence of common germline mutations in several genes engaged in the repair of DNA double-strand break by homologous recombination in patients with triple-negative breast cancers and hereditary non-triple-negative breast cancers. Tumors deficient in this type of DNA damage repair are known to be especially sensitive to DNA cross-linking agents (e.g., platinum drugs and to poly(ADP-ribose polymerase (PARP inhibitors.Genetic testing was performed for 36 common germline mutations in genes engaged in the repair of DNA by homologous recombination, i.e., BRCA1, BRCA2, CHEK2, NBN, ATM, PALB2, BARD1, and RAD51D, in 202 consecutive patients with triple-negative breast cancers and hereditary non-triple-negative breast cancers.Thirty five (22.2% of 158 patients in the triple-negative group carried mutations in genes involved in DNA repair by homologous recombination, while 10 (22.7% of the 44 patients in the hereditary non-triple-negative group carried such mutations. Mutations in BRCA1 were most frequent in patients with triple-negative breast cancer (18.4%, and mutations in CHEK2 were most frequent in patients with hereditary non-triple-negative breast cancers (15.9%. In addition, in the triple-negative group, mutations in CHEK2, NBN, and ATM (3.8% combined were found, while mutations in BRCA1, NBN, and PALB2 (6.8% combined were identified in the hereditary non-triple-negative group.Identifying mutations in genes engaged in DNA damage repair by homologous recombination other than BRCA1/2 can substantially increase the proportion of patients with triple-negative breast cancer and hereditary non-triple-negative breast cancer who may be eligible for therapy using PARP inhibitors and platinum drugs.

  9. MGB probe assay for rapid detection of mtDNA11778 mutation in the Chinese LHON patients by real-time PCR

    Institute of Scientific and Technical Information of China (English)

    Jian-yong WANG; Yang-shun GU; Jing WANG; Yi TONG; Ying WANG; Jun-bing SHAO; Ming QI

    2008-01-01

    Objective:Leber's hereditary optic neuropathY (LHON)is a maternally inherited degeneration of the optic nerve caused by point mutations of mitochondrial DNA(mtDNA).Many unsolved questions regarding the penetrance and pathophysiological mechanism of LHON demand efficient and reliable mutation testing.This study aims to develop a minor groove binder(MGB) probe assay for rapid detection of mtDNA11778 mutation and heteroplasmy in Chinese LHON patients by real-time polymerase chain reaction(PCR).Methods:Forty-eight patients suspected of having LHON and their maternal relatives underwent a molecular genetic evaluation,with 20 normal individuals as a control group at the same time.A real-time PCR involving two MGB probes was used to detect the mtDNA 11778 mutation and heteroplasmy.A linear standard curve was obtained by pUCmLHONG and pUCmLHONA clones.Results:All 48 LHON patients and their matemal relatives were positive for mtDNA 11778 mutation in our assay,27 heteroplasmic and 21 homoplasmic.Eighteen cases did not show an occurrence of the disease,while 9 developed the disease among the 27 heteroplasmic mutation cases.Eleven did not show an occurrence of the disease,while 10 cases developed the disease among 21 homoplasmic mutation cases.There was a significant difierence in the incidence between the heteroplasmic and the homoplasmic mutation types.The time needed for running a real-time PCR assay was only 80 min.Conclusion:This real-time PCR assay is a rapid,reliable method for mtDNA mutation detection as well as heteroplasmy quantification.Detecting this ratio is very important for predicting phenotypic expression of unaffected carriers.

  10. Response to immunotherapy in a patient with adult onset Leigh syndrome and T9176C mtDNA mutation.

    Science.gov (United States)

    Chuquilin, Miguel; Govindarajan, Raghav; Peck, Dawn; Font-Montgomery, Esperanza

    2016-09-01

    Leigh syndrome is a mitochondrial disease caused by mutations in different genes, including ATP6A for which no known therapy is available. We report a case of adult-onset Leigh syndrome with response to immunotherapy. A twenty year-old woman with baseline learning difficulties was admitted with progressive behavioral changes, diplopia, headaches, bladder incontinence, and incoordination. Brain MRI and PET scan showed T2 hyperintensity and increased uptake in bilateral basal ganglia, respectively. Autoimmune encephalitis was suspected and she received plasmapheresis with clinical improvement. She was readmitted 4 weeks later with dysphagia and aspiration pneumonia. Plasmapheresis was repeated with resolution of her symptoms. Given the multisystem involvement and suggestive MRI changes, genetic testing was done, revealing a homoplasmic T9176C ATPase 6 gene mtDNA mutation. Monthly IVIG provided clinical improvement with worsening when infusions were delayed. Leigh syndrome secondary to mtDNA T9176C mutations could have an autoimmune mechanism that responds to immunotherapy. PMID:27408822

  11. Automated extraction of DNA from blood and PCR setup using a Tecan Freedom EVO liquid handler for forensic genetic STR typing of reference samples.

    Science.gov (United States)

    Stangegaard, Michael; Frøslev, Tobias G; Frank-Hansen, Rune; Hansen, Anders J; Morling, Niels

    2011-04-01

    We have implemented and validated automated protocols for DNA extraction and PCR setup using a Tecan Freedom EVO liquid handler mounted with the Te-MagS magnetic separation device (Tecan, Männedorf, Switzerland). The protocols were validated for accredited forensic genetic work according to ISO 17025 using the Qiagen MagAttract DNA Mini M48 kit (Qiagen GmbH, Hilden, Germany) from fresh whole blood and blood from deceased individuals. The workflow was simplified by returning the DNA extracts to the original tubes minimizing the risk of misplacing samples. The tubes that originally contained the samples were washed with MilliQ water before the return of the DNA extracts. The PCR was setup in 96-well microtiter plates. The methods were validated for the kits: AmpFℓSTR Identifiler, SGM Plus and Yfiler (Applied Biosystems, Foster City, CA), GenePrint FFFL and PowerPlex Y (Promega, Madison, WI). The automated protocols allowed for extraction and addition of PCR master mix of 96 samples within 3.5h. In conclusion, we demonstrated that (1) DNA extraction with magnetic beads and (2) PCR setup for accredited, forensic genetic short tandem repeat typing can be implemented on a simple automated liquid handler leading to the reduction of manual work, and increased quality and throughput. PMID:21609694

  12. Mutations in mitochondrial DNA associated with hypertension%高血压相关的线粒体DNA突变

    Institute of Scientific and Technical Information of China (English)

    薛凌; 陈红; 孟燕子; 王燕; 卢中秋; 吕建新; 管敏鑫

    2011-01-01

    线粒体DNA(mtDNA)突变是高血压发病的分子机制之一.已经报道的与原发性高血压相关的mtDNA突变包括:tRNAMetA4435G,tRNAMet/tRNAGln A4401G,tRNAIleA4263G,T4291C和A4295G突变.这些高血压相关的mtDNA突变改变了相应的线粒体tRNA的结构,导致线粒体tRNA的代谢障碍.而线粒体tRNAs的代谢缺陷则影响蛋白质合成,造成氧化磷酸化缺陷,降低ATP的合成,增加活性氧的产生.因此,线粒体的功能缺陷可能在高血压的发生发展中起一定的作用.mtDNA突变发病的组织特异性则可能与线粒体tRNAs的代谢以及核修饰基因相关.目前发现的这些高血压相关的mtDNA突变则应该作为今后高血压诊断的遗传风险因子,高血压相关的线粒体功能缺陷的深入研究也将进一步诠释母系遗传高血压的分子致病机制,为高血压的预防、控制和治疗提供依据,文章对高血压相关的mtDNA突变进行了综述.%Mutations in mitochondria! DNA (mtDNA) are one of the molecular bases of hypertension. Among these, the tKNAMel A4435G, tRNAMe7tRNAGto A4401Q tRNA11' A4263Q T4291C and A4295G mutations have been reported to be associated with essential hypertension. These mutations alter the structure of the corresponding mitochondrial tRNAs and cause failures in tRNA metabolism. These shortages of these tRNAs lead to an impairment of mitochondrial protein synthesis and a failure in the oxidative phosphorylation function. These result in a deficit in ATP synthesis and an increase of generation of reactive oxygen species. As a result, these mitochondrial dysfunctions may contribute to the development of hypertension. Furthermore, the tissue specificity of these pathogenic mtDNA mutations might be associated with tRNA metabolism and nuclear modifier genes. These mtDNA mutations should be considered as inherited risk factors for future molecular diagnosis. Thus, these findings provide new insights into the molecular mechanism, management and

  13. Two families with Leber's hereditary optic neuropathy carrying G11778A and T14502C mutations with haplogroup H2a2a1 in mitochondrial DNA.

    Science.gov (United States)

    Qiao, Chen; Wei, Tanwei; Hu, Bo; Peng, Chunyan; Qiu, Xueping; Wei, Li; Yan, Ming

    2015-08-01

    The mitochondrial haplogroup has been reported to affect the clinical expression of Leber's hereditary optic neuropathy (LHON). The present study aimed to investigate the interaction between mutations and the haplogroup of mitochondrial DNA (mtDNA) in families. Two unrelated families with LHON were enrolled in the study, and clinical, genetic and molecular characterizations were determined in the affected and unaffected family members. Polymerase chain reaction direct sequencing was performed using 24 pairs of overlapping primers for whole mtDNA to screen for mutations and haplogroup. Bioinformatics analysis was performed to evaluate the pathogenic effect of these mtDNA mutations and the haplogroup. The G11778A mutation was identified in the two families. In addition, the members of family 2 exhibited the T14502C mutation and those in family 1 exhibited the T3394C and T14502C mutations, which were regarded as secondary mutations. The penetrance of visual loss in families 1 and 2 were 30.8 and 33.3%, respectively. In addition, the two families were found to be in the H2a2a1 haplogroup. In this limited sample size, it was demonstrated that the H2a2a1 haplogroup had a possible protective effect against LHON. Additional modifying factors, including environmental factors, lifestyle, estrogen levels and nuclear genes may also be important in LHON.

  14. Mutation by DNA shuffling of 5-enolpyruvylshikimate-3-phosphate synthase from Malus domestica for improved glyphosate resistance.

    Science.gov (United States)

    Tian, Yong-Sheng; Xu, Jing; Peng, Ri-He; Xiong, Ai-Sheng; Xu, Hu; Zhao, Wei; Fu, Xiao-Yan; Han, Hong-Juan; Yao, Quan-Hong

    2013-09-01

    A new 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Malus domestica (MdEPSPS) was cloned and characterized by rapid amplification of cDNA ends to identify an EPSPS gene appropriate for the development of transgenic glyphosate-tolerant plants. However, wild-type MdEPSPS is not suitable for the development of transgenic glyphosate-tolerant plants because of its poor glyphosate resistance. Thus, we performed DNA shuffling on MdEPSPS, and one highly glyphosate-resistant mutant with mutations in eight amino acids (N63D, N86S, T101A, A187T, D230G, H317R, Y399R and C413A.) was identified after five rounds of DNA shuffling and screening. Among the eight amino acid substitutions on this mutant, only two residue changes (T101A and A187T) were identified by site-directed mutagenesis as essential and additive in altering glyphosate resistance, which was further confirmed by kinetic analyses. The single-site A187T mutation has also never been previously reported as an important residue for glyphosate resistance. Furthermore, transgenic rice was used to confirm the potential of MdEPSPS mutant in developing glyphosate-resistant crops.

  15. Protective effect of curcumin and chlorophyllin against DNA mutation induced by cyclophosphamide or benzo[a]pyrene

    Energy Technology Data Exchange (ETDEWEB)

    Ibrahim, M.A.; Elbehairy, A.M.; Ghoneim, M.A.; Amer, H.A. [Cairo Univ., Giza (Egypt). Biochemistry Dept. and Biotechnology Center

    2007-03-15

    The current study was carried out to evaluate the potency of curcumin and chlorophyllin as natural antioxidants to reduce the oxidative stress markers induced by cyclophosphamide (CP) and benzo[a]pyrene [B(a)P] which were used as free radical inducers. For this purpose, 126 male albino rats were used. The animals were assigned into 4 main groups: negative control group; oxidant-treated group (subdivided into two subgroups: cyclophosphamide- treated group and benzo[a]pyrene-treated group); curcumin-treated group; and chlorophyllin-treated group. Liver samples were collected after two days post the oxidant inoculation and at the end of the experimental period (10 weeks). These samples were examined for determination of liver microsomal malondialdehyde (MDA), DNA fragmentation, restriction fragment length polymorphism (RFLP) and 8-hydroxy deoxyguanosine (8-OHdG) concentration. Both CP and B(a)P caused increments in DNA fragmentation percentages, liver microsomal MDA, concentration of 8-OHdG and induced point mutation. Treatment of rats with either curcumin or chlorophyllin revealed lower DNA fragmentation percentages, liver microsomal MDA concentration, concentration of 8-OHdG and prevented induction of mutations, i. e., reversed the oxidative stress induced by CP and B(a)P and proved that they were capable of protecting rats against the oxidative damage evoked by these oxidants. (orig.)

  16. Genome-wide profiling identifies a DNA methylation signature that associates with TET2 mutations in diffuse large B-cell lymphoma

    DEFF Research Database (Denmark)

    Asmar, Fazila; Punj, Vasu; Christensen, Jesper Aagaard;

    2013-01-01

    The discovery that the Ten-Eleven Translocation (TET) hydroxylases cause DNA demethylation has fundamentally changed the notion of how DNA methylation is regulated. Clonal analysis of the hematopoetic stem cell compartment suggests that TET2 mutations can be early events in hematologic cancers...

  17. A novel, double mutation in DNA gyrase A of Escherichia coli conferring resistance to quinolone antibiotics.

    OpenAIRE

    Truong, Q C; Nguyen Van, J C; Shlaes, D; Gutmann, L; Moreau, N J

    1997-01-01

    A spontaneous Escherichia coli mutant, named Q3, resistant to nalidixic acid was obtained from a previously described clinical isolate of E. coli, Q2, resistant to fluoroquinolones but susceptible to nalidixic acid (E. Cambau, F. Bordon, E. Collatz, and L. Gutmann, Antimicrob. Agents Chemother. 37:1247-1252, 1993). Q3 harbored the mutation Asp82Gly in addition to the Gly81Asp mutation of Q2. The different mutations leading to Gly81Asp, Asp82Gly, and Gly81AspAsp82Gly were introduced into the g...

  18. Previously Unclassified Mutation of mtDNA m.3472T>C: Evidence of Pathogenicity in Leber's Hereditary Optic Neuropathy.

    Science.gov (United States)

    Sheremet, N L; Nevinitsyna, T A; Zhorzholadze, N V; Ronzina, I A; Itkis, Y S; Krylova, T D; Tsygankova, P G; Malakhova, V A; Zakharova, E Y; Tokarchuk, A V; Panteleeva, A A; Karger, E M; Lyamzaev, K G; Avetisov, S E

    2016-07-01

    Leber's hereditary optic neuropathy (LHON) refers to a group of mitochondrial diseases and is characterized by defects of the mitochondrial electron transport chain and decreased level of oxidative phosphorylation. The list of LHON primary mtDNA mutations is regularly updated. In this study, we describe the homoplasmic nucleotide substitution m.3472T>C in the MT-ND1 (NADH-ubiquinone oxidoreductase chain 1) gene and specific changes in cell metabolism in a patient with LHON and his asymptomatic sister. To confirm the presence of mutation-related mitochondrial dysfunction, respiration of skin fibroblasts and platelets from the patient and his sister was studied, as well as the mitochondrial potential and production of reactive oxygen species in the skin fibroblasts. In addition, based on characteristics of the toxic effect of paraquat, a new approach was developed for detecting the functional activity of complex I of the mitochondrial respiratory chain.

  19. Detection of the mtDNA 14484 mutation on an African-specific haplotype: Implications about its role in causing Leber hereditary optic neuropathy

    Energy Technology Data Exchange (ETDEWEB)

    Torroni, A.; Petrozzi, M.; Terracina, M. [Universita` di Roma (Italy)] [and others

    1996-07-01

    Leber hereditary optic neuropathy (LHON) is a maternally transmitted disease whose primary clinical manifestation is acute or subacute bilateral loss of central vision leading to central scotoma and blindness. To date, LHON has been associated with 18 mtDNA missense mutations, even though, for many of these mutations, it remains unclear whether they cause the disease, contribute to the pathology, or are nonpathogenic mtDNA polymorphisms. On the basis of numerous criteria, which include the specificity for LHON, the frequency in the general population, and the penetrance within affected pedigrees, the detection of associated defects in the respiratory chain, mutations at three nucleotide positions (nps), 11778 (G{r_arrow}A), 3460 (G{r_arrow}A), and 14484 (T{r_arrow}C) have been classified as high-risk and primary LHON mutations. Overall, these three mutations encompass {ge}90% of the LHON cases. 29 refs., 1 fig.

  20. The exome sequencing identified the mutation in YARS2 encoding the mitochondrial tyrosyl-tRNA synthetase as a nuclear modifier for the phenotypic manifestation of Leber's hereditary optic neuropathy-associated mitochondrial DNA mutation.

    Science.gov (United States)

    Jiang, Pingping; Jin, Xiaofen; Peng, Yanyan; Wang, Meng; Liu, Hao; Liu, Xiaoling; Zhang, Zengjun; Ji, Yanchun; Zhang, Juanjuan; Liang, Min; Zhao, Fuxin; Sun, Yan-Hong; Zhang, Minglian; Zhou, Xiangtian; Chen, Ye; Mo, Jun Qin; Huang, Taosheng; Qu, Jia; Guan, Min-Xin

    2016-02-01

    Leber's hereditary optic neuropathy (LHON) is the most common mitochondrial disorder. Nuclear modifier genes are proposed to modify the phenotypic expression of LHON-associated mitochondrial DNA (mtDNA) mutations. By using an exome sequencing approach, we identified a LHON susceptibility allele (c.572G>T, p.191Gly>Val) in YARS2 gene encoding mitochondrial tyrosyl-tRNA synthetase, which interacts with m.11778G>A mutation to cause visual failure. We performed functional assays by using lymphoblastoid cell lines derived from members of Chinese families (asymptomatic individuals carrying m.11778G>A mutation, or both m.11778G>A and heterozygous p.191Gly>Val mutations and symptomatic subjects harboring m.11778G>A and homozygous p.191Gly>Val mutations) and controls lacking these mutations. The 191Gly>Val mutation reduced the YARS2 protein level in the mutant cells. The aminoacylated efficiency and steady-state level of tRNA(Tyr) were markedly decreased in the cell lines derived from patients both carrying homozygous YARS2 p.191Gly>Val and m.11778G>A mutations. The failure in tRNA(Tyr) metabolism impaired mitochondrial translation, especially for polypeptides with high content of tyrosine codon such as ND4, ND5, ND6 and COX2 in cells lines carrying homozygous YARS2 p.191Gly>Val and m.11778G>A mutations. The YARS2 p.191Gly>Val mutation worsened the respiratory phenotypes associated with m.11778G>A mutation, especially reducing activities of complexes I and IV. The respiratory deficiency altered the efficiency of mitochondrial ATP synthesis and increased the production of reactive oxygen species. Thus, mutated YARS2 aggravates mitochondrial dysfunctions associated with the m.11778G>A mutation, exceeding the threshold for the expression of blindness phenotype. Our findings provided new insights into the pathophysiology of LHON that were manifested by interaction between mtDNA mutation and mutated nuclear-modifier YARS2. PMID:26647310

  1. The exome sequencing identified the mutation in YARS2 encoding the mitochondrial tyrosyl-tRNA synthetase as a nuclear modifier for the phenotypic manifestation of Leber's hereditary optic neuropathy-associated mitochondrial DNA mutation.

    Science.gov (United States)

    Jiang, Pingping; Jin, Xiaofen; Peng, Yanyan; Wang, Meng; Liu, Hao; Liu, Xiaoling; Zhang, Zengjun; Ji, Yanchun; Zhang, Juanjuan; Liang, Min; Zhao, Fuxin; Sun, Yan-Hong; Zhang, Minglian; Zhou, Xiangtian; Chen, Ye; Mo, Jun Qin; Huang, Taosheng; Qu, Jia; Guan, Min-Xin

    2016-02-01

    Leber's hereditary optic neuropathy (LHON) is the most common mitochondrial disorder. Nuclear modifier genes are proposed to modify the phenotypic expression of LHON-associated mitochondrial DNA (mtDNA) mutations. By using an exome sequencing approach, we identified a LHON susceptibility allele (c.572G>T, p.191Gly>Val) in YARS2 gene encoding mitochondrial tyrosyl-tRNA synthetase, which interacts with m.11778G>A mutation to cause visual failure. We performed functional assays by using lymphoblastoid cell lines derived from members of Chinese families (asymptomatic individuals carrying m.11778G>A mutation, or both m.11778G>A and heterozygous p.191Gly>Val mutations and symptomatic subjects harboring m.11778G>A and homozygous p.191Gly>Val mutations) and controls lacking these mutations. The 191Gly>Val mutation reduced the YARS2 protein level in the mutant cells. The aminoacylated efficiency and steady-state level of tRNA(Tyr) were markedly decreased in the cell lines derived from patients both carrying homozygous YARS2 p.191Gly>Val and m.11778G>A mutations. The failure in tRNA(Tyr) metabolism impaired mitochondrial translation, especially for polypeptides with high content of tyrosine codon such as ND4, ND5, ND6 and COX2 in cells lines carrying homozygous YARS2 p.191Gly>Val and m.11778G>A mutations. The YARS2 p.191Gly>Val mutation worsened the respiratory phenotypes associated with m.11778G>A mutation, especially reducing activities of complexes I and IV. The respiratory deficiency altered the efficiency of mitochondrial ATP synthesis and increased the production of reactive oxygen species. Thus, mutated YARS2 aggravates mitochondrial dysfunctions associated with the m.11778G>A mutation, exceeding the threshold for the expression of blindness phenotype. Our findings provided new insights into the pathophysiology of LHON that were manifested by interaction between mtDNA mutation and mutated nuclear-modifier YARS2.

  2. UV-induced mutations in epidermal cells of mice defective in DNA polymerase η and/or ι.

    Science.gov (United States)

    Kanao, Rie; Yokoi, Masayuki; Ohkumo, Tsuyoshi; Sakurai, Yasutaka; Dotsu, Kantaro; Kura, Shinobu; Nakatsu, Yoshimichi; Tsuzuki, Teruhisa; Masutani, Chikahide; Hanaoka, Fumio

    2015-05-01

    Xeroderma pigmentosum variant (XP-V) is a human rare inherited recessive disease, predisposed to sunlight-induced skin cancer, which is caused by deficiency in DNA polymerase η (Polη). Polη catalyzes accurate translesion synthesis (TLS) past pyrimidine dimers, the most prominent UV-induced lesions. DNA polymerase ι (Polι) is a paralog of Polη that has been suggested to participate in TLS past UV-induced lesions, but its function in vivo remains uncertain. We have previously reported that Polη-deficient and Polη/Polι double-deficient mice showed increased susceptibility to UV-induced carcinogenesis. Here, we investigated UV-induced mutation frequencies and spectra in the epidermal cells of Polη- and/or Polι-deficient mice. While Polη-deficient mice showed significantly higher UV-induced mutation frequencies than wild-type mice, Polι deficiency did not influence the frequencies in the presence of Polη. Interestingly, the frequencies in Polη/Polι double-deficient mice were statistically lower than those in Polη-deficient mice, although they were still higher than those of wild-type mice. Sequence analysis revealed that most of the UV-induced mutations in Polη-deficient and Polη/Polι double-deficient mice were base substitutions at dipyrimidine sites. An increase in UV-induced mutations at both G:C and A:T pairs associated with Polη deficiency suggests that Polη contributes to accurate TLS past both thymine- and cytosine-containing dimers in vivo. A significant decrease in G:C to A:T transition in Polη/Polι double-deficient mice when compared with Polη-deficient mice suggests that Polι is involved in error-prone TLS past cytosine-containing dimers when Polη is inactivated.

  3. A Simple Oligonucleotide Biochip Capable of Rapidly Detecting Known Mitochondrial DNA Mutations in Chinese Patients with Leber’S Hereditary Optic Neuropathy (LHON

    Directory of Open Access Journals (Sweden)

    Wei-Dong Du

    2011-01-01

    Full Text Available Leber's hereditary optic neuropathy (LHON is a maternally transmitted disease. Clinically, no efficient assay protocols have been available. In this study, we aimed to develop an oligonucleotide biochip specialized for detection of known base substitution mutations in mitochondrial DNA causing LHON and to investigate frequencies of LHON relevant variants in Anhui region of China. Thirty-two pairs of oligonucleotide probes matched with the mutations potentially linked to LHON were covalently immobilized. Cy5-lablled targets were amplified from blood DNA samples by a multiplex PCR method. Two kinds of primary mutations 11778 G > A and 14484 T > C from six confirmed LHON patients were interrogated to validate this biochip format. Further, fourteen Chinese LHON pedigrees and twenty-five unrelated healthy individuals were investigated by the LHON biochip, direct sequencing and pyrosequencing, respectively. The biochip was found to be able efficiently to discriminate homoplasmic and heteroplasmic mtDNA mutations in LHON. Biochip analysis revealed that twelve of eighteen LHON symptomatic cases from the 14 Chinese pedigree harbored the mutations either 11778G > A, 14484T > C or 3460G > A, respectively, accounting for 66.7%. The mutation 11778G > A in these patients was homoplasmic and prevalent (55.5%, 10 of 18 cases. The mutations 3460G > A and 3394T > C were found to co-exist in one LHON case. The mutation 13708G > A appeared in one LHON pedigree. Smaller amount of sampling and reaction volume, easier target preparation, fast and high-throughput were the main advantages of the biochip over direct DNA sequencing and pyrosequencing. Our findings suggested that primary mutations of 11778G > A, 14484T > C or 3460G > A are main variants of mtDNA gene leading to LHON in China. The biochip would easily be implemented in clinical diagnosis.

  4. A simple oligonucleotide biochip capable of rapidly detecting known mitochondrial DNA mutations in Chinese patients with Leber's hereditary optic neuropathy (LHON).

    Science.gov (United States)

    Du, Wei-Dong; Chen, Gang; Cao, Hui-Min; Jin, Qing-Hui; Liao, Rong-Feng; He, Xiang-Cheng; Chen, Da-Ben; Huang, Shu-Ren; Zhao, Hui; Lv, Yong-Mei; Tang, Hua-Yang; Tang, Xian-Fa; Wang, Yong-Qing; Sun, Song; Zhao, Jian-Long; Zhang, Xue-Jun

    2011-01-01

    Leber's hereditary optic neuropathy (LHON) is a maternally transmitted disease. Clinically, no efficient assay protocols have been available. In this study, we aimed to develop an oligonucleotide biochip specialized for detection of known base substitution mutations in mitochondrial DNA causing LHON and to investigate frequencies of LHON relevant variants in Anhui region of China. Thirty-two pairs of oligonucleotide probes matched with the mutations potentially linked to LHON were covalently immobilized. Cy5-lablled targets were amplified from blood DNA samples by a multiplex PCR method. Two kinds of primary mutations 11778 G > A and 14484 T > C from six confirmed LHON patients were interrogated to validate this biochip format. Further, fourteen Chinese LHON pedigrees and twenty-five unrelated healthy individuals were investigated by the LHON biochip, direct sequencing and pyrosequencing, respectively. The biochip was found to be able efficiently to discriminate homoplasmic and heteroplasmic mtDNA mutations in LHON. Biochip analysis revealed that twelve of eighteen LHON symptomatic cases from the 14 Chinese pedigree harbored the mutations either 11778G > A, 14484T > C or 3460G A, respectively, accounting for 66.7%. The mutation 11778G > A in these patients was homoplasmic and prevalent (55.5%, 10 of 18 cases). The mutations 3460G > A and 3394T > C were found to co-exist in one LHON case. The mutation 13708G > A appeared in one LHON pedigree. Smaller amount of sampling and reaction volume, easier target preparation, fast and high-throughput were the main advantages of the biochip over direct DNA sequencing and pyrosequencing. Our findings suggested that primary mutations of 11778G > A, 14484T > C or 3460G > A are main variants of mtDNA gene leading to LHON in China. The biochip would easily be implemented in clinical diagnosis.

