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Sample records for dna mutations generated

  1. Common Mitochondrial DNA Mutations generated through DNA-mediated Charge Transport#

    Science.gov (United States)

    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 = 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 cells incubated with the rhodium complex, DNA mutations in a portion of the mitochondrial genome were assessed via manual sequencing. The mutational pattern is consistent with dG to dT transversions in the repetitive guanine tracts. Significantly, the mutational pattern found overlaps oxidative damage hot spots seen previously. These mutations are found within conserved sequence block II, a critical regulatory element involved in DNA replication, and these have been identified as sites of low oxidation potential to which oxidative damage is funneled. Based upon this mutational analysis and its correspondence to sites of long range oxidative damage, we infer a critical role for DNA charge transport in generating these mutations and, thus, in regulating mitochondrial DNA replication under oxidative stress. PMID:19128037

  2. Next-generation sequencing reveals DGUOK mutations in adult patients with mitochondrial DNA multiple deletions

    Science.gov (United States)

    Garone, Caterina; Bordoni, Andreina; Gutierrez Rios, Purificacion; Calvo, Sarah E.; Ripolone, Michela; Ranieri, Michela; Rizzuti, Mafalda; Villa, Luisa; Magri, Francesca; Corti, Stefania; Bresolin, Nereo; Mootha, Vamsi K.; Moggio, Maurizio; DiMauro, Salvatore; Comi, Giacomo P.; Sciacco, Monica

    2012-01-01

    The molecular diagnosis of mitochondrial disorders still remains elusive in a large proportion of patients, but advances in next generation sequencing are significantly improving our chances to detect mutations even in sporadic patients. Syndromes associated with mitochondrial DNA multiple deletions are caused by different molecular defects resulting in a wide spectrum of predominantly adult-onset clinical presentations, ranging from progressive external ophthalmoplegia to multi-systemic disorders of variable severity. The mutations underlying these conditions remain undisclosed in half of the affected subjects. We applied next-generation sequencing of known mitochondrial targets (MitoExome) to probands presenting with adult-onset mitochondrial myopathy and harbouring mitochondrial DNA multiple deletions in skeletal muscle. We identified autosomal recessive mutations in the DGUOK gene (encoding mitochondrial deoxyguanosine kinase), which has previously been associated with an infantile hepatocerebral form of mitochondrial DNA depletion. Mutations in DGUOK occurred in five independent subjects, representing 5.6% of our cohort of patients with mitochondrial DNA multiple deletions, and impaired both muscle DGUOK activity and protein stability. Clinical presentations were variable, including mitochondrial myopathy with or without progressive external ophthalmoplegia, recurrent rhabdomyolysis in a young female who had received a liver transplant at 9 months of age and adult-onset lower motor neuron syndrome with mild cognitive impairment. These findings reinforce the concept that mutations in genes involved in deoxyribonucleotide metabolism can cause diverse clinical phenotypes and suggest that DGUOK should be screened in patients harbouring mitochondrial DNA deletions in skeletal muscle. PMID:23043144

  3. Generation and Characterization of Induced Pluripotent Stem Cells from Patients with mtDNA Mutations.

    Science.gov (United States)

    Hämäläinen, Riikka H; Suomalainen, Anu

    2016-01-01

    Generation of induced pluripotent stem cells from patient cells has revolutionized disease modeling in recent years. One research area, where disease models have previously been scarce, is disorders with mutations in mitochondrial DNA. These are a common cause for human disease and often cause very tissue specific phenotypes with vast clinical heterogeneity. iPS technology has now opened up new possibilities for mechanistic studies of these diseases.

  4. Performance of amplicon-based next generation DNA sequencing for diagnostic gene mutation profiling in oncopathology.

    Science.gov (United States)

    Sie, Daoud; Snijders, Peter J F; Meijer, Gerrit A; Doeleman, Marije W; van Moorsel, Marinda I H; van Essen, Hendrik F; Eijk, Paul P; Grünberg, Katrien; van Grieken, Nicole C T; Thunnissen, Erik; Verheul, Henk M; Smit, Egbert F; Ylstra, Bauke; Heideman, Daniëlle A M

    2014-10-01

    Next generation DNA sequencing (NGS) holds promise for diagnostic applications, yet implementation in routine molecular pathology practice requires performance evaluation on DNA derived from routine formalin-fixed paraffin-embedded (FFPE) tissue specimens. The current study presents a comprehensive analysis of TruSeq Amplicon Cancer Panel-based NGS using a MiSeq Personal sequencer (TSACP-MiSeq-NGS) for somatic mutation profiling. TSACP-MiSeq-NGS (testing 212 hotspot mutation amplicons of 48 genes) and a data analysis pipeline were evaluated in a retrospective learning/test set approach (n = 58/n = 45 FFPE-tumor DNA samples) against 'gold standard' high-resolution-melting (HRM)-sequencing for the genes KRAS, EGFR, BRAF and PIK3CA. Next, the performance of the validated test algorithm was assessed in an independent, prospective cohort of FFPE-tumor DNA samples (n = 75). In the learning set, a number of minimum parameter settings was defined to decide whether a FFPE-DNA sample is qualified for TSACP-MiSeq-NGS and for calling mutations. The resulting test algorithm revealed 82% (37/45) compliance to the quality criteria and 95% (35/37) concordant assay findings for KRAS, EGFR, BRAF and PIK3CA with HRM-sequencing (kappa = 0.92; 95% CI = 0.81-1.03) in the test set. Subsequent application of the validated test algorithm to the prospective cohort yielded a success rate of 84% (63/75), and a high concordance with HRM-sequencing (95% (60/63); kappa = 0.92; 95% CI = 0.84-1.01). TSACP-MiSeq-NGS detected 77 mutations in 29 additional genes. TSACP-MiSeq-NGS is suitable for diagnostic gene mutation profiling in oncopathology.

  5. NSD1 mutations generate a genome-wide DNA methylation signature.

    LENUS (Irish Health Repository)

    Choufani, S

    2015-12-22

    Sotos syndrome (SS) represents an important human model system for the study of epigenetic regulation; it is an overgrowth\\/intellectual disability syndrome caused by mutations in a histone methyltransferase, NSD1. As layered epigenetic modifications are often interdependent, we propose that pathogenic NSD1 mutations have a genome-wide impact on the most stable epigenetic mark, DNA methylation (DNAm). By interrogating DNAm in SS patients, we identify a genome-wide, highly significant NSD1(+\\/-)-specific signature that differentiates pathogenic NSD1 mutations from controls, benign NSD1 variants and the clinically overlapping Weaver syndrome. Validation studies of independent cohorts of SS and controls assigned 100% of these samples correctly. This highly specific and sensitive NSD1(+\\/-) signature encompasses genes that function in cellular morphogenesis and neuronal differentiation, reflecting cardinal features of the SS phenotype. The identification of SS-specific genome-wide DNAm alterations will facilitate both the elucidation of the molecular pathophysiology of SS and the development of improved diagnostic testing.

  6. 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.

  7. 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)

    Kim, Seung Tae; Lee, Won-Suk; 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

    2015-11-24

    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.

  8. 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.

  9. DNA polymerases eta and theta function in the same genetic pathway to generate mutations at A/T during somatic hypermutation of Ig genes.

    Science.gov (United States)

    Masuda, Keiji; Ouchida, Rika; Hikida, Masaki; Kurosaki, Tomohiro; Yokoi, Masayuki; Masutani, Chikahide; Seki, Mineaki; Wood, Richard D; Hanaoka, Fumio; O-Wang, Jiyang

    2007-06-15

    Somatic hypermutation of the Ig genes requires the activity of multiple DNA polymerases to ultimately introduce mutations at both A/T and C/G base pairs. Mice deficient for DNA polymerase eta (POLH) exhibited an approximately 80% reduction of the mutations at A/T, whereas absence of polymerase (POLQ) resulted in approximately 20% reduction of both A/T and C/G mutations. To investigate whether the residual A/T mutations observed in the absence of POLH are generated by POLQ and how these two polymerases might cooperate or compete with each other to generate A/T mutations, here we have established mice deficient for both POLH and POLQ. Polq(-/-)Polh(-/-) mice, however, did not show a further decrease of A/T mutations as compared with Polh(-/-) mice, suggesting that POLH and POLQ function in the same genetic pathway in the generation of these mutations. Frequent misincorporation of nucleotides, in particular opposite template T, is a known feature of POLH, but the efficiency of extension beyond the misincorporation differs significantly depending on the nature of the mispairing. Remarkably, we found that POLQ catalyzed extension more efficiently than POLH from all types of mispaired termini opposite A or T. Moreover, POLQ was able to extend mispaired termini generated by POLH albeit at a relatively low efficiency. These results reveal genetic and biochemical interactions between POLH and POLQ and suggest that POLQ might cooperate with POLH to generate some of the A/T mutations during the somatic hypermutation of Ig genes.

  10. Pms2 and uracil-DNA glycosylases act jointly in the mismatch repair pathway to generate Ig gene mutations at A-T base pairs.

    Science.gov (United States)

    Girelli Zubani, Giulia; Zivojnovic, Marija; De Smet, Annie; Albagli-Curiel, Olivier; Huetz, François; Weill, Jean-Claude; Reynaud, Claude-Agnès; Storck, Sébastien

    2017-04-03

    During somatic hypermutation (SHM) of immunoglobulin genes, uracils introduced by activation-induced cytidine deaminase are processed by uracil-DNA glycosylase (UNG) and mismatch repair (MMR) pathways to generate mutations at G-C and A-T base pairs, respectively. Paradoxically, the MMR-nicking complex Pms2/Mlh1 is apparently dispensable for A-T mutagenesis. Thus, how detection of U:G mismatches is translated into the single-strand nick required for error-prone synthesis is an open question. One model proposed that UNG could cooperate with MMR by excising a second uracil in the vicinity of the U:G mismatch, but it failed to explain the low impact of UNG inactivation on A-T mutagenesis. In this study, we show that uracils generated in the G1 phase in B cells can generate equal proportions of A-T and G-C mutations, which suggests that UNG and MMR can operate within the same time frame during SHM. Furthermore, we show that Ung -/- Pms2 -/- mice display a 50% reduction in mutations at A-T base pairs and that most remaining mutations at A-T bases depend on two additional uracil glycosylases, thymine-DNA glycosylase and SMUG1. These results demonstrate that Pms2/Mlh1 and multiple uracil glycosylases act jointly, each one with a distinct strand bias, to enlarge the immunoglobulin gene mutation spectrum from G-C to A-T bases. © 2017 Girelli Zubani et al.

  11. Selective identification of somatic mutations in pancreatic cancer cells through a combination of next-generation sequencing of plasma DNA using molecular barcodes and a bioinformatic variant filter.

    Science.gov (United States)

    Kukita, Yoji; Ohkawa, Kazuyoshi; Takada, Ryoji; Uehara, Hiroyuki; Katayama, Kazuhiro; Kato, Kikuya

    2018-01-01

    The accuracy of next-generation sequencing (NGS) for detecting tumor-specific mutations in plasma DNA is hindered by errors introduced during PCR/sequencing, base substitutions caused by DNA damage, and pre-existing mutations in normal cells that are present at a low frequency. Here, we performed NGS of genes related to pancreatic cancer (comprising 2.8 kb of genomic DNA) in plasma DNA (average 4.5 ng) using molecular barcodes. The average number of sequenced molecules was 900, and the sequencing depth per molecule was 100 or more. We also developed a bioinformatic variant filter, called CV78, to remove variants that were not considered to be tumor-specific, i.e., those that are either absent or occur at low frequencies in the Catalogue of Somatic Mutations in Cancer database. In a cohort comprising 57 pancreatic cancer patients and 12 healthy individuals, sequencing initially identified variants in 31 (54%) and 5 (42%), respectively, whereas after applying the CV78 filter, 19 (33%) and zero were variant-positive. In a validation cohort consisting of 86 patients with pancreatic cancer and 20 patients with intraductal papillary mucinous neoplasm (IPMN), 62 (72%) with pancreatic cancer patients and 10 (50%) IPMN patients were initially variant positive. After CV78 filtering, these values were reduced to 32 (37%) and 1 (5%), respectively. The variant allele frequency of filtered variants in plasma ranged from 0.25% to 76.1%. Therefore, combining NGS and molecular barcodes with subsequent filtering is likely to eliminate most non-tumor-specific mutations.

  12. The role of mitochondrial DNA mutations in mammalian aging.

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    Gregory C Kujoth

    2007-02-01

    Full Text Available Mitochondrial DNA (mtDNA accumulates both base-substitution mutations and deletions with aging in several tissues in mammals. Here, we examine the evidence supporting a causative role for mtDNA mutations in mammalian aging. We describe and compare human diseases and mouse models associated with mitochondrial genome instability. We also discuss potential mechanisms for the generation of these mutations and the means by which they may mediate their pathological consequences. Strategies for slowing the accumulation and attenuating the effects of mtDNA mutations are discussed.

  13. Assessment of the quality of DNA from various formalin-fixed paraffin-embedded (FFPE) tissues and the use of this DNA for next-generation sequencing (NGS) with no artifactual mutation.

    Science.gov (United States)

    Einaga, Naoki; Yoshida, Akio; Noda, Hiroko; Suemitsu, Masaaki; Nakayama, Yuki; Sakurada, Akihisa; Kawaji, Yoshiko; Yamaguchi, Hiromi; Sasaki, Yasushi; Tokino, Takashi; Esumi, Mariko

    2017-01-01

    Formalin-fixed, paraffin-embedded (FFPE) tissues used for pathological diagnosis are valuable for studying cancer genomics. In particular, laser-capture microdissection of target cells determined by histopathology combined with FFPE tissue section immunohistochemistry (IHC) enables precise analysis by next-generation sequencing (NGS) of the genetic events occurring in cancer. The result is a new strategy for a pathological tool for cancer diagnosis: 'microgenomics'. To more conveniently and precisely perform microgenomics, we revealed by systematic analysis the following three details regarding FFPE DNA compared with paired frozen tissue DNA. 1) The best quality of FFPE DNA is obtained by tissue fixation with 10% neutral buffered formalin for 1 day and heat treatment of tissue lysates at 95°C for 30 minutes. 2) IHC staining of FFPE tissues decreases the quantity and quality of FFPE DNA to one-fourth, and antigen retrieval (at 120°C for 15 minutes, pH 6.0) is the major reason for this decrease. 3) FFPE DNA prepared as described herein is sufficient for NGS. For non-mutated tissue specimens, no artifactual mutation occurs during FFPE preparation, as shown by precise comparison of NGS of FFPE DNA and paired frozen tissue DNA followed by validation. These results demonstrate that even FFPE tissues used for routine clinical diagnosis can be utilized to obtain reliable NGS data if appropriate conditions of fixation and validation are applied.

  14. DNA evolved to minimize frameshift mutations

    OpenAIRE

    Agoni, Valentina

    2013-01-01

    Point mutations can surely be dangerous but what is worst than to lose the reading frame?! Does DNA evolved a strategy to try to limit frameshift mutations?! Here we investigate if DNA sequences effectively evolved a system to minimize frameshift mutations analyzing the transcripts of proteins with high molecular weights.

  15. Chimeric proteins for detection and quantitation of DNA mutations, DNA sequence variations, DNA damage and DNA mismatches

    Science.gov (United States)

    McCutchen-Maloney, Sandra L.

    2002-01-01

    Chimeric proteins having both DNA mutation binding activity and nuclease activity are synthesized by recombinant technology. The proteins are of the general formula A-L-B and B-L-A where A is a peptide having DNA mutation binding activity, L is a linker and B is a peptide having nuclease activity. The chimeric proteins are useful for detection and identification of DNA sequence variations including DNA mutations (including DNA damage and mismatches) by binding to the DNA mutation and cutting the DNA once the DNA mutation is detected.

  16. Roles of Mitochondrial DNA Mutations in Stem Cell Ageing

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    Tianhong Su

    2018-03-01

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

  17. Detection and quantitation of single nucleotide polymorphisms, DNA sequence variations, DNA mutations, DNA damage and DNA mismatches

    Science.gov (United States)

    McCutchen-Maloney, Sandra L.

    2002-01-01

    DNA mutation binding proteins alone and as chimeric proteins with nucleases are used with solid supports to detect DNA sequence variations, DNA mutations and single nucleotide polymorphisms. The solid supports may be flow cytometry beads, DNA chips, glass slides or DNA dips sticks. DNA molecules are coupled to solid supports to form DNA-support complexes. Labeled DNA is used with unlabeled DNA mutation binding proteins such at TthMutS to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by binding which gives an increase in signal. Unlabeled DNA is utilized with labeled chimeras to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by nuclease activity of the chimera which gives a decrease in signal.

  18. Chronic radiation exposure: possibility of studying mutation process in generations based on the established DNA bank of exposed individuals and their offspring

    International Nuclear Information System (INIS)

    Rusinova, Galina G.; Adamova, Galina V.; Dudchenko, Natalya N.; Azizova, Tamara V.; Kurbatov, Andrey V.

    2002-01-01

    Data were summarized on the DNA Bank establishment for workers of the Mayak nuclear facility in Southern Ural, who were exposed to different doses of chronic radiation from γ -rays during the first years of the enterprise operations (1948-1958) and their families. Some workers were exposed to combined radiation (external + internal radiation from incorporated 239 Pu). The DNA Bank was established to store the unique genetic material from these individuals and their offspring for future risk estimation of the late consequences of radiation exposure using modern molecular-genetic technologies. Today, DNA Bank contains genetic material from 1,500 individuals and 218 families. The computer database was generated for the DNA Bank. It included individual medical-demographic, occupational descriptions and doses, quantitative and qualitative DNA data. Literature data on radiation-induced genome instability (variability of hypervariable areas) were also analyzed. Prospects of the DNA Bank establishment are also presented. The work is carried out on extension of the DNA Bank of exposed individuals and their offspring

  19. Fidelity and mutational spectrum of Pfu DNA polymerase on a human mitochondrial DNA sequence.

    Science.gov (United States)

    André, P; Kim, A; Khrapko, K; Thilly, W G

    1997-08-01

    The study of rare genetic changes in human tissues requires specialized techniques. Point mutations at fractions at or below 10(-6) must be observed to discover even the most prominent features of the point mutational spectrum. PCR permits the increase in number of mutant copies but does so at the expense of creating many additional mutations or "PCR noise". Thus, each DNA sequence studied must be characterized with regard to the DNA polymerase and conditions used to avoid interpreting a PCR-generated mutation as one arising in human tissue. The thermostable DNA polymerase derived from Pyrococcus furiosus designated Pfu has the highest fidelity of any DNA thermostable polymerase studied to date, and this property recommends it for analyses of tissue mutational spectra. Here, we apply constant denaturant capillary electrophoresis (CDCE) to separate and isolate the products of DNA amplification. This new strategy permitted direct enumeration and identification of point mutations created by Pfu DNA polymerase in a 96-bp low melting domain of a human mitochondrial sequence despite the very low mutant fractions generated in the PCR process. This sequence, containing part of the tRNA glycine and NADH dehydrogenase subunit 3 genes, is the target of our studies of mitochondrial mutagenesis in human cells and tissues. Incorrectly synthesized sequences were separated from the wild type as mutant/wild-type heteroduplexes by sequential enrichment on CDCE. An artificially constructed mutant was used as an internal standard to permit calculation of the mutant fraction. Our study found that the average error rate (mutations per base pair duplication) of Pfu was 6.5 x 10(-7), and five of its more frequent mutations (hot spots) consisted of three transversions (GC-->TA, AT-->TA, and AT-->CG), one transition (AT-->GC), and one 1-bp deletion (in an AAAAAA sequence). To achieve an even higher sensitivity, the amount of Pfu-induced mutants must be reduced.

  20. DNA mismatch repair preferentially protects genes from mutation.

    Science.gov (United States)

    Belfield, Eric J; Ding, Zhong Jie; Jamieson, Fiona J C; Visscher, Anne M; Zheng, Shao Jian; Mithani, Aziz; Harberd, Nicholas P

    2018-01-01

    Mutation is the source of genetic variation and fuels biological evolution. Many mutations first arise as DNA replication errors. These errors subsequently evade correction by cellular DNA repair, for example, by the well-known DNA mismatch repair (MMR) mechanism. Here, we determine the genome-wide effects of MMR on mutation. We first identify almost 9000 mutations accumulated over five generations in eight MMR-deficient mutation accumulation (MA) lines of the model plant species, Arabidopsis thaliana We then show that MMR deficiency greatly increases the frequency of both smaller-scale insertions and deletions (indels) and of single-nucleotide variant (SNV) mutations. Most indels involve A or T nucleotides and occur preferentially in homopolymeric (poly A or poly T) genomic stretches. In addition, we find that the likelihood of occurrence of indels in homopolymeric stretches is strongly related to stretch length, and that this relationship causes ultrahigh localized mutation rates in specific homopolymeric stretch regions. For SNVs, we show that MMR deficiency both increases their frequency and changes their molecular mutational spectrum, causing further enhancement of the GC to AT bias characteristic of organisms with normal MMR function. Our final genome-wide analyses show that MMR deficiency disproportionately increases the numbers of SNVs in genes, rather than in nongenic regions of the genome. This latter observation indicates that MMR preferentially protects genes from mutation and has important consequences for understanding the evolution of genomes during both natural selection and human tumor growth. © 2018 Belfield et al.; Published by Cold Spring Harbor Laboratory Press.

  1. Next generation DNA led technologies

    CERN Document Server

    Jyothsna, G; Kashyap, Amita

    2016-01-01

    This brief highlights advances in DNA technologies and their wider applications. DNA is the source of life and has been studied since a generation, but very little is known as yet. Several sophisticated technologies of the current era have laid their foundations on the principle of DNA based mechanisms. DNA based technologies are bringing a new revolution of Advanced Science and Technology. Forensic Investigation, Medical Diagnosis, Paternity Disputes, Individual Identity, Health insurance, Motor Insurance have incorporated the DNA testing and profiling technologies for settling the issues.

  2. Mitochondrial DNA exhibits resistance to induced point and deletion mutations

    Science.gov (United States)

    Valente, William J.; Ericson, Nolan G.; Long, Alexandra S.; White, Paul A.; Marchetti, Francesco; Bielas, Jason H.

    2016-01-01

    The accumulation of somatic mitochondrial DNA (mtDNA) mutations contributes to the pathogenesis of human disease. Currently, mitochondrial mutations are largely considered results of inaccurate processing of its heavily damaged genome. However, mainly from a lack of methods to monitor mtDNA mutations with sufficient sensitivity and accuracy, a link between mtDNA damage and mutation has not been established. To test the hypothesis that mtDNA-damaging agents induce mtDNA mutations, we exposed MutaTMMouse mice to benzo[a]pyrene (B[a]P) or N-ethyl-N-nitrosourea (ENU), daily for 28 consecutive days, and quantified mtDNA point and deletion mutations in bone marrow and liver using our newly developed Digital Random Mutation Capture (dRMC) and Digital Deletion Detection (3D) assays. Surprisingly, our results demonstrate mutagen treatment did not increase mitochondrial point or deletion mutation frequencies, despite evidence both compounds increase nuclear DNA mutations and demonstrated B[a]P adduct formation in mtDNA. These findings contradict models of mtDNA mutagenesis that assert the elevated rate of mtDNA mutation stems from damage sensitivity and abridged repair capacity. Rather, our results demonstrate induced mtDNA damage does not readily convert into mutation. These findings suggest robust mitochondrial damage responses repress induced mutations after mutagen exposure. PMID:27550180

  3. Generation of an induced pluripotent stem cell (iPSC line from a 40-year-old patient with the A8344G mutation of mitochondrial DNA and MERRF (myoclonic epilepsy with ragged red fibers syndrome

    Directory of Open Access Journals (Sweden)

    Yu-Ting Wu

    2018-03-01

    Full Text Available Mitochondrial defects are associated with clinical manifestations from common diseases to rare genetic disorders. Myoclonus epilepsy associated with ragged-red fibers (MERRF syndrome results from an A to G transition at nucleotide position 8344 in the tRNALys gene of mitochondrial DNA (mtDNA and is characterized by myoclonus, myopathy and severe neurological symptoms. In this study, Sendai reprogramming method was used to generate an iPS cell line carrying the A8344G mutation of mtDNA from a MERRF patient. This patient-specific iPSC line expressed pluripotent stem cell markers, possessed normal karyotype, and displayed the capability to differentiate into mature cells in three germ layers.