  5. Translesion DNA polymerases Pol , Pol , Pol , Pol and Rev1 are not essential for repeat-induced point mutation in Neurospora crassa

    Indian Academy of Sciences (India)

    Ranjan Tamuli; C Ravindran; Durgadas P Kasbekar

    2006-12-01

    Pol , Pol , Pol , Pol and Rev1 are specialized DNA polymerases that are able to synthesize DNA across a damaged template. DNA synthesis by such translesion polymerases can be mutagenic due to the miscoding nature of most damaged nucleotides. In fact, many mutational and hypermutational processes in systems ranging from yeast to mammals have been traced to the activity of such polymerases. We show however, that the translesion polymerases are dispensable for repeat-induced point mutation (RIP) in Neurospora crassa. Additionally, we demonstrate that the upr-1 gene, which encodes the catalytic subunit of Pol , is a highly polymorphic locus in Neurospora.

  6. ATRX mutations and glioblastoma: Impaired DNA damage repair, alternative lengthening of telomeres, and genetic instability.

    Science.gov (United States)

    Koschmann, Carl; Lowenstein, Pedro R; Castro, Maria G

    2016-05-01

    Alpha thalassemia/mental retardation syndrome X-linked (ATRX) is mutated in nearly a third of pediatric glioblastoma (GBM) patients. We developed an animal model of ATRX-deficient GBM. Using this model combined with analysis of multiple human glioma genome-wide datasets, we determined that ATRX mutation leads to genetic instability, impaired non-homologous end joining, and alternate lengthening of telomeres (ALT). PMID:27314101

  7. CpG dinucleotides and the mutation rate of non-CpG DNA

    OpenAIRE

    Walser, Jean-Claude; Ponger, Loïc; Furano, Anthony V

    2008-01-01

    The neutral mutation rate is equal to the base substitution rate when the latter is not affected by natural selection. Differences between these rates may reveal that factors such as natural selection, linkage, or a mutator locus are affecting a given sequence. We examined the neutral base substitution rate by measuring the sequence divergence of ∼30,000 pairs of inactive orthologous L1 retrotransposon sequences interspersed throughout the human and chimpanzee genomes. In contrast to other st...

  8. Detection of Somatic Mutations by High-Resolution DNA Melting (HRM) Analysis in Multiple Cancers

    OpenAIRE

    Jesus Gonzalez-Bosquet; Jacob Calcei; Wei, Jun S.; Montserrat Garcia-Closas; Sherman, Mark E.; Stephen Hewitt; Joseph Vockley; Jolanta Lissowska; Yang, Hannah P.; Javed Khan; Stephen Chanock

    2011-01-01

    Identification of somatic mutations in cancer is a major goal for understanding and monitoring the events related to cancer initiation and progression. High resolution melting (HRM) curve analysis represents a fast, post-PCR high-throughput method for scanning somatic sequence alterations in target genes. The aim of this study was to assess the sensitivity and specificity of HRM analysis for tumor mutation screening in a range of tumor samples, which included 216 frozen pediatric small rounde...

  9. 5-hydroxymethylcytosine marks regions with reduced mutation frequency in human DNA

    Science.gov (United States)

    Tomkova, Marketa; McClellan, Michael; Kriaucionis, Skirmantas; Schuster-Boeckler, Benjamin

    2016-01-01

    CpG dinucleotides are the main mutational hot-spot in most cancers. The characteristic elevated C>T mutation rate in CpG sites has been related to 5-methylcytosine (5mC), an epigenetically modified base which resides in CpGs and plays a role in transcription silencing. In brain nearly a third of 5mCs have recently been found to exist in the form of 5-hydroxymethylcytosine (5hmC), yet the effect of 5hmC on mutational processes is still poorly understood. Here we show that 5hmC is associated with an up to 53% decrease in the frequency of C>T mutations in a CpG context compared to 5mC. Tissue specific 5hmC patterns in brain, kidney and blood correlate with lower regional CpG>T mutation frequency in cancers originating in the respective tissues. Together our data reveal global and opposing effects of the two most common cytosine modifications on the frequency of cancer causing somatic mutations in different cell types. DOI: http://dx.doi.org/10.7554/eLife.17082.001 PMID:27183007

  10. Structural and Mutational Analysis of Escherichia coli AlkB Provides Insight into Substrate Specificity and DNA Damage Searching

    Energy Technology Data Exchange (ETDEWEB)

    Holland, P.; Hollis, T

    2010-01-01

    In Escherichia coli, cytotoxic DNA methyl lesions on the N1 position of purines and N3 position of pyrimidines are primarily repaired by the 2-oxoglutarate (2-OG) iron(II) dependent dioxygenase, AlkB. AlkB repairs 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) lesions, but it also repairs 1-methylguanine (1-meG) and 3-methylthymine (3-meT) at a much less efficient rate. How the AlkB enzyme is able to locate and identify methylated bases in ssDNA has remained an open question. We determined the crystal structures of the E. coli AlkB protein holoenzyme and the AlkB-ssDNA complex containing a 1-meG lesion. We coupled this to site-directed mutagenesis of amino acids in and around the active site, and tested the effects of these mutations on the ability of the protein to bind both damaged and undamaged DNA, as well as catalyze repair of a methylated substrate. A comparison of our substrate-bound AlkB-ssDNA complex with our unliganded holoenzyme reveals conformational changes of residues within the active site that are important for binding damaged bases. Site-directed mutagenesis of these residues reveals novel insight into their roles in DNA damage recognition and repair. Our data support a model that the AlkB protein utilizes at least two distinct conformations in searching and binding methylated bases within DNA: a 'searching' mode and 'repair' mode. Moreover, we are able to functionally separate these modes through mutagenesis of residues that affect one or the other binding state. Finally, our mutagenesis experiments show that amino acid D135 of AlkB participates in both substrate specificity and catalysis.

  11. Structural and mutational analysis of Escherichia coli AlkB provides insight into substrate specificity and DNA damage searching.

    Directory of Open Access Journals (Sweden)

    Paul J Holland

    Full Text Available BACKGROUND: In Escherichia coli, cytotoxic DNA methyl lesions on the N1 position of purines and N3 position of pyrimidines are primarily repaired by the 2-oxoglutarate (2-OG iron(II dependent dioxygenase, AlkB. AlkB repairs 1-methyladenine (1-meA and 3-methylcytosine (3-meC lesions, but it also repairs 1-methylguanine (1-meG and 3-methylthymine (3-meT at a much less efficient rate. How the AlkB enzyme is able to locate and identify methylated bases in ssDNA has remained an open question. METHODOLOGY/PRINCIPAL FINDINGS: We determined the crystal structures of the E. coli AlkB protein holoenzyme and the AlkB-ssDNA complex containing a 1-meG lesion. We coupled this to site-directed mutagenesis of amino acids in and around the active site, and tested the effects of these mutations on the ability of the protein to bind both damaged and undamaged DNA, as well as catalyze repair of a methylated substrate. CONCLUSIONS/SIGNIFICANCE: A comparison of our substrate-bound AlkB-ssDNA complex with our unliganded holoenzyme reveals conformational changes of residues within the active site that are important for binding damaged bases. Site-directed mutagenesis of these residues reveals novel insight into their roles in DNA damage recognition and repair. Our data support a model that the AlkB protein utilizes at least two distinct conformations in searching and binding methylated bases within DNA: a "searching" mode and "repair" mode. Moreover, we are able to functionally separate these modes through mutagenesis of residues that affect one or the other binding state. Finally, our mutagenesis experiments show that amino acid D135 of AlkB participates in both substrate specificity and catalysis.

  12. Mutational analysis of an archaeal minichromosome maintenance protein exterior hairpin reveals critical residues for helicase activity and DNA binding

    Directory of Open Access Journals (Sweden)

    Brewster Aaron S

    2010-08-01

    Full Text Available Abstract Background The mini-chromosome maintenance protein (MCM complex is an essential replicative helicase for DNA replication in Archaea and Eukaryotes. While the eukaryotic complex consists of six homologous proteins (MCM2-7, the archaeon Sulfolobus solfataricus has only one MCM protein (ssoMCM, six subunits of which form a homohexamer. We have recently reported a 4.35Å crystal structure of the near full-length ssoMCM. The structure reveals a total of four β-hairpins per subunit, three of which are located within the main channel or side channels of the ssoMCM hexamer model generated based on the symmetry of the N-terminal Methanothermobacter thermautotrophicus (mtMCM structure. The fourth β-hairpin, however, is located on the exterior of the hexamer, near the exit of the putative side channels and next to the ATP binding pocket. Results In order to better understand this hairpin's role in DNA binding and helicase activity, we performed a detailed mutational and biochemical analysis of nine residues on this exterior β-hairpin (EXT-hp. We examined the activities of the mutants related to their helicase function, including hexamerization, ATPase, DNA binding and helicase activities. The assays showed that some of the residues on this EXT-hp play a role for DNA binding as well as for helicase activity. Conclusions These results implicate several current theories regarding helicase activity by this critical hexameric enzyme. As the data suggest that EXT-hp is involved in DNA binding, the results reported here imply that the EXT-hp located near the exterior exit of the side channels may play a role in contacting DNA substrate in a manner that affects DNA unwinding.

  13. Does Ataxia Telangiectasia Mutated (ATM) protect testicular and germ cell DNA integrity by regulating the redox status?

    Science.gov (United States)

    Godschalk, Roger W L; Vanhees, Kimberly; Maas, Lou; Drittij, Marie-Jose; Pachen, Daniëlle; van Doorn-Khosrovani, Sahar van Waalwijk; van Schooten, Frederik J; Haenen, Guido R M M

    2016-08-01

    A balanced redox homeostasis in the testis is essential for genetic integrity of sperm. Reactive oxygen species can disturb this balance by oxidation of glutathione, which is regenerated using NADPH, formed by glucose-6-phosphate dehydrogenase (G6PDH). G6PDH is regulated by the Ataxia Telangiectasia Mutated (Atm) protein. Therefore, we studied the redox status and DNA damage in testes and sperm of mice that carried a deletion in Atm. The redox status in heterozygote mice, reflected by glutathione levels and antioxidant capacity, was lower than in wild type mice, and in homozygotes the redox status was even lower. The redox status correlated with oxidative DNA damage that was highest in mice that carried Atm deletions. Surprisingly, G6PDH activity was highest in homozygotes carrying the deletion. These data indicate that defective Atm reduces the redox homeostasis of the testis and genetic integrity of sperm by regulating glutathione levels independently from G6PDH activity. PMID:27318254

  14. Transfection with extracellularly UV-damaged DNA induces human and rat cells to express a mutator phenotype towards parvovirus H-1

    Energy Technology Data Exchange (ETDEWEB)

    Dinsart, C.; Cornelis, J.J.; Klein, B.; van der Eb, A.J.; Rommelaere, J.

    1984-02-01

    Human and rat cells transfected with UV-irradiated linear double-stranded DNA from calf thymus displayed a mutator activity. This phenotype was identified by growing a lytic thermosensitive single-stranded DNA virus (parvovirus H-1) in those cells and determining viral reversion frequencies. Likewise, exogenous UV-irradiated closed circular DNAs, either double-stranded (simian virus 40) or single-stranded (phi X174), enhanced the ability of recipient cells to mutate parvovirus H-1. The magnitude of mutator activity expression increased along with the number of UV lesions present in the inoculated DNA up to a saturation level. Unirradiated DNA displayed little inducing capacity, irrespective of whether it was single or double stranded. Deprivation of a functional replication origin did not impede UV-irradiated simian virus 40 DNA from providing rat and human cells with a mutator function. Our data suggest that in mammalian cells a trans-acting mutagenic signal might be generated from UV-irradiated DNA without the necessity for damaged DNA to replicate.

  15. Does tumorigenesis select for or against mutations of the DNA repair-associated genes BRCA2 and MRE11?: Considerations from somatic mutations in microsatellite unstable (MSI gastrointestinal cancers

    Directory of Open Access Journals (Sweden)

    Elghalbzouri-Maghrani Elhaam

    2006-01-01

    Full Text Available Abstract Background The BRCA2 and MRE11 proteins participate in the repair of double-strand DNA breaks by homologous recombination. Germline BRCA2 mutations predispose to ovarian, breast and pancreatic cancer, while a germline MRE11 mutation is associated with an ataxia telangiectasia-like disorder. Somatic mutations of BRCA2 are rare in typical sporadic cancers. In tumors having microsatellite instability (MSI, somatic truncating mutations in a poly [A] tract of BRCA2 are reported on occasion. Results We analyzed gastrointestinal MSI cancers by whole gene BRCA2 sequencing, finding heterozygous truncating mutations in seven (47% of 15 patients. There was no cellular functional defect in RAD51 focus-formation in three heterozygously mutated lines studied, although other potential functions of the BRCA2 protein could still be affected. A prior report of mutations in primary MSI tumors affecting the IVS5-(5–15 poly [T] tract of the MRE11 gene was confirmed and extended by analysis of the genomic sequence and protein expression in MSI cancer cell lines. Statistical analysis of the published MRE11 mutation rate in MSI tumors did not provide evidence for a selective pressure favoring biallelic mutations at this repeat. Conclusion Perhaps conflicting with common suspicions, the data are not compatible with selective pressures during tumorigenesis promoting the functional loss of BRCA2 and MRE11 in MSI tumors. Instead, these data fit closely with an absence of selective pressures acting on BRCA2 and MRE11 gene status during tumorigenesis.

  16. MUTATIONS THAT PROBE THE COOPERATIVE ASSEMBLY OF O6-ALKYLGUANINE-DNA ALKYLTRANSFERASE (AGT) COMPLEXES†

    OpenAIRE

    Adams, Claire A.; Fried, Michael G.

    2011-01-01

    O6-alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O6-alkylguanine and O4-alkylthymine adducts present in DNA that has been exposed to alkylating agents. AGT binds DNA cooperatively and models of cooperative complexes predict that residues 1–7 of one protein molecule and 163–169 of a neighboring protein are closely juxtaposed. To test these models we used directed mutagenesis to substitute triplets of alanine for triplets of native residues across these two sequences. Six of eight d...

  17. Strikingly different penetrance of LHON in two Chinese families with primary mutation G11778A is independent of mtDNA haplogroup background and secondary mutation G13708A

    Energy Technology Data Exchange (ETDEWEB)

    Wang Huawei [Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223 (China)]|[Laboratory for Conservation and Utilization of Bio-resource, Yunnan University, Kunming 650091 (China); Jia Xiaoyun; Ji Yanli [State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060 (China); Kong Qingpeng [State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223 (China); Zhang Qingjiong [State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060 (China)], E-mail: qingjiongzhang@yahoo.com; Yao Yonggang [Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223 (China)]|[State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223 (China)], E-mail: ygyaozh@yahoo.com; Zhang Yaping [Laboratory for Conservation and Utilization of Bio-resource, Yunnan University, Kunming 650091 (China)]|[State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223 (China)

    2008-08-25

    The penetrance of Leber's hereditary optic neuropathy (LHON) in families with primary mitochondrial DNA (mtDNA) mutations is very complex. Matrilineal and nuclear genetic background, as well as environmental factors, have been reported to be involved in different affected pedigrees. Here we describe two large Chinese families that show a striking difference in the penetrance of LHON, in which 53.3% and 15.0% of members were affected (P < 0.02), respectively. Analysis of the complete mtDNA genome of the two families revealed the presence of the primary mutation G11778A and several other variants suggesting the same haplogroup status G2a. The family with higher penetrance contained a previously described secondary mutation G13708A, which presents a polymorphism in normal Chinese samples and does not affect in vivo mitochondrial oxidative metabolism as described in a previous study. Evolutionary analysis failed to indicate any putatively pathogenic mutation that cosegregated with G11778A in these two pedigrees. Our results suggest that the variable penetrance of LHON in the two Chinese families is independent of both their mtDNA haplotype background and a secondary mutation G13708A. As a result, it is likely that unknown nuclear gene involvement and/or other factors contribute to the strikingly different penetrance of LHON.

  18. Evidence for interplay among yeast replicative DNA polymerases alpha, delta and epsilon from studies of exonuclease and polymerase active site mutations

    Directory of Open Access Journals (Sweden)

    Pavlov Youri I

    2004-05-01

    Full Text Available Abstract Background DNA polymerase ε (Pol ε is essential for S-phase replication, DNA damage repair and checkpoint control in yeast. A pol2-Y831A mutation leading to a tyrosine to alanine change in the Pol ε active site does not cause growth defects and confers a mutator phenotype that is normally subtle but strong in a mismatch repair-deficient strain. Here we investigate the mechanism responsible for the mutator effect. Results Purified four-subunit Y831A Pol ε turns over more deoxynucleoside triphosphates to deoxynucleoside monophosphates than does wild-type Pol ε, suggesting altered coordination between the polymerase and exonuclease active sites. The pol2-Y831A mutation suppresses the mutator effect of the pol2-4 mutation in the exonuclease active site that abolishes proofreading by Pol ε, as measured in haploid strain with the pol2-Y831A,4 double mutation. Analysis of mutation rates in diploid strains reveals that the pol2-Y831A allele is recessive to pol2-4. In addition, the mutation rates of strains with the pol2-4 mutation in combination with active site mutator mutations in Pol δ and Pol α suggest that Pol ε may proofread certain errors made by Pol α and Pol δ during replication in vivo. Conclusions Our data suggest that Y831A replacement in Pol ε reduces replication fidelity and its participation in chromosomal replication, but without eliminating an additional function that is essential for viability. This suggests that other polymerases can substitute for certain functions of polymerase ε.

  19. Statistical Mutation Calling from Sequenced Overlapping DNA Pools in TILLING Experiments

    Directory of Open Access Journals (Sweden)

    Comai Luca

    2011-07-01

    Full Text Available Abstract Background TILLING (Targeting induced local lesions IN genomes is an efficient reverse genetics approach for detecting induced mutations in pools of individuals. Combined with the high-throughput of next-generation sequencing technologies, and the resolving power of overlapping pool design, TILLING provides an efficient and economical platform for functional genomics across thousands of organisms. Results We propose a probabilistic method for calling TILLING-induced mutations, and their carriers, from high throughput sequencing data of overlapping population pools, where each individual occurs in two pools. We assign a probability score to each sequence position by applying Bayes' Theorem to a simplified binomial model of sequencing error and expected mutations, taking into account the coverage level. We test the performance of our method on variable quality, high-throughput sequences from wheat and rice mutagenized populations. Conclusions We show that our method effectively discovers mutations in large populations with sensitivity of 92.5% and specificity of 99.8%. It also outperforms existing SNP detection methods in detecting real mutations, especially at higher levels of coverage variability across sequenced pools, and in lower quality short reads sequence data. The implementation of our method is available from: http://www.cs.ucdavis.edu/filkov/CAMBa/.

  20. A fully automated 384 capillary array for DNA sequencer. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Li, Qingbo; Kane, T

    2003-03-20

    Phase I SpectruMedix has successfully developed an automatic 96-capillary array DNA prototype based on the multiplexed capillary electrophoresis system originated from Ames Laboratory-USDOE, Iowa State University. With computer control of all steps involved in a 96-capillary array running cycle, the prototype instrument (the SCE9600) is now capable of sequencing 450 base pairs (bp) per capillary, or 48,000 bp per instrument run within 2 hrs. Phase II of this grant involved the advancement of the core 96 capillary technologies, as well as designing a high density 384 capillary prototype. True commercialization of the 96 capillary instrument involved finalization of the gel matrix, streamlining the instrument hardware, creating a more reliable capillary cartridge, and further advancement of the data processing software. Together these silos of technology create a truly commercializable product (the SCE9610) capable of meeting the operation needs of the sequencing centers.

  1. De novo COX2 mutation in a LHON family of Caucasian origin: implication for the role of mtDNA polymorphism in human pathology.

    Science.gov (United States)

    Zhadanov, Sergey I; Atamanov, Vasiliy V; Zhadanov, Nikolay I; Schurr, Theodore G

    2006-01-01

    Recent studies suggest that certain mutations with phylogeographic importance as haplogroup markers may also influence the phenotypic expression of particular mitochondrial disorders. One such disorder, Leber's hereditary optic neuropathy (LHON), demonstrates a clear expression bias in mtDNAs belonging to haplogroup J, a West Eurasian maternal lineage defined by polymorphic markers that have been called 'secondary' disease mutations. In this report, we present evidence for a de novo heteroplasmic COX2 mutation associated with a LHON clinical phenotype. This particular mutation-at nucleotide position 7,598-occurs in West Eurasian haplogroup H, the most common maternal lineage among individuals of European descent, whereas previous studies have detected this mutation only in East Eurasian haplogroup E. A review of the available mtDNA sequence data indicates that the COX2 7598 mutation occurs as a homoplasic event at the tips of these phylogenetic branches, suggesting that it could be a variant that is rapidly eliminated by selection. This finding points to the potential background influence of polymorphisms on the expression of mild deleterious mutations such as LHON mtDNA defects and further highlights the difficulties in distinguishing deleterious mtDNA changes from neutral polymorphisms and their significance in the development of mitochondriopathies.

  2. Analyses of point mutation repair and allelic heterogeneity generated by CRISPR/Cas9 and single-stranded DNA oligonucleotides.

    Science.gov (United States)

    Bialk, Pawel; Sansbury, Brett; Rivera-Torres, Natalia; Bloh, Kevin; Man, Dula; Kmiec, Eric B

    2016-09-09

    The repair of a point mutation can be facilitated by combined activity of a single-stranded oligonucleotide and a CRISPR/Cas9 system. While the mechanism of action of combinatorial gene editing remains to be elucidated, the regulatory circuitry of nucleotide exchange executed by oligonucleotides alone has been largely defined. The presence of the appropriate CRISPR/Cas9 system leads to an enhancement in the frequency of gene editing directed by single-stranded DNA oligonucleotides. While CRISPR/Cas9 executes double-stranded DNA cleavage efficiently, closure of the broken chromosomes is dynamic, as varying degrees of heterogeneity of the cleavage products appear to accompany the emergence of the corrected base pair. We provide a detailed analysis of allelic variance at and surrounding the target site. In one particular case, we report sequence alteration directed by a distinct member of the same gene family. Our data suggests that single-stranded DNA molecules may influence DNA junction heterogeneity created by CRISPR/Cas9.

  3. Analyses of point mutation repair and allelic heterogeneity generated by CRISPR/Cas9 and single-stranded DNA oligonucleotides.

    Science.gov (United States)

    Bialk, Pawel; Sansbury, Brett; Rivera-Torres, Natalia; Bloh, Kevin; Man, Dula; Kmiec, Eric B

    2016-01-01

    The repair of a point mutation can be facilitated by combined activity of a single-stranded oligonucleotide and a CRISPR/Cas9 system. While the mechanism of action of combinatorial gene editing remains to be elucidated, the regulatory circuitry of nucleotide exchange executed by oligonucleotides alone has been largely defined. The presence of the appropriate CRISPR/Cas9 system leads to an enhancement in the frequency of gene editing directed by single-stranded DNA oligonucleotides. While CRISPR/Cas9 executes double-stranded DNA cleavage efficiently, closure of the broken chromosomes is dynamic, as varying degrees of heterogeneity of the cleavage products appear to accompany the emergence of the corrected base pair. We provide a detailed analysis of allelic variance at and surrounding the target site. In one particular case, we report sequence alteration directed by a distinct member of the same gene family. Our data suggests that single-stranded DNA molecules may influence DNA junction heterogeneity created by CRISPR/Cas9. PMID:27609304

  4. Mathematical modelling of the automated FADU assay for the quantification of DNA strand breaks and their repair in human peripheral mononuclear blood cells

    International Nuclear Information System (INIS)

    Cells continuously undergo DNA damage from exogenous agents like irradiation or genotoxic chemicals or from endogenous radicals produced by normal cellular metabolic activities. DNA strand breaks are one of the most common genotoxic lesions and they can also arise as intermediates of DNA repair activity. Unrepaired DNA damage can lead to genomic instability, which can massively compromise the health status of organisms. Therefore it is important to measure and quantify DNA damage and its repair. We have previously published an automated method for measuring DNA strand breaks based on fluorimetric detection of alkaline DNA unwinding [1], and here we present a mathematical model of the FADU assay, which enables to an analytic expression for the relation between measured fluorescence and the number of strand breaks. Assessment of the formation and also the repair of DNA strand breaks is a crucial functional parameter to investigate genotoxicity in living cells. A reliable and convenient method to quantify DNA strand breakage is therefore of significant importance for a wide variety of scientific fields, e.g. toxicology, pharmacology, epidemiology and medical sciences

  5. Congenital encephalomyopathy and adult-onset myopathy and diabetes mellitus: Different phenotypic associations of a new heteroplasmic mtDNA tRNA glutamic acid mutation

    Energy Technology Data Exchange (ETDEWEB)

    Hanna, M.G.; Nelson, I.; Sweeney, M.G.; Cooper, J.M.; Watkins, P.J.; Morgan-Hughes, J.A.; Harding, A.E. [Kings College Hospital, London (United Kingdom)

    1995-05-01

    We report the clinical, biochemical, and molecular genetic findings in a family with an unusual mitochondrial disease phenotype harboring a novel mtDNA tRNA glutamic acid mutation at position 14709. The proband and his sister presented with congenital myopathy and mental retardation and subsequently developed cerebellar ataxia. Other family members had either adult-onset diabetes mellitus with muscle weakness or adult-onset diabetes mellitus alone. Ragged-red and cytochrome c oxidase (COX)-negative fibers were present in muscle biopsies. Biochemical studies of muscle mitochondria showed reduced complex I and IV activities. The mtDNA mutation was heteroplasmic in blood and muscle in all matrilineal relatives analyzed. Primary myoblast, but not fibroblast, cultures containing high proportions of mutant mtDNA exhibited impaired mitochondrial translation. These observations indicate that mtDNA tRNA point mutations should be considered in the differential diagnosis of congenital myopathy. In addition they illustrate the diversity of phenotypes associated with this mutation in the same family and further highlight the association between mtDNA mutations and diabetes mellitus. 43 refs., 4 figs., 1 tab.

  6. Genetic mutation analysis of HBV covalently closed circular DNA in peripheral blood mononuclear cells from chronic hepatitis B patients with nucleos(tide analog-resistant mutations in serum virions

    Directory of Open Access Journals (Sweden)

    Zhong-bin LI

    2012-06-01

    Full Text Available Objective  To analyze the characteristics of genetic mutations in reverse-transcriptase (RT domain of HBV covalently closed circular DNA (cccDNA in peripheral blood mononuclear cells (PBMCs obtained from chronic hepatitis B (CHB patients with drug-resistant mutations in serum virions during nucleoside/nucleotide analog (NA therapy. Methods  A total of 30 CHB patients admitted to 302 Hospital of PLA from July 2010 to August 2011 were included in this study. All the patients were confirmed to harbor the drug-resistant mutations in serum virions during an NA therapy longer than 6 months. Total DNA was extracted from PBMCs isolated from 30 whole blood samples at the same time point as that of serum analysis. Plasmid-safe ATP-dependent DNase (PSAD digestion in combination with rolling circle amplification and gap-spanning semi-nested PCR were used to amplify the RT region of HBV cccDNA. NA-resistant-associated mutations were analyzed at nine sites. Results  HBV cccDNA was efficiently amplified in 16 out of 30 (53.3% PBMC samples, and the detection rate was not correlated with HBeAg-positive rate, serum ALT level or HBV DNA load. Five of 16 (31.3% patients were sustained to have genotype B HBV infection, and 11 of 16 (68.8% were of genotype C HBV infection, and the result was consistent with the genotyping results using serum HBV. Different from drug-resistant mutations detected in the serum virions, the viruses detected in HBV cccDNA of 16 PBMC samples were all wild-type viruses without NA-resistant-associated mutations in RT region. Conclusions  During NA antiviral treatment, if drug-resistant mutations occur in serum HBV DNA of CHB patients, the dominant species of HBV cccDNA in PBMCs from the same patient is still the original wild-type strains. It is speculated that PBMCs might be the potential "repository" of HBV wild-type strain in vivo.