  4. Replicative DNA polymerase mutations in cancer☆

    Science.gov (United States)

    Heitzer, Ellen; Tomlinson, Ian

    2014-01-01

    Three DNA polymerases — Pol α, Pol δ and Pol ɛ — are essential for DNA replication. After initiation of DNA synthesis by Pol α, Pol δ or Pol ɛ take over on the lagging and leading strand respectively. Pol δ and Pol ɛ perform the bulk of replication with very high fidelity, which is ensured by Watson–Crick base pairing and 3′exonuclease (proofreading) activity. Yeast models have shown that mutations in the exonuclease domain of Pol δ and Pol ɛ homologues can cause a mutator phenotype. Recently, we identified germline exonuclease domain mutations (EDMs) in human POLD1 and POLE that predispose to ‘polymerase proofreading associated polyposis’ (PPAP), a disease characterised by multiple colorectal adenomas and carcinoma, with high penetrance and dominant inheritance. Moreover, somatic EDMs in POLE have also been found in sporadic colorectal and endometrial cancers. Tumors with EDMs are microsatellite stable and show an ‘ultramutator’ phenotype, with a dramatic increase in base substitutions. PMID:24583393

  5. Replicative DNA polymerase mutations in cancer.

    Science.gov (United States)

    Heitzer, Ellen; Tomlinson, Ian

    2014-02-01

    Three DNA polymerases - Pol α, Pol δ and Pol ɛ - are essential for DNA replication. After initiation of DNA synthesis by Pol α, Pol δ or Pol ɛ take over on the lagging and leading strand respectively. Pol δ and Pol ɛ perform the bulk of replication with very high fidelity, which is ensured by Watson-Crick base pairing and 3'exonuclease (proofreading) activity. Yeast models have shown that mutations in the exonuclease domain of Pol δ and Pol ɛ homologues can cause a mutator phenotype. Recently, we identified germline exonuclease domain mutations (EDMs) in human POLD1 and POLE that predispose to 'polymerase proofreading associated polyposis' (PPAP), a disease characterised by multiple colorectal adenomas and carcinoma, with high penetrance and dominant inheritance. Moreover, somatic EDMs in POLE have also been found in sporadic colorectal and endometrial cancers. Tumors with EDMs are microsatellite stable and show an 'ultramutator' phenotype, with a dramatic increase in base substitutions. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  6. 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

  7. Markov chain for estimating human mitochondrial DNA mutation pattern

    Science.gov (United States)

    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.

  8. 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.

  9. Circulating cell-free DNA mutation patterns in early and late stage colon and pancreatic cancer.

    Science.gov (United States)

    Vietsch, Eveline E; Graham, Garrett T; McCutcheon, Justine N; Javaid, Aamir; Giaccone, Giuseppe; Marshall, John L; Wellstein, Anton

    2017-12-01

    Cancer is a heterogeneous disease harboring diverse subclonal populations that can be discriminated by their DNA mutations. Environmental pressure selects subclones that ultimately drive disease progression and tumor relapse. Circulating cell-free DNA (ccfDNA) can be used to approximate the mutational makeup of cancer lesions and can serve as a marker for monitoring disease progression at the molecular level without the need for invasively acquired samples from primary or metastatic lesions. This potential for molecular analysis makes ccfDNA attractive for the study of clonal evolution and for uncovering emerging therapeutic resistance or sensitivity. We assessed ccfDNA from colon and pancreatic adenocarcinoma patients using next generation sequencing of 56 cancer-associated genes at the time of primary resectable disease and metastatic progression and compared this to the mutational patterns of the primary tumor. 28%-47% of non-synonymous mutations in the primary tumors were also detected in the ccfDNA while 71%-78% mutations found in ccfDNA were not detected in the primary tumors. ccfDNA collected at the time of progression harbored 3-5 new mutations not detected in ccfDNA at the earlier collection time points. We conclude that incorporation of ccfDNA analysis provides crucial insights into the changing molecular makeup of progressive colon and pancreatic cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Compound mitochondrial DNA mutations in a neurological patient ...

    Indian Academy of Sciences (India)

    Compound mitochondrial DNA mutations in a neurological patient with ataxia, myoclonus and deafness. Ji Hoon Park, Bo Ram Yoon, Hye Jin Kim, Phil Hyu Lee, Byung-Ok Choi and Ki Wha Chung. J. Genet. 93, 173–177. Table 1. Variations from the whole mtDNA sequence in the AMDF patient. Mutation. Report. Locus/ ...

  11. Presence of a consensus DNA motif at nearby DNA sequence of the mutation susceptible CG nucleotides.

    Science.gov (United States)

    Chowdhury, Kaushik; Kumar, Suresh; Sharma, Tanu; Sharma, Ankit; Bhagat, Meenakshi; Kamai, Asangla; Ford, Bridget M; Asthana, Shailendra; Mandal, Chandi C

    2018-01-10

    Complexity in tissues affected by cancer arises from somatic mutations and epigenetic modifications in the genome. The mutation susceptible hotspots present within the genome indicate a non-random nature and/or a position specific selection of mutation. An association exists between the occurrence of mutations and epigenetic DNA methylation. This study is primarily aimed at determining mutation status, and identifying a signature for predicting mutation prone zones of tumor suppressor (TS) genes. Nearby sequences from the top five positions having a higher mutation frequency in each gene of 42 TS genes were selected from a cosmic database and were considered as mutation prone zones. The conserved motifs present in the mutation prone DNA fragments were identified. Molecular docking studies were done to determine putative interactions between the identified conserved motifs and enzyme methyltransferase DNMT1. Collective analysis of 42 TS genes found GC as the most commonly replaced and AT as the most commonly formed residues after mutation. Analysis of the top 5 mutated positions of each gene (210 DNA segments for 42 TS genes) identified that CG nucleotides of the amino acid codons (e.g., Arginine) are most susceptible to mutation, and found a consensus DNA "T/AGC/GAGGA/TG" sequence present in these mutation prone DNA segments. Similar to TS genes, analysis of 54 oncogenes not only found CG nucleotides of the amino acid Arg as the most susceptible to mutation, but also identified the presence of similar consensus DNA motifs in the mutation prone DNA fragments (270 DNA segments for 54 oncogenes) of oncogenes. Docking studies depicted that, upon binding of DNMT1 methylates to this consensus DNA motif (C residues of CpG islands), mutation was likely to occur. Thus, this study proposes that DNMT1 mediated methylation in chromosomal DNA may decrease if a foreign DNA segment containing this consensus sequence along with CG nucleotides is exogenously introduced to dividing

  12. 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.

  13. DNA mutation motifs in the genes associated with inherited diseases.

    Directory of Open Access Journals (Sweden)

    Michal Růžička

    Full Text Available Mutations in human genes can be responsible for inherited genetic disorders and cancer. Mutations can arise due to environmental factors or spontaneously. It has been shown that certain DNA sequences are more prone to mutate. These sites are termed hotspots and exhibit a higher mutation frequency than expected by chance. In contrast, DNA sequences with lower mutation frequencies than expected by chance are termed coldspots. Mutation hotspots are usually derived from a mutation spectrum, which reflects particular population where an effect of a common ancestor plays a role. To detect coldspots/hotspots unaffected by population bias, we analysed the presence of germline mutations obtained from HGMD database in the 5-nucleotide segments repeatedly occurring in genes associated with common inherited disorders, in particular, the PAH, LDLR, CFTR, F8, and F9 genes. Statistically significant sequences (mutational motifs rarely associated with mutations (coldspots and frequently associated with mutations (hotspots exhibited characteristic sequence patterns, e.g. coldspots contained purine tract while hotspots showed alternating purine-pyrimidine bases, often with the presence of CpG dinucleotide. Using molecular dynamics simulations and free energy calculations, we analysed the global bending properties of two selected coldspots and two hotspots with a G/T mismatch. We observed that the coldspots were inherently more flexible than the hotspots. We assume that this property might be critical for effective mismatch repair as DNA with a mutation recognized by MutSα protein is noticeably bent.

  14. DNA mutation motifs in the genes associated with inherited diseases.

    Science.gov (United States)

    Růžička, Michal; Kulhánek, Petr; Radová, Lenka; Čechová, Andrea; Špačková, Naďa; Fajkusová, Lenka; Réblová, Kamila

    2017-01-01

    Mutations in human genes can be responsible for inherited genetic disorders and cancer. Mutations can arise due to environmental factors or spontaneously. It has been shown that certain DNA sequences are more prone to mutate. These sites are termed hotspots and exhibit a higher mutation frequency than expected by chance. In contrast, DNA sequences with lower mutation frequencies than expected by chance are termed coldspots. Mutation hotspots are usually derived from a mutation spectrum, which reflects particular population where an effect of a common ancestor plays a role. To detect coldspots/hotspots unaffected by population bias, we analysed the presence of germline mutations obtained from HGMD database in the 5-nucleotide segments repeatedly occurring in genes associated with common inherited disorders, in particular, the PAH, LDLR, CFTR, F8, and F9 genes. Statistically significant sequences (mutational motifs) rarely associated with mutations (coldspots) and frequently associated with mutations (hotspots) exhibited characteristic sequence patterns, e.g. coldspots contained purine tract while hotspots showed alternating purine-pyrimidine bases, often with the presence of CpG dinucleotide. Using molecular dynamics simulations and free energy calculations, we analysed the global bending properties of two selected coldspots and two hotspots with a G/T mismatch. We observed that the coldspots were inherently more flexible than the hotspots. We assume that this property might be critical for effective mismatch repair as DNA with a mutation recognized by MutSα protein is noticeably bent.

  15. Polymorphism and mutation analysis of genomic DNA on cancer

    International Nuclear Information System (INIS)

    Ohta, Tsutomu

    2003-01-01

    DNA repair is a universal process in living cells that maintains the structural integrity of chromosomal DNA molecules in face of damage. A deficiency in DNA damage repair is associated with an increased cancer risk by increasing a mutation frequency of cancer-related genes. Variation in DNA repair capacity may be genetically determined. Therefore, we searched single-nucleotide polymorphisms (SNPs) in major DNA repair genes. This led to the finding of 600 SNPs and mutations including many novel SNPs in Japanese population. Case-control studies to explore the contribution of the SNPs in DNA repair genes to the risk of lung cancer revealed that five SNPs are associated with lung carcinogenesis. One of these SNPs is found in RAD54L gene, which is involved in double-strand DNA repair. We analyzed and reported activities of Rad54L protein with SNP and mutations. (authors)

  16. 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. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Performance of mitochondrial DNA mutations detecting early stage cancer

    International Nuclear Information System (INIS)

    Jakupciak, John P; Srivastava, Sudhir; Sidransky, David; O'Connell, Catherine D; Maragh, Samantha; Markowitz, Maura E; Greenberg, Alissa K; Hoque, Mohammad O; Maitra, Anirban; Barker, Peter E; Wagner, Paul D; Rom, William N

    2008-01-01

    Mutations in the mitochondrial genome (mtgenome) have been associated with cancer and many other disorders. These mutations can be point mutations or deletions, or admixtures (heteroplasmy). The detection of mtDNA mutations in body fluids using resequencing microarrays, which are more sensitive than other sequencing methods, could provide a strategy to measure mutation loads in remote anatomical sites. We determined the mtDNA mutation load in the entire mitochondrial genome of 26 individuals with different early stage cancers (lung, bladder, kidney) and 12 heavy smokers without cancer. MtDNA was sequenced from three matched specimens (blood, tumor and body fluid) from each cancer patient and two matched specimens (blood and sputum) from smokers without cancer. The inherited wildtype sequence in the blood was compared to the sequences present in the tumor and body fluid, detected using the Affymetrix Genechip ® Human Mitochondrial Resequencing Array 1.0 and supplemented by capillary sequencing for noncoding region. Using this high-throughput method, 75% of the tumors were found to contain mtDNA mutations, higher than in our previous studies, and 36% of the body fluids from these cancer patients contained mtDNA mutations. Most of the mutations detected were heteroplasmic. A statistically significantly higher heteroplasmy rate occurred in tumor specimens when compared to both body fluid of cancer patients and sputum of controls, and in patient blood compared to blood of controls. Only 2 of the 12 sputum specimens from heavy smokers without cancer (17%) contained mtDNA mutations. Although patient mutations were spread throughout the mtDNA genome in the lung, bladder and kidney series, a statistically significant elevation of tRNA and ND complex mutations was detected in tumors. Our findings indicate comprehensive mtDNA resequencing can be a high-throughput tool for detecting mutations in clinical samples with potential applications for cancer detection, but it is

  18. Performance of mitochondrial DNA mutations detecting early stage cancer

    Directory of Open Access Journals (Sweden)

    Wagner Paul D

    2008-10-01

    Full Text Available Abstract Background Mutations in the mitochondrial genome (mtgenome have been associated with cancer and many other disorders. These mutations can be point mutations or deletions, or admixtures (heteroplasmy. The detection of mtDNA mutations in body fluids using resequencing microarrays, which are more sensitive than other sequencing methods, could provide a strategy to measure mutation loads in remote anatomical sites. Methods We determined the mtDNA mutation load in the entire mitochondrial genome of 26 individuals with different early stage cancers (lung, bladder, kidney and 12 heavy smokers without cancer. MtDNA was sequenced from three matched specimens (blood, tumor and body fluid from each cancer patient and two matched specimens (blood and sputum from smokers without cancer. The inherited wildtype sequence in the blood was compared to the sequences present in the tumor and body fluid, detected using the Affymetrix Genechip® Human Mitochondrial Resequencing Array 1.0 and supplemented by capillary sequencing for noncoding region. Results Using this high-throughput method, 75% of the tumors were found to contain mtDNA mutations, higher than in our previous studies, and 36% of the body fluids from these cancer patients contained mtDNA mutations. Most of the mutations detected were heteroplasmic. A statistically significantly higher heteroplasmy rate occurred in tumor specimens when compared to both body fluid of cancer patients and sputum of controls, and in patient blood compared to blood of controls. Only 2 of the 12 sputum specimens from heavy smokers without cancer (17% contained mtDNA mutations. Although patient mutations were spread throughout the mtDNA genome in the lung, bladder and kidney series, a statistically significant elevation of tRNA and ND complex mutations was detected in tumors. Conclusion Our findings indicate comprehensive mtDNA resequencing can be a high-throughput tool for detecting mutations in clinical samples with

  19. Improving the Performance of Somatic Mutation Identification by Recovering Circulating Tumor DNA Mutations.

    Science.gov (United States)

    Fu, Yu; Jovelet, Cécile; Filleron, Thomas; Pedrero, Marion; Motté, Nelly; Boursin, Yannick; Luo, Yufei; Massard, Christophe; Campone, Mario; Levy, Christelle; Diéras, Véronique; Bachelot, Thomas; Garrabey, Julie; Soria, Jean-Charles; Lacroix, Ludovic; André, Fabrice; Lefebvre, Celine

    2016-10-15

    DNA extracted from cancer patients' whole blood may contain somatic mutations from circulating tumor DNA (ctDNA) fragments. In this study, we introduce cmDetect, a computational method for the systematic identification of ctDNA mutations using whole-exome sequencing of a cohort of tumor and corresponding peripheral whole-blood samples. Through the analysis of simulated data, we demonstrated an increase in sensitivity in calling somatic mutations by combining cmDetect to two widely used mutation callers. In a cohort of 93 breast cancer metastatic patients, cmDetect identified ctDNA mutations in 54% of the patients and recovered somatic mutations in cancer genes EGFR, PIK3CA, and TP53 We further showed that cmDetect detected ctDNA in 89% of patients with confirmed mutated cell-free tumor DNA by plasma analyses (n = 9) within 46 pan-cancer patients. Our results prompt immediate consideration of the use of this method as an additional step in somatic mutation calling using whole-exome sequencing data with blood samples as controls. Cancer Res; 76(20); 5954-61. ©2016 AACR. ©2016 American Association for Cancer Research.

  20. The role of DNA polymerase {iota} in UV mutational spectra

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Jun-Hyuk [Division of Biology, Beckman Research Institute, City of Hope, Duarte, CA 91010 (United States); Besaratinia, Ahmad [Division of Biology, Beckman Research Institute, City of Hope, Duarte, CA 91010 (United States); Lee, Dong-Hyun [Division of Biology, Beckman Research Institute, City of Hope, Duarte, CA 91010 (United States); Lee, Chong-Soon [Department of Biochemistry, College of Natural Sciences, Yeungnam University, Gyongsan 712-749 (Korea, Republic of); Pfeifer, Gerd P. [Division of Biology, Beckman Research Institute, City of Hope, Duarte, CA 91010 (United States)]. E-mail: gpfeifer@coh.org

    2006-07-25

    UVB (280-320 nm) and UVC (200-280 nm) irradiation generate predominantly cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts in DNA. CPDs are thought to be responsible for most of the UV-induced mutations. Thymine-thymine CPDs, and probably also CPDs containing cytosine, are replicated in vivo in a largely accurate manner by a DNA polymerase {eta} (Pol {eta}) dependent process. Pol {eta} is a DNA damage-tolerant and error-prone DNA polymerase encoded by the POLH (XPV) gene in humans. Another member of the Y family of error-prone DNA polymerases is POLI encoding DNA polymerase iota (Pol {iota}). In order to clarify the specific role of Pol {iota} in UV mutagenesis, we have used an siRNA knockdown approach in combination with a supF shuttle vector which replicates in mammalian cells, similar as we have previously done for Pol {eta}. Synthetic RNA duplexes were used to efficiently inhibit Pol {iota} expression in 293T cells. The supF shuttle vector was irradiated with 254 nm UVC and replicated in 293T cells in presence of anti-Pol {iota} siRNA. Surprisingly, there was a consistent reduction of recovered plasmid from cells with Pol {iota} knockdown and this was independent of UV irradiation of the plasmid. The supF mutant frequency was unchanged in the siRNA knockdown cells relative to control cells confirming that Pol {iota} does not play an important role in UV mutagenesis. UV-induced supF mutants were sequenced from siRNA-treated cells and controls. Neither the type of mutations nor their distribution along the supF gene were significantly different between controls and siRNA knockdown cells and were predominantly C to T and CC to TT transitions at dipyrimidine sites. These results show that Pol {iota} has no significant role in UV lesion bypass and mutagenesis in vivo and provides some initial data suggesting that this polymerase may be involved in replication of extrachromosomal DNA.

  1. 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.

  2. Near-ultraviolet mutation of transforming DNA irradiated in vivo

    International Nuclear Information System (INIS)

    Cabrera-Juarez, E.; Setlow, J.K.

    1981-01-01

    Our previous work has demonstrated that whereas near-UV radiation is not a mutagen for Haemophilus influenzae cells, it does induce mutations in purified transforming DNA. In order to test various hypotheses concerning this difference. We have irradiated cells at 334 and 365 nm, then lysed them and assayed the DNA for induced mutations and for inactivation of transforming ability. The inactivation was only a little lower than observed with highly purified transforming DNA. The DNA irradiated in vivo was mutated at both wavelengths, but with considerably lower efficiency than was purified DNA. Neither incubation of the cells after irradiation and before lysis nor freezing and thawing the cells significantly changed the amount of mutation. It is concluded that there is some protection of the DNA against premutational lesions by the in vivo environment, but that it is not enough to account for the total lack of mutation of the cells. A probable explanation of this lack of cell mutation is that lethal lesions in the cells are induced much more readily than premutational lesions. (orig.)

  3. Germline Mutations in DNA Repair Genes in Lung Adenocarcinoma.

    Science.gov (United States)

    Parry, Erin M; Gable, Dustin L; Stanley, Susan E; Khalil, Sara E; Antonescu, Valentin; Florea, Liliana; Armanios, Mary

    2017-11-01

    Although lung cancer is generally thought to be environmentally provoked, anecdotal familial clustering has been reported, suggesting that there may be genetic susceptibility factors. We systematically tested whether germline mutations in eight candidate genes may be risk factors for lung adenocarcinoma. We studied lung adenocarcinoma cases for which germline sequence data had been generated as part of The Cancer Genome Atlas project but had not been previously analyzed. We selected eight genes, ATM serine/threonine kinase gene (ATM), BRCA2, DNA repair associated gene (BRCA2), checkpoint kinase 2 gene (CHEK2), EGFR, parkin RBR E3 ubiquitin protein ligase gene (PARK2), telomerase reverse transcriptase gene (TERT), tumor protein p53 gene (TP53), and Yes associated protein 1 gene (YAP1), on the basis of prior anecdotal association with lung cancer or genome-wide association studies. Among 555 lung adenocarcinoma cases, we detected 14 pathogenic mutations in five genes; they occurred at a frequency of 2.5% and represented an OR of 66 (95% confidence interval: 33-125, p mutations fell most commonly in ATM (50%), followed by TP53, BRCA2, EGFR, and PARK2. Most (86%) of these variants had been reported in other familial cancer syndromes. Another 12 cases (2%) carried ultrarare variants that were predicted to be deleterious by three protein prediction programs; these most frequently involved ATM and BRCA2. A subset of patients with lung adenocarcinoma, at least 2.5% to 4.5%, carry germline variants that have been linked to cancer risk in Mendelian syndromes. The genes fall most frequently in DNA repair pathways. Our data indicate that patients with lung adenocarcinoma, similar to other solid tumors, include a subset of patients with inherited susceptibility. Copyright © 2017 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

  4. An overview about mitochondrial DNA mutations in ovarian cancer

    African Journals Online (AJOL)

    Iyer Mahalaxmi

    2017-07-29

    Jul 29, 2017 ... High susceptibility of mtDNA to mutation through ROS leads to inefficient repair system and lack of protec- tive histones. The network of mitochondria is extremely dynamic which is precisely regulated during stressful conditions. This muta- tions in mtDNA due to increased accumulation results in ageing of.

  5. The role of mitochondrial DNA mutations in aging and sarcopenia: Implications for the mitochondrial vicious cycle theory of aging

    OpenAIRE

    Hiona, Asimina; Leeuwenburgh, Christiaan

    2007-01-01

    Aging is associated with a progressive loss of skeletal muscle mass and strength and the mechanisms mediating these effects likely involve mitochondrial DNA (mtDNA) mutations, mitochondrial dysfunction and the activation of mitochondrial mediated apoptosis. Because the mitochondrial genome is densely packed and close to the main generator of reactive oxygen species (ROS) in the cell, the electron transport chain (ETC), an important role for mtDNA mutations in aging has been proposed. Point mu...

  6. A comprehensive characterization of mitochondrial DNA mutations in glioblastoma multiforme.

    Science.gov (United States)

    Vidone, Michele; Clima, Rosanna; Santorsola, Mariangela; Calabrese, Claudia; Girolimetti, Giulia; Kurelac, Ivana; Amato, Laura Benedetta; Iommarini, Luisa; Trevisan, Elisa; Leone, Marco; Soffietti, Riccardo; Morra, Isabella; Faccani, Giuliano; Attimonelli, Marcella; Porcelli, Anna Maria; Gasparre, Giuseppe

    2015-06-01

    Glioblastoma multiforme (GBM) is the most malignant brain cancer in adults, with a poor prognosis, whose molecular stratification still represents a challenge in pathology and clinics. On the other hand, mitochondrial DNA (mtDNA) mutations have been found in most tumors as modifiers of the bioenergetics state, albeit in GBM a characterization of the mtDNA status is lacking to date. Here, a characterization of the burden of mtDNA mutations in GBM samples was performed. First, investigation of tumor-specific vs. non tumor-specific mutations was carried out with the MToolBox bioinformatics pipeline by analyzing 45 matched tumor/blood samples, from whole genome or whole exome sequencing datasets obtained from The Cancer Genome Atlas (TCGA) consortium. Additionally, the entire mtDNA sequence was obtained in a dataset of 104 fresh-frozen GBM samples. Mitochondrial mutations with potential pathogenic interest were prioritized based on heteroplasmic fraction, nucleotide variability, and in silico prediction of pathogenicity. A preliminary biochemical analysis of the activity of mitochondrial respiratory complexes was also performed on fresh-frozen GBM samples. Although a high number of mutations was detected, we report that the large majority of them does not pass the prioritization filters. Therefore, a relatively limited burden of pathogenic mutations is indeed carried by GBM, which did not appear to determine a general impairment of the respiratory chain. This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. The application of estrogen receptor-1 mutations' detection through circulating tumor dna in breast cancer

    Directory of Open Access Journals (Sweden)

    Binliang Liu

    2017-01-01

    Full Text Available Breast cancer is the most common cancer in women worldwide. Endocrine therapy is the cornerstone of treatment for patients with hormone receptor-positive advanced breast cancer. Unfortunately, although most patients initially respond to endocrine treatment, they will eventually acquire resistance to endocrine therapy. The mechanisms of endocrine resistance are complicated. In particular, the estrogen receptor-1 (ESR1 mutation has been recognized as an important topic in recent years. Mutation of ESR1 leads to complete aromatase inhibitor resistance and partial resistance to estrogen receptor agonists and antagonists. Therefore, during clinical treatment, it is of great importance to continuously monitor ESR1 mutations before and after endocrine therapy. Conventional tissue biopsies have unavoidable disadvantages, and therefore, the use of circulating tumor DNA (ctDNA has become more prevalent because it is noninvasive and convenient, has excellent sensitivity, and can quickly assess the overall situation of the tumor. The current methods for detecting ctDNA ESR1 mutations mainly include droplet digital polymerase chain reaction and next-generation sequencing techniques. Based on their advantages and disadvantages, we can establish an initial ESR1 mutation monitoring system. However, developing robust methods to monitor ESR1 mutation, detecting endocrine drug resistance, and evaluating prognoses for guiding clinical treatment strategies require long-term exploration. In this review, we will summarize recent concepts and advancements regarding ESR1 mutation monitoring, ctDNA detection technology, and their application in endocrine therapy of breast cancer.