  7. Mitochondrial DNA point 13731 mutation in spinocerebellar ataxia%线粒体DNA13731点突变与脊髓小脑性共济失调相关性研究

    Institute of Scientific and Technical Information of China (English)

    王栋慧; 王进

    2009-01-01

    Objective To study the possible relationship between the point mutation in mitochondrial DNA (mtDNA) and the progression spinocerebellar ataxia(SCA).Methods Polymerase chain reaction(PCR) was used to amplify the mtDNA segments of these patients and their relatives individuals,40 volunteers. The mtDNA segment lied in the above mtDNA ND5 gene. For PCR products of rhe mtDNA segment,single strand conformation polymorphism(SSCP) was executed to detect mutations and the abnormal segments were sequenced.Results We had found a new mtDNA mutation in segments of mtDNA point 13731(T>C),was identified in 1 patient and 1 presymptomatic relatives.Conclusion A new point mutation of detected mitochondrial DNA may be lated to SCA.%目的 探索线粒体DNA(mtDNA)突变位点与脊髓小脑性共济失调(SCA)的关系.方法 采用聚合酶链反应(PCR)对基因确诊的四个SCA家系10例患者及其亲属共34例与40例健康对照的线粒体ND5基因片段进行扩增,扩增产物进行单链构象多态性分析(SSCP),对SSCP出现异常的样本进行相应mtDNA片段测序.结果 在一家系的1名确诊患者及1名症状前患者检测到mtDNA13731(T>C)点突变.结论 脊髓小脑性共济失调的发生、发展可能与mtDNA突变有关.

  8. DNA Variations in Oculocutaneous Albinism: An Updated Mutation List and Current Outstanding Issues in Molecular Diagnostics

    OpenAIRE

    Simeonov, Dimitre R.; Wang, Xinjing; Wang, Chen; Sergeev, Yuri; Dolinska, Monika; Bower, Matthew; Fischer, Roxanne; Winer, David; Dubrovsky, Genia; BALOG, JOAN Z.; Huizing, Marjan; Hart, Rachel; Zein, Wadih M.; William A Gahl; Brooks, Brian P.

    2013-01-01

    Oculocutaneous albinism (OCA) is a rare genetic disorder of melanin synthesis that results in hypopigmented hair, skin, and eyes. There are four types of OCA, caused by mutations in TYR (OCA-1), OCA2 (OCA-2), TYRP1 (OCA-3), or SLC45A2 (OCA-4). Here we report 22 novel mutations; 14 from a cohort of 61 patients seen as part of the NIH OCA Natural History Study and 8 from a prior study at the University of Minnesota. We also include a comprehensive list of almost 600 previously reported OCA muta...

  9. A filter paper-based microdevice for low-cost, rapid, and automated DNA extraction and amplification from diverse sample types.

    Science.gov (United States)

    Gan, Wupeng; Zhuang, Bin; Zhang, Pengfei; Han, Junping; Li, Cai-Xia; Liu, Peng

    2014-10-01

    A plastic microfluidic device that integrates a filter disc as a DNA capture phase was successfully developed for low-cost, rapid and automated DNA extraction and PCR amplification from various raw samples. The microdevice was constructed by sandwiching a piece of Fusion 5 filter, as well as a PDMS (polydimethylsiloxane) membrane, between two PMMA (poly(methyl methacrylate)) layers. An automated DNA extraction from 1 μL of human whole blood can be finished on the chip in 7 minutes by sequentially aspirating NaOH, HCl, and water through the filter. The filter disc containing extracted DNA was then taken out directly for PCR. On-chip DNA purification from 0.25-1 μL of human whole blood yielded 8.1-21.8 ng of DNA, higher than those obtained using QIAamp® DNA Micro kits. To realize DNA extraction from raw samples, an additional sample loading chamber containing a filter net with an 80 μm mesh size was designed in front of the extraction chamber to accommodate sample materials. Real-world samples, including whole blood, dried blood stains on Whatman® 903 paper, dried blood stains on FTA™ cards, buccal swabs, saliva, and cigarette butts, can all be processed in the system in 8 minutes. In addition, multiplex amplification of 15 STR (short tandem repeat) loci and Sanger-based DNA sequencing of the 520 bp GJB2 gene were accomplished from the filters that contained extracted DNA from blood. To further prove the feasibility of integrating this extraction method with downstream analyses, "in situ" PCR amplifications were successfully performed in the DNA extraction chamber following DNA purification from blood and blood stains without DNA elution. Using a modified protocol to bond the PDMS and PMMA, our plastic PDMS devices withstood the PCR process without any leakage. This study represents a significant step towards the practical application of on-chip DNA extraction methods, as well as the development of fully integrated genetic analytical systems.

  10. Automated extraction of DNA from blood and PCR setup using a Tecan Freedom EVO liquid handler for forensic genetic STR typing of reference samples

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Frøslev, Tobias G; Frank-Hansen, Rune;

    2011-01-01

    We have implemented and validated automated protocols for DNA extraction and PCR setup using a Tecan Freedom EVO liquid handler mounted with the Te-MagS magnetic separation device (Tecan, Männedorf, Switzerland). The protocols were validated for accredited forensic genetic work according to ISO...... 17025 using the Qiagen MagAttract DNA Mini M48 kit (Qiagen GmbH, Hilden, Germany) from fresh whole blood and blood from deceased individuals. The workflow was simplified by returning the DNA extracts to the original tubes minimizing the risk of misplacing samples. The tubes that originally contained...... the samples were washed with MilliQ water before the return of the DNA extracts. The PCR was setup in 96-well microtiter plates. The methods were validated for the kits: AmpFlSTR Identifiler, SGM Plus and Yfiler (Applied Biosystems, Foster City, CA), GenePrint FFFL and PowerPlex Y (Promega, Madison, WI...

  11. Ionizing radiation-induced mutation of human cells with different DNA repair capacities

    Energy Technology Data Exchange (ETDEWEB)

    Amundson, S.A.; Chen, D.J.

    1994-12-31

    We have observed significant differences in the response to ionizing radiation of two closely related human cell lines, and now compare the effects on these lines of both low and intermediate LET radiation. Compared to TK6, WTK1 has an enhanced X-ray survival, and is also more resistant to cell killing by {alpha}-particles. The hprt locus is more mutable in WTK1 than in TK6 by both X-rays and {alpha}-particles. WTK1 is also more mutable by {alpha}-particles than by X-rays at the hprt locus. X-ray-induced mutation at the heterozygous tk locus in WTK1 is about 25 fold higher than in TK6, while {alpha}-particle-induced mutation is nearly 50 fold higher at this locus. Also, the slowly growing tk- mutants, which comprise the majority of spontaneous and X-ray-induced tk- mutants of TK6, were not induced significantly by {alpha}-particles. Previously, we showed that TK6 has a reduced capacity for recombination compared with WTK1, and therefore, these results indicate that recombinational repair may contribute to both cell survival and mutation-induction following exposure to ionizing radiation. Such a mechanism may aid cell survival, but could also result in increased deleterious effects such as the unmasking of recessive mutations in cancer suppresser genes.

  12. Early cardiac involvement in children carrying the A3243G mtDNA mutation.

    NARCIS (Netherlands)

    Wortmann, S.B.; Rodenburg, R.J.T.; Backx, A.P.C.M.; Schmitt, E.; Smeitink, J.A.M.; Morava, E.

    2007-01-01

    The phenotypic spectrum of the mitochondrial A3243G DKA mutation is highly variable, particularly when occuring in childhood. In contrast to the classical presentation in adulthood (MELAS syndrome; mitochondria! myopathy, encephalopathy, lactic acidosis and stroke-like episodes) children show a diff

  13. Centrifugal LabTube platform for fully automated DNA purification and LAMP amplification based on an integrated, low-cost heating system.

    Science.gov (United States)

    Hoehl, Melanie M; Weißert, Michael; Dannenberg, Arne; Nesch, Thomas; Paust, Nils; von Stetten, Felix; Zengerle, Roland; Slocum, Alexander H; Steigert, Juergen

    2014-06-01

    This paper introduces a disposable battery-driven heating system for loop-mediated isothermal DNA amplification (LAMP) inside a centrifugally-driven DNA purification platform (LabTube). We demonstrate LabTube-based fully automated DNA purification of as low as 100 cell-equivalents of verotoxin-producing Escherichia coli (VTEC) in water, milk and apple juice in a laboratory centrifuge, followed by integrated and automated LAMP amplification with a reduction of hands-on time from 45 to 1 min. The heating system consists of two parallel SMD thick film resistors and a NTC as heating and temperature sensing elements. They are driven by a 3 V battery and controlled by a microcontroller. The LAMP reagents are stored in the elution chamber and the amplification starts immediately after the eluate is purged into the chamber. The LabTube, including a microcontroller-based heating system, demonstrates contamination-free and automated sample-to-answer nucleic acid testing within a laboratory centrifuge. The heating system can be easily parallelized within one LabTube and it is deployable for a variety of heating and electrical applications.

  14. Somatic point mutations in mtDNA control region are influenced by genetic background and associated with healthy aging: a GEHA study

    DEFF Research Database (Denmark)

    Rose, Giuseppina; Romeo, Giuseppe; Dato, Serena;

    2010-01-01

    Tissue specific somatic mutations occurring in the mtDNA control region have been proposed to provide a survival advantage. Data on twins and on relatives of long-lived subjects suggested that the occurrence/accumulation of these mutations may be genetically influenced. To further investigate....... We found a significant correlation of the mtDNA control region heteroplasmy between sibs, confirming a genetic influence on this phenomenon. Furthermore, many subjects showed heteroplasmy due to mutations different from the C150T transition. In these cases heteroplasmy was correlated within sibpairs...... in Finnish and northern Italian samples, but not in southern Italians. This suggested that the genetic contribution to control region mutations may be population specific. Finally, we observed a possible correlation between heteroplasmy and Hand Grip strength, one of the best markers of physical performance...

  15. Difficult diagnosis of the fragile X syndrome made possible by direct detection of DNA mutations.

    OpenAIRE

    Tarleton, J; Wong, S.; Heitz, D.; Schwartz, C.

    1992-01-01

    Genetic recombination near the fragile X locus (Xq27.3) has frequently been a problem in linkage studies of families in which the fragile X is segregating. This case report illustrates the resolution of a difficult situation in a fragile X family for whom cytogenetic studies were inconclusive and where recombination had twice confounded attempts at prenatal DNA diagnosis by RFLP analysis. Using a newly developed DNA probe, StB12.3, for direct detection of DNA instability in the fragile X locu...

  16. A case of neuromyelitis optica harboring both anti-aquaporin-4 antibodies and a pathogenic mitochondrial DNA mutation for Leber's hereditary optic neuropathy.

    Science.gov (United States)

    Shiraishi, Wataru; Hayashi, Shintaro; Kamada, Takashi; Isobe, Noriko; Yamasaki, Ryo; Murai, Hiroyuki; Ohyagi, Yasumasa; Kira, Jun-ichi

    2014-02-01

    We report the first case of definite neuromyelitis optica (NMO) with a pathogenic mitochondrial DNA (mtDNA) mutation for Leber's hereditary optic neuropathy (LHON) (G11778A point mutation). A 36-year-old Japanese woman had experienced recurrent neurological symptoms originating from involvements of the optic nerves and spinal cord. She finally lost her bilateral vision, and spastic paraparesis and sensory disturbances below the T6 level remained despite intensive immunotherapies. Brain and spinal magnetic resonance imaging (MRI) revealed T2-high-intensity lesions in the optic nerves and thoracic spinal cord, but no lesions in the brain. A blood examination revealed positivity for both anti-aquaproin-4 antibodies and an LHON mtDNA mutation.

  17. STN1 OB Fold Mutation Alters DNA Binding and Affects Selective Aspects of CST Function

    Science.gov (United States)

    Bhattacharjee, Anukana; Stewart, Jason; Chaiken, Mary; Price, Carolyn M.

    2016-01-01

    Mammalian CST (CTC1-STN1-TEN1) participates in multiple aspects of telomere replication and genome-wide recovery from replication stress. CST resembles Replication Protein A (RPA) in that it binds ssDNA and STN1 and TEN1 are structurally similar to RPA2 and RPA3. Conservation between CTC1 and RPA1 is less apparent. Currently the mechanism underlying CST action is largely unknown. Here we address CST mechanism by using a DNA-binding mutant, (STN1 OB-fold mutant, STN1-OBM) to examine the relationship between DNA binding and CST function. In vivo, STN1-OBM affects resolution of endogenous replication stress and telomere duplex replication but telomeric C-strand fill-in and new origin firing after exogenous replication stress are unaffected. These selective effects indicate mechanistic differences in CST action during resolution of different replication problems. In vitro binding studies show that STN1 directly engages both short and long ssDNA oligonucleotides, however STN1-OBM preferentially destabilizes binding to short substrates. The finding that STN1-OBM affects binding to only certain substrates starts to explain the in vivo separation of function observed in STN1-OBM expressing cells. CST is expected to engage DNA substrates of varied length and structure as it acts to resolve different replication problems. Since STN1-OBM will alter CST binding to only some of these substrates, the mutant should affect resolution of only a subset of replication problems, as was observed in the STN1-OBM cells. The in vitro studies also provide insight into CST binding mechanism. Like RPA, CST likely contacts DNA via multiple OB folds. However, the importance of STN1 for binding short substrates indicates differences in the architecture of CST and RPA DNA-protein complexes. Based on our results, we propose a dynamic DNA binding model that provides a general mechanism for CST action at diverse forms of replication stress. PMID:27690379

  18. Heart tissue of harlequin (hq)/Big Blue mice has elevated reactive oxygen species without significant impact on the frequency and nature of point mutations in nuclear DNA

    Energy Technology Data Exchange (ETDEWEB)

    Crabbe, Rory A. [Department of Biology, University of Western Ontario, London, Ontario, N6A 5B7 (Canada); Hill, Kathleen A., E-mail: khill22@uwo.ca [Department of Biology, University of Western Ontario, London, Ontario, N6A 5B7 (Canada)

    2010-09-10

    Age is a major risk factor for heart disease, and cardiac aging is characterized by elevated mitochondrial reactive oxygen species (ROS) with compromised mitochondrial and nuclear DNA integrity. To assess links between increased ROS levels and mutations, we examined in situ levels of ROS and cII mutation frequency, pattern and spectrum in the heart of harlequin (hq)/Big Blue mice. The hq mouse is a model of premature aging with mitochondrial dysfunction and increased risk of oxidative stress-induced heart disease with the means for in vivo mutation detection. The hq mutation produces a significant downregulation in the X-linked apoptosis-inducing factor gene (Aif) impairing both the antioxidant and oxidative phosphorylation functions of AIF. Brain and skin of hq disease mice have elevated frequencies of point mutations in nuclear DNA and histopathology characterized by cell loss. Reports of associated elevations in ROS in brain and skin have mixed results. Herein, heart in situ ROS levels were elevated in hq disease compared to AIF-proficient mice (p < 0.0001) yet, mutation frequency and pattern were similar in hq disease, hq carrier and AIF-proficient mice. Heart cII mutations were also assessed 15 days following an acute exposure to an exogenous ROS inducer (10 mg paraquat/kg). Acute paraquat exposure with a short mutant manifestation period was insufficient to elevate mutation frequency or alter mutation pattern in the post-mitotic heart tissue of AIF-proficient mice. Paraquat induction of ROS requires mitochondrial complex I and thus is likely compromised in hq mice. Results of this preliminary survey and the context of recent literature suggest that determining causal links between AIF deficiency and the premature aging phenotypes of specific tissues is better addressed with assay of mitochondrial ROS and large-scale changes in mitochondrial DNA in specific cell types.

  19. Rapid detection of ERG11 gene mutations in clinical Candida albicans isolates with reduced susceptibility to fluconazole by rolling circle amplification and DNA sequencing

    Directory of Open Access Journals (Sweden)

    Ellis David

    2009-08-01

    Full Text Available Abstract Background Amino acid substitutions in the target enzyme Erg11p of azole antifungals contribute to clinically-relevant azole resistance in Candida albicans. A simple molecular method for rapid detection of ERG11 gene mutations would be an advantage as a screening tool to identify potentially-resistant strains and to track their movement. To complement DNA sequencing, we developed a padlock probe and rolling circle amplification (RCA-based method to detect a series of mutations in the C. albicans ERG11 gene using "reference" azole-resistant isolates with known mutations. The method was then used to estimate the frequency of ERG11 mutations and their type in 25 Australian clinical C. albicans isolates with reduced susceptibility to fluconazole and in 23 fluconazole-susceptible isolates. RCA results were compared DNA sequencing. Results The RCA assay correctly identified all ERG11 mutations in eight "reference" C. albicans isolates. When applied to 48 test strains, the RCA method showed 100% agreement with DNA sequencing where an ERG11 mutation-specific probe was used. Of 20 different missense mutations detected by sequencing in 24 of 25 (96% isolates with reduced fluconazole susceptibility, 16 were detected by RCA. Five missense mutations were detected by both methods in 18 of 23 (78% fluconazole-susceptible strains. DNA sequencing revealed that mutations in non-susceptible isolates were all due to homozygous nucleotide changes. With the exception of the mutations leading to amino acid substitution E266D, those in fluconazole-susceptible strains were heterozygous. Amino acid substitutions common to both sets of isolates were D116E, E266D, K128T, V437I and V488I. Substitutions unique to isolates with reduced fluconazole susceptibility were G464 S (n = 4 isolates, G448E (n = 3, G307S (n = 3, K143R (n = 3 and Y123H, S405F and R467K (each n = 1. DNA sequencing revealed a novel substitution, G450V, in one isolate. Conclusion The sensitive RCA

  20. Genetic signatures from amplification profiles characterize DNA mutation in somatic and radiation-induced sports of chrysanthemum

    International Nuclear Information System (INIS)

    The chrysanthemum (Dendranthema grandiflora Tzvelev.) cultivars 'Dark Charm', 'Salmon Charm', 'Coral Charm' and 'Dark Bronze Charm' are either radiation-induced mutants or spontaneous sports of 'Charm' and constitute a family or series of plants that primarily differ in flower color. These cultivars, which were difficult to differentiate genetically by DNA amplification fingerprinting (DAF), were easily identified by using arbitrary signatures from amplification profiles (ASAP). Genomic DNA was first amplified with three standard octamer arbitrary primers, all of which produced monomorphic profiles. Products from each of these DNA fingerprints were subsequently reamplified using four minihairpin decamer primers. The 12 primer combinations produced signatures containing approximately 37% polymorphic character loci, which were used to estimate genetic relationships between cultivars. Forty-six (32%) unique amplification products were associated with individual cultivars. The number of ASAP polymorphisms detected provided an estimate of the mutation rate in the mutant cultivars, ranging from 0.03% to 1.6% of nucleotide changes within an average of 18 kb of arbitrary amplified DAF sequence. The ASAP technique permits the clear genetic identification of somatic mutants and radiation-induced sports that are genetically highly homogeneous and should facilitate marker assisted breeding and protection of plant breeders rights of varieties or cultivars

  1. Alternative mechanisms of telomere lengthening: permissive mutations, DNA repair proteins and tumorigenic progression

    OpenAIRE

    Sandy Gocha1, April Renee; Harris, Julia; Groden, Joanna

    2012-01-01

    Telomeres protect chromosome termini to maintain genomic stability and regulate cellular lifespan. Maintenance of telomere length is required for neoplastic cells after the acquisition of mutations that deregulate cell cycle control and increase cellular proliferation, and can occur through expression of the enzyme telomerase or in a telomerase-independent manner termed alternative lengthening of telomeres (ALT). The precise mechanisms that govern the activation of ALT or telomerase in tumor ...

  2. Separating the effects of mutation and selection in producing DNA skew in bacterial chromosomes

    Directory of Open Access Journals (Sweden)

    Morton Brian R

    2007-10-01

    Full Text Available Abstract Background Many bacterial chromosomes display nucleotide asymmetry, or skew, between the leading and lagging strands of replication. Mutational differences between these strands result in an overall pattern of skew that is centered about the origin of replication. Such a pattern could also arise from selection coupled with a bias for genes coded on the leading strand. The relative contributions of selection and mutation in producing compositional skew are largely unknown. Results We describe a model to quantify the contribution of mutational differences between the leading and lagging strands in producing replication-induced skew. When the origin and terminus of replication are known, the model can be used to estimate the relative accumulation of G over C and of A over T on the leading strand due to replication effects in a chromosome with bidirectional replication arms. The model may also be implemented in a maximum likelihood framework to estimate the locations of origin and terminus. We find that our estimations for the origin and terminus agree very well with the location of genes that are thought to be associated with the replication origin. This indicates that our model provides an accurate, objective method of determining the replication arms and also provides support for the hypothesis that these genes represent an ancestral cluster of origin-associated genes. Conclusion The model has several advantages over other methods of analyzing genome skew. First, it quantifies the role of mutation in generating skew so that its effect on composition, for example codon bias, can be assessed. Second, it provides an objective method for locating origin and terminus, one that is based on chromosome-wide accumulation of leading vs lagging strand nucleotide differences. Finally, the model has the potential to be utilized in a maximum likelihood framework in order to analyze the effect of chromosome rearrangements on nucleotide composition.

  3. A DNA polymerase mutation that suppresses the segregation bias of an ARS plasmid in Saccharomyces cerevisiae.

    OpenAIRE

    Houtteman, S W; Elder, R T

    1993-01-01

    Yeast autonomously replicating sequence (ARS) plasmids exhibit an unusual segregation pattern during mitosis. While the nucleus divides equally into mother and daughter cells, all copies of the ARS plasmid will often remain in the mother cell. A screen was designed to isolate mutations that suppress this segregation bias. A plasmid with a weak ARS (wARS) that displayed an extremely high segregation bias was constructed. When cells were grown under selection for the wARS plasmid, the resulting...

  4. Specific UV-induced mutation spectrum in the p53 gene of skin tumors from DNA-repair-deficient xeroderma pigmentosum patients

    International Nuclear Information System (INIS)

    The UV component of sunlight is the major carcinogen involved in the etiology of skin cancers. The authors have studied the rare, hereditary syndrome xeroderma pigmentosum (XP), which is characterized by a very high incidence of cutaneous tumors on exposed skin at an early age, probably due to a deficiency in excision repair of UV-induced lesions. It is interesting to determine the UV mutation spectrum in XP skin tumors in order to correlate the absence of repair of specific DNA lesions and the initiation of skin tumors. The p53 gene is frequently mutated in human cancers and represents a good target for studying mutation spectra since there are >100 potential sites for phenotypic mutations. Using reverse transcription-PCR and single-strand conformation polymorphism to analyze >40 XP skin tumors (mainly basal and squamous cell carcinomas), the authors have found that 40% (17 out of 43) contained at least one point mutation on the p53 gene. All the mutations were located at dipyrimidine sites, essentially at CC sequences, which are hot spots for UV-induced DNA lesions. Sixty-one percent of these mutations were tandem CC → TT mutations considered to be unique to UV-induced lesions; these mutations are not observed in internal human tumors. All the mutations, except two, must be due to translesion synthesis of unrepaired dipyrimidine lesions left on the nontranscribed strand. These results show the existence of preferential repair of UV lesions [either pyrimidine dimers or pyrimidine-pyrimidone (6-4) photoproducts] on the transcribed strand in human tissues

  5. Interfacing click chemistry with automated oligonucleotide synthesis for the preparation of fluorescent DNA probes containing internal xanthene and cyanine dyes.

    Science.gov (United States)

    Astakhova, I Kira; Wengel, Jesper

    2013-01-14

    Double-labeled oligonucleotide probes containing fluorophores interacting by energy-transfer mechanisms are essential for modern bioanalysis, molecular diagnostics, and in vivo imaging techniques. Although bright xanthene and cyanine dyes are gaining increased prominence within these fields, little attention has thus far been paid to probes containing these dyes internally attached, a fact which is mainly due to the quite challenging synthesis of such oligonucleotide probes. Herein, by using 2'-O-propargyl uridine phosphoramidite and a series of xanthenes and cyanine azide derivatives, we have for the first time performed solid-phase copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click labeling during the automated phosphoramidite oligonucleotide synthesis followed by postsynthetic click reactions in solution. We demonstrate that our novel strategy is rapid and efficient for the preparation of novel oligonucleotide probes containing internally positioned xanthene and cyanine dye pairs and thus represents a significant step forward for the preparation of advanced fluorescent oligonucleotide probes. Furthermore, we demonstrate that the novel xanthene and cyanine labeled probes display unusual and very promising photophysical properties resulting from energy-transfer interactions between the fluorophores controlled by nucleic acid assembly. Potential benefits of using these novel fluorescent probes within, for example, molecular diagnostics and fluorescence microscopy include: Considerable Stokes shifts (40-110 nm), quenched fluorescence of single-stranded probes accompanied by up to 7.7-fold light-up effect of emission upon target DNA/RNA binding, remarkable sensitivity to single-nucleotide mismatches, generally high fluorescence brightness values (FB up to 26), and hence low limit of target detection values (LOD down to <5 nM).

  6. Semi-automated high-throughput fluorescent intercalator displacement-based discovery of cytotoxic DNA binding agents from a large compound library.

    Science.gov (United States)

    Glass, Lateca S; Bapat, Aditi; Kelley, Mark R; Georgiadis, Millie M; Long, Eric C

    2010-03-01

    High-throughput fluorescent intercalator displacement (HT-FID) was adapted to the semi-automated screening of a commercial compound library containing 60,000 molecules resulting in the discovery of cytotoxic DNA-targeted agents. Although commercial libraries are routinely screened in drug discovery efforts, the DNA binding potential of the compounds they contain has largely been overlooked. HT-FID led to the rapid identification of a number of compounds for which DNA binding properties were validated through demonstration of concentration-dependent DNA binding and increased thermal melting of A/T- or G/C-rich DNA sequences. Selected compounds were assayed further for cell proliferation inhibition in glioblastoma cells. Seven distinct compounds emerged from this screening procedure that represent structures unknown previously to be capable of targeting DNA leading to cell death. These agents may represent structures worthy of further modification to optimally explore their potential as cytotoxic anti-cancer agents. In addition, the general screening strategy described may find broader impact toward the rapid discovery of DNA targeted agents with biological activity.

  7. Study on 4977-bp deletion mutation of mitochondrial DNA in lung cancer

    Institute of Scientific and Technical Information of China (English)

    DAI Ji-gang; XIAO Ying-bin; MIN Jia-xin; ZHANG Guo-qiang; YAO Ke; ZHOU Ren-jie

    2005-01-01

    Objective: To study the 4977-bp deletion of mitochondiral DNA in lung cancer, adjacent normal tissue and health lung and its significance in the development of cancer. Methods: Thirty-seven matched lung cancer/adjacent histologically normal and 20 "true" normal lung tissue samples from patients without lung cancer were analyzed by long PCR technique. Results: Mitochondrial DNA 4977-bp deletion was detected in 54. 1% (20/37) of lung cancers, 59.5% (22/37) of adjacent normal and 30.0% (6/30) of"true" normal lung tissues. The correlation of 4977-bp deletion with age and smoking factors was present in our data. Conclusion: Mitochondrial DNA 4977-bp deletion is not specific to lung cancer and unlikely to play an important role in carcinogenesis, and may only reflect the environmental and genetic influences during tumor progression.

  8. Strikingly different penetrance of LHON in two Chinese families with primary mutation G11778A is independent of mtDNA haplogroup background and secondary mutation G13708A.

    Science.gov (United States)

    Wang, Hua-Wei; Jia, Xiaoyun; Ji, Yanli; Kong, Qing-Peng; Zhang, Qingjiong; Yao, Yong-Gang; Zhang, Ya-Ping

    2008-08-25

    The penetrance of Leber's hereditary optic neuropathy (LHON) in families with primary mitochondrial DNA (mtDNA) mutations is very complex. Matrilineal and nuclear genetic background, as well as environmental factors, have been reported to be involved in different affected pedigrees. Here we describe two large Chinese families that show a striking difference in the penetrance of LHON, in which 53.3% and 15.0% of members were affected (PLHON in the two Chinese families is independent of both their mtDNA haplotype background and a secondary mutation G13708A. As a result, it is likely that unknown nuclear gene involvement and/or other factors contribute to the strikingly different penetrance of LHON.

  9. Prevalence of mutations in HBV DNA polymerase gene associated with nucleos(tide resistance in treatment-naive patients with Chronic Hepatitis B in Central China

    Directory of Open Access Journals (Sweden)

    Youyun Zhao

    2016-04-01

    Full Text Available Abstract Objective There are a lot of disagreements in the studies on hepatitis B virus (HBV DNA polymerase mutation rate associated with nucleos(tide analogues (NAs in treatment-naive chronic hepatitis B (CHB patients. This is the first study aimed to investigate the prevalence of spontaneous HBV resistance mutations in Central China. Methods This study included treatment-naive patients with CHB from June 2012 to May 2015 receiving care at the Institute of Liver Disease in Central China. All patients completed a questionnaire covering different aspects, such as family medical history, course of liver disease, medication history, alcohol use, among others. Mutations in HBV DNA polymerase associated with NAs resistance were detected using INNO-LiPA assay. Results 269 patients were infected with HBV genotype B (81.4%, C (17.9%, and both B and C (0.7%. Mutations in HBV DNA polymerase were detected in 24 patients (8.9% including rtM204I/V (n = 6, rtN236T (n = 5, rtM250V (n = 2, rtL180M (n = 2, rtT184G (n = 1, rtM207I (n = 1, rtS202I (n = 1, rtM204V/I & rtL180M (n = 5, and rtM204I & rtM250V (n = 1. Conclusion Spontaneous HBV resistance mutations in HBV DNA polymerase were found in treatment-naive patients with CHB in Central China. These findings suggest that we should analyze HBV DNA polymerase resistance mutation associated with NAs before giving antiviral therapy such as lamivudine (LAM, adefovir (ADV, and telbivudine (LdT.

  10. Structure-guided mutational analysis of the OB, HhH, and BRCT domains of Escherichia coli DNA ligase.

    Science.gov (United States)

    Wang, Li Kai; Nair, Pravin A; Shuman, Stewart

    2008-08-22

    NAD(+)-dependent DNA ligases (LigAs) are ubiquitous in bacteria and essential for growth. LigA enzymes have a modular structure in which a central catalytic core composed of nucleotidyltransferase and oligonucleotide-binding (OB) domains is linked via a tetracysteine zinc finger to distal helix-hairpin-helix (HhH) and BRCT (BRCA1-like C-terminal) domains. The OB and HhH domains contribute prominently to the protein clamp formed by LigA around nicked duplex DNA. Here we conducted a structure-function analysis of the OB and HhH domains of Escherichia coli LigA by alanine scanning and conservative substitutions, entailing 43 mutations at 22 amino acids. We thereby identified essential functional groups in the OB domain that engage the DNA phosphodiester backbone flanking the nick (Arg(333)); penetrate the minor grove and distort the nick (Val(383) and Ile(384)); or stabilize the OB fold (Arg(379)). The essential constituents of the HhH domain include: four glycines (Gly(455), Gly(489), Gly(521), Gly(553)), which bind the phosphate backbone across the minor groove at the outer margins of the LigA-DNA interface; Arg(487), which penetrates the minor groove at the outer margin on the 3 (R)-OH side of the nick; and Arg(446), which promotes protein clamp formation via contacts to the nucleotidyltransferase domain. We find that the BRCT domain is required in its entirety for effective nick sealing and AMP-dependent supercoil relaxation.

  11. 线粒体DNA突变合并肾脏损害%Mitochondrial DNA mutation and renal involvement

    Institute of Scientific and Technical Information of China (English)

    谢红浪; 许书添; 何群鹏; 杨柳; 郭锦洲; 桂兰兰; 陈惠萍; 刘志红

    2012-01-01

    目的:探讨3例线粒体病合并慢性肾脏病(CKD)患者的临床特点及预后. 方法:2011年5月至2012年9月在南京军区南京总医院全军肾脏病研究所住院的CKD患者中共3例经线粒体DNA(mtDNA)基因检测确诊线粒体病,分析其肾外受累及肾脏损害的临床表现和肾活检病理特点,并观察其预后. 结果:(1)3例患者中2例青少年(14岁),另1例30岁,全部患者均消瘦(体质量指数13 ~ 17 kg/m2).例1生长发育迟缓,例3为早产儿.例1、例2有糖尿病家族史,例2有尿毒症家族史.(2)3例患者病程3~7月,临床均表现为水肿、蛋白尿和胱抑素C增高,肾小管损伤明显,均无高血压和镜下血尿.例1和例3组织学示局灶节段性肾小球硬化(FSGS),例2肾小球轻度系膜增生伴小动脉节段透明变性.(3)3例患者中糖尿病(2例)、高乳酸血症(2例)、听力消失或下降(3例)、癫痫发作(1例)、脑梗塞(1例)、智力障碍(2例)、视野缺失(1例),心律失常(1例).(4)3例患者均经基因测序并证实mtDNA 3243 A>G突变位点,例2母亲和弟弟也检出与患者相同突变位点.(5)经治疗3例均停免疫抑制剂,补充辅酶Q10和左卡尼汀等治疗,蛋白尿部分缓解,血清肌酐稳定. 结论:本文首次在国内报道3例伴肾脏损害的线粒体病患者,提示这类患者并非罕见,临床除肾脏受累外均伴明显的心脏和中枢神经系统损害.临床医师应加强对此类疾病的认识.%Objective : Mitochondrial cytopathy is a heterogeneous disease with multiple organ system involvement. We report 3 mitochondrial cytopathy patients with mitochondrial DNA (mtDNA) point mutation and renal involvement. Methodology: Clinical feature and family history were collected. Renal biopsy was performed. DNA was isolated from peripheral blood leukocytes of the patients. Polymerase chain reaction was performed to amplify the mtDNA. Sequencing analysis was used to detect the presence of any point mutation of mtDNA

  12. Evaluation of AgClNPs@SBA-15/IL nanoparticle-induced oxidative stress and DNA mutation in Escherichia coli.

    Science.gov (United States)

    Karimi, Farrokh; Dabbagh, Somayyeh; Alizadeh, Sina; Rostamnia, Sadegh

    2016-08-01

    The bactericidal effects of silver nanoparticles have been demonstrated in the past years. Recently, the new antimicrobial compounds of silver nanoparticles with different formulations have been developed. In this work, AgClNPs@SBA-15/IL as a new compound of Ag nanoparticles, was synthesized and characterized by XRD, TEM, SEM, FTIR, and EDX. The antibacterial activity and the molecular mechanism effects of AgClNPs@SBA-15/IL nanoparticles (SNPs) on Escherichia coli DH5α cells were investigated by analyzing the growth inhibitory, H2O2 level, catalase activity, DNA mutation, and plasmid copy number following treatment with AgClNPs@SBA-15/IL nanoparticles. In experimental results, the minimum inhibitory concentration (MIC) was observed in 75 μg/ml and the antibacterial efficacy (ABE) in CFU analysis was estimated 95.3 %. In bacterial cells treated with 75 and 100 μg/ml, H2O2 level significantly increased and catalase activity decreased compared with control. The random amplified polymorphic DNA (RAPD) was used to evaluate the effect of AgClNPs@SBA-15/IL nanoparticles in DNA damages and mutation in E. coli genome. RADP-PCR results indicated different banding patterns including appearance or disappearance of bands and differences in their intensity. Cluster analysis of the RAPD-PCR results based on genetic similarity showed genetic difference between E. coli cells treated with AgClNPs@SBA-15/IL nanoparticles, and control and phylogenetic tree were divided to two clusters. Plasmid copy number analysis indicated that after 8 h incubation of E. coli cells with 50, 75, and 100 μg/ml AgClNPs@SBA-15/IL nanoparticles, copy number of pET21a (+) significantly decreased compared with control which indicating DNA replication inhibition by Ag nanoparticles. In conclusion, the results of this study indicated that AgClNPs@SBA-15/IL nanoparticles can be used as an effective bactericidal agent against bacterial cells. PMID:27209037

  13. DNA methylation and somatic mutations converge on cell cycle and define similar evolutionary histories in brain tumors

    Science.gov (United States)

    Johnson, Brett E.; Hong, Chibo; Hamilton, Emily G.; Bell, Robert J.A.; Smirnov, Ivan V.; Reis, Gerald F.; Phillips, Joanna J.; Barnes, Michael J.; Idbaih, Ahmed; Alentorn, Agusti; Kloezeman, Jenneke J.; Lamfers, Martine L. M.; Bollen, Andrew W.; Taylor, Barry S.; Molinaro, Annette M.; Olshen, Adam B.; Chang, Susan M.; Song, Jun S.; Costello, Joseph F.

    2015-01-01

    Summary The evolutionary history of tumor cell populations can be reconstructed from patterns of genetic alterations. In contrast to stable genetic events, epigenetic states are reversible and sensitive to the microenvironment, prompting the question whether epigenetic information can similarly be used to discover tumor phylogeny. We examined the spatial and temporal dynamics of DNA methylation in a cohort of low-grade gliomas and their patient-matched recurrences. Genes transcriptionally upregulated through promoter hypomethylation during malignant progression to high-grade glioblastoma were enriched in cell cycle function, evolving in parallel with genetic alterations that deregulate the G1/S cell cycle checkpoint. Moreover, phyloepigenetic relationships robustly recapitulated phylogenetic patterns inferred from somatic mutations. These findings highlight widespread co-dependency of genetic and epigenetic events throughout brain tumor evolution. PMID:26373278

  14. DNA Methylation and Somatic Mutations Converge on the Cell Cycle and Define Similar Evolutionary Histories in Brain Tumors.

    Science.gov (United States)

    Mazor, Tali; Pankov, Aleksandr; Johnson, Brett E; Hong, Chibo; Hamilton, Emily G; Bell, Robert J A; Smirnov, Ivan V; Reis, Gerald F; Phillips, Joanna J; Barnes, Michael J; Idbaih, Ahmed; Alentorn, Agusti; Kloezeman, Jenneke J; Lamfers, Martine L M; Bollen, Andrew W; Taylor, Barry S; Molinaro, Annette M; Olshen, Adam B; Chang, Susan M; Song, Jun S; Costello, Joseph F

    2015-09-14

    The evolutionary history of tumor cell populations can be reconstructed from patterns of genetic alterations. In contrast to stable genetic events, epigenetic states are reversible and sensitive to the microenvironment, prompting the question whether epigenetic information can similarly be used to discover tumor phylogeny. We examined the spatial and temporal dynamics of DNA methylation in a cohort of low-grade gliomas and their patient-matched recurrences. Genes transcriptionally upregulated through promoter hypomethylation during malignant progression to high-grade glioblastoma were enriched in cell cycle function, evolving in parallel with genetic alterations that deregulate the G1/S cell cycle checkpoint. Moreover, phyloepigenetic relationships robustly recapitulated phylogenetic patterns inferred from somatic mutations. These findings highlight widespread co-dependency of genetic and epigenetic events throughout brain tumor evolution. PMID:26373278

  15. Malignant chondroblastoma presenting as a recurrent pelvic tumor with DNA aneuploidy and p53 mutation as supportive evidence of malignancy

    Energy Technology Data Exchange (ETDEWEB)

    Ostrowski, M.L. [Department of Pathology and Laboratory Medicine, Baylor College of Medicine, The Methodist Hospital and Texas Children' s Hospital, Houston, Texas (United States); Department of Pathology and Laboratory Medicine, Houston, TX (United States). Methodist Hospital; Johnson, M.E. [Department of Orthopedic Surgery, Baylor College of Medicine, The Methodist Hospital and Texas Children' s Hospital, Houston, Texas (United States); Truong, L.D.; Hicks, M.J.; Spjut, H.J. [Department of Pathology and Laboratory Medicine, Baylor College of Medicine, The Methodist Hospital and Texas Children' s Hospital, Houston, Texas (United States); Smith, F.E. [Department of Oncology, Baylor College of Medicine, The Methodist Hospital and Texas Children' s Hospital, Houston, Texas (United States)

    1999-11-01

    We report a rare case of malignant chondroblastoma, which presented in a 47-year-old man as a recurrent tumor, 18 years following wide excision of a typical pelvic chondroblastoma. Radiologic studies of the recurrent tumor showed a large, lytic, destructive lesion of the right pelvic bones and femur, with a pathologic fracture of the latter, a large pelvic soft tissue mass, and multiple pulmonary metastases. Biopsy tissue showed typical features of chondroblastoma, but also increased nuclear atypia, hyperchromasia, and pleomorphism, compared to the original tumor, and, most significantly, abnormal mitotic figures. Immunohistochemical studies of the recurrent tumor revealed p53 mutation and extensive proliferative activity, and flow cytometric studies showed DNA aneuploidy, none of which was present in the original tumor. The patient received chemotherapy and radiation, but died of disease eight months after presentation. We also review chondroblastoma in general, to assign this unusual lesion to a tumor subtype. (orig.)

  16. Obtaining insurance after DNA diagnostics : A survey among hypertrophic cardiomyopathy mutation carriers

    NARCIS (Netherlands)

    Christiaans, Imke; Kok, Tjitske M.; Van Langen, Irene M.; Birnie, Erwin; Bonsel, Gouke J.; Wilde, Arthur A. M.; Smets, Ellen M. A.

    2010-01-01

    Hypertrophic cardiomyopathy (HCM) is a common hereditary heart disease associated with increased mortality. Disclosure of DNA test results may have social implications such as low access to insurance. In the Netherlands, insurance companies are restricted in the use of genetic information of their c

  17. Obtaining insurance after DNA diagnostics: a survey among hypertrophic cardiomyopathy mutation carriers

    NARCIS (Netherlands)

    I. Christiaans; T.M. Kok; I.M. van Langen; E. Birnie; G.J. Bonsel; A.A.M. Wilde; E.M.A. Smets

    2010-01-01

    Hypertrophic cardiomyopathy (HCM) is a common hereditary heart disease associated with increased mortality. Disclosure of DNA test results may have social implications such as low access to insurance. In the Netherlands, insurance companies are restricted in the use of genetic information of their c

  18. Desmin common mutation is associated with multi-systemic disease manifestations and depletion of mitochondria and mitochondrial DNA

    Directory of Open Access Journals (Sweden)

    Elizabeth eMcCormick

    2015-06-01

    Full Text Available Desmin (DES is a major muscle scaffolding protein that also functions to anchor mitochondria. Pathogenic DES mutations, however, have not previously been recognized as a cause of multi-systemic mitochondrial disease. Here, we describe a 45-year-old man who presented to The Children’s Hospital of Philadelphia Mitochondrial-Genetics Diagnostic Clinic for evaluation of progressive cardiac, neuromuscular, gastrointestinal, and mood disorders. Muscle biopsy at age 45 was remarkable for cytoplasmic bodies, as well as ragged red fibers and SDH positive/COX negative fibers that were suggestive of a mitochondrial myopathy. Muscle also showed significant reductions in mitochondrial content (16% of control mean for citrate synthase activity and mitochondrial DNA (35% of control mean. His family history was significant for cardiac conduction defects and myopathy in multiple maternal relatives. Multiple single gene and panel-based sequencing studies were unrevealing. Whole exome sequencing identified a known pathogenic p.S13F mutation in DES that had previously been associated with desmin-related myopathy. Desmin-related myopathy is an autosomal dominant disorder characterized by right ventricular hypertrophic cardiomyopathy, myopathy, and arrhythmias. However, neuropathy, gastrointestinal dysfunction, and depletion of both mitochondria and mitochondrial DNA have not previously been widely recognized in this disorder. Recognition that mitochondrial dysfunction occurs in desmin-related myopathy clarifies the basis for the multi-systemic manifestations, as are typical of primary mitochondrial disorders. Understanding the mitochondrial pathophysiology of desmin-related myopathy highlights the possibility of new therapies for the otherwise untreatable and often fatal class of disease. We postulate that drug treatments aimed at improving mitochondrial biogenesis or reducing oxidative stress may be effective therapies to ameliorate the effects of desmin

  19. PhyloBot: A Web Portal for Automated Phylogenetics, Ancestral Sequence Reconstruction, and Exploration of Mutational Trajectories

    Science.gov (United States)

    Hanson-Smith, Victor; Johnson, Alexander

    2016-01-01

    The method of phylogenetic ancestral sequence reconstruction is a powerful approach for studying evolutionary relationships among protein sequence, structure, and function. In particular, this approach allows investigators to (1) reconstruct and “resurrect” (that is, synthesize in vivo or in vitro) extinct proteins to study how they differ from modern proteins, (2) identify key amino acid changes that, over evolutionary timescales, have altered the function of the protein, and (3) order historical events in the evolution of protein function. Widespread use of this approach has been slow among molecular biologists, in part because the methods require significant computational expertise. Here we present PhyloBot, a web-based software tool that makes ancestral sequence reconstruction easy. Designed for non-experts, it integrates all the necessary software into a single user interface. Additionally, PhyloBot provides interactive tools to explore evolutionary trajectories between ancestors, enabling the rapid generation of hypotheses that can be tested using genetic or biochemical approaches. Early versions of this software were used in previous studies to discover genetic mechanisms underlying the functions of diverse protein families, including V-ATPase ion pumps, DNA-binding transcription regulators, and serine/threonine protein kinases. PhyloBot runs in a web browser, and is available at the following URL: http://www.phylobot.com. The software is implemented in Python using the Django web framework, and runs on elastic cloud computing resources from Amazon Web Services. Users can create and submit jobs on our free server (at the URL listed above), or use our open-source code to launch their own PhyloBot server. PMID:27472806

  20. PhyloBot: A Web Portal for Automated Phylogenetics, Ancestral Sequence Reconstruction, and Exploration of Mutational Trajectories.

    Science.gov (United States)

    Hanson-Smith, Victor; Johnson, Alexander

    2016-07-01

    The method of phylogenetic ancestral sequence reconstruction is a powerful approach for studying evolutionary relationships among protein sequence, structure, and function. In particular, this approach allows investigators to (1) reconstruct and "resurrect" (that is, synthesize in vivo or in vitro) extinct proteins to study how they differ from modern proteins, (2) identify key amino acid changes that, over evolutionary timescales, have altered the function of the protein, and (3) order historical events in the evolution of protein function. Widespread use of this approach has been slow among molecular biologists, in part because the methods require significant computational expertise. Here we present PhyloBot, a web-based software tool that makes ancestral sequence reconstruction easy. Designed for non-experts, it integrates all the necessary software into a single user interface. Additionally, PhyloBot provides interactive tools to explore evolutionary trajectories between ancestors, enabling the rapid generation of hypotheses that can be tested using genetic or biochemical approaches. Early versions of this software were used in previous studies to discover genetic mechanisms underlying the functions of diverse protein families, including V-ATPase ion pumps, DNA-binding transcription regulators, and serine/threonine protein kinases. PhyloBot runs in a web browser, and is available at the following URL: http://www.phylobot.com. The software is implemented in Python using the Django web framework, and runs on elastic cloud computing resources from Amazon Web Services. Users can create and submit jobs on our free server (at the URL listed above), or use our open-source code to launch their own PhyloBot server.

  1. PhyloBot: A Web Portal for Automated Phylogenetics, Ancestral Sequence Reconstruction, and Exploration of Mutational Trajectories.

    Science.gov (United States)

    Hanson-Smith, Victor; Johnson, Alexander

    2016-07-01

    The method of phylogenetic ancestral sequence reconstruction is a powerful approach for studying evolutionary relationships among protein sequence, structure, and function. In particular, this approach allows investigators to (1) reconstruct and "resurrect" (that is, synthesize in vivo or in vitro) extinct proteins to study how they differ from modern proteins, (2) identify key amino acid changes that, over evolutionary timescales, have altered the function of the protein, and (3) order historical events in the evolution of protein function. Widespread use of this approach has been slow among molecular biologists, in part because the methods require significant computational expertise. Here we present PhyloBot, a web-based software tool that makes ancestral sequence reconstruction easy. Designed for non-experts, it integrates all the necessary software into a single user interface. Additionally, PhyloBot provides interactive tools to explore evolutionary trajectories between ancestors, enabling the rapid generation of hypotheses that can be tested using genetic or biochemical approaches. Early versions of this software were used in previous studies to discover genetic mechanisms underlying the functions of diverse protein families, including V-ATPase ion pumps, DNA-binding transcription regulators, and serine/threonine protein kinases. PhyloBot runs in a web browser, and is available at the following URL: http://www.phylobot.com. The software is implemented in Python using the Django web framework, and runs on elastic cloud computing resources from Amazon Web Services. Users can create and submit jobs on our free server (at the URL listed above), or use our open-source code to launch their own PhyloBot server. PMID:27472806

  2. Somatic Mutations Are Not Observed by Exome Sequencing of Lymphocyte DNA from Monozygotic Twins Discordant for Congenital Hypothyroidism due to Thyroid Dysgenesis

    Science.gov (United States)

    Magne, Fabien; Serpa, Roman; Van Vliet, Guy; Samuels, Mark E.; Deladoëy, Johnny

    2016-01-01

    Background/Aims Congenital primary hypothyroidism (CH) is a rare pediatric disorder estimated to occur in about 1: 2,500 live births. Approximately half of these cases entail ectopic thyroid tissue, which is believed to result from a migration defect during embryogenesis. Approximately 3% of CH cases are explained by mutation(s) in known genes, most of which are transcription factors implicated in the embryology of the thyroid gland. Surprisingly, monozygotic (MZ) twins are usually discordant for CH due to thyroid dysgenesis, suggesting that most cases are not caused by transmitted genetic variation. One possible explanation is somatic mutation in genes involved in thyroid migration occurring after zygotic twinning. Such mutations should be observed only in the affected twin. Methods To test the hypothesis of somatic mutation, we performed whole exome sequencing of DNA from three pairs of MZ twins discordant for CH with ectopic glands. Results We found no somatic mutations exclusive to any of the three affected twins or in any of the unaffected twins. Conclusion Either somatic mutations are not significant for the etiology of CH or else such mutations lie outside regions of the genome accessible by exome sequencing technology. PMID:25277881

  3. A versatile-deployable bacterial detection system for food and environmental safety based on LabTube-automated DNA purification, LabReader-integrated amplification, readout and analysis.

    Science.gov (United States)

    Hoehl, Melanie M; Bocholt, Eva Schulte; Kloke, Arne; Paust, Nils; von Stetten, Felix; Zengerle, Roland; Steigert, Juergen; Slocum, Alexander H

    2014-06-01

    Contamination of foods is a public health hazard that episodically causes thousands of deaths and sickens millions worldwide. To ensure food safety and quality, rapid, low-cost and easy-to-use detection methods are desirable. Here, the LabSystem is introduced for integrated, automated DNA purification, amplification and detection. It consists of a disposable, centrifugally driven DNA purification platform (LabTube) and a low-cost UV/vis-reader (LabReader). For demonstration of the LabSystem in the context of food safety, purification of Escherichia coli (non-pathogenic E. coli and pathogenic verotoxin-producing E. coli (VTEC)) in water and milk and the product-spoiler Alicyclobacillus acidoterrestris (A. acidoterrestris) in apple juice was integrated and optimized in the LabTube. Inside the LabReader, the purified DNA was amplified, readout and analyzed using both qualitative isothermal loop-mediated DNA amplification (LAMP) and quantitative real-time PCR. For the LAMP-LabSystem, the combined detection limits for purification and amplification of externally lysed VTEC and A. acidoterrestris are 10(2)-10(3) cell-equivalents. In the PCR-LabSystem for E. coli cells, the quantification limit is 10(2) cell-equivalents including LabTube-integrated lysis. The demonstrated LabSystem only requires a laboratory centrifuge (to operate the disposable, fully closed LabTube) and a low-cost LabReader for DNA amplification, readout and analysis. Compared with commercial DNA amplification devices, the LabReader improves sensitivity and specificity by the simultaneous readout of four wavelengths and the continuous readout during temperature cycling. The use of a detachable eluate tube as an interface affords semi-automation of the LabSystem, which does not require specialized training. It reduces the hands-on time from about 50 to 3 min with only two handling steps: sample input and transfer of the detachable detection tube.

  4. A Point Mutation in DNA Polymerase β (POLB) Gene Is Associated with Increased Progesterone Receptor (PR) Expression and Intraperitoneal Metastasis in Gastric Cancer

    Science.gov (United States)

    Tan, Xiaohui; Wu, Xiaoling; Ren, Shuyang; Wang, Hongyi; Li, Zhongwu; Alshenawy, Weaam; Li, Wenmei; Cui, Jiantao; Luo, Guangbin; Siegel, Robert S.; Fu, Sidney W.; Lu, Youyong

    2016-01-01

    Increased expression of progesterone receptor (PR) has been reported in gastric cancer (GC). We have previously identified a functional T889C point mutation in DNA polymerase beta (POLB), a DNA repair gene in GC. To provide a detailed analysis of molecular changes associated with the mutation, human cDNA microarrays focusing on 18 signal transduction pathways were used to analyze differential gene expression profiles between GC tissues with T889C mutant in POLB gene and those with wild type. Among the differentially expressed genes, notably, PR was one of the significantly up-regulated genes in T889C mutant POLB tissues, which were subsequently confirmed in POLB gene transfected AGS cell line. Interestingly, patients with T889C mutation and PR positivity were associated with higher incidence of intraperitoneal metastasis (IM). In vitro studies indicate that PR expression was upregulated in AGS cell line when transfected with T889C mutant expression vector. Cotransfection of T889C mutant allele and PR gene induced cell migration in the cell line. These data demonstrated that T889C mutation-associated PR overexpression results in increased IM. Therefore, T889C mutation-associated PR overexpression may serve as a biomarker for an adverse prognosis for human GC.