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

    International Nuclear Information System (INIS)

    Fu, Liang-Yu; Wang, Guang-Zhong; Ma, Bin-Guang; Zhang, Hong-Yu

    2011-01-01

    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.

  9. Somatic mtDNA mutations in lung tissues of pesticide-exposed fruit growers.

    Science.gov (United States)

    Wang, Cheng-Ye; Zhao, Zhong-Bao

    2012-01-27

    Some pesticides have been considered potential chemical mutagens and their widespread use involves the assessment of their potentially hazardous effects. The mitochondrial genome is especially prone to DNA damage and thus can serve as a biomarker to monitor the genotoxicity of pesticides to human DNA. We performed a screening for somatic mutations in lung tissues from pesticide-exposed fruit growers, by direct comparing the entire mtDNA sequences of the lung tissue and the matched peripheral blood from the same individual. A phylogenetic approach and a high standard procedure were utilized to avoid potential errors in data generation and analysis. We observed a significantly increased frequency of mtDNA somatic mutations in lung tissues which had been exposed to pesticides multiple times by inhalation, and the potential biological significance of these mutations was further discussed. The samples represented in this observational study, which has multiple exposures to pesticides, experience a significant greater incidence of mtDNA mutations, suggesting that multiple exposures to pesticides could damage human mtDNA and cause somatic mutations. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  10. Direct nanomaterial-DNA contact effects on DNA and mutation induction.

    Science.gov (United States)

    Thongkumkoon, P; Sangwijit, K; Chaiwong, C; Thongtem, S; Singjai, P; Yu, L D

    2014-04-07

    The toxicity of nanomaterials has been well known, but mechanisms involved have been little known. This study was aimed at looking at direct interaction between nanomaterials and naked DNA for some fundamental understanding. Two different types of nanomaterials, carbon nanotubes (CNTs) and tungsten trioxide (WO₃) nanoplates, were simply mixed with naked DNA plasmid, respectively, in two different contact modes, dry or wet (in solution), for varied time periods. DNA topological forms were analyzed for changes using gel electrophoresis and fluoro-spectrometry. The nanomaterial-contacted DNA was transferred into bacteria Escherichia coli (E. coli) cells for mutation observation. Certain types and degrees of DNA damage were observed, such as single strand break and double strand break, and bacterial mutation was confirmed. The DNA damage increased with the contacting time in an exponential manner and increased more rapidly in the initial stage for the wet contact. The nanomaterials-contacted DNA transferred bacteria had about less than 10% survival but almost 100% mutation for the surviving cells. The CNTs were more offensive than the metal oxide nanomaterials. The mutation spectrum from the DNA sequencing analysis showed that DNA point mutation was dominated by transversion, which was dominated by guanine changes in the wet contact condition while by cytosine changes in the dry contact condition. The point mutation occurrence in the wet contact was more than in the dry contact, confirming the wet contact more active and thus dangerous than dry contact. This experiment, although as a model study, revealed that direct simple contacts between nanomaterials and DNA could cause DNA changes and thus induce mutations which might potentially lead to cancers, diseases and genetic changes. This could be a mechanism for nanomaterial genotoxicity to the cells and also provided a caution to applications in using nanomaterials for DNA delivery. Copyright © 2014 Elsevier Ireland

  11. 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

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

    Science.gov (United States)

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

    2016-03-01

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

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

    International Nuclear Information System (INIS)

    Mendelsohn, M.L.

    1985-01-01

    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

  14. Reduced local mutation density in regulatory DNA of cancer genomes is linked to DNA repair.

    Science.gov (United States)

    Polak, Paz; Lawrence, Michael S; Haugen, Eric; Stoletzki, Nina; Stojanov, Petar; Thurman, Robert E; Garraway, Levi A; Mirkin, Sergei; Getz, Gad; Stamatoyannopoulos, John A; Sunyaev, Shamil R

    2014-01-01

    Carcinogenesis and neoplastic progression are mediated by the accumulation of somatic mutations. Here we report that the local density of somatic mutations in cancer genomes is highly reduced specifically in accessible regulatory DNA defined by DNase I hypersensitive sites. This reduction is independent of any known factors influencing somatic mutation density and is observed in diverse cancer types, suggesting a general mechanism. By analyzing individual cancer genomes, we show that the reduced local mutation density within regulatory DNA is linked to intact global genome repair machinery, with nearly complete abrogation of the hypomutation phenomenon in individual cancers that possess mutations in components of the nucleotide excision repair system. Together, our results connect chromatin structure, gene regulation and cancer-associated somatic mutation.

  15. The role of mitochondrial DNA mutations in aging and sarcopenia: implications for the mitochondrial vicious cycle theory of aging.

    Science.gov (United States)

    Hiona, Asimina; Leeuwenburgh, Christiaan

    2008-01-01

    Aging is associated with a progressive loss of skeletal muscle mass and strength and the mechanisms mediating these effects likely involve mitochondrial DNA (mtDNA) mutations, mitochondrial dysfunction and the activation of mitochondrial-mediated apoptosis. Because the mitochondrial genome is densely packed and close to the main generator of reactive oxygen species (ROS) in the cell, the electron transport chain (ETC), an important role for mtDNA mutations in aging has been proposed. Point mutations and deletions in mtDNA accumulate with age in a wide variety of tissues in mammals, including humans, and often coincide with significant tissue dysfunction. Here, we examine the evidence supporting a causative role for mtDNA mutations in aging and sarcopenia. We review experimental outcomes showing that mtDNA mutations, leading to mitochondrial dysfunction and possibly apoptosis, are causal to the process of sarcopenia. Moreover, we critically discuss and dispute an important part of the mitochondrial 'vicious cycle' theory of aging which proposes that accumulation of mtDNA mutations may lead to an enhanced mitochondrial ROS production and ever increasing oxidative stress which ultimately leads to tissue deterioration and aging. Potential mechanism(s) by which mtDNA mutations may mediate their pathological consequences in skeletal muscle are also discussed.

  16. Stalled replication forks generate a distinct mutational signature in yeast

    DEFF Research Database (Denmark)

    Larsen, Nicolai B.; Liberti, Sascha E.; Vogel, Ivan

    2017-01-01

    Proliferating cells acquire genome alterations during the act of DNA replication. This leads to mutation accumulation and somatic cell mosaicism in multicellular organisms, and is also implicated as an underlying cause of aging and tumorigenesis. The molecular mechanisms of DNA replication-associ...

  17. Mitochondrial DNA mutation load in a family with the m.8344A>G point mutation and lipomas

    DEFF Research Database (Denmark)

    Jeppesen, Tina Dysgaard; Al-Hashimi, Noor; Duno, Morten

    2017-01-01

    Studies have shown that difference in mtDNA mutation load among tissues is a result of postnatal modification. We present five family members with the m.8344A>G with variable phenotypes but uniform intrapersonal distribution of mutation load, indicating that there is no postnatal modification of mt......DNA mutation load in this genotype....

  18. Oxidative Stress Is Not a Major Contributor to Somatic Mitochondrial DNA Mutations

    Science.gov (United States)

    Itsara, Leslie S.; Kennedy, Scott R.; Fox, Edward J.; Yu, Selina; Hewitt, Joshua J.; Sanchez-Contreras, Monica; Cardozo-Pelaez, Fernando; Pallanck, Leo J.

    2014-01-01

    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. PMID:24516391

  19. Replication Errors Made During Oogenesis Lead to Detectable De Novo mtDNA Mutations in Zebrafish Oocytes with a Low mtDNA Copy Number.

    Science.gov (United States)

    Otten, Auke B C; Stassen, Alphons P M; Adriaens, Michiel; Gerards, Mike; Dohmen, Richard G J; Timmer, Adriana J; Vanherle, Sabina J V; Kamps, Rick; Boesten, Iris B W; Vanoevelen, Jo M; Muller, Marc; Smeets, Hubert J M

    2016-12-01

    Of all pathogenic mitochondrial DNA (mtDNA) mutations in humans, ∼25% is de novo, although the occurrence in oocytes has never been directly assessed. We used next-generation sequencing to detect point mutations directly in the mtDNA of 3-15 individual mature oocytes and three somatic tissues from eight zebrafish females. Various statistical and biological filters allowed reliable detection of de novo variants with heteroplasmy ≥1.5%. In total, we detected 38 de novo base substitutions, but no insertions or deletions. These 38 de novo mutations were present in 19 of 103 mature oocytes, indicating that ∼20% of the mature oocytes carry at least one de novo mutation with heteroplasmy ≥1.5%. This frequency of de novo mutations is close to that deducted from the reported error rate of polymerase gamma, the mitochondrial replication enzyme, implying that mtDNA replication errors made during oogenesis are a likely explanation. Substantial variation in the mutation prevalence among mature oocytes can be explained by the highly variable mtDNA copy number, since we previously reported that ∼20% of the primordial germ cells have a mtDNA copy number of ≤73 and would lead to detectable mutation loads. In conclusion, replication errors made during oogenesis are an important source of de novo mtDNA base substitutions and their location and heteroplasmy level determine their significance. Copyright © 2016 by the Genetics Society of America.

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

    NARCIS (Netherlands)

    Sijmons, Rolf H.; Hofstra, Robert M. W.

    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

  1. 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.

  2. Compound mitochondrial DNA mutations in a neurological patient ...

    Indian Academy of Sciences (India)

    Mitochondrial diseases caused by mitochondrial dysfunction are, clinically and genetically, ... revealed that two mutation sites are highly conserved among different species (H. sapiens: NC_012920.1, YP_003024029.1; E. cabal- lus: NC_001640.1 ... was then used to transform Escherichia coli (DH5α). Plas- mid DNA was ...

  3. Compound mitochondrial DNA mutations in a neurological patient ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Genetics; Volume 93; Issue 1. Compound mitochondrial DNA mutations in a neurological patient with ataxia, myoclonus and deafness. Ji Hoon Park Bo Ram Yoon Hye Jin Kim Phil Hyu Lee Byung-Ok Choi Ki Wha Chung. Research Note Volume 93 Issue 1 April 2014 pp 173-177 ...

  4. Carrier molecules and extraction of circulating tumor DNA for next generation sequencing in colorectal cancer.

    Science.gov (United States)

    Beránek, Martin; Sirák, Igor; Vošmik, Milan; Petera, Jiří; Drastíková, Monika; Palička, Vladimír

    The aims of the study were: i) to compare circulating tumor DNA (ctDNA) yields obtained by different manual extraction procedures, ii) to evaluate the addition of various carrier molecules into the plasma to improve ctDNA extraction recovery, and iii) to use next generation sequencing (NGS) technology to analyze KRAS, BRAF, and NRAS somatic mutations in ctDNA from patients with metastatic colorectal cancer. Venous blood was obtained from patients who suffered from metastatic colorectal carcinoma. For plasma ctDNA extraction, the following carriers were tested: carrier RNA, polyadenylic acid, glycogen, linear acrylamide, yeast tRNA, salmon sperm DNA, and herring sperm DNA. Each extract was characterized by quantitative real-time PCR and next generation sequencing. The addition of polyadenylic acid had a significant positive effect on the amount of ctDNA eluted. The sequencing data revealed five cases of ctDNA mutated in KRAS and one patient with a BRAF mutation. An agreement of 86% was found between tumor tissues and ctDNA. Testing somatic mutations in ctDNA seems to be a promising tool to monitor dynamically changing genotypes of tumor cells circulating in the body. The optimized process of ctDNA extraction should help to obtain more reliable sequencing data in patients with metastatic colorectal cancer.