  5. Novel Point Mutations and A8027G Polymorphism in Mitochondrial-DNA-Encoded Cytochrome c Oxidase II Gene in Mexican Patients with Probable Alzheimer Disease

    Directory of Open Access Journals (Sweden)

    Verónica Loera-Castañeda

    2014-01-01

    Full Text Available Mitochondrial dysfunction has been thought to contribute to Alzheimer disease (AD pathogenesis through the accumulation of mitochondrial DNA mutations and net production of reactive oxygen species (ROS. Mitochondrial cytochrome c-oxidase plays a key role in the regulation of aerobic production of energy and is composed of 13 subunits. The 3 largest subunits (I, II, and III forming the catalytic core are encoded by mitochondrial DNA. The aim of this work was to look for mutations in mitochondrial cytochrome c-oxidase gene II (MTCO II in blood samples from probable AD Mexican patients. MTCO II gene was sequenced in 33 patients with diagnosis of probable AD. Four patients (12% harbored the A8027G polymorphism and three of them were early onset (EO AD cases with familial history of the disease. In addition, other four patients with EOAD had only one of the following point mutations: A8003C, T8082C, C8201T, or G7603A. Neither of the point mutations found in this work has been described previously for AD patients, and the A8027G polymorphism has been described previously; however, it hasn’t been related to AD. We will need further investigation to demonstrate the role of the point mutations of mitochondrial DNA in the pathogenesis of AD.

  6. Novel Point Mutations and A8027G Polymorphism in Mitochondrial-DNA-Encoded Cytochrome c Oxidase II Gene in Mexican Patients with Probable Alzheimer Disease

    Science.gov (United States)

    Loera-Castañeda, Verónica; Sandoval-Ramírez, Lucila; Pacheco Moisés, Fermín Paul; Macías-Islas, Miguel Ángel; Alatorre Jiménez, Moisés Alejandro; González-Renovato, Erika Daniela; Cortés-Enríquez, Fernando; Célis de la Rosa, Alfredo; Velázquez-Brizuela, Irma E.

    2014-01-01

    Mitochondrial dysfunction has been thought to contribute to Alzheimer disease (AD) pathogenesis through the accumulation of mitochondrial DNA mutations and net production of reactive oxygen species (ROS). Mitochondrial cytochrome c-oxidase plays a key role in the regulation of aerobic production of energy and is composed of 13 subunits. The 3 largest subunits (I, II, and III) forming the catalytic core are encoded by mitochondrial DNA. The aim of this work was to look for mutations in mitochondrial cytochrome c-oxidase gene II (MTCO II) in blood samples from probable AD Mexican patients. MTCO II gene was sequenced in 33 patients with diagnosis of probable AD. Four patients (12%) harbored the A8027G polymorphism and three of them were early onset (EO) AD cases with familial history of the disease. In addition, other four patients with EOAD had only one of the following point mutations: A8003C, T8082C, C8201T, or G7603A. Neither of the point mutations found in this work has been described previously for AD patients, and the A8027G polymorphism has been described previously; however, it hasn't been related to AD. We will need further investigation to demonstrate the role of the point mutations of mitochondrial DNA in the pathogenesis of AD. PMID:24701363

  7. Multiple symmetric lipomas with high levels of mtDNA with the tRNA(Lys) A-->G(8344) mutation as the only manifestation of disease in a carrier of myoclonus epilepsy and ragged-red fibers (MERRF) syndrome.

    OpenAIRE

    Holme, E; Larsson, N G; Oldfors, A; Tulinius, M; Sahlin, P; Stenman, G

    1993-01-01

    We have investigated the morphology, cytogenetics, and the fraction of mtDNA with the tRNA(Lys) A-->G(8344) mutation in three lipomas in a carrier of this mutation. The son of the patient had myoclonus epilepsy and ragged-red fibers syndrome. The fraction of mtDNA with the tRNA(Lys) mutation varied between 62% and 80% in cultured skin fibroblasts, lymphocytes, normal adipose tissue, and muscle. In the three lipomas the mean fraction of mutated mtDNA was 90%, 94%, and 94%. Ultrastructural exam...

  8. Gain-of-function mutations of Ptpn11 (Shp2) cause aberrant mitosis and increase susceptibility to DNA damage-induced malignancies.

    Science.gov (United States)

    Liu, Xia; Zheng, Hong; Li, Xiaobo; Wang, Siying; Meyerson, Howard J; Yang, Wentian; Neel, Benjamin G; Qu, Cheng-Kui

    2016-01-26

    Gain-of-function (GOF) mutations of protein tyrosine phosphatase nonreceptor type 11 Ptpn11 (Shp2), a protein tyrosine phosphatase implicated in multiple cell signaling pathways, are associated with childhood leukemias and solid tumors. The underlying mechanisms are not fully understood. Here, we report that Ptpn11 GOF mutations disturb mitosis and cytokinesis, causing chromosomal instability and greatly increased susceptibility to DNA damage-induced malignancies. We find that Shp2 is distributed to the kinetochore, centrosome, spindle midzone, and midbody, all of which are known to play critical roles in chromosome segregation and cytokinesis. Mouse embryonic fibroblasts with Ptpn11 GOF mutations show a compromised mitotic checkpoint. Centrosome amplification and aberrant mitosis with misaligned or lagging chromosomes are significantly increased in Ptpn11-mutated mouse and patient cells. Abnormal cytokinesis is also markedly increased in these cells. Further mechanistic analyses reveal that GOF mutant Shp2 hyperactivates the Polo-like kinase 1 (Plk1) kinase by enhancing c-Src kinase-mediated tyrosine phosphorylation of Plk1. This study provides novel insights into the tumorigenesis associated with Ptpn11 GOF mutations and cautions that DNA-damaging treatments in Noonan syndrome patients with germ-line Ptpn11 GOF mutations could increase the risk of therapy-induced malignancies.

  9. Simultaneous occurrence of the 11778 (ND4) and the 9438 (COX III) mtDNA mutations in Leber hereditary optic neuropathy: Molecular, biochemical, and clinical findings

    Energy Technology Data Exchange (ETDEWEB)

    Oostra, R.J.; Bleeker-Wagemakers, E.M.; Zwart, R. [Ophthalmic Research Institute, Amsterdam (Netherlands)] [and others

    1995-10-01

    Three mtDNA point mutations at nucleotide position (np) 3460, at np 11778 and at np 14484, are thought to be of primary importance in the pathogenesis of Leber hereditary optic neuropathy (LHON), a maternally inherited disease characterized by subacute central vision loss. These mutations are present in genes coding for subunits of complex I (NADH dehydrogenase) of the respiratory chain, occur exclusively in LHON maternal pedigrees, and have never been reported to occur together. Johns and Neufeld postulated that an mtDNA mutation at np 9438, in the gene coding for one of the subunits (COX III) of complex IV (cytochrome c oxidase), was also of primary importance. Johns and Neufeld (1993) found this mutation, which changed a conserved glycine to a serine, in 5 unrelated LHON probands who did not carry one of the presently known primary mutations, but they did not find it in 400 controls. However, the role of this sequence variant has been questioned in the Journal when it has been found to occur in apparently healthy African and Cuban individuals. Subsequently, Johns et al. described this mutation in two Cuban individuals presenting with optic and peripheral neuropathy. 22 refs., 1 fig., 1 tab.

  10. Application of a Novel and Automated Branched DNA in Situ Hybridization Method for the Rapid and Sensitive Localization of mRNA Molecules in Plant Tissues

    Directory of Open Access Journals (Sweden)

    Andrew J. Bowling

    2014-04-01

    Full Text Available Premise of the study: A novel branched DNA detection technology, RNAscope in situ hybridization (ISH, originally developed for use on human clinical and animal tissues, was adapted for use in plant tissue in an attempt to overcome some of the limitations associated with traditional ISH assays. Methods and Results: Zea mays leaf tissue was formaldehyde fixed and paraffin embedded (FFPE and then probed with the RNAscope ISH assay for two endogenous genes, phosphoenolpyruvate carboxylase (PEPC and phosphoenolpyruvate carboxykinase (PEPCK. Results from both manual and automated methods showed tissue- and cell-specific mRNA localization patterns expected from these well-studied genes. Conclusions: RNAscope ISH is a sensitive method that generates high-quality, easily interpretable results from FFPE plant tissues. Automation of the RNAscope method on the Ventana Discovery Ultra platform allows significant advantages for repeatability, reduction in variability, and flexibility of workflow processes.

  11. Digital PCR for quantification of recurrent and potentially actionable somatic mutations in circulating free DNA from patients with diffuse large B-cell lymphoma.

    Science.gov (United States)

    Camus, Vincent; Sarafan-Vasseur, Nasrin; Bohers, Elodie; Dubois, Sydney; Mareschal, Sylvain; Bertrand, Philippe; Viailly, Pierre-Julien; Ruminy, Philippe; Maingonnat, Catherine; Lemasle, Emilie; Stamatoullas, Aspasia; Picquenot, Jean-Michel; Cornic, Marie; Beaussire, Ludivine; Bastard, Christian; Frebourg, Thierry; Tilly, Hervé; Jardin, Fabrice

    2016-09-01

    Diffuse large B-cell lymphoma (DLBCL) is an aggressive and heterogeneous malignancy harboring frequent targetable activating somatic mutations. Emerging evidence suggests that circulating cell-free DNA (cfDNA) can be used to detect somatic variants in DLBCL using Next-Generation Sequencing (NGS) experiments. In this proof-of-concept study, we chose to develop simple and valuable digital PCR (dPCR) assays for the detection of recurrent exportin-1 (XPO1) E571K, EZH2 Y641N, and MYD88 L265P mutations in DLBCL patients, thereby identifying patients most likely to potentially benefit from targeted therapies. We demonstrated that our dPCR assays were sufficiently sensitive to detect rare XPO1, EZH2, and MYD88 mutations in plasma cfDNA, with a sensitivity of 0.05%. cfDNA somatic mutation detection by dPCR seems to be a promising technique in the management of DLBCL, in addition to NGS experiments. PMID:26883583

  12. Impact of a novel homozygous mutation in nicotinamide nucleotide transhydrogenase on mitochondrial DNA integrity in a case of familial glucocorticoid deficiency

    Directory of Open Access Journals (Sweden)

    Yasuko Fujisawa

    2015-06-01

    General significance: By studying a family affected with a novel point mutation in the NNT gene, a gene–dose response was found for various mitochondrial outcomes providing for novel insights into the role of NNT in the maintenance of mtDNA integrity beyond that described for preventing oxidative stress.

  13. DNA Glycosylases Involved in Base Excision Repair May Be Associated with Cancer Risk in BRCA1 and BRCA2 Mutation Carriers

    DEFF Research Database (Denmark)

    Osorio, Ana; Milne, Roger L; Kuchenbaecker, Karoline;

    2014-01-01

    Single Nucleotide Polymorphisms (SNPs) in genes involved in the DNA Base Excision Repair (BER) pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of th...

  14. DNA Glycosylases Involved in Base Excision Repair May Be Associated with Cancer Risk in BRCA1 and BRCA2 Mutation Carriers

    NARCIS (Netherlands)

    Osorio, Ana; Milne, Roger L.; Kuchenbaecker, Karoline; Vaclova, Tereza; Pita, Guillermo; Alonso, Rosario; Peterlongo, Paolo; Blanco, Ignacio; de la Hoya, Miguel; Duran, Mercedes; Diez, Orland; Ramon y Cajal, Teresa; Konstantopoulou, Irene; Martinez-Bouzas, Cristina; Conejero, Raquel Andres; Soucy, Penny; McGuffog, Lesley; Barrowdale, Daniel; Lee, Andrew; Arver, Brita; Rantala, Johanna; Loman, Niklas; Ehrencrona, Hans; Olopade, Olufunmilayo I.; Beattie, Mary S.; Domchek, Susan M.; Nathanson, Katherine; Rebbeck, Timothy R.; Arun, Banu K.; Karlan, Beth Y.; Walsh, Christine; Lester, Jenny; John, Esther M.; Whittemore, Alice S.; Daly, Mary B.; Southey, Melissa; Hopper, John; Terry, Mary B.; Buys, Saundra S.; Janavicius, Ramunas; Dorfling, Cecilia M.; van Rensburg, Elizabeth J.; Steele, Linda; Neuhausen, Susan L.; Ding, Yuan Chun; Hansen, Thomas V. O.; Jonson, Lars; Ejlertsen, Bent; Gerdes, Anne-Marie; Infante, Mar; Herraez, Belen; Moreno, Leticia Thais; Weitzel, Jeffrey N.; Herzog, Josef; Weeman, Kisa; Manoukian, Siranoush; Peissel, Bernard; Zaffaroni, Daniela; Scuvera, Giulietta; Bonanni, Bernardo; Mariette, Frederique; Volorio, Sara; Viel, Alessandra; Varesco, Liliana; Papi, Laura; Ottini, Laura; Tibiletti, Maria Grazia; Radice, Paolo; Yannoukakos, Drakoulis; Garber, Judy; Ellis, Steve; Frost, Debra; Platte, Radka; Fineberg, Elena; Evans, Gareth; Lalloo, Fiona; Izatt, Louise; Eeles, Ros; Adlard, Julian; Davidson, Rosemarie; Cole, Trevor; Eccles, Diana; Cook, Jackie; Hodgson, Shirley; Brewer, Carole; Tischkowitz, Marc; Douglas, Fiona; Porteous, Mary; Side, Lucy; Walker, Lisa; Morrison, Patrick; Donaldson, Alan; Kennedy, John; Foo, Claire; Godwin, Andrew K.; Schmutzler, Rita Katharina; Wappenschmidt, Barbara; Rhiem, Kerstin; Engel, Christoph; Meindl, Alfons; Ditsch, Nina; Arnold, Norbert; Plendl, Hans Joerg; Niederacher, Dieter; Sutter, Christian; Wang-Gohrke, Shan; Steinemann, Doris; Preisler-Adams, Sabine; Kast, Karin; Varon-Mateeva, Raymonda; Gehrig, Andrea; Stoppa-Lyonnet, Dominique; Sinilnikova, Olga M.; Mazoyer, Sylvie; Damiola, Francesca; Poppe, Bruce; Claes, Kathleen; Piedmonte, Marion; Tucker, Kathy; Backes, Floor; Rodriguez, Gustavo; Brewster, Wendy; Wakeley, Katie; Rutherford, Thomas; Caldes, Trinidad; Nevanlinna, Heli; Aittomaki, Kristiina; Rookus, Matti A.; van Os, Theo A. M.; van der Kolk, Lizet; de Lange, J. L.; Meijers-Heijboer, Hanne E. J.; van der Hout, A. H.; van Asperen, Christi J.; Gomez Garcia, Encarna B.; Hoogerbrugge, Nicoline; Collee, J. Margriet; van Deurzen, Carolien H. M.; van der Luijt, Rob B.; Devilee, Peter; Olah, Edith; Lazaro, Conxi; Teule, Alex; Menendez, Mireia; Jakubowska, Anna; Cybulski, Cezary; Gronwald, Jacek; Lubinski, Jan; Durda, Katarzyna; Jaworska-Bieniek, Katarzyna; Johannsson, Oskar Th; Maugard, Christine; Montagna, Marco; Tognazzo, Silvia; Teixeira, Manuel R.; Healey, Sue; Olswold, Curtis; Guidugli, Lucia; Lindor, Noralane; Slager, Susan; Szabo, Csilla I.; Vijai, Joseph; Robson, Mark; Kauff, Noah; Zhang, Liying; Rau-Murthy, Rohini; Fink-Retter, Anneliese; Singer, Christian F.; Rappaport, Christine; Kaulich, Daphne Geschwantler; Pfeiler, Georg; Tea, Muy-Kheng; Berger, Andreas; Phelan, Catherine M.; Greene, Mark H.; Mai, Phuong L.; Lejbkowicz, Flavio; Andrulis, Irene; Mulligan, Anna Marie; Glendon, Gord; Toland, Amanda Ewart; Bojesen, Anders; Pedersen, Inge Sokilde; Sunde, Lone; Thomassen, Mads; Kruse, Torben A.; Jensen, Uffe Birk; Friedman, Eitan; Laitman, Yael; Shimon, Shani Paluch; Simard, Jacques; Easton, Douglas F.; Offit, Kenneth; Couch, Fergus J.; Chenevix-Trench, Georgia; Antoniou, Antonis C.; Benitez, Javier

    2014-01-01

    Single Nucleotide Polymorphisms (SNPs) in genes involved in the DNA Base Excision Repair (BER) pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of the c

  15. Somatic mutations in glioblastoma are associated with methylguanine-DNA methyltransferase methylation

    OpenAIRE

    McDonald, Kerrie L.; Tabone, Tania; Nowak, Anna K; Erber, Wendy N.

    2015-01-01

    The high level of methylguanine-DNA methyltransferase (MGMT) in glioblastoma is responsible for resistance to alkylating agents, such as temozolomide (TMZ). In glioblastomas with a methylated MGMT promoter, MGMT deficiency is presumed, resulting in an enhanced effect of TMZ. The aim of the present study was to investigate whether genomic alterations work synergistically with MGMT methylation status and contribute to the response to treatment and overall prognosis in glioblastoma. The current ...

  16. Detection of BRAF Mutations Using a Fully Automated Platform and Comparison with High Resolution Melting, Real-Time Allele Specific Amplification, Immunohistochemistry and Next Generation Sequencing Assays, for Patients with Metastatic Melanoma.

    Directory of Open Access Journals (Sweden)

    Alexandre Harlé

    Full Text Available Metastatic melanoma is a severe disease with one of the highest mortality rate in skin diseases. Overall survival has significantly improved with immunotherapy and targeted therapies. Kinase inhibitors targeting BRAF V600 showed promising results. BRAF genotyping is mandatory for the prescription of anti-BRAF therapies.Fifty-nine formalin-fixed paraffin-embedded melanoma samples were assessed using High-Resolution-Melting (HRM PCR, Real-time allele-specific amplification (RT-ASA PCR, Next generation sequencing (NGS, immunohistochemistry (IHC and the fully-automated molecular diagnostics platform IdyllaTM. Sensitivity, specificity, positive predictive value and negative predictive value were calculated using NGS as the reference standard to compare the different assays.BRAF mutations were found in 28(47.5%, 29(49.2%, 31(52.5%, 29(49.2% and 27(45.8% samples with HRM, RT-ASA, NGS, IdyllaTM and IHC respectively. Twenty-six (81.2% samples were found bearing a c.1799T>A (p.Val600Glu mutation, three (9.4% with a c.1798_1799delinsAA (p.Val600Lys mutation and one with c.1789_1790delinsTC (p.Leu597Ser mutation. Two samples were found bearing complex mutations.HRM appears the less sensitive assay for the detection of BRAF V600 mutations. The RT-ASA, IdyllaTM and IHC assays are suitable for routine molecular diagnostics aiming at the prescription of anti-BRAF therapies. IdyllaTM assay is fully-automated and requires less than 2 minutes for samples preparation and is the fastest of the tested assays.

  17. Hypersensitivity to mutation and sister-chromatid-exchange induction in CHO cell mutants defective in incising DNA containing UV lesions

    International Nuclear Information System (INIS)

    Five UV-sensitive mutant strains of CHO cells representing different genetic complementation groups were analyzed for their ability to perform the incision step of nucleotide excision repair after UV exposure. The assay utilized inhibitors of DNA synthesis to accumulate the short-lived strand breaks resulting from repair incisions. After 6 J/m2, each of the mutants showed 2, the rate in AA8 was similar to that at 6 J/m2, but the rates in the mutants were significantly higher (approx. 20% of the rate of AA8). Thus by this incision assay the mutants were phenotypically indistinguishable. Each of the mutants were hypersensitive to mutation induction at both the hprt and aprt loci by a factor of 10, and in the one strain tested ouabain resistance was induced sevenfold more efficiently than in AA8 cells. Sister chromatid exchange was also induced with sevenfold increased efficiency in the two mutant strains examined. Thus, here CHO mutants resemble xeroderma pigmentosum cells in terms of their incision defects and their hypersensitivity to DNA damage by UV

  18. 紫外线致DNA突变的快速测定研究%A Fast Determination of DNA Mutation Induced by Ultraviolet Radiation

    Institute of Scientific and Technical Information of China (English)

    陆峰; 刘荔荔; 张晓芳; 吴玉田

    2001-01-01

    Electrophoresis, chromatography, immunoassay, sequencing and other time consuming approaches have been developed to determine DNA base mismatching, oxidative lesion or strand breaks. Sometimes,however, only qualitative information is enough to decide whether mutation has happened to DNA and its extent.Convolution spectrometry (CS), a new technique to discover ultrafine difference on ultraviolet (UV) absorption of different substances, is originally employed to find out any subtle mutation of DNA induced by UV radiation. Mutative DNA is compared with ego criteria based on the spectra of the former DNA, any difference is quantitatively expressed by dispersion (δ). Visible changes cannot be observed on second -derivative spectra until the mutation gets δup to 11.48%. Dimethyl sulfoxide is an intensifier of UV 254 nm induced DNA mutation and protector at 365 nm,which is simply confirmed by increasing and decreasing δ. Every convolution procedure takes less than 1 min. Convolution spectrometry provides a fast, simple, sensitive and inexpensive alternative to determine DNA mutation, and to screen anti-mutational medicines.%毛细管电泳、变性色谱、免疫吸附、DNA测序方法等已广泛用于DNA碱基错配、氧化缺失、链断裂等变化的检测,但有时如进行药物筛选时,只需定性地检测DNA是否变化或变化程度.本文采用褶合光谱法定性地检测紫外线致DNA变化程度,将突变后DNA的褶合光谱与未变异前DNA自身标准比较,并以差谱值δ量化地表示突变程度.当褶合光谱δ高达11.48%时,才能从二阶导数光谱上发现差异,表明方法的灵敏度远远高于前者.加入DMSO后,溶液在254nm照射时,δ升高,表现为DNA变异诱导剂;溶液在365 nm照射时,δ降低,表现为DNA变异保护剂.褶合光谱法快速、简便、灵敏、经济,可以作为检测DNA突变、筛选抗突变药物的一种新型方法.

  19. Identification of BRCA1/2 founder mutations in Southern Chinese breast cancer patients using gene sequencing and high resolution DNA melting analysis.

    Directory of Open Access Journals (Sweden)

    Ava Kwong

    Full Text Available BACKGROUND: Ethnic variations in breast cancer epidemiology and genetics have necessitated investigation of the spectra of BRCA1 and BRCA2 mutations in different populations. Knowledge of BRCA mutations in Chinese populations is still largely unknown. We conducted a multi-center study to characterize the spectra of BRCA mutations in Chinese breast and ovarian cancer patients from Southern China. METHODOLOGY/PRINCIPAL FINDINGS: A total of 651 clinically high-risk breast and/or ovarian cancer patients were recruited from the Hong Kong Hereditary Breast Cancer Family Registry from 2007 to 2011. Comprehensive BRCA1 and BRCA2 mutation screening was performed using bi-directional sequencing of all coding exons of BRCA1 and BRCA2. Sequencing results were confirmed by in-house developed full high resolution DNA melting (HRM analysis. Among the 451 probands analyzed, 69 (15.3% deleterious BRCA mutations were identified, comprising 29 in BRCA1 and 40 in BRCA2. The four recurrent BRCA1 mutations (c.470_471delCT, c.3342_3345delAGAA, c.5406+1_5406+3delGTA and c.981_982delAT accounted for 34.5% (10/29 of all BRCA1 mutations in this cohort. The four recurrent BRCA2 mutations (c.2808_2811delACAA, c.3109C>T, c.7436_7805del370 and c.9097_9098insA accounted for 40% (16/40 of all BRCA2 mutations. Haplotype analysis was performed to confirm 1 BRCA1 and 3 BRCA2 mutations are putative founder mutations. Rapid HRM mutation screening for a panel of the founder mutations were developed and validated. CONCLUSION: In this study, our findings suggest that BRCA mutations account for a substantial proportion of hereditary breast/ovarian cancer in Southern Chinese population. Knowing the spectrum and frequency of the founder mutations in this population will assist in the development of a cost-effective rapid screening assay, which in turn facilitates genetic counseling and testing for the purpose of cancer risk assessment.

  20. The mtDNA NARP mutation activates the actin-Nrf2 signaling of antioxidant defenses

    International Nuclear Information System (INIS)

    An efficient handling of superoxides by antioxidant defenses is a crucial issue for cells with respiratory chain deficient mitochondria. We used human cultured skin fibroblasts to delineate the mechanism controlling the expression of antioxidant defenses in the case of a severe ATPase deficiency resulting from an 8993T>G mutation in the mitochondrial ATPase6 gene. We observed the nuclear translocation of the transcription factor Nrf2 associated with thinning of the actin stress fibers. The mobilization of the Nrf2 signaling pathway could be mimicked by a chemical blockade of the ATPase with a specific inhibitor, oligomycin. Interestingly enough, Nrf2 nuclear translocation was not observed in the case of a severe cytochrome oxidase deficiency, indicating that studying the status of this signaling pathway could throw some light on the importance of the oxidative insult associated with different respiratory chain defects

  1. A de novo frameshift mutation in chromodomain helicase DNA-binding domain 8 (CHD8): A case report and literature review.

    Science.gov (United States)

    Merner, Nancy; Forgeot d'Arc, Baudouin; Bell, Scott C; Maussion, Gilles; Peng, Huashan; Gauthier, Julie; Crapper, Liam; Hamdan, Fadi F; Michaud, Jacques L; Mottron, Laurent; Rouleau, Guy A; Ernst, Carl

    2016-05-01

    Mutations in chromodomain helicase DNA-binding domain 8 (CHD8) have been identified in independent genotyping studies of autism spectrum disorder. To better understand the phenotype associated with CHD8 mutations, we genotyped all CHD8 exons in carefully assessed cohorts of autism (n = 142), schizophrenia (SCZ; n = 143), and intellectual disability (ID; n = 94). We identified one frameshift mutation, seven non-synonymous variants, and six synonymous variants. The frameshift mutation, p.Asn2092Lysfs*2, which creates a premature stop codon leading to the loss of 212 amino acids of the protein, was from an autism case on whom we present multiple clinical assessments and pharmacological treatments spanning more than 10 years. RNA and protein analysis support a model where the transcript generated from the mutant allele results in haploinsufficiency of CHD8. This case report supports the association of CHD8 mutations with classical autism, macrocephaly, infantile hypotonia, speech delay, lack of major ID, and psychopathology in late adolescence caused by insufficient dosage of CHD8. Review of 16 other CHD8 mutation cases suggests that clinical features and their severity vary considerably across individuals; however, these data support a CHD8 mutation syndrome, further highlighting the importance of genomic medicine to guide clinical assessment and treatment. PMID:26789910

  2. Sequence analysis of lacZ~- mutations induced by ion beam irradiation in double-stranded M13mp18DNA

    Institute of Scientific and Technical Information of China (English)

    杨剑波; 吴李君; 李莉; 吴家道; 余增亮; 许智宏

    1997-01-01

    While M13mpl8 double-stranded DNA was irradiated with ion beam, and transfected into E. coli JM103, a decrease of transfecting activity was discovered. The lacZ-mutation frequency at 20% survival could reach (3.6-16.8) × 104, about 2.3-10 times that of unirradiated M13DNA. Altogether, 27 lacZ~ mutants were select-ed, 10 of which were used for sequencing. 7 of the sequenced mutants show base changes in 250-bp region examined (the remaining 3 mutants probably have base changes outside the regions sequenced). 5 of the base-changed mutants contain more than one mutational base sites (some of them even have 5-6 mutational base sites in 250-bp region ex-amined) ; this dense distribution of base changes in polysites has seldom been seen in X-rays, γ-rays or UV induced DNA mutations. Our experiments also showed that the types of base changes include transitions( 50 % ), transversions (45% ) and deletion (5% ); no addition or duplication was observed. The transitions were mainly C→T and A→G; the transversion

  3. DNA damage-processing in E. coli: on-going protein synthesis is required for fixation of UV-induced lethality and mutation.

    Science.gov (United States)

    Burger, Amanda; Raymer, Jenny; Bockrath, R

    2002-10-01

    UV irradiation of E. coli produces photoproducts in the DNA genome. In consequence, some bacteria lose viability (colony-forming ability) or remain viable as mutant cells. However, the end-points of viability inactivation (lethality) or mutation are determined by cellular processes that act on the UV-damaged DNA. We have investigated the in vivo time course for processes that deal with cyclobutane pyrimidine dimers (CPD) which can be specifically removed by photoreactivation (PR). At different times during post-UV incubation, samples were challenged with PR and assayed for viability or mutation. We used excision-defective E. coli B/r cells and worked under yellow light to avoid background PR. During post-UV incubation (0-100min) in fully supplemented defined medium, inactivation and mutation were initially significantly reversed by PR but the extent of this reversal decreased during continued incubation defining "fixation" of lethality or mutation, respectively. In contrast, if protein synthesis was restricted during the post-UV incubation, no fixation developed. When chloramphenicol was added to inhibit protein synthesis after 30min of supplemented post-UV incubation, at a time sufficient for expression of UV-induced protein(s), fixation of lethality or mutation was still annulled (no change in the effectiveness of PR developed). Lethality fixation did progress when protein synthesis was restricted and the cells were incubated in the presence of puromycin or were either clpP or clpX defective. We discuss these and related results to suggest (1) on-going protein synthesis is required in the fixation process for lethality and mutation to sustain an effective level of a hypothetical protein sensitive to ClpXP proteolysis and (2) this protein plays a critical role in the process leading to exchange between Pol III activity and alternative polymerase activities required as each cell deals with damage in template DNA.

  4. [The analysis of mitochondrial DNA haplogroups and variants for Leber's hereditary optic neuropathy in Chinese families carrying the m.14484T >C mutation].

    Science.gov (United States)

    Meng, Xiangjuan; Zhu, Jinping; Gao, Min; Zhang, Sai; Zhao, Fuxin; Zhang, Juanjuan; Liu, Xiaoling; Wei, Qiping; Tong, Yi; Zhang, Minglian; Qu, Jia; Guan, Minxin

    2014-04-01

    The m.14484T>C mutation in mitochondrial ND6 gene (MT-ND6) is a primary mutation underlying the development of Leber's hereditary optic neuropathy (LHON) , but by itself not enough to cause visual loss. To explore the role of mitochondrial haplogroups on the expression of LHON for the people carrying the m.14484T>C mutation, we performed systematic and extended mutational screening of MT-ND6 gene in a cohort of 1177 Han Chinese patients with LHON. A total of 67 affected subjects carried the homoplasmic m.14484T>C mutation, accounting for 5.7% of this LHON population. The penetrances of optic neuropathy among 51 pedigrees carrying the m.14484T>C mutation ranged from 5.6% to 100.0%, with the average of 21.5%. The sequence analysis of entire mitochondrial genomes of 51 probands exhibited distinct sets of polymorphisms belonging to 18 Eastern Asian haplogroups. The frequencies of haplogroup A and haplogroup F were sig-nificantly less in the LHON mtDNA samples than those in 106 Chinese controls. On the other hand, the haplogroup M10a accounted for 9.8% of the patient's mtDNA samples but was absent in 106 Chinese controls. Strikingly, the average pene-trance (46.13%) of optic neuropathy for the pedigrees carrying mitochondrial haplogroup M10a was higher than those car-rying other mtDNA haplogroups. These observations indicated that mitochondrial haplogroup M10a may increase the risk of visual loss.

  5. Mutations in the DNA-binding domain of NR2E3 affect in vivo dimerization and interaction with CRX.

    Directory of Open Access Journals (Sweden)

    Raphael Roduit

    Full Text Available BACKGROUND: NR2E3 (PNR is an orphan nuclear receptor essential for proper photoreceptor determination and differentiation. In humans, mutations in NR2E3 have been associated with the recessively inherited enhanced short wavelength sensitive (S- cone syndrome (ESCS and, more recently, with autosomal dominant retinitis pigmentosa (adRP. NR2E3 acts as a suppressor of the cone generation program in late mitotic retinal progenitor cells. In adult rod photoreceptors, NR2E3 represses cone-specific gene expression and acts in concert with the transcription factors CRX and NRL to activate rod-specific genes. NR2E3 and CRX have been shown to physically interact in vitro through their respective DNA-binding domains (DBD. The DBD also contributes to homo- and heterodimerization of nuclear receptors. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed NR2E3 homodimerization and NR2E3/CRX complex formation in an in vivo situation by Bioluminescence Resonance Energy Transfer (BRET(2. NR2E3 wild-type protein formed homodimers in transiently transfected HEK293T cells. NR2E3 homodimerization was impaired in presence of disease-causing mutations in the DBD, except for the p.R76Q and p.R104W mutant proteins. Strikingly, the adRP-linked p.G56R mutant protein interacted with CRX with a similar efficiency to that of NR2E3 wild-type and p.R311Q proteins. In contrast, all other NR2E3 DBD-mutant proteins did not interact with CRX. The p.G56R mutant protein was also more effective in abolishing the potentiation of rhodospin gene transactivation by the NR2E3 wild-type protein. In addition, the p.G56R mutant enhanced the transrepression of the M- and S-opsin promoter, while all other NR2E3 DBD-mutants did not. CONCLUSIONS/SIGNIFICANCE: These results suggest different disease mechanisms in adRP- and ESCS-patients carrying NR2E3 mutations. Titration of CRX by the p.G56R mutant protein acting as a repressor in trans may account for the severe clinical phenotype in adRP patients.

  6. The DNA Ligase IV Syndrome R278H Mutation Impairs B Lymphopoiesis via Error-Prone Nonhomologous End-Joining.

    Science.gov (United States)

    Park, Jihye; Welner, Robert S; Chan, Mei-Yee; Troppito, Logan; Staber, Philipp B; Tenen, Daniel G; Yan, Catherine T

    2016-01-01

    Hypomorphic mutations in the nonhomologous end-joining (NHEJ) DNA repair protein DNA ligase IV (LIG4) lead to immunodeficiency with varying severity. In this study, using a murine knock-in model, we investigated the mechanisms underlying abnormalities in class switch recombination (CSR) associated with the human homozygous Lig4 R278H mutation. Previously, we found that despite the near absence of Lig4 end-ligation activity and severely reduced mature B cell numbers, Lig4(R278H/R278H) (Lig4(R/R)) mice exhibit only a partial CSR block, producing near normal IgG1 and IgE but substantially reduced IgG3, IgG2b, and IgA serum levels. In this study, to address the cause of these abnormalities, we assayed CSR in Lig4(R/R) B cells generated via preassembled IgH and IgK V region exons (HL). This revealed that Lig4(R278H) protein levels while intact exhibited a higher turnover rate during activation of switching to IgG3 and IgG2b, as well as delays in CSR kinetics associated with defective proliferation during activation of switching to IgG1 and IgE. Activated Lig4(R/R)HL B cells consistently accumulated high frequencies of activation-induced cytidine deaminase-dependent IgH locus chromosomal breaks and translocations and were more prone to apoptosis, effects that appeared to be p53-independent, as p53 deficiency did not markedly influence these events. Importantly, NHEJ instead of alternative end-joining (A-EJ) was revealed as the predominant mechanism catalyzing robust CSR. Defective CSR was linked to failed NHEJ and residual A-EJ access to unrepaired double-strand breaks. These data firmly demonstrate that Lig4(R278H) activity renders NHEJ to be more error-prone, and they predict increased error-prone NHEJ activity and A-EJ suppression as the cause of the defective B lymphopoiesis in Lig4 patients. PMID:26608917

  7. Mitochondrial DNA from colorectal cancer cells induces the mutation of NIH3T3 cells%大肠癌线粒体DNA诱导NIH3T3细胞D-环突变

    Institute of Scientific and Technical Information of China (English)

    姬宏莉; 姬宏娟; 宋卫兵; 马永全; 杜芳; 王辉; 肖冰

    2011-01-01

    Objective To investigate the mtDNA mutation in NIH3T3 cell, which was transfected with mutated mtDNA from colorectal cancer cell line. Methods Recombinant eukaryotic vector, expressing plasmid of the mutated mtDNA, was transfected into NIH3T3 cell using Lipofection 2000 TM and screened by G418. The mutated mtDNA of transfected NIH3T3 cell was detected by PCR. Results Mutation and polymorphism was observed in NIH3T3 cell transfected with mutated mtDNA. Conclusion MtDNA mutations in colorectal cancer cell affects NIH3T3 cell mtDNA loci, though further study is required.%目的 观察突变的大肠癌细胞线粒体DNA(mtDNA)转染 NIH3T3 细胞后转染细胞mtDNA D-环突变特性.方法 通过脂质体法(Lipofection 2000TM) 将大肠癌细胞突变的 mtDNA 真核表达载体转染 NIH3T3细胞,利用 G418 抗性筛选克隆细胞;用PCR法检测转染细胞线粒体突变情况.结果 突变mtDNA 导致转染细胞 mtDNA 的突变和多态性变化.结论 突变的大肠癌 mtDNA可致NIH3T3细胞mtDNA位点变化,但具体的机制和过程有待于进一步研究.

  8. DNA glycosylases involved in base excision repair may be associated with cancer risk in BRCA1 and BRCA2 mutation carriers.

    Science.gov (United States)

    Osorio, Ana; Milne, Roger L; Kuchenbaecker, Karoline; Vaclová, Tereza; Pita, Guillermo; Alonso, Rosario; Peterlongo, Paolo; Blanco, Ignacio; de la Hoya, Miguel; Duran, Mercedes; Díez, Orland; Ramón Y Cajal, Teresa; Konstantopoulou, Irene; Martínez-Bouzas, Cristina; Andrés Conejero, Raquel; Soucy, Penny; McGuffog, Lesley; Barrowdale, Daniel; Lee, Andrew; Swe-Brca; Arver, Brita; Rantala, Johanna; Loman, Niklas; Ehrencrona, Hans; Olopade, Olufunmilayo I; Beattie, Mary S; Domchek, Susan M; Nathanson, Katherine; Rebbeck, Timothy R; Arun, Banu K; Karlan, Beth Y; Walsh, Christine; Lester, Jenny; John, Esther M; Whittemore, Alice S; Daly, Mary B; Southey, Melissa; Hopper, John; Terry, Mary B; Buys, Saundra S; Janavicius, Ramunas; Dorfling, Cecilia M; van Rensburg, Elizabeth J; Steele, Linda; Neuhausen, Susan L; Ding, Yuan Chun; Hansen, Thomas V O; Jønson, Lars; Ejlertsen, Bent; Gerdes, Anne-Marie; Infante, Mar; Herráez, Belén; Moreno, Leticia Thais; Weitzel, Jeffrey N; Herzog, Josef; Weeman, Kisa; Manoukian, Siranoush; Peissel, Bernard; Zaffaroni, Daniela; Scuvera, Giulietta; Bonanni, Bernardo; Mariette, Frederique; Volorio, Sara; Viel, Alessandra; Varesco, Liliana; Papi, Laura; Ottini, Laura; Tibiletti, Maria Grazia; Radice, Paolo; Yannoukakos, Drakoulis; Garber, Judy; Ellis, Steve; Frost, Debra; Platte, Radka; Fineberg, Elena; Evans, Gareth; Lalloo, Fiona; Izatt, Louise; Eeles, Ros; Adlard, Julian; Davidson, Rosemarie; Cole, Trevor; Eccles, Diana; Cook, Jackie; Hodgson, Shirley; Brewer, Carole; Tischkowitz, Marc; Douglas, Fiona; Porteous, Mary; Side, Lucy; Walker, Lisa; Morrison, Patrick; Donaldson, Alan; Kennedy, John; Foo, Claire; Godwin, Andrew K; Schmutzler, Rita Katharina; Wappenschmidt, Barbara; Rhiem, Kerstin; Engel, Christoph; Meindl, Alfons; Ditsch, Nina; Arnold, Norbert; Plendl, Hans Jörg; Niederacher, Dieter; Sutter, Christian; Wang-Gohrke, Shan; Steinemann, Doris; Preisler-Adams, Sabine; Kast, Karin; Varon-Mateeva, Raymonda; Gehrig, Andrea; Stoppa-Lyonnet, Dominique; Sinilnikova, Olga M; Mazoyer, Sylvie; Damiola, Francesca; Poppe, Bruce; Claes, Kathleen; Piedmonte, Marion; Tucker, Kathy; Backes, Floor; Rodríguez, Gustavo; Brewster, Wendy; Wakeley, Katie; Rutherford, Thomas; Caldés, Trinidad; Nevanlinna, Heli; Aittomäki, Kristiina; Rookus, Matti A; van Os, Theo A M; van der Kolk, Lizet; de Lange, J L; Meijers-Heijboer, Hanne E J; van der Hout, A H; van Asperen, Christi J; Gómez Garcia, Encarna B; Hoogerbrugge, Nicoline; Collée, J Margriet; van Deurzen, Carolien H M; van der Luijt, Rob B; Devilee, Peter; Hebon; Olah, Edith; Lázaro, Conxi; Teulé, Alex; Menéndez, Mireia; Jakubowska, Anna; Cybulski, Cezary; Gronwald, Jacek; Lubinski, Jan; Durda, Katarzyna; Jaworska-Bieniek, Katarzyna; Johannsson, Oskar Th; Maugard, Christine; Montagna, Marco; Tognazzo, Silvia; Teixeira, Manuel R; Healey, Sue; Investigators, Kconfab; Olswold, Curtis; Guidugli, Lucia; Lindor, Noralane; Slager, Susan; Szabo, Csilla I; Vijai, Joseph; Robson, Mark; Kauff, Noah; Zhang, Liying; Rau-Murthy, Rohini; Fink-Retter, Anneliese; Singer, Christian F; Rappaport, Christine; Geschwantler Kaulich, Daphne; Pfeiler, Georg; Tea, Muy-Kheng; Berger, Andreas; Phelan, Catherine M; Greene, Mark H; Mai, Phuong L; Lejbkowicz, Flavio; Andrulis, Irene; Mulligan, Anna Marie; Glendon, Gord; Toland, Amanda Ewart; Bojesen, Anders; Pedersen, Inge Sokilde; Sunde, Lone; Thomassen, Mads; Kruse, Torben A; Jensen, Uffe Birk; Friedman, Eitan; Laitman, Yael; Shimon, Shani Paluch; Simard, Jacques; Easton, Douglas F; Offit, Kenneth; Couch, Fergus J; Chenevix-Trench, Georgia; Antoniou, Antonis C; Benitez, Javier

    2014-04-01

    Single Nucleotide Polymorphisms (SNPs) in genes involved in the DNA Base Excision Repair (BER) pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of the components of the BER pathway, PARP1 (poly ADP ribose polymerase), and both BRCA1 and BRCA2. In the present study, we have performed a comprehensive analysis of 18 genes involved in BER using a tagging SNP approach in a large series of BRCA1 and BRCA2 mutation carriers. 144 SNPs were analyzed in a two stage study involving 23,463 carriers from the CIMBA consortium (the Consortium of Investigators of Modifiers of BRCA1 and BRCA2). Eleven SNPs showed evidence of association with breast and/or ovarian cancer at p<0.05 in the combined analysis. Four of the five genes for which strongest evidence of association was observed were DNA glycosylases. The strongest evidence was for rs1466785 in the NEIL2 (endonuclease VIII-like 2) gene (HR: 1.09, 95% CI (1.03-1.16), p = 2.7 × 10(-3)) for association with breast cancer risk in BRCA2 mutation carriers, and rs2304277 in the OGG1 (8-guanine DNA glycosylase) gene, with ovarian cancer risk in BRCA1 mutation carriers (HR: 1.12 95%CI: 1.03-1.21, p = 4.8 × 10(-3)). DNA glycosylases involved in the first steps of the BER pathway may be associated with cancer risk in BRCA1/2 mutation carriers and should be more comprehensively studied.

  9. Validation of Next-Generation Sequencing of Entire Mitochondrial Genomes and the Diversity of Mitochondrial DNA Mutations in Oral Squamous Cell Carcinoma.

    Directory of Open Access Journals (Sweden)

    Anita Kloss-Brandstätter

    Full Text Available Oral squamous cell carcinoma (OSCC is mainly caused by smoking and alcohol abuse and shows a five-year survival rate of ~50%. We aimed to explore the variation of somatic mitochondrial DNA (mtDNA mutations in primary oral tumors, recurrences and metastases.We performed an in-depth validation of mtDNA next-generation sequencing (NGS on an Illumina HiSeq 2500 platform for its application to cancer tissues, with the goal to detect low-level heteroplasmies and to avoid artifacts. Therefore we genotyped the mitochondrial genome (16.6 kb from 85 tissue samples (tumors, recurrences, resection edges, metastases and blood collected from 28 prospectively recruited OSCC patients applying both Sanger sequencing and high-coverage NGS (~35,000 reads per base.We observed a strong correlation between Sanger sequencing and NGS in estimating the mixture ratio of heteroplasmies (r = 0.99; p10% were predominant. Four out of six patients who developed a local tumor recurrence showed mutations in the recurrence that had also been observed in the primary tumor. Three out of five patients, who had tumor metastases in the lymph nodes of their necks, shared mtDNA mutations between primary tumors and lymph node metastases. The percentage of mutation heteroplasmy increased from the primary tumor to lymph node metastases.We conclude that Sanger sequencing is valid for heteroplasmy quantification for heteroplasmies ≥10% and that NGS is capable of reliably detecting and quantifying heteroplasmies down to the 1%-level. The finding of shared mutations between primary tumors, recurrences and metastasis indicates a clonal origin of malignant cells in oral cancer.

  10. DNA glycosylases involved in base excision repair may be associated with cancer risk in BRCA1 and BRCA2 mutation carriers.

    Directory of Open Access Journals (Sweden)

    Ana Osorio

    2014-04-01

    Full Text Available Single Nucleotide Polymorphisms (SNPs in genes involved in the DNA Base Excision Repair (BER pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of the components of the BER pathway, PARP1 (poly ADP ribose polymerase, and both BRCA1 and BRCA2. In the present study, we have performed a comprehensive analysis of 18 genes involved in BER using a tagging SNP approach in a large series of BRCA1 and BRCA2 mutation carriers. 144 SNPs were analyzed in a two stage study involving 23,463 carriers from the CIMBA consortium (the Consortium of Investigators of Modifiers of BRCA1 and BRCA2. Eleven SNPs showed evidence of association with breast and/or ovarian cancer at p<0.05 in the combined analysis. Four of the five genes for which strongest evidence of association was observed were DNA glycosylases. The strongest evidence was for rs1466785 in the NEIL2 (endonuclease VIII-like 2 gene (HR: 1.09, 95% CI (1.03-1.16, p = 2.7 × 10(-3 for association with breast cancer risk in BRCA2 mutation carriers, and rs2304277 in the OGG1 (8-guanine DNA glycosylase gene, with ovarian cancer risk in BRCA1 mutation carriers (HR: 1.12 95%CI: 1.03-1.21, p = 4.8 × 10(-3. DNA glycosylases involved in the first steps of the BER pathway may be associated with cancer risk in BRCA1/2 mutation carriers and should be more comprehensively studied.

  11. cDNA analyses of CAPN3 enhance mutation detection and reveal a low prevalence of LGMD2A patients in Denmark

    DEFF Research Database (Denmark)

    Duno, M.; Sveen, M.L.; Schwartz, M.;

    2008-01-01

    Calpainopathy or limb-girdle muscular dystrophy type 2A (LGMD2A) is generally recognized as the most prevalent form of recessive LGMD and is caused by mutations in the CAPN3 gene. Out of a cohort of 119 patients fulfilling clinical criteria for LGMD2, referred to our neuromuscular clinic, 46 were......, only a single heterozygous mutation could be identified both at the genomic level and on full-length CAPN3 cDNA. All three patients exhibited a highly abnormal western blot for calpain-3 and clinical characteristics of LGMD2A. Only three of the genetically confirmed LGMD2A patients were of Danish...

  12. Chloroplast DNA diversity in wild and cultivated species of rice (Genus Oryza, section Oryza). Cladistic-mutation and genetic-distance analysis.

    Science.gov (United States)

    Dally, A M; Second, G

    1990-08-01

    Using a novel nonaqueous procedure, chloroplast DNA was isolated from 318 individual adult rice plants, representing 247 accessions and the breadth of the diversity in section Oryza of genus Oryza. Among them, 32 different cpDNA restriction patterns were distinguished using the restriction endonucleases EcoRI and AvaI, and they were further characterized by restriction with BamHI, HindIII, SmaI, PstI, and BstEII enzymes. The differences in the electrophoretic band patterns were parsimoniously interpreted as being the result of 110 mutations, including 47 restriction site mutations. The relationships between band patterns were studied by a cladistic analysis based on shared mutations and by the computation of genetic distances based on shared bands. The deduced relationships were compared with earlier taxonomical studies. The maternal parents for BC genome allotetraploids were deduced. Within species, cpDNA diversity was found larger in those species with an evolutionary history of recent introgression and/or allotetraploidization. Occasional paternal inheritance and recombination of cpDNA in rice was suggested.

  13. Automated direct sequencing of the iduronate-2 sulfatase gene reveals a vast spectrum of mutations causing Hunter syndrome (mucopolysaccharidosis type II) and a {open_quotes}hot spot{close_quotes} at R468

    Energy Technology Data Exchange (ETDEWEB)

    Whitley, C.B; Jonsson, J.J.; Aronovich, E.L. [Univ. of Minnesota Medical School, Minneapolis, MN (United States)

    1994-09-01

    Hunter syndrome is an X-linked recessive, lethal disease resulting from deficiency of iduronate-2-sulfatase (IDS) catalytic activity. Because of low reproductive fitness, most affected individuals are expected to have new mutations. Most of such defects are anticipated to be single base pair (bp) changes; however, several previous studies utilizing Southern analysis of RT-PCR have identified numerous large gene deletions in patients having the {open_quotes}severe form{close_quotes} with neurologic disease. To investigate the spectrum of IDS mutations, we have developed a method of automated direct sequencing of RT-PCR products representing the entire IDS coding region. Of 19 patients studied by this approach, only 1 had an IDS coding region which did not contain a mutation; 1 had a single bp insertion; 1 had a 2 bp deletion; and 13 had single-base substitutions. Of the 13 having single base substitutions, 2 resulted in aberrant splicing. Only 1 patient had a complete gene deletion; in view of previous reports, there was a surprising lack of major gene deletions. Notably, a CpG dinucleotide at R468 was identified as a {open_quotes}hot spot{close_quotes} for mutation. Five unrelated individuals had substitutions at this site which thus accounted for 28% of all mutations in this series: R468W (3 patients) and R468Q (2 patients). MspI digestion provided a method of rapid diagnosis and determination of heterozygote status for such R468 mutations. Genotype-phenotype correlations in this R468 group are not yet possible because of confounding information, i.e., there are both {open_quotes}mild{close_quotes} and {open_quotes}severe{close_quotes} patients in this group and some have co-existing neurologic diseases. This approach of gene sequencing appears to be necessary, and sufficient, to characterize the vast spectrum of mutations in Hunter syndrome.

  14. Effects of space flight on DNA mutation and secondary metabolites of licorice (Glycyrrhiza uralensis Fisch.)

    Institute of Scientific and Technical Information of China (English)

    GAO WenYuan; LI KeFeng; YAN Shuo; GAO XiuMei; HU LiMin

    2009-01-01

    Licorice (Glycyrrhiza uralensis Fisch.) seeds were flown on a recoverable satellite for 18 days(the average radiation dose in the flight recovery module was 0.102 mGy/d, the distance from flight apogee to earth was 350 km, gravity 10~(-6)). After returning to earth, the seeds were germinated and grown to maturity. The parallel ground-based seeds were also planted under the same conditions. The leaves of licorice were used for inter-simple sequence repeat (ISSR) analysis and the two main secondary metabolites in one-year-old roots were analyzed by high performance liquid chromatography (HPLC).Among 22 random primers used in this experiment, 6 primers generated different DNA band types. Analysis of HPLC showed that the content of glycyrrhizic acid (GA) and liquiritin (LQ) in the roots from seeds flown in space was respectively 2.19, 1.18 times higher than that of the control group. The results demonstrated that the extraterrestrial environment induced mutagenic effects on licorice and affected its secondary metabolites. These changes indicated that extraterrestrial orbit is possible means of breeding of licorice so as to preserve this endangered medicinal plant.

  15. Mulibrey nanism: Two novel mutations in a child identified by Array CGH and DNA sequencing.

    Science.gov (United States)

    Mozzillo, Enza; Cozzolino, Carla; Genesio, Rita; Melis, Daniela; Frisso, Giulia; Orrico, Ada; Lombardo, Barbara; Fattorusso, Valentina; Discepolo, Valentina; Della Casa, Roberto; Simonelli, Francesca; Nitsch, Lucio; Salvatore, Francesco; Franzese, Adriana

    2016-08-01

    In childhood, several rare genetic diseases have overlapping symptoms and signs, including those regarding growth alterations, thus the differential diagnosis is sometimes difficult. The proband, aged 3 years, was suspected to have Silver-Russel syndrome because of intrauterine growth retardation, postnatal growth retardation, typical facial dysmorphic features, macrocephaly, body asymmetry, and bilateral fifth finger clinodactyly. Other features were left atrial and ventricular enlargement and patent foramen ovale. Total X-ray skeleton showed hypoplasia of the twelfth rib bilaterally and of the coccyx, slender long bones with thick cortex, and narrow medullary channels. The genetic investigation did not confirm Silver-Russel syndrome. At the age of 5 the patient developed an additional sign: hepatomegaly. Array CGH revealed a 147 kb deletion (involving TRIM 37 and SKA2 genes) on one allele of chromosome 17, inherited from his mother. These results suggested Mulibrey nanism. The clinical features were found to fit this hypothesis. Sequencing of the TRIM 37 gene showed a single base change at a splicing locus, inherited from his father that provoked a truncated protein. The combined use of Array CGH and DNA sequencing confirmed diagnosis of Mulibrey nanism. The large deletion involving the SKA2 gene, along with the increased frequency of malignant tumours in mulibrey patients, suggests closed monitoring for cancer of our patient and his mother. Array CGH should be performed as first tier test in all infants with multiple anomalies. The clinician should reconsider the clinical features when the genetics suggests this. © 2016 Wiley Periodicals, Inc.

  16. Effects of space flight on DNA mutation and secondary metabolites of licorice (Glycyrrhiza uralensis Fisch.)

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Licorice (Glycyrrhiza uralensis Fisch.) seeds were flown on a recoverable satellite for 18 days(the average radiation dose in the flight recovery module was 0.102 mGy/d, the distance from flight apogee to earth was 350 km, gravity 10-6). After returning to earth, the seeds were germinated and grown to maturity. The parallel ground-based seeds were also planted under the same conditions. The leaves of licorice were used for inter-simple sequence repeat (ISSR) analysis and the two main secondary me-tabolites in one-year-old roots were analyzed by high performance liquid chromatography (HPLC). Among 22 random primers used in this experiment, 6 primers generated different DNA band types. Analysis of HPLC showed that the content of glycyrrhizic acid (GA) and liquiritin (LQ) in the roots from seeds flown in space was respectively 2.19, 1.18 times higher than that of the control group. The results demonstrated that the extraterrestrial environment induced mutagenic effects on licorice and affected its secondary metabolites. These changes indicated that extraterrestrial orbit is possible means of breeding of licorice so as to preserve this endangered medicinal plant.