  5. Mutations in DNA polymerase eta are not detected in squamous cell carcinoma of the skin.

    Science.gov (United States)

    Glick, Eitan; White, Lisa M; Elliott, Nathan A; Berg, Daniel; Kiviat, Nancy B; Loeb, Lawrence A

    2006-11-01

    The major etiological agent in skin cancer is exposure to UV-irradiation and the concomitant DNA damage. UV-induced DNA lesions, such as thymine dimers, block DNA synthesis by the major DNA polymerases and inhibit the progression of DNA replication. Bypass of thymine dimers and related lesions is dependent on the translesion polymerase DNA polymerase eta (Poleta). In the inherited disorder, xeroderma pigmentosum variant (XPV), inactivation of Poleta results in extreme sensitivity to UV light and a marked increase in the incidence of skin cancer. Here, we tested the hypothesis that somatic mutations and/or polymorphisms in the POLH gene that encodes Poleta are associated with the induction of UV-dependent skin cancers. We sequenced the exonic regions of the Poleta open reading frame in DNA from 17 paired samples of squamous cell skin carcinoma and adjacent histologically normal tissue. We analyzed approximately 120,000 nucleotides and detected no mutations in POLH in the tumors. However, we identified 6 different single-nucleotide polymorphisms, 3 of them previously undocumented, which were present in both the tumor and paired normal tissue. We conclude that neither mutations nor polymorphisms in the coding regions of POLH are required for the generation of human skin squamous cell carcinoma.

  6. DNA polymerase η mutational signatures are found in a variety of different types of cancer.

    Science.gov (United States)

    Rogozin, Igor B; Goncearenco, Alexander; Lada, Artem G; De, Subhajyoti; Yurchenko, Vyacheslav; Nudelman, German; Panchenko, Anna R; Cooper, David N; Pavlov, Youri I

    2018-02-15

    DNA polymerase (pol) η is a specialized error-prone polymerase with at least two quite different and contrasting cellular roles: to mitigate the genetic consequences of solar UV irradiation, and promote somatic hypermutation in the variable regions of immunoglobulin genes. Misregulation and mistargeting of pol η can compromise genome integrity. We explored whether the mutational signature of pol η could be found in datasets of human somatic mutations derived from normal and cancer cells. A substantial excess of single and tandem somatic mutations within known pol η mutable motifs was noted in skin cancer as well as in many other types of human cancer, suggesting that somatic mutations in A:T bases generated by DNA polymerase η are a common feature of tumorigenesis. Another peculiarity of pol ηmutational signatures, mutations in YCG motifs, led us to speculate that error-prone DNA synthesis opposite methylated CpG dinucleotides by misregulated pol η in tumors might constitute an additional mechanism of cytosine demethylation in this hypermutable dinucleotide.

  7. Single Amino Acid Mutation Controls Hole Transfer Dynamics in DNA-Methyltransferase HhaI Complexes.

    Science.gov (United States)

    Corbella, Marina; Voityuk, Alexander A; Curutchet, Carles

    2015-09-17

    Different mutagenic effects are generated by DNA oxidation that implies the formation of radical cation states (so-called holes) on purine nucleobases. The interaction of DNA with proteins may protect DNA from oxidative damage owing to hole transfer (HT) from the stack to aromatic amino acids. However, how protein binding affects HT dynamics in DNA is still poorly understood. Here, we report a computational study of HT in DNA complexes with methyltransferase HhaI with the aim of elucidating the molecular factors that explain why long-range DNA HT is inhibited when the glutamine residue inserted in the double helix is mutated into a tryptophan. We combine molecular dynamics, quantum chemistry, and kinetic Monte Carlo simulations and find that protein binding stabilizes the energies of the guanine radical cation states and significantly impacts the corresponding electronic couplings, thus determining the observed behavior, whereas the formation of a tryptophan radical leads to less efficient HT.

  8. Optimal control of gene mutation in DNA replication.

    Science.gov (United States)

    Yu, Juanyi; Li, Jr-Shin; Tarn, Tzyh-Jong

    2012-01-01

    We propose a molecular-level control system view of the gene mutations in DNA replication from the finite field concept. By treating DNA sequences as state variables, chemical mutagens and radiation as control inputs, one cell cycle as a step increment, and the measurements of the resulting DNA sequence as outputs, we derive system equations for both deterministic and stochastic discrete-time, finite-state systems of different scales. Defining the cost function as a summation of the costs of applying mutagens and the off-trajectory penalty, we solve the deterministic and stochastic optimal control problems by dynamic programming algorithm. In addition, given that the system is completely controllable, we find that the global optimum of both base-to-base and codon-to-codon deterministic mutations can always be achieved within a finite number of steps.

  9. 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

    2014-01-01

    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. PMID:24602056

  10. Inspecting Targeted Deep Sequencing of Whole Genome Amplified DNA Versus Fresh DNA for Somatic Mutation Detection: A Genetic Study in Myelodysplastic Syndrome Patients.

    Science.gov (United States)

    Palomo, Laura; Fuster-Tormo, Francisco; Alvira, Daniel; Ademà, Vera; Armengol, María Pilar; Gómez-Marzo, Paula; de Haro, Nuri; Mallo, Mar; Xicoy, Blanca; Zamora, Lurdes; Solé, Francesc

    2017-08-01

    Whole genome amplification (WGA) has become an invaluable method for preserving limited samples of precious stock material and has been used during the past years as an alternative tool to increase the amount of DNA before library preparation for next-generation sequencing. Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic stem cell disorders characterized by presenting somatic mutations in several myeloid-related genes. In this work, targeted deep sequencing has been performed on four paired fresh DNA and WGA DNA samples from bone marrow of MDS patients, to assess the feasibility of using WGA DNA for detecting somatic mutations. The results of this study highlighted that, in general, the sequencing and alignment statistics of fresh DNA and WGA DNA samples were similar. However, after variant calling and when considering variants detected at all frequencies, there was a high level of discordance between fresh DNA and WGA DNA (overall, a higher number of variants was detected in WGA DNA). After proper filtering, a total of three somatic mutations were detected in the cohort. All somatic mutations detected in fresh DNA were also identified in WGA DNA and validated by whole exome sequencing.

  11. Analytical validation of BRAF mutation testing from circulating free DNA using the amplification refractory mutation testing system.

    Science.gov (United States)

    Aung, Kyaw L; Donald, Emma; Ellison, Gillian; Bujac, Sarah; Fletcher, Lynn; Cantarini, Mireille; Brady, Ged; Orr, Maria; Clack, Glen; Ranson, Malcolm; Dive, Caroline; Hughes, Andrew

    2014-05-01

    BRAF mutation testing from circulating free DNA (cfDNA) using the amplification refractory mutation testing system (ARMS) holds potential as a surrogate for tumor mutation testing. Robust assay validation is needed to establish the optimal clinical matrix for measurement and cfDNA-specific mutation calling criteria. Plasma- and serum-derived cfDNA samples from 221 advanced melanoma patients were analyzed for BRAF c.1799T>A (p.V600E) mutation using ARMS in two stages in a blinded fashion. cfDNA-specific mutation calling criteria were defined in stage 1 and validated in stage 2. cfDNA concentrations in serum and plasma, and the sensitivities and specificities of BRAF mutation detection in these two clinical matrices were compared. Sensitivity of BRAF c.1799T>A (p.V600E) mutation detection in cfDNA was increased by using mutation calling criteria optimized for cfDNA (these criteria were adjusted from those used for archival tumor biopsies) without compromising specificity. Sensitivity of BRAF mutation detection in serum was 44% (95% CI, 35% to 53%) and in plasma 52% (95% CI, 43% to 61%). Specificity was 96% (95% CI, 90% to 99%) in both matrices. Serum contains significantly higher total cfDNA than plasma, whereas the proportion of tumor-derived mutant DNA was significantly higher in plasma. Using mutation calling criteria optimized for cfDNA improves sensitivity of BRAF c.1799T>A (p.V600E) mutation detection. The proportion of tumor-derived cfDNA in plasma was significantly higher than in serum. Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  12. Clustered Mutation Signatures Reveal that Error-Prone DNA Repair Targets Mutations to Active Genes.

    Science.gov (United States)

    Supek, Fran; Lehner, Ben

    2017-07-27

    Many processes can cause the same nucleotide change in a genome, making the identification of the mechanisms causing mutations a difficult challenge. Here, we show that clustered mutations provide a more precise fingerprint of mutagenic processes. Of nine clustered mutation signatures identified from >1,000 tumor genomes, three relate to variable APOBEC activity and three are associated with tobacco smoking. An additional signature matches the spectrum of translesion DNA polymerase eta (POLH). In lymphoid cells, these mutations target promoters, consistent with AID-initiated somatic hypermutation. In solid tumors, however, they are associated with UV exposure and alcohol consumption and target the H3K36me3 chromatin of active genes in a mismatch repair (MMR)-dependent manner. These regions normally have a low mutation rate because error-free MMR also targets H3K36me3 chromatin. Carcinogens and error-prone repair therefore redistribute mutations to the more important regions of the genome, contributing a substantial mutation load in many tumors, including driver mutations. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. The influence of calf thymus DNA and deoxyribonucleosides on the induction of different mutation types in Drosophila

    International Nuclear Information System (INIS)

    Ondrej, M.

    1975-01-01

    The influence of an exogenous DNA on the induction of mutations by X rays was compared with the influence of an equimolar mixture of four deoxyribonucleosides. Pre-treatment and post-treatment with the calf thymus DNA did not influence mutation frequency in the specific loci dp, b, cn and bw as well as Minute mutations induced in the Drosophila sperm by X radiation. Pre-treatment with the equimolar mixture of four deoxyribonucleosides increased the frequency of the Minutes but did not affect mutation frequency in the loci dp, b, cn, bw. The equimolar mixture of nucleosides alone induced a low frequency of Minute mutations in the Drosophila sperm. DNA alone induced a low frequency of recessive lethals. These lethals arose as mosaics of small sectors of the gonads of the F 1 females and were revealed as late as in the F 3 generation. (author)

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

    Science.gov (United States)

    Moehrle, Bettina M; Geiger, Hartmut

    2016-10-01

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

  15. Fidelity and Mutational Spectrum of Pfu DNA Polymerase on a Human Mitochondrial DNA Sequence

    Science.gov (United States)

    André, Paulo; Kim, Andrea; Khrapko, Konstantin; Thilly, William G.

    1997-01-01

    The study of rare genetic changes in human tissues requires specialized techniques. Point mutations at fractions at or below 10−6 must be observed to discover even the most prominent features of the point mutational spectrum. PCR permits the increase in number of mutant copies but does so at the expense of creating many additional mutations or “PCR noise”. Thus, each DNA sequence studied must be characterized with regard to the DNA polymerase and conditions used to avoid interpreting a PCR-generated mutation as one arising in human tissue. The thermostable DNA polymerase derived from Pyrococcus furiosus designated Pfu has the highest fidelity of any DNA thermostable polymerase studied to date, and this property recommends it for analyses of tissue mutational spectra. Here, we apply constant denaturant capillary electrophoresis (CDCE) to separate and isolate the products of DNA amplification. This new strategy permitted direct enumeration and identification of point mutations created by Pfu DNA polymerase in a 96-bp low melting domain of a human mitochondrial sequence despite the very low mutant fractions generated in the PCR process. This sequence, containing part of the tRNA glycine and NADH dehydrogenase subunit 3 genes, is the target of our studies of mitochondrial mutagenesis in human cells and tissues. Incorrectly synthesized sequences were separated from the wild type as mutant/wild-type heteroduplexes by sequential enrichment on CDCE. An artificially constructed mutant was used as an internal standard to permit calculation of the mutant fraction. Our study found that the average error rate (mutations per base pair duplication) of Pfu was 6.5 × 10−7, and five of its more frequent mutations (hot spots) consisted of three transversions (GC → TA, AT → TA, and AT → CG), one transition (AT → GC), and one 1-bp deletion (in an AAAAAA sequence). To achieve an even higher sensitivity, the amount of Pfu-induced mutants must be

  16. Single-Molecule Counting of Point Mutations by Transient DNA Binding

    Science.gov (United States)

    Su, Xin; Li, Lidan; Wang, Shanshan; Hao, Dandan; Wang, Lei; Yu, Changyuan

    2017-03-01

    High-confidence detection of point mutations is important for disease diagnosis and clinical practice. Hybridization probes are extensively used, but are hindered by their poor single-nucleotide selectivity. Shortening the length of DNA hybridization probes weakens the stability of the probe-target duplex, leading to transient binding between complementary sequences. The kinetics of probe-target binding events are highly dependent on the number of complementary base pairs. Here, we present a single-molecule assay for point mutation detection based on transient DNA binding and use of total internal reflection fluorescence microscopy. Statistical analysis of single-molecule kinetics enabled us to effectively discriminate between wild type DNA sequences and single-nucleotide variants at the single-molecule level. A higher single-nucleotide discrimination is achieved than in our previous work by optimizing the assay conditions, which is guided by statistical modeling of kinetics with a gamma distribution. The KRAS c.34 A mutation can be clearly differentiated from the wild type sequence (KRAS c.34 G) at a relative abundance as low as 0.01% mutant to WT. To demonstrate the feasibility of this method for analysis of clinically relevant biological samples, we used this technology to detect mutations in single-stranded DNA generated from asymmetric RT-PCR of mRNA from two cancer cell lines.

  17. R248Q mutation--Beyond p53-DNA binding.

    Science.gov (United States)

    Ng, Jeremy W K; Lama, Dilraj; Lukman, Suryani; Lane, David P; Verma, Chandra S; Sim, Adelene Y L

    2015-12-01

    R248 in the DNA binding domain (DBD) of p53 interacts directly with the minor groove of DNA. Earlier nuclear magnetic resonance (NMR) studies indicated that the R248Q mutation resulted in conformation changes in parts of DBD far from the mutation site. However, how information propagates from the mutation site to the rest of the DBD is still not well understood. We performed a series of all-atom molecular dynamics (MD) simulations to dissect sterics and charge effects of R248 on p53-DBD conformation: (i) wild-type p53 DBD; (ii) p53 DBD with an electrically neutral arginine side-chain; (iii) p53 DBD with R248A; (iv) p53 DBD with R248W; and (v) p53 DBD with R248Q. Our results agree well with experimental observations of global conformational changes induced by the R248Q mutation. Our simulations suggest that both charge- and sterics are important in the dynamics of the loop (L3) where the mutation resides. We show that helix 2 (H2) dynamics is altered as a result of a change in the hydrogen bonding partner of D281. In turn, neighboring L1 dynamics is altered: in mutants, L1 predominantly adopts the recessed conformation and is unable to interact with the major groove of DNA. We focused our attention the R248Q mutant that is commonly found in a wide range of cancer and observed changes at the zinc-binding pocket that might account for the dominant negative effects of R248Q. Furthermore, in our simulations, the S6/S7 turn was more frequently solvent exposed in R248Q, suggesting that there is a greater tendency of R248Q to partially unfold and possibly lead to an increased aggregation propensity. Finally, based on the observations made in our simulations, we propose strategies for the rescue of R248Q mutants. © 2015 Wiley Periodicals, Inc.

  18. Artifactual mutations resulting from DNA lesions limit detection levels in ultrasensitive sequencing applications

    Science.gov (United States)

    Arbeithuber, Barbara; Makova, Kateryna D.; Tiemann-Boege, Irene

    2016-01-01

    The need in cancer research or evolutionary biology to detect rare mutations or variants present at very low frequencies (DNA lesions introduce important error sources in ultrasensitive technologies such as single molecule PCR (smPCR) applications (e.g. droplet-digital PCR), or next-generation sequencing (NGS) based methods. Using templates with known amplifiable lesions (8-oxoguanine, deaminated 5-methylcytosine, uracil, and DNA heteroduplexes), we assessed with smPCR and duplex sequencing that templates with these lesions were amplified very efficiently by proofreading polymerases (except uracil), leading to G->T, and to a lesser extent, to unreported G->C substitutions at 8-oxoguanine lesions, and C->T transitions in amplified uracil containing templates. Long heat incubations common in many DNA extraction protocols significantly increased the number of G->T substitutions. Moreover, in ∼50-80% smPCR reactions we observed the random amplification preference of only one of both DNA strands explaining the known ‘PCR jackpot effect’, with the result that a lesion became indistinguishable from a true mutation or variant. Finally, we showed that artifactual mutations derived from uracil and 8-oxoguanine could be significantly reduced by DNA repair enzymes. PMID:27477585

  19. The dnaE173 mutator mutation confers on the alpha subunit of Escherichia coli DNA polymerase III a capacity for highly processive DNA synthesis and stable binding to primer/template DNA.

    Science.gov (United States)

    Yanagihara, Fusamitsu; Yoshida, Shohei; Sugaya, Yutaka; Maki, Hisaji

    2007-08-01

    The strong mutator mutation dnaE173 which causes an amino-acid substitution in the alpha subunit of DNA polymerase III is unique in its ability to induce sequence-substitution mutations. We showed previously that multiple biochemical properties of DNA polymerase III holoenzyme of Escherichia coli are simultaneously affected by the dnaE173 mutation. These effects include a severely reduced proofreading capacity, an increased resistance to replication-pausing on the template DNA, a capability to readily promote strand-displacement DNA synthesis, a reduced rate of DNA chain elongation, and an ability to catalyze highly processive DNA synthesis in the absence of the beta-clamp subunit. Here we show that, in contrast to distributive DNA synthesis exhibited by wild-type alpha subunit, the dnaE173 mutant form of alpha subunit catalyzes highly processive DNA chain elongation without the aid of the beta-clamp. More surprisingly, the dnaE173 alpha subunit appeared to form a stable complex with primer/template DNA, while no such affinity was detected with wild-type alpha subunit. We consider that the highly increased affinity of alpha subunit for primer/template DNA is the basis for the pleiotropic effects of the dnaE173 mutation on DNA polymerase III, and provides a clue to the molecular mechanisms underlying sequence substitution mutagenesis.

  20. Targeted DNA sequencing and in situ mutation analysis using mobile phone microscopy

    Science.gov (United States)

    Kühnemund, Malte; Wei, Qingshan; Darai, Evangelia; Wang, Yingjie; Hernández-Neuta, Iván; Yang, Zhao; Tseng, Derek; Ahlford, Annika; Mathot, Lucy; Sjöblom, Tobias; Ozcan, Aydogan; Nilsson, Mats

    2017-01-01

    Molecular diagnostics is typically outsourced to well-equipped centralized laboratories, often far from the patient. We developed molecular assays and portable optical imaging designs that permit on-site diagnostics with a cost-effective mobile-phone-based multimodal microscope. We demonstrate that targeted next-generation DNA sequencing reactions and in situ point mutation detection assays in preserved tumour samples can be imaged and analysed using mobile phone microscopy, achieving a new milestone for tele-medicine technologies.

  1. 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.

  2. The next generation of targeted mutation discovery

    NARCIS (Netherlands)

    Harakalova, M.

    2013-01-01

    Sequencing technologies (NGS) now allows efficient analysis of the complete protein-coding regions of genomes (exomes) for multiple samples in a single sequencing run. In Chapter 2, we present our results with a genomic DNA pooling strategy for rare variant discovery on a NGS platform. The high

  3. 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.

  4. cDNA sequencing improves the detection of P53 missense mutations in colorectal cancer

    International Nuclear Information System (INIS)

    Szybka, Malgorzata; Kordek, Radzislaw; Zakrzewska, Magdalena; Rieske, Piotr; Pasz-Walczak, Grazyna; Kulczycka-Wojdala, Dominika; Zawlik, Izabela; Stawski, Robert; Jesionek-Kupnicka, Dorota; Liberski, Pawel P

    2009-01-01

    Recently published data showed discrepancies beteween P53 cDNA and DNA sequencing in glioblastomas. We hypothesised that similar discrepancies may be observed in other human cancers. To this end, we analyzed 23 colorectal cancers for P53 mutations and gene expression using both DNA and cDNA sequencing, real-time PCR and immunohistochemistry. We found P53 gene mutations in 16 cases (15 missense and 1 nonsense). Two of the 15 cases with missense mutations showed alterations based only on cDNA, and not DNA sequencing. Moreover, in 6 of the 15 cases with a cDNA mutation those mutations were difficult to detect in the DNA sequencing, so the results of DNA analysis alone could be misinterpreted if the cDNA sequencing results had not also been available. In all those 15 cases, we observed a higher ratio of the mutated to the wild type template by cDNA analysis, but not by the DNA analysis. Interestingly, a similar overexpression of P53 mRNA was present in samples with and without P53 mutations. In terms of colorectal cancer, those discrepancies might be explained under three conditions: 1, overexpression of mutated P53 mRNA in cancer cells as compared with normal cells; 2, a higher content of cells without P53 mutation (normal cells and cells showing K-RAS and/or APC but not P53 mutation) in samples presenting P53 mutation; 3, heterozygous or hemizygous mutations of P53 gene. Additionally, for heterozygous mutations unknown mechanism(s) causing selective overproduction of mutated allele should also be considered. Our data offer new clues for studying discrepancy in P53 cDNA and DNA sequencing analysis

  5. 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

  6. "First generation" automated DNA sequencing technology.

    Science.gov (United States)

    Slatko, Barton E; Kieleczawa, Jan; Ju, Jingyue; Gardner, Andrew F; Hendrickson, Cynthia L; Ausubel, Frederick M

    2011-10-01

    Beginning in the 1980s, automation of DNA sequencing has greatly increased throughput, reduced costs, and enabled large projects to be completed more easily. The development of automation technology paralleled the development of other aspects of DNA sequencing: better enzymes and chemistry, separation and imaging technology, sequencing protocols, robotics, and computational advancements (including base-calling algorithms with quality scores, database developments, and sequence analysis programs). Despite the emergence of high-throughput sequencing platforms, automated Sanger sequencing technology remains useful for many applications. This unit provides background and a description of the "First-Generation" automated DNA sequencing technology. It also includes protocols for using the current Applied Biosystems (ABI) automated DNA sequencing machines. © 2011 by John Wiley & Sons, Inc.

  7. 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.

  8. Novel FOXC2 Mutation in Hereditary Distichiasis Impairs DNA-Binding Activity and Transcriptional Activation.

    Science.gov (United States)

    Zhang, Leilei; He, Jie; Han, Bing; Lu, Linna; Fan, Jiayan; Zhang, He; Ge, Shengfang; Zhou, Yixiong; Jia, Renbing; Fan, Xianqun

    2016-01-01

    Distichiasis presents as double rows of eyelashes arising from aberrant differentiation of the meibomian glands of the eyelids, and it may be sporadic or hereditary. FOXC2 gene mutations in hereditary distichiasis are rarely reported. Here, we examined two generations of a Chinese family with hereditary distichiasis but without lymphedema or other features of LD syndrome. The FOXC2 gene was amplified and sequenced in all family members. Subcellular localization and luciferase assays were performed to assess the activity of the mutant FOXC2 protein. Clinical examinations showed distichiasis, lower eyelid ectropion, congenital ptosis and photophobia in all affected individuals. Sequence analysis revealed a novel frameshift mutation, c.964_965insG, in the coding region of the FOXC2 gene. This mutation caused protein truncation due to the presence of a premature stop codon. A fluorescence assay showed that this mutation did not change the nuclear localization of the protein. However, it impaired DNA-binding activity and decreased transcriptional activation. This is the first report of a FOXC2 mutation in hereditary distichiasis in the Chinese population. The findings of our study expand the FOXC2 mutation spectrum and contribute to the understanding of the genotype-phenotype correlation of this disease.

  9. Infantile presentation of the mtDNA A3243G tRNA(Leu (UUR)) mutation.

    NARCIS (Netherlands)

    Okhuijsen-Kroes, E.J.; Trijbels, J.M.F.; Sengers, R.C.A.; Mariman, E.C.M.; Heuvel, L.P.W.J. van den; Wendel, U.A.H.; Koch, G.; Smeitink, J.A.M.