  17. Parkinson's disease brain mitochondria have impaired respirasome assembly, age-related increases in distribution of oxidative damage to mtDNA and no differences in heteroplasmic mtDNA mutation abundance

    Directory of Open Access Journals (Sweden)

    Keeney Paula M

    2009-09-01

    Full Text Available Abstract Background Sporadic Parkinson's disease (sPD is a nervous system-wide disease that presents with a bradykinetic movement disorder and is frequently complicated by depression and cognitive impairment. sPD likely has multiple interacting causes that include increased oxidative stress damage to mitochondrial components and reduced mitochondrial bioenergetic capacity. We analyzed mitochondria from postmortem sPD and CTL brains for evidence of oxidative damage to mitochondrial DNA (mtDNA, heteroplasmic mtDNA point mutations and levels of electron transport chain proteins. We sought to determine if sPD brains possess any mtDNA genotype-respiratory phenotype relationships. Results Treatment of sPD brain mtDNA with the mitochondrial base-excision repair enzyme 8-oxyguanosine glycosylase-1 (hOGG1 inhibited, in an age-dependent manner, qPCR amplification of overlapping ~2 kbase products; amplification of CTL brain mtDNA showed moderate sensitivity to hOGG1 not dependent on donor age. hOGG1 mRNA expression was not different between sPD and CTL brains. Heteroplasmy analysis of brain mtDNA using Surveyor nuclease® showed asymmetric distributions and levels of heteroplasmic mutations across mtDNA but no patterns that statistically distinguished sPD from CTL. sPD brain mitochondria displayed reductions of nine respirasome proteins (respiratory complexes I-V. Reduced levels of sPD brain mitochondrial complex II, III and V, but not complex I or IV proteins, correlated closely with rates of NADH-driven electron flow. mtDNA levels and PGC-1α expression did not differ between sPD and CTL brains. Conclusion PD brain mitochondria have reduced mitochondrial respiratory protein levels in complexes I-V, implying a generalized defect in respirasome assembly. These deficiencies do not appear to arise from altered point mutational burden in mtDNA or reduction of nuclear signaling for mitochondrial biogenesis, implying downstream etiologies. The origin of age

  18. Mulibrey nanism: Two novel mutations in a child identified by Array CGH and DNA sequencing.

    Science.gov (United States)

    Mozzillo, Enza; Cozzolino, Carla; Genesio, Rita; Melis, Daniela; Frisso, Giulia; Orrico, Ada; Lombardo, Barbara; Fattorusso, Valentina; Discepolo, Valentina; Della Casa, Roberto; Simonelli, Francesca; Nitsch, Lucio; Salvatore, Francesco; Franzese, Adriana

    2016-08-01

    In childhood, several rare genetic diseases have overlapping symptoms and signs, including those regarding growth alterations, thus the differential diagnosis is sometimes difficult. The proband, aged 3 years, was suspected to have Silver-Russel syndrome because of intrauterine growth retardation, postnatal growth retardation, typical facial dysmorphic features, macrocephaly, body asymmetry, and bilateral fifth finger clinodactyly. Other features were left atrial and ventricular enlargement and patent foramen ovale. Total X-ray skeleton showed hypoplasia of the twelfth rib bilaterally and of the coccyx, slender long bones with thick cortex, and narrow medullary channels. The genetic investigation did not confirm Silver-Russel syndrome. At the age of 5 the patient developed an additional sign: hepatomegaly. Array CGH revealed a 147 kb deletion (involving TRIM 37 and SKA2 genes) on one allele of chromosome 17, inherited from his mother. These results suggested Mulibrey nanism. The clinical features were found to fit this hypothesis. Sequencing of the TRIM 37 gene showed a single base change at a splicing locus, inherited from his father that provoked a truncated protein. The combined use of Array CGH and DNA sequencing confirmed diagnosis of Mulibrey nanism. The large deletion involving the SKA2 gene, along with the increased frequency of malignant tumours in mulibrey patients, suggests closed monitoring for cancer of our patient and his mother. Array CGH should be performed as first tier test in all infants with multiple anomalies. The clinician should reconsider the clinical features when the genetics suggests this. © 2016 Wiley Periodicals, Inc. PMID:27256967

  19. Mitochondrial DNA m.3242G > A mutation, an under diagnosed cause of hypertrophic cardiomyopathy and renal tubular dysfunction?

    NARCIS (Netherlands)

    Wortmann, S.B.; Champion, M.P.; Heuvel, L.P. van den; Barth, H.; Trutnau, B.; Craig, K.; Lammens, M.M.; Schreuder, M.F.; Taylor, R.W.; Smeitink, J.A.M.; Wevers, R.A.; Rodenburg, R.J.T.; Morava, E.

    2012-01-01

    We present two new patients with the recently described mitochondrial m.3242G > A mutation. Although the mutation is situated next to the well known m.3243A > G mutation, the most common alteration associated with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episode

  20. An isothermal primer extension method for whole genome amplification of fresh and degraded DNA: applications in comparative genomic hybridization, genotyping and mutation screening.

    Science.gov (United States)

    Lee, Cheryl I P; Leong, Siew Hong; Png, Adrian E H; Choo, Keng Wah; Syn, Christopher; Lim, Dennis T H; Law, Hai Yang; Kon, Oi Lian

    2006-01-01

    We describe a protocol that uses a bioinformatically optimized primer in an isothermal whole genome amplification (WGA) reaction. Overnight incubation at 37 degrees C efficiently generates several hundred- to several thousand-fold increases in input DNA. The amplified product retains reasonably faithful quantitative representation of unamplified whole genomic DNA (gDNA). We provide protocols for applying this isothermal primer extension WGA protocol in three different techniques of genomic analysis: comparative genomic hybridization (CGH), genotyping at simple tandem repeat (STR) loci and screening for single base mutations in a common monogenic disorder, beta-thalassemia. gDNA extracted from formalin-fixed paraffin-embedded (FFPE) tissues can also be amplified with this protocol.

  1. 10例MELAS综合征中线粒体DNA A3243G点突变的检测%Detection of A3243G point mutation in mitochondrial DNA from 10 cases of MELAS

    Institute of Scientific and Technical Information of China (English)

    王朝霞; 刘淑萍; 杨艳玲; 袁云; 吴丽娟; 戚豫; 陈清棠

    2002-01-01

    目的对10例MELAS型线粒体脑肌病患者进行线粒体DNA A3243G点突变的检测。方法 用PCR-限制性内切酶分析法(restriction analysis),检测10例MELAS患者及其8名母系亲属的肌肉和/或外周血细胞中有无mtDNA的A3243G点突变,并进行突变型mtDNA的定量。结果 在10例患者的肌肉和/血细胞中,均检测到A3243点突变。突变型mtDNA的比例在血细胞(7例)中为10.8%-47.8%,在肌肉(5例)中为39.4%-67.7%。有2例患者同时进行了肌肉和血细胞标本的检测,突变型mtDNA的比例肌肉组织均高于血细胞。在血细胞中,年轻患者的突变型比例通常较高。在1个家系中可证实为母系遗传。但在3例先证者的母亲及2例先证者的同胞均未检测到此突变。 结论 10例MELAS综合征患者均携有mtDNA A3243G点突变。在6个家庭中,只有1个家庭可证实为母系遗传,另外5个家庭中此突变可能为散发性,提示在中国人MELAS的发病机制中,mtDNA A3243G点突变为新生突变的居多。%Objective To search for A3243G point mutations in mitochondrial DNA (mtDNA) from 10 cases of mitochondrial encephalomyopathy, lactic acidosis and strokelike episodes (MELAS).Methods Using PCR-restriction analysis, we investigated A3243G point mutations in mtDNA of muscle and/or blood cells from 10 patients and their 8 maternal relatives. We also quantitated the A3243G mtDNA in samples harboring the mutation.Results A3243G point mutations were identified in all muscle and/or blood samples from 10 MELAS patients. The proportion of mutant mtDNA was 10.8%-47.8% in blood (7 cases), and 39.4%-67.7% in muscle (5 cases). This ratio was invariably higher in muscle than in blood from two patients whose blood and muscle samples were both available. Younger patients usually carried higher proportions of A3243G mutant mtDNA in blood. Eight maternal relatives from 6 families were also examined. Maternal transmission of the disease could be

  2. The SWI/SNF Subunit INI1 Contains an N-Terminal Winged Helix DNA Binding Domain that Is a Target for Mutations in Schwannomatosis.

    Science.gov (United States)

    Allen, Mark D; Freund, Stefan M V; Zinzalla, Giovanna; Bycroft, Mark

    2015-07-01

    SWI/SNF complexes use the energy of ATP hydrolysis to remodel chromatin. In mammals they play a central role in regulating gene expression during differentiation and proliferation. Mutations in SWI/SNF subunits are among the most frequent gene alterations in cancer. The INI1/hSNF5/SMARCB1 subunit is mutated in both malignant rhabdoid tumor, a highly aggressive childhood cancer, and schwannomatosis, a tumor-predisposing syndrome characterized by mostly benign tumors of the CNS. Here, we show that mutations in INI1 that cause schwannomatosis target a hitherto unidentified N-terminal winged helix DNA binding domain that is also present in the BAF45a/PHF10 subunit of the SWI/SNF complex. The domain is structurally related to the SKI/SNO/DAC domain, which is found in a number of metazoan chromatin-associated proteins. PMID:26073604

  3. Comparative synchronous fluorescence spectrophotometry and 32P-postlabeling analysis of PAH-DNA adducts in human lung and the relationship to TP53 mutations

    DEFF Research Database (Denmark)

    Andreassen, Åshild; Kure, Elin H.; Nielsen, Per Sabro;

    1996-01-01

    Polycyclic aromatic hydrocarbon (PAH)-DNA adducts were studied in human lung from 39 lung cancer patients by synchronous fluorescence spectrophotometric (SFS) and 32P-postlabeling assays. Regression analysis of the samples failed to detect any correlation between benzo[a]pyrene-diolepoxide (BPDE......)-DNA adducts detected by SFS and the BPDE co-migrating spot detected by 32P-postlabeling. We have also analyzed the relationship between adduct levels and TP53 mutations. By postlabeling diagonal radioactive zone (DRZ) adducts were detected in 37 of 39 (95%) lung tissues from lung cancer patients...

  4. Analysis of p53 gene mutations in human gliomas by polymerase chain reaction-based single-strand conformation polymorphism and DNA sequencing.

    Science.gov (United States)

    Sarkar, F H; Kupsky, W J; Li, Y W; Sreepathi, P

    1994-03-01

    Mutations in the p53 gene have been recognized in brain tumors, and clonal expansion of p53 mutant cells has been shown to be associated with glioma progression. However, studies on the p53 gene have been limited by the need for frozen tissues. We have developed a method utilizing polymerase chain reaction (PCR) for the direct analysis of p53 mutation by single-strand conformation polymorphism (SSCP) and by direct DNA sequencing of the p53 gene using a single 10-microns paraffin-embedded tissue section. We applied this method to screen for p53 gene mutations in exons 5-8 in human gliomas utilizing paraffin-embedded tissues. Twenty paraffin blocks containing tumor were selected from surgical specimens from 17 different adult patients. Tumors included six anaplastic astrocytomas (AAs), nine glioblastomas (GBs), and two mixed malignant gliomas (MMGs). The tissue section on the stained glass slide was used to guide microdissection of an unstained adjacent tissue section to ensure > 90% of the tumor cell population for p53 mutational analysis. Simultaneously, microdissection of the tissue was also carried out to obtain normal tissue from adjacent areas as a control. Mutations in the p53 gene were identified in 3 of 17 (18%) patients by PCR-SSCP analysis and subsequently confirmed by PCR-based DNA sequencing. Mutations in exon 5 resulting in amino acid substitution were found in one thalamic AA (codon 158, CGC > CTT: Arg > Leu) and one cerebral hemispheric GB (codon 151, CCG > CTG: Pro > Leu).(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Lambda YES: a multifunctional cDNA expression vector for the isolation of genes by complementation of yeast and Escherichia coli mutations.

    OpenAIRE

    Elledge, S J; Mulligan, J T; Ramer, S W; Spottswood, M; Davis, R W

    1991-01-01

    This work describes a multifunctional phage lambda expression vector system, lambda YES, designed to facilitate gene isolation from eukaryotes by complementation of Escherichia coli and Saccharomyces cerevisiae mutations. lambda YES vectors have a selection for cDNA inserts using an oligo adaptor strategy and are capable of expressing genes in both E. coli and S. cerevisiae. They also allow conversion from phage lambda to plasmid clones by using the cre-lox site-specific recombination system,...

  6. Severe manifestation of Leber's hereditary optic neuropathy due to 11778G>A mtDNA mutation in a female with hypoestrogenism due to Perrault syndrome.

    Science.gov (United States)

    Badura-Stronka, Magdalena; Wawrocka, Anna; Zawieja, Krzysztof; Silska, Sylwia; Krawczyński, Maciej Robert

    2013-11-01

    Perrault syndrome (PS) is a rare autosomal recessive condition with ovarian dysgenesis, hearing deficit and neurological abnormalities in female patients. The molecular basis of the syndrome is heterogeneous, mutations in the HSD17B4 gene have been identified in one family and mutations in the HARS2 gene have been found in another one. We have excluded pathogenic changes in the HSD17B4 gene and in the HARS2 gene by a direct sequencing of all coding exons in a female with clinical hallmarks of PS, ataxia and mild mental retardation. In addition, the patient suffers from severe Leber's hereditary optic neuropathy (LHON) due to 11778G>A mtDNA mutation. This case is the first reported patient with PS and LHON. Possible influence of hypoestrogenism on the manifestation of optic neuropathy in this patient is discussed in the context of recent findings concerning the crucial role of estrogens in supporting the vision capacity in LHON-related mtDNA mutation carriers.

  7. Visual recovery in a man with the rare combination of mtDNA 11778 LHON mutation and a MS-like disease after mitoxantrone therapy.

    Science.gov (United States)

    Buhmann, C; Gbadamosi, J; Heesen, C

    2002-10-01

    We describe a young man with prognostic unfavourable homoplasmatic mitochondrial DNA(mt DNA) 11778 Leber's hereditary optic neuropathy (LHON) point mutation and confirmed multiple sclerosis (MS). This combination of LHON and MS-like disease is rare in both sexes, and in men has been described in only a few case reports. In a 4-year follow-up during immunosuppressive therapy with mitoxantrone, we found a remarkable time delayed visual recovery 12 months after acute onset of rapid sequential bilateral subtotal visual loss followed by episodes of isolated acute demyelinative optic neuropathy. Visual recovery to such extent after this latency is uncommon in both mtDNA 11778 LHON mutation and optic neuritis (ON) in MS. Relapses in visual deterioration must be considered as extremely rare in LHON. This case might support the hypothesis of an immunological pathogenetic factor in combined LHON and MS, and possibly in LHON alone. We suggest a search for the LHON mutation in MS patients with predominant visual impairment, independent of patients' gender.

  8. Mutation-Induced Population Shift in the MexR Conformational Ensemble Disengages DNA Binding: A Novel Mechanism for MarR Family Derepression.

    Science.gov (United States)

    Anandapadamanaban, Madhanagopal; Pilstål, Robert; Andresen, Cecilia; Trewhella, Jill; Moche, Martin; Wallner, Björn; Sunnerhagen, Maria

    2016-08-01

    MexR is a repressor of the MexAB-OprM multidrug efflux pump operon of Pseudomonas aeruginosa, where DNA-binding impairing mutations lead to multidrug resistance (MDR). Surprisingly, the crystal structure of an MDR-conferring MexR mutant R21W (2.19 Å) presented here is closely similar to wild-type MexR. However, our extended analysis, by molecular dynamics and small-angle X-ray scattering, reveals that the mutation stabilizes a ground state that is deficient of DNA binding and is shared by both mutant and wild-type MexR, whereas the DNA-binding state is only transiently reached by the more flexible wild-type MexR. This population shift in the conformational ensemble is effected by mutation-induced allosteric coupling of contact networks that are independent in the wild-type protein. We propose that the MexR-R21W mutant mimics derepression by small-molecule binding to MarR proteins, and that the described allosteric model based on population shifts may also apply to other MarR family members. PMID:27427478

  9. Differential radiosensitivity phenotypes of DNA-PKcs mutations affecting NHEJ and HRR systems following irradiation with gamma-rays or very low fluences of alpha particles.

    Directory of Open Access Journals (Sweden)

    Yu-Fen Lin

    Full Text Available We have examined cell-cycle dependence of chromosomal aberration induction and cell killing after high or low dose-rate γ irradiation in cells bearing DNA-PKcs mutations in the S2056 cluster, the T2609 cluster, or the kinase domain. We also compared sister chromatid exchanges (SCE production by very low fluences of α-particles in DNA-PKcs mutant cells, and in homologous recombination repair (HRR mutant cells including Rad51C, Rad51D, and Fancg/xrcc9. Generally, chromosomal aberrations and cell killing by γ-rays were similarly affected by mutations in DNA-PKcs, and these mutant cells were more sensitive in G1 than in S/G2 phase. In G1-irradiated DNA-PKcs mutant cells, both chromosome- and chromatid-type breaks and exchanges were in excess than wild-type cells. For cells irradiated in late S/G2 phase, mutant cells showed very high yields of chromatid breaks compared to wild-type cells. Few exchanges were seen in DNA-PKcs-null, Ku80-null, or DNA-PKcs kinase dead mutants, but exchanges in excess were detected in the S2506 or T2609 cluster mutants. SCE induction by very low doses of α-particles is resulted from bystander effects in cells not traversed by α-particles. SCE seen in wild-type cells was completely abolished in Rad51C- or Rad51D-deficient cells, but near normal in Fancg/xrcc9 cells. In marked contrast, very high levels of SCEs were observed in DNA-PKcs-null, DNA-PKcs kinase-dead and Ku80-null mutants. SCE induction was also abolished in T2609 cluster mutant cells, but was only slightly reduced in the S2056 cluster mutant cells. Since both non-homologous end-joining (NHEJ and HRR systems utilize initial DNA lesions as a substrate, these results suggest the possibility of a competitive interference phenomenon operating between NHEJ and at least the Rad51C/D components of HRR; the level of interaction between damaged DNA and a particular DNA-PK component may determine the level of interaction of such DNA with a relevant HRR component.

  10. A point mutation in the DNA-binding domain of HPV-2 E2 protein increases its DNA-binding capacity and reverses its transcriptional regulatory activity on the viral early promoter

    Directory of Open Access Journals (Sweden)

    Gao Chen

    2012-02-01

    Full Text Available Abstract Background The human papillomavirus (HPV E2 protein is a multifunctional DNA-binding protein. The transcriptional activity of HPV E2 is mediated by binding to its specific binding sites in the upstream regulatory region of the HPV genomes. Previously we reported a HPV-2 variant from a verrucae vulgaris patient with huge extensive clustered cutaneous, which have five point mutations in its E2 ORF, L118S, S235P, Y287H, S293R and A338V. Under the control of HPV-2 LCR, co-expression of the mutated HPV E2 induced an increased activity on the viral early promoter. In the present study, a series of mammalian expression plasmids encoding E2 proteins with one to five amino acid (aa substitutions for these mutations were constructed and transfected into HeLa, C33A and SiHa cells. Results CAT expression assays indicated that the enhanced promoter activity was due to the co-expressions of the E2 constructs containing A338V mutation within the DNA-binding domain. Western blots analysis demonstrated that the transiently transfected E2 expressing plasmids, regardless of prototype or the A338V mutant, were continuously expressed in the cells. To study the effect of E2 mutations on its DNA-binding activity, a serial of recombinant E2 proteins with various lengths were expressed and purified. Electrophoresis mobility shift assays (EMSA showed that the binding affinity of E2 protein with A338V mutation to both an artificial probe with two E2 binding sites or HPV-2 and HPV-16 promoter-proximal LCR sequences were significantly stronger than that of the HPV-2 prototype E2. Furthermore, co-expression of the construct containing A338V mutant exhibited increased activities on heterologous HPV-16 early promoter P97 than that of prototype E2. Conclusions These results suggest that the mutation from Ala to Val at aa 338 is critical for E2 DNA-binding and its transcriptional regulation.

  11. Monitoring of epidermal growth factor receptor tyrosine kinase inhibitor-sensitizing and resistance mutations in the plasma DNA of patients with advanced non-small cell lung cancer during treatment with erlotinib

    DEFF Research Database (Denmark)

    Sorensen, Boe S; Wu, Lin; Wei, Wen;

    2014-01-01

    BACKGROUND: The feasibility of monitoring epidermal growth factor receptor (EGFR) mutations in plasma DNA from patients with advanced non-small cell lung cancer (NSCLC) during treatment with erlotinib and its relation to disease progression was investigated. METHODS: The amount of EGFR-mutant DNA...... was tested in plasma DNA from patients with advanced NSCLC with allele-specific polymerase chain reaction assays. Blood samples from 23 patients with adenocarcinoma of NSCLC that carried tyrosine kinase inhibitor-sensitizing EGFR mutations were taken immediately before treatment with erlotinib. Additional...... blood samples were taken at timed intervals until erlotinib treatment was withdrawn. RESULTS: The amount of plasma DNA with sensitizing EGFR mutations was found to be reduced after the first cycle of erlotinib treatment in 22 of 23 patients (96%). No patients presented with the resistant T790M mutation...

  12. A Non-Invasive Droplet Digital PCR (ddPCR) Assay to Detect Paternal CFTR Mutations in the Cell-Free Fetal DNA (cffDNA) of Three Pregnancies at Risk of Cystic Fibrosis via Compound Heterozygosity

    OpenAIRE

    Debrand, Emmanuel; Lykoudi, Alexandra; Bradshaw, Elizabeth; Allen, Stephanie K.

    2015-01-01

    Introduction Non-invasive prenatal diagnosis (NIPD) makes use of cell-free fetal DNA (cffDNA) in the mother’s bloodstream as an alternative to invasive sampling methods such as amniocentesis or CVS, which carry a 0.5–1% risk of fetal loss. We describe a droplet digital PCR (ddPCR) assay designed to inform the testing options for couples whose offspring are at risk of suffering from cystic fibrosis via compound heterozygosity. By detecting the presence or absence of the paternal mutation in th...

  13. The DNA ND4 12026 A to G mutation of mitochondria and type 2 diabetes%线粒体DNA ND4 12026基因突变与糖尿病

    Institute of Scientific and Technical Information of China (English)

    秦跃娟; 于永春; 史京衡; 吴松华

    2001-01-01

    目的研究线粒体DNA ND4 12026位点在中国上海地区非肥胖2型糖尿病患者中的突变情况,并探讨该位点突变的临床特征。方法采用PCR-SSCP、PCR-RFLP及PCR产物直接测序等技术检测线粒体DNA、ND4片段的突变情况。结果发现286例糖尿病患者血样中有12例患者mtDNA ND4基因上12026位点存在AtoG的点突变,导致异亮氨酸错义突变成缬氨酸,而在242例非糖尿病对照组个体中仅1例存在同样突变。对该位点突变的临床分析发现起病年龄低,58%有家族史,需要胰岛素治疗。结论12026位点A to G的突变可能与糖尿病的发生有关,并有一定的临床特征。%Objective To investigate the prevalence of mutation at position 12026 of mitochondrial ND4 gene in Chinese diabetes and analyze the clinical characteristics of these patients. Methods Using PCR-SSCP, PCR-RFLP and PCR product direct sequencing technique, we analyzed the mutation of mitochondrial DNA(mtDNA) ND4 gene.Results A to G mutation at position 12026 of mitochondrial DNA ND4 were found in 12 diabetics (12/286) and one in the control subjects, which was resulted from misplacement of isoleucine by valine. The patients with mutation exhibited characteristics of early onset, more family histories(58 % ) and needed insulin therapy more frequently than the patients without the mutation. Conclusion We infer that the mutation at position 12026 is related to the development of diabetes. (Shanghai Med J, 2001,24: 210-213)

  14. Heterozygous PALB2 c.1592delT mutation channels DNA double-strand break repair into error-prone pathways in breast cancer patients.

    Science.gov (United States)

    Obermeier, K; Sachsenweger, J; Friedl, T W P; Pospiech, H; Winqvist, R; Wiesmüller, L

    2016-07-21

    Hereditary heterozygous mutations in a variety of DNA double-strand break (DSB) repair genes have been associated with increased breast cancer risk. In the Finnish population, PALB2 (partner and localizer of BRCA2) represents a major susceptibility gene for female breast cancer, and so far, only one mutation has been described, c.1592delT, which leads to a sixfold increased disease risk. PALB2 is thought to participate in homologous recombination (HR). However, the effect of the Finnish founder mutation on DSB repair has not been investigated. In the current study, we used a panel of lymphoblastoid cell lines (LCLs) derived from seven heterozygous female PALB2 c.1592delT mutation carriers with variable health status and six wild-type matched controls. The results of our DSB repair analysis showed that the PALB2 mutation causes specific changes in pathway usage, namely increases in error-prone single-strand annealing (SSA) and microhomology-mediated end-joining (MMEJ) compared with wild-type LCLs. These data indicated haploinsufficiency regarding the suppression of error-prone DSB repair in PALB2 mutation carriers. To the contrary, neither reduced HR activities, nor impaired RAD51 filament assembly, nor sensitization to PARP inhibition were consistently observed. Expression of truncated mutant versus wild-type PALB2 verified a causal role of PALB2 c.1592delT in the shift to error-prone repair. Discrimination between healthy and malignancy-presenting PALB2 mutation carriers revealed a pathway shift particularly in the breast cancer patients, suggesting interaction of PALB2 c.1592delT with additional genomic lesions. Interestingly, the studied PALB2 mutation was associated with 53BP1 accumulation in the healthy mutation carriers but not the patients, and 53BP1 was limiting for error-prone MMEJ in patients but not in healthy carriers. Our study identified a rise in error-prone DSB repair as a potential threat to genomic integrity in heterozygous PALB2 mutation carriers

  15. Low-dose DNA damage and replication stress responses quantified by optimized automated single-cell image analysis

    DEFF Research Database (Denmark)

    Mistrik, Martin; Oplustilova, Lenka; Lukas, Jiri;

    2009-01-01

    advantages and applicability of this technique. Our present data on assessment of low radiation doses, repair kinetics, spontaneous DNA damage in cancer cells, as well as constitutive and replication stress-induced HR events and their dependence on upstream factors within the DDR machinery document the......Maintenance of genome integrity is essential for homeostasis and survival as impaired DNA damage response (DDR) may predispose to grave pathologies such as neurodegenerative and immunodeficiency syndromes, cancer and premature aging. Therefore, accurate assessment of DNA damage caused by...... environmental or metabolic genotoxic insults is critical for contemporary biomedicine. The available physical, flow cytometry and sophisticated scanning approaches to DNA damage estimation each have some drawbacks such as insufficient sensitivity, limitation to analysis of cells in suspension, or high costs and...

  16. Discovery and characterization of artifactual mutations in deep coverage targeted capture sequencing data due to oxidative DNA damage during sample preparation.

    Science.gov (United States)

    Costello, Maura; Pugh, Trevor J; Fennell, Timothy J; Stewart, Chip; Lichtenstein, Lee; Meldrim, James C; Fostel, Jennifer L; Friedrich, Dennis C; Perrin, Danielle; Dionne, Danielle; Kim, Sharon; Gabriel, Stacey B; Lander, Eric S; Fisher, Sheila; Getz, Gad

    2013-04-01

    As researchers begin probing deep coverage sequencing data for increasingly rare mutations and subclonal events, the fidelity of next generation sequencing (NGS) laboratory methods will become increasingly critical. Although error rates for sequencing and polymerase chain reaction (PCR) are well documented, the effects that DNA extraction and other library preparation steps could have on downstream sequence integrity have not been thoroughly evaluated. Here, we describe the discovery of novel C > A/G > T transversion artifacts found at low allelic fractions in targeted capture data. Characteristics such as sequencer read orientation and presence in both tumor and normal samples strongly indicated a non-biological mechanism. We identified the source as oxidation of DNA during acoustic shearing in samples containing reactive contaminants from the extraction process. We show generation of 8-oxoguanine (8-oxoG) lesions during DNA shearing, present analysis tools to detect oxidation in sequencing data and suggest methods to reduce DNA oxidation through the introduction of antioxidants. Further, informatics methods are presented to confidently filter these artifacts from sequencing data sets. Though only seen in a low percentage of reads in affected samples, such artifacts could have profoundly deleterious effects on the ability to confidently call rare mutations, and eliminating other possible sources of artifacts should become a priority for the research community.

  17. Application of DNA based marker mutations for improvement of cereals and other sexually reproduced crop plants. Proceedings of a final research co-ordination meeting

    International Nuclear Information System (INIS)

    The Co-ordinated Research Programme (CRP) on the Application of DNA Based Marker Mutations for Improvement of Cereals and Other Sexually Reproduced Crop Plants represents the first of three CRPs dealing with the application of molecular markers to mutations and plant breeding and was implemented between 1992 and 1996. A second companion CRP entitled Use of Novel DNA Fingerprinting Techniques for the Detection and Characterization of Genetic Variation in Vegetatively Propagated Crops devoted to the application of molecular markers in vegetatively propagated crops species was implemented between 1993 and 1997. One positive consequence of these two CRPs has been the implementation of a third CRP entitled Radioactively Labeled DNA Probes for Crop Improvement, which began in 1995 and aims to provide enabling technologies, in the form of probes and primers, to laboratories in developing countries. The rapid development of molecular marker technologies has also resulted in a dramatic increase in request from developing Member States for technical co-operation projects utilizing molecular markers to improve local varieties for biotic and abiotic stresses and other traits of relevance. With the intensified use of induced mutations in genetic studies, it will be important to continue the important work of understanding induced mutations at the molecular level. Evidence of the progress made in implementing molecular marker technologies in laboratories around the world is presented in this publication, which contains the results presented by the participants at the fourth and final Research Co-ordination Meeting of the CRP held in Vienna, 4-8 November 1996. The FAO and IAEA wish to express their sincere appreciation to the participants of the meeting for their work during the project period resulting in the summary and scientific reports presented in this publication

  18. A UV-sensitive syndrome patient with a specific CSA mutation reveals separable roles for CSA in response to UV and oxidative DNA damage.

    Science.gov (United States)

    Nardo, Tiziana; Oneda, Roberta; Spivak, Graciela; Vaz, Bruno; Mortier, Laurent; Thomas, Pierre; Orioli, Donata; Laugel, Vincent; Stary, Anne; Hanawalt, Philip C; Sarasin, Alain; Stefanini, Miria

    2009-04-14

    UV-sensitive syndrome (UV(S)S) is a recently-identified autosomal recessive disorder characterized by mild cutaneous symptoms and defective transcription-coupled repair (TC-NER), the subpathway of nucleotide excision repair (NER) that rapidly removes damage that can block progression of the transcription machinery in actively-transcribed regions of DNA. Cockayne syndrome (CS) is another genetic disorder with sun sensitivity and defective TC-NER, caused by mutations in the CSA or CSB genes. The clinical hallmarks of CS include neurological/developmental abnormalities and premature aging. UV(S)S is genetically heterogeneous, in that it appears in individuals with mutations in CSB or in a still-unidentified gene. We report the identification of a UV(S)S patient (UV(S)S1VI) with a novel mutation in the CSA gene (p.trp361cys) that confers hypersensitivity to UV light, but not to inducers of oxidative damage that are notably cytotoxic in cells from CS patients. The defect in UV(S)S1VI cells is corrected by expression of the WT CSA gene. Expression of the p.trp361cys-mutated CSA cDNA increases the resistance of cells from a CS-A patient to oxidative stress, but does not correct their UV hypersensitivity. These findings imply that some mutations in the CSA gene may interfere with the TC-NER-dependent removal of UV-induced damage without affecting its role in the oxidative stress response. The differential sensitivity toward oxidative stress might explain the difference between the range and severity of symptoms in CS and the mild manifestations in UV(s)S patients that are limited to skin photosensitivity without precocious aging or neurodegeneration.

  19. High throughput detection of Coxiella burnetii by real-time PCR with internal control system and automated DNA preparation

    Directory of Open Access Journals (Sweden)

    Kramme Stefanie

    2008-05-01

    Full Text Available Abstract Background Coxiella burnetii is the causative agent of Q-fever, a widespread zoonosis. Due to its high environmental stability and infectivity it is regarded as a category B biological weapon agent. In domestic animals infection remains either asymptomatic or presents as infertility or abortion. Clinical presentation in humans can range from mild flu-like illness to acute pneumonia and hepatitis. Endocarditis represents the most common form of chronic Q-fever. In humans serology is the gold standard for diagnosis but is inadequate for early case detection. In order to serve as a diagnostic tool in an eventual biological weapon attack or in local epidemics we developed a real-time 5'nuclease based PCR assay with an internal control system. To facilitate high-throughput an automated extraction procedure was evaluated. Results To determine the minimum number of copies that are detectable at 95% chance probit analysis was used. Limit of detection in blood was 2,881 copies/ml [95%CI, 2,188–4,745 copies/ml] with a manual extraction procedure and 4,235 copies/ml [95%CI, 3,143–7,428 copies/ml] with a fully automated extraction procedure, respectively. To demonstrate clinical application a total of 72 specimens of animal origin were compared with respect to manual and automated extraction. A strong correlation between both methods was observed rendering both methods suitable. Testing of 247 follow up specimens of animal origin from a local Q-fever epidemic rendered real-time PCR more sensitive than conventional PCR. Conclusion A sensitive and thoroughly evaluated real-time PCR was established. Its high-throughput mode may show a useful approach to rapidly screen samples in local outbreaks for other organisms relevant for humans or animals. Compared to a conventional PCR assay sensitivity of real-time PCR was higher after testing samples from a local Q-fever outbreak.

  20. 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)

    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 (CdCl2) and cadmium sulphate (CdSO4) 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

  1. Only male matrilineal relatives with Leber's hereditary optic neuropathy in a large Chinese family carrying the mitochondrial DNA G11778A mutation

    International Nuclear Information System (INIS)

    We report here the characterization of a five-generation large Chinese family with Leber's hereditary optic neuropathy (LHON). Very strikingly, six affected individuals of 38 matrilineal relatives (17 females/21 males) are exclusively males in this Chinese family. These matrilineal relatives in this family exhibited late-onset/progressive visual impairment with a wide range of severity, ranging from blindness to normal vision. The age of onset in visual impairment varies from 17 to 30 years. Sequence analysis of the complete mitochondrial genome in this pedigree revealed the presence of the G11778A mutation in ND4 gene and 29 other variants. This mitochondrial genome belongs to the Southern Chinese haplogroup B5b. We showed that the G11778A mutation is present at near homoplasmy in matrilineal relatives of this Chinese family but not in 164 Chinese controls. Incomplete penetrance of LHON in this family indicates the involvement of modulatory factors in the phenotypic expression of visual dysfunction associated with the G11778A mutation. However, none of other mtDNA variants are evolutionarily conserved and implicated to have significantly functional consequence. Thus, nuclear modifier gene(s) or environmental factor(s) seem to account for the penetrance and phenotypic variability of LHON in this Chinese family carrying the G11778A mutation

  2. Determination of a new collagen type I alpha 2 gene point mutation which causes a Gly640 Cys substitution in osteogenesis imperfecta and prenatal diagnosis by DNA hybridisation.

    Science.gov (United States)

    Gomez-Lira, M; Sangalli, A; Pignatti, P F; Digilio, M C; Giannotti, A; Carnevale, E; Mottes, M

    1994-01-01

    The molecular defect responsible for a sporadic case of extremely severe (type II/III) osteogenesis imperfecta was investigated. The mutation site was localised in the collagen type I pro alpha 2 mRNA molecules produced by the proband's skin fibroblasts by chemical cleavage of mismatch in heteroduplex nucleic acids. Reverse transcription-polymerase chain reaction DNA amplification, followed by cloning and sequencing, showed heterozygosity for a G to T transversion in the first nucleotide of exon 37 of the COL1A2 gene, which led to a cysteine for glycine substitution at position 640 of the triple helical domain. This newly characterised mutation is localised in a domain which contains several milder mutations, confirming that glycine substitutions within the alpha 2(I) chain do not follow a linear gradient pattern for genotype to phenotype correlations. In a subsequent pregnancy, absence of the G2327T mutation in the fetus was shown by allele specific oligonucleotide hybridisation to the trophoblast derived fibroblast mRNA after reverse transcription and in vitro amplification. (The nucleotide number assigned to the mutant base was inferred from the numbering system devised by the Osteogenesis Imperfecta Analysis Consortium (The OIAC Newsletter, 1 April 1994).) Images PMID:7891382

  3. The Cerebro-oculo-facio-skeletal Syndrome Point Mutation F231L in the ERCC1 DNA Repair Protein Causes Dissociation of the ERCC1-XPF Complex*

    Science.gov (United States)

    Faridounnia, Maryam; Wienk, Hans; Kovačič, Lidija; Folkers, Gert E.; Jaspers, Nicolaas G. J.; Kaptein, Robert; Hoeijmakers, Jan H. J.; Boelens, Rolf

    2015-01-01

    The ERCC1-XPF heterodimer, a structure-specific DNA endonuclease, is best known for its function in the nucleotide excision repair (NER) pathway. The ERCC1 point mutation F231L, located at the hydrophobic interaction interface of ERCC1 (excision repair cross-complementation group 1) and XPF (xeroderma pigmentosum complementation group F), leads to severe NER pathway deficiencies. Here, we analyze biophysical properties and report the NMR structure of the complex of the C-terminal tandem helix-hairpin-helix domains of ERCC1-XPF that contains this mutation. The structures of wild type and the F231L mutant are very similar. The F231L mutation results in only a small disturbance of the ERCC1-XPF interface, where, in contrast to Phe231, Leu231 lacks interactions stabilizing the ERCC1-XPF complex. One of the two anchor points is severely distorted, and this results in a more dynamic complex, causing reduced stability and an increased dissociation rate of the mutant complex as compared with wild type. These data provide a biophysical explanation for the severe NER deficiencies caused by this mutation. PMID:26085086

  4. SNPase-ARMS qPCR: Ultrasensitive Mutation-Based Detection of Cell-Free Tumor DNA in Melanoma Patients

    OpenAIRE

    Stadler, Julia; Eder, Johanna; Pratscher, Barbara; Brandt, Sabine; Schneller, Doris; Müllegger, Robert; Vogl, Claus; Trautinger, Franz; Brem, Gottfried; Burgstaller, Joerg P.

    2015-01-01

    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 appar...

  5. Semi-automated extraction of microbial DNA from feces for qPCR and phylogenetic microarray analysis

    NARCIS (Netherlands)

    Nylund, L.; Heilig, G.H.J.; Salminen, S.; Vos, de W.M.; Satokari, R.M.

    2010-01-01

    The human gastrointestinal tract (GI-tract) harbors a complex microbial ecosystem, largely composed of so far uncultured species, which can be detected only by using techniques such as PCR and by different hybridization techniques including phylogenetic microarrays. Manual DNA extraction from feces

  6. Clinical and molecular features of an infant patient affected by Leigh Disease associated to m.14459G > A mitochondrial DNA mutation: a case report

    Directory of Open Access Journals (Sweden)

    Moggio Maurizio

    2011-07-01

    Full Text Available Abstract Background Leigh Syndrome (LS is a severe neurodegenerative disorder characterized by bilateral symmetrical necrotic lesions in the basal ganglia and brainstem. Onset is in early infancy and prognosis is poor. Causative mutations have been disclosed in mitochondrial DNA and nuclear genes affecting respiratory chain subunits and assembly factors. Case presentation Here we report the clinical and molecular features of a 15-month-old female LS patient. Direct sequencing of her muscle-derived mtDNA revealed the presence of two apparently homoplasmic variants: the novel m.14792C > G and the already known m.14459G > A resulting in p.His16Asp change in cytochrome b (MT-CYB and p.Ala72Val substitution in ND6 subunit, respectively. The m.14459G > A was heteroplasmic in the mother's blood-derived DNA. Conclusions The m.14459G > A might lead to LS, complicated LS or Leber Optic Hereditary Neuropathy. A comprehensive re-evaluation of previously described 14459G > A-mutated patients does not explain this large clinical heterogeneity.

  7. Collateral damage: Spread of repeat-induced point mutation from a duplicated DNA sequence into an adjoining single-copy gene in Neurospora crassa

    Indian Academy of Sciences (India)

    Meenal Vyas; Durgadas P Kasbekar

    2005-02-01

    Repeat-induced point mutation (RIP) is an unusual genome defense mechanism that was discovered in Neurospora crassa. RIP occurs during a sexual cross and induces numerous G : C to A : T mutations in duplicated DNA sequences and also methylates many of the remaining cytosine residues. We measured the susceptibility of the erg-3 gene, present in single copy, to the spread of RIP from duplications of adjoining sequences. Genomic segments of defined length (1, 1.5 or 2 kb) and located at defined distances (0, 0.5, 1 or 2 kb) upstream or downstream of the erg-3 open reading frame (ORF) were amplified by polymerase chain reaction (PCR), and the duplications were created by transformation of the amplified DNA. Crosses were made with the duplication strains and the frequency of erg-3 mutant progeny provided a measure of the spread of RIP from the duplicated segments into the erg-3 gene. Our results suggest that ordinarily RIP-spread does not occur. However, occasionally the mechanism that confines RIP to the duplicated segment seems to fail (frequency 0.1–0.8%) and then RIP can spread across as much as 1 kb of unduplicated DNA. Additionally, the bacterial hph gene appeared to be very susceptible to the spread of RIP-associated cytosine methylation.

  8. Interfacing click chemistry with automated oligonucleotide synthesis for the preparation of fluorescent DNA probes containing internal xanthene and cyanine dyes

    DEFF Research Database (Denmark)

    Astakhova, I Kira; Wengel, Jesper

    2013-01-01

    for the first time performed solid-phase copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click labeling during the automated phosphoramidite oligonucleotide synthesis followed by postsynthetic click reactions in solution. We demonstrate that our novel strategy is rapid and efficient for the preparation...... of novel oligonucleotide probes containing internally positioned xanthene and cyanine dye pairs and thus represents a significant step forward for the preparation of advanced fluorescent oligonucleotide probes. Furthermore, we demonstrate that the novel xanthene and cyanine labeled probes display unusual...... and very promising photophysical properties resulting from energy-transfer interactions between the fluorophores controlled by nucleic acid assembly. Potential benefits of using these novel fluorescent probes within, for example, molecular diagnostics and fluorescence microscopy include: Considerable...

  9. Novel mutations and DNA-based screening in non-Jewish carriers of Tay-Sachs disease.

    OpenAIRE

    Akerman, B R; Natowicz, M R; Kaback, M M; Loyer, M.; Campeau, E; Gravel, R A

    1997-01-01

    We have evaluated the feasibility of using PCR-based mutation screening for non-Jewish enzyme-defined carriers identified through Tay-Sachs disease-prevention programs. Although Tay-Sachs mutations are rare in the general population, non-Jewish individuals may be screened as spouses of Jewish carriers or as relatives of probands. In order to define a panel of alleles that might account for the majority of mutations in non-Jewish carriers, we investigated 26 independent alleles from 20 obligat...

  10. Mating-type suppression of the DNA-repair defect of the yeast rad6 delta mutation requires the activity of genes in the RAD52 epistasis group.

    Science.gov (United States)

    Yan, Y X; Schiestl, R H; Prakash, L

    1995-06-01

    The RAD6 gene of Saccharomyces cerevisiae is required for post-replication repair of UV-damaged DNA, UV mutagenesis, and sporulation. Here, we show that the radiation sensitivity of a MATa rad6 delta strain can be suppressed by the MAT alpha 2 gene carried on a multicopy plasmid. The a1-alpha 2 suppression is specific to the RAD6 pathway, as mutations in genes required for nucleotide excision repair or for recombinational repair do not show such mating-type suppression. The a1-alpha 2 suppression of the rad6 delta mutation requires the activity of the RAD52 group of genes, suggesting that suppression occurs by channelling of post-replication gaps present in the rad6 delta mutant into the RAD52 recombinational repair pathway. The a1-alpha 2 repressor could mediate this suppression via an enhancement in the expression, or the activity, of recombination genes.

  11. Mutation of the mouse Syce1 gene disrupts synapsis and suggests a link between synaptonemal complex structural components and DNA repair.

    Directory of Open Access Journals (Sweden)

    Ewelina Bolcun-Filas

    2009-02-01

    Full Text Available In mammals, the synaptonemal complex is a structure required to complete crossover recombination. Although suggested by cytological work, in vivo links between the structural proteins of the synaptonemal complex and the proteins of the recombination process have not previously been made. The central element of the synaptonemal complex is traversed by DNA at sites of recombination and presents a logical place to look for interactions between these components. There are four known central element proteins, three of which have previously been mutated. Here, we complete the set by creating a null mutation in the Syce1 gene in mouse. The resulting disruption of synapsis in these animals has allowed us to demonstrate a biochemical interaction between the structural protein SYCE2 and the repair protein RAD51. In normal meiosis, this interaction may be responsible for promoting homologous synapsis from sites of recombination.

  12. Structural and mutational analyses of Deinococcus radiodurans UvrA2 provide insight into DNA binding and damage recognition by UvrAs.

    Science.gov (United States)

    Timmins, Joanna; Gordon, Elspeth; Caria, Sofia; Leonard, Gordon; Acajjaoui, Samira; Kuo, Mei-Shiue; Monchois, Vincent; McSweeney, Sean

    2009-04-15

    UvrA proteins are key actors in DNA damage repair and play an essential role in prokaryotic nucleotide excision repair (NER), a pathway that is unique in its ability to remove a broad spectrum of DNA lesions. Understanding the DNA binding and damage recognition activities of the UvrA family is a critical component for establishing the molecular basis of this process. Here we report the structure of the class II UvrA2 from Deinococcus radiodurans in two crystal forms. These structures, coupled with mutational analyses and comparison with the crystal structure of class I UvrA from Bacillus stearothermophilus, suggest a previously unsuspected role for the identified insertion domains of UvrAs in both DNA binding and damage recognition. Taken together, the available information suggests a model for how UvrA interacts with DNA and thus sheds new light on the molecular mechanisms underlying the role of UvrA in the early steps of NER.

  13. A pedigree of Leber's hereditary optic neuropathy with mtDNA14484 mutation%伴有mtDNA14484位点突变的Leber病一大家系

    Institute of Scientific and Technical Information of China (English)

    童绎; 王影; 姜枫; 刘斌; 张守康; 杨薇

    2008-01-01

    Objective To analyze a pedigree of Leber' s hereditary optic neuropathy, and its penetrance, anticipation, and spontaneous eyesight improvement, and its relationship with mitochondrial DNA mutation. Methods Eighteen members in the tinnily were undergone routine visual check. Five cases were taken visual evoked potential and visual field examination. DNA sequencing was performed on6 eases to check the mtDNA11778,3460 and 14484 loci.Results (1)The offsprings from the first wife in the first generation showed decreased acuity of the two eyes, which was optic atrophy identified by funduseopy. (2) The mtDNA had mutation at position 14484, but not at positions 11778 and 3460. Conclusion The pedigree showed a typical maternal inheritance of Leber' s hereditary optic neuropathy. It was eaused by mtDNA 14484 mutation.%目的 研究Leber病一大家系的遗传因素,探讨其外显率、遗传早现、自发视力恢复,以及与mtDNA的关联性.方法 对家系中18例患者进行调查,并行视力、眼底等常规检查;5例患者作了视觉诱发电位(visual evoked potential,VEP)及视野检查;应用聚合酶链反应对6例患者外周血液进行mtDNA11778、3460及144843个位点检查.结果 (1)本家系第Ⅰ代娶二妻,前妻后代皆无明显诱因出现双眼视力下降,经眼底镜检查符合视神经萎缩诊断.后妻无眼病,其后代均无眼病.(2)6例患者的mtDNA检查显示11778、3460位点未发现突变,而在14484位点出现同质性突变.结论 该家系Leber病呈典型母系遗传,该病的临床表现可能与mtDNA14484位点突变密切关联.

  14. The mitochondrial ND1 m.3337G>A mutation associated to multiple mitochondrial DNA deletions in a patient with Wolfram syndrome and cardiomyopathy

    Energy Technology Data Exchange (ETDEWEB)

    Mezghani, Najla [Laboratoire de Genetique Moleculaire Humaine, Faculte de Medecine de Sfax, Universite de Sfax (Tunisia); Mnif, Mouna [Service d' endocrinologie, C.H.U. Habib Bourguiba de Sfax (Tunisia); Mkaouar-Rebai, Emna, E-mail: emna_mkaouar@mail2world.com [Laboratoire de Genetique Moleculaire Humaine, Faculte de Medecine de Sfax, Universite de Sfax (Tunisia); Kallel, Nozha [Service d' endocrinologie, C.H.U. Habib Bourguiba de Sfax (Tunisia); Salem, Ikhlass Haj [Laboratoire de Genetique Moleculaire Humaine, Faculte de Medecine de Sfax, Universite de Sfax (Tunisia); Charfi, Nadia; Abid, Mohamed [Service d' endocrinologie, C.H.U. Habib Bourguiba de Sfax (Tunisia); Fakhfakh, Faiza [Laboratoire de Genetique Moleculaire Humaine, Faculte de Medecine de Sfax, Universite de Sfax (Tunisia)

    2011-07-29

    Highlights: {yields} We reported a patient with Wolfram syndrome and dilated cardiomyopathy. {yields} We detected the ND1 mitochondrial m.3337G>A mutation in 3 tested tissues (blood leukocytes, buccal mucosa and skeletal muscle). {yields} Long-range PCR amplification revealed the presence of multiple mitochondrial deletions in the skeletal muscle. {yields} The deletions remove several tRNA and protein-coding genes. -- Abstract: Wolfram syndrome (WFS) is a rare hereditary disorder also known as DIDMOAD (diabetes insipidus, diabetes mellitus, optic atrophy, and deafness). It is a heterogeneous disease and full characterization of all clinical and biological features of this disorder is difficult. The wide spectrum of clinical expression, affecting several organs and tissues, and the similarity in phenotype between patients with Wolfram syndrome and those with certain types of respiratory chain diseases suggests mitochondrial DNA (mtDNA) involvement in Wolfram syndrome patients. We report a Tunisian patient with clinical features of moderate Wolfram syndrome including diabetes, dilated cardiomyopathy and neurological complications. The results showed the presence of the mitochondrial ND1 m.3337G>A mutation in almost homoplasmic form in 3 tested tissues of the proband (blood leukocytes, buccal mucosa and skeletal muscle). In addition, the long-range PCR amplifications revealed the presence of multiple deletions of the mitochondrial DNA extracted from the patient's skeletal muscle removing several tRNA and protein-coding genes. Our study reported a Tunisian patient with clinical features of moderate Wolfram syndrome associated with cardiomyopathy, in whom we detected the ND1 m.3337G>A mutation with mitochondrial multiple deletions.

  15. Elevated incidence of polyp formation in APC(Min/⁺Msh2⁻/⁻ mice is independent of nitric oxide-induced DNA mutations.

    Directory of Open Access Journals (Sweden)

    Antoaneta Belcheva

    Full Text Available Gut microbiota has been linked to a number of human diseases including colon cancer. However, the mechanism through which gut bacteria influence colon cancer development and progression remains unclear. Perturbation of the homeostasis between the host immune system and microbiota leads to inflammation and activation of macrophages which produce large amounts of nitric oxide that acts as a genotoxic effector molecule to suppress bacterial growth. However, nitric oxide also has genotoxic effects to host cells by producing mutations that can predispose to colon cancer development. The major DNA lesions caused by nitric oxide are 8oxoG and deamination of deoxycytosine bases. Cellular glycosylases that belong to the base excision repair pathway have been demonstrated to repair these mutations. Recent evidence suggests that the mismatch repair pathway (MMR might also repair nitric oxide-induced DNA damage. Since deficiency in MMR predisposes to colon cancer, we hypothesized that MMR-deficient colon epithelial cells are incapable of repairing nitric-oxide induced genetic lesions that can promote colon cancer. Indeed, we found that the MMR pathway repairs nitric oxide-induced DNA mutations in cell lines. To test whether nitric oxide promotes colon cancer, we genetically ablated the inducible nitric oxide synthase (iNOS or inhibited iNOS activity in the APC(Min/+Msh2(-/- mouse model of colon cancer. However, despite the fact that nitric oxide production was strongly reduced in the colon using both approaches, colon cancer incidence was not affected. These data show that nitric oxide and iNOS do not promote colon cancer in APC(Min/+Msh2(-/- mice.

  16. The mitochondrial ND1 m.3337G>A mutation associated to multiple mitochondrial DNA deletions in a patient with Wolfram syndrome and cardiomyopathy

    International Nuclear Information System (INIS)

    Highlights: → We reported a patient with Wolfram syndrome and dilated cardiomyopathy. → We detected the ND1 mitochondrial m.3337G>A mutation in 3 tested tissues (blood leukocytes, buccal mucosa and skeletal muscle). → Long-range PCR amplification revealed the presence of multiple mitochondrial deletions in the skeletal muscle. → The deletions remove several tRNA and protein-coding genes. -- Abstract: Wolfram syndrome (WFS) is a rare hereditary disorder also known as DIDMOAD (diabetes insipidus, diabetes mellitus, optic atrophy, and deafness). It is a heterogeneous disease and full characterization of all clinical and biological features of this disorder is difficult. The wide spectrum of clinical expression, affecting several organs and tissues, and the similarity in phenotype between patients with Wolfram syndrome and those with certain types of respiratory chain diseases suggests mitochondrial DNA (mtDNA) involvement in Wolfram syndrome patients. We report a Tunisian patient with clinical features of moderate Wolfram syndrome including diabetes, dilated cardiomyopathy and neurological complications. The results showed the presence of the mitochondrial ND1 m.3337G>A mutation in almost homoplasmic form in 3 tested tissues of the proband (blood leukocytes, buccal mucosa and skeletal muscle). In addition, the long-range PCR amplifications revealed the presence of multiple deletions of the mitochondrial DNA extracted from the patient's skeletal muscle removing several tRNA and protein-coding genes. Our study reported a Tunisian patient with clinical features of moderate Wolfram syndrome associated with cardiomyopathy, in whom we detected the ND1 m.3337G>A mutation with mitochondrial multiple deletions.

  17. Sequence of DNA flanking the exons of the HEXA gene, and identification of mutations in Tay-Sachs disease.

    OpenAIRE

    Triggs-Raine, B L; Akerman, B R; Clarke, J T; Gravel, R A

    1991-01-01

    The rapid identification of mutations causing Tay-Sachs disease requires the capacity to readily screen the regions of the HEXA gene most likely to be affected by mutation. We have sequenced the portions of the introns flanking each of the 14 HEXA exons in order to specify oligonucleotide primers for the PCR-dependent amplification of each exon and splice-junction sequence. The amplified products were analyzed, by electrophoresis in nondenaturing polyacrylamide gels, for the presence of eithe...

  18. Genome-wide SNP scan of pooled DNA reveals nonsense mutation in FGF20 in the scaleless line of featherless chickens

    Directory of Open Access Journals (Sweden)

    Wells Kirsty L

    2012-06-01

    Full Text Available Abstract Background Scaleless (sc/sc chickens carry a single recessive mutation that causes a lack of almost all body feathers, as well as foot scales and spurs, due to a failure of skin patterning during embryogenesis. This spontaneous mutant line, first described in the 1950s, has been used extensively to explore the tissue interactions involved in ectodermal appendage formation in embryonic skin. Moreover, the trait is potentially useful in tropical agriculture