    2001-01-01

    Mitochondrial DNA (mtDNA) disorders are clinically very heterogeneous, ranging from single organ involvement to severe multisystem disease. One of the most frequently observed mtDNA mutations is the A-to-G transition at position 3243 of the tRNA(Leu (UUR)) gene. This mutation is often related to

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

    International Nuclear Information System (INIS)

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

    1992-01-01

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

  11. Tumor volume determines the feasibility of cell-free DNA sequencing for mutation detection in non-small cell lung cancer.

    Science.gov (United States)

    Ohira, Tatsuo; Sakai, Kazuko; Matsubayashi, Jun; Kajiwara, Naohiro; Kakihana, Masatoshi; Hagiwara, Masaru; Hibi, Masaaki; Yoshida, Koichi; Maeda, Junichi; Ohtani, Keishi; Nagao, Toshitaka; Nishio, Kazuto; Ikeda, Norihiko

    2016-11-01

    Next-generation sequencing (NGS) and digital PCR technologies allow analysis of the mutational profile of circulating cell-free DNA (cfDNA) in individuals with advanced lung cancer. We have now evaluated the feasibility of cfDNA sequencing for mutation detection in patients with non-small cell lung cancer at earlier stages. A total of 150 matched tumor and serum samples were collected from non-small cell lung cancer patients at stages IA-IIIA. Amplicon sequencing with DNA extracted from tumor tissue detected frequent mutations in EGFR (37% of patients), TP53 (39%), and KRAS (10%), consistent with previous findings. In contrast, NGS of cfDNA identified only EGFR, TP53, and PIK3CA mutations in three, five, and one patient, respectively, even though adequate amounts of cfDNA were extracted (median of 4936 copies/mL serum). Next-generation sequencing showed a high accuracy (98.8%) compared with droplet digital PCR for cfDNA mutation detection, suggesting that the low frequency of mutations in cfDNA was not due to a low assay sensitivity. Whereas the yield of cfDNA did not differ among tumor stages, the cfDNA mutations were detected in seven patients at stages IIA-IIIA and at T2b or T3. Tumor volume was significantly higher in the cfDNA mutation-positive patients than in the negative patients at stages T2b-T4 (159.1 ± 58.0 vs. 52.5 ± 9.9 cm 3 , P = 0.014). Our results thus suggest that tumor volume is a determinant of the feasibility of mutation detection with cfDNA as the analyte. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  12. 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.

  13. Oxidatively generated base damage to cellular DNA

    Energy Technology Data Exchange (ETDEWEB)

    Cadet, Jean [Laboratoire ' Lesions des Acides Nucleiques' , SCIB-UMR-E no.3 - CEA/UJF, Institut nano-sciences et Cryogenie, CEA/Grenoble, F-38054 Grenoble Cedex 9 (France); Departement de Medecine Nucleaire et Radiobiologie, Faculte de medecine de des sciences de la sante, Universite de Sherbrooke, Sherbrooke, Quebec, J1H 5N4 (Canada); Douki, Thierry; Ravanat, Jean-Luc [Laboratoire ' Lesions des Acides Nucleiques' , SCIB-UMR-E no.3 - CEA/UJF, Institut nano-sciences et Cryogenie, CEA/Grenoble, F-38054 Grenoble Cedex 9 (France)

    2010-07-01

    Search for the formation of oxidatively base damage in cellular DNA has been a matter of debate for more than 40 years due to the lack of accurate methods for the measurement of the lesions. HPLC associated with either tandem mass spectrometry (MS/MS) or electrochemical detector (ECD) together with optimized DNA extraction conditions constitutes a relevant analytical approach. This has allowed the accurate measurement of oxidatively generated single and clustered base damage in cellular DNA following exposure to acute oxidative stress conditions mediated by ionizing radiation. UVA light and one-electron oxidants. In this review the formation of 11 single base lesions that is accounted for by reactions of singlet oxygen, hydroxyl radical or high intensity UVC laser pulses with nucleobases is discussed on the basis of the mechanisms available from model studies. In addition several clustered lesions were found to be generated in cellular DNA as the result of one initial radical hit on either a vicinal base or the 2-deoxyribose. Information on nucleo-base modifications that are formed upon addition of reactive aldehydes arising from the breakdown of lipid hydroperoxides is also provided. (authors)

  14. Geant4-DNA simulations using complex DNA geometries generated by the DnaFabric tool

    Science.gov (United States)

    Meylan, S.; Vimont, U.; Incerti, S.; Clairand, I.; Villagrasa, C.

    2016-07-01

    Several DNA representations are used to study radio-induced complex DNA damages depending on the approach and the required level of granularity. Among all approaches, the mechanistic one requires the most resolved DNA models that can go down to atomistic DNA descriptions. The complexity of such DNA models make them hard to modify and adapt in order to take into account different biological conditions. The DnaFabric project was started to provide a tool to generate, visualise and modify such complex DNA models. In the current version of DnaFabric, the models can be exported to the Geant4 code to be used as targets in the Monte Carlo simulation. In this work, the project was used to generate two DNA fibre models corresponding to two DNA compaction levels representing the hetero and the euchromatin. The fibres were imported in a Geant4 application where computations were performed to estimate the influence of the DNA compaction on the amount of calculated DNA damage. The relative difference of the DNA damage computed in the two fibres for the same number of projectiles was found to be constant and equal to 1.3 for the considered primary particles (protons from 300 keV to 50 MeV). However, if only the tracks hitting the DNA target are taken into account, then the relative difference is more important for low energies and decreases to reach zero around 10 MeV. The computations were performed with models that contain up to 18,000 DNA nucleotide pairs. Nevertheless, DnaFabric will be extended to manipulate multi-scale models that go from the molecular to the cellular levels.

  15. 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. Copyright © 2016. Published by Elsevier B.V.

  16. A novel mutation in mitochondrial DNA in a patient with diabetes, deafness and proteinuria.

    Science.gov (United States)

    Adema, A Y; Janssen, M C H; van der Heijden, J W

    2016-12-01

    Maternally inherited deafness and diabetes (MIDD) is characterised by a defect in insulin secretion and bilateral hearing impairment. The m.3243A>G mutation is the most reported in mitochondrial DNA (mtDNA) causing MIDD, although other, rare, mtDNA point mutations have also been mentioned. We report on a 28-year-old Caucasian woman with a history of diabetes, kidney disease, deafness, diarrhoea, myopathy and fatigue. The diagnosis of mitochondrial disease was made in this patient, which resulted from a novel 09155A>G mutation in the mtDNA. As far as we know, this mutation has never been described before as causing MIDD.

  17. Self-cytoplasmic DNA upregulates the mutator enzyme APOBEC3A leading to chromosomal DNA damage.

    Science.gov (United States)

    Suspène, Rodolphe; Mussil, Bianka; Laude, Hélène; Caval, Vincent; Berry, Noémie; Bouzidi, Mohamed S; Thiers, Valérie; Wain-Hobson, Simon; Vartanian, Jean-Pierre

    2017-04-07

    Foreign and self-cytoplasmic DNA are recognized by numerous DNA sensor molecules leading to the production of type I interferons. Such DNA agonists should be degraded otherwise cells would be chronically stressed. Most human APOBEC3 cytidine deaminases can initiate catabolism of cytoplasmic mitochondrial DNA. Using the human myeloid cell line THP-1 with an interferon inducible APOBEC3A gene, we show that cytoplasmic DNA triggers interferon α and β production through the RNA polymerase III transcription/RIG-I pathway leading to massive upregulation of APOBEC3A. By catalyzing C→U editing in single stranded DNA fragments, the enzyme prevents them from re-annealing so attenuating the danger signal. The price to pay is chromosomal DNA damage in the form of CG→TA mutations and double stranded DNA breaks which, in the context of chronic inflammation, could drive cells down the path toward cancer. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  18. Exploring the common molecular basis for the universal DNA mutation bias: revival of Löwdin mutation model.

    Science.gov (United States)

    Fu, Liang-Yu; Wang, Guang-Zhong; Ma, Bin-Guang; Zhang, Hong-Yu

    2011-06-10

    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 Löwdin 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, Löwdin model offers a common explanation for the observed universal mutation bias and thus has broad biological implications. Copyright © 2011 Elsevier Inc. All rights reserved.

  19. RANDNA: a random DNA sequence generator.

    Science.gov (United States)

    Piva, Francesco; Principato, Giovanni

    2006-01-01

    Monte Carlo simulations are useful to verify the significance of data. Genomic regularities, such as the nucleotide correlations or the not uniform distribution of the motifs throughout genomic or mature mRNA sequences, exist and their significance can be checked by means of the Monte Carlo test. The test needs good quality random sequences in order to work, moreover they should have the same nucleotide distribution as the sequences in which the regularities have been found. Random DNA sequences are also useful to estimate the background score of an alignment, that is a threshold below which the resulting score is merely due to chance. We have developed RANDNA, a free software which allows to produce random DNA or RNA sequences setting both their length and the percentage of nucleotide composition. Sequences having the same nucleotide distribution of exonic, intronic or intergenic sequences can be generated. Its graphic interface makes it possible to easily set the parameters that characterize the sequences being produced and saved in a text format file. The pseudo-random number generator function of Borland Delphi 6 is used, since it guarantees a good randomness, a long cycle length and a high speed. We have checked the quality of sequences generated by the software, by means of well-known tests, both by themselves and versus genuine random sequences. We show the good quality of the generated sequences. The software, complete with examples and documentation, is freely available to users from: http://www.introni.it/en/software.

  20. Ultra-deep sequencing of mouse mitochondrial DNA: mutational patterns and their origins.

    Directory of Open Access Journals (Sweden)

    Adam Ameur

    2011-03-01

    Full Text Available Somatic mutations of mtDNA are implicated in the aging process, but there is no universally accepted method for their accurate quantification. We have used ultra-deep sequencing to study genome-wide mtDNA mutation load in the liver of normally- and prematurely-aging mice. Mice that are homozygous for an allele expressing a proof-reading-deficient mtDNA polymerase (mtDNA mutator mice have 10-times-higher point mutation loads than their wildtype siblings. In addition, the mtDNA mutator mice have increased levels of a truncated linear mtDNA molecule, resulting in decreased sequence coverage in the deleted region. In contrast, circular mtDNA molecules with large deletions occur at extremely low frequencies in mtDNA mutator mice and can therefore not drive the premature aging phenotype. Sequence analysis shows that the main proportion of the mutation load in heterozygous mtDNA mutator mice and their wildtype siblings is inherited from their heterozygous mothers consistent with germline transmission. We found no increase in levels of point mutations or deletions in wildtype C57Bl/6N mice with increasing age, thus questioning the causative role of these changes in aging. In addition, there was no increased frequency of transversion mutations with time in any of the studied genotypes, arguing against oxidative damage as a major cause of mtDNA mutations. Our results from studies of mice thus indicate that most somatic mtDNA mutations occur as replication errors during development and do not result from damage accumulation in adult life.

  1. Role of Mitochondrial DNA Mutations in Cellular Vulnerability to Mitochondria-Specific Environmental Toxins

    National Research Council Canada - National Science Library

    Hirsch, Etienne C

    2005-01-01

    In recent years, growing evidence has shown that mutations of mitochondrial DNA (mtDNA) are an important cause of mitochondrial disorders in humans, and have been associated with common neurodegenerative disorders, aging and cancers...

  2. Detection of a mosaic PIK3CA mutation in dental DNA from a child with megalencephaly capillary malformation syndrome.

    Science.gov (United States)

    McDermott, John H; Byers, Helen; Clayton-Smith, Jill

    2016-01-01

    The megalencephaly capillary malformation syndrome (MCAP, OMIM 602501) is known to be associated with mosaic mutations in PIK3CA occurring during embryogenesis. Standard sequencing technologies are relatively poor at indentifying sequence changes that only affect a small percentage of cells, and the mutations are frequently not identified in lymphocyte DNA, with biopsies of the affected tissues often being required to detect mosaic mutations. Such invasive procedures are not always acceptable to parents. We describe the case of a patient in whom we were able to confirm a causative PIK3CA mutation, first found thorugh next-generation sequencing, in several tissue types including a secondary tooth. As part of this work, we were also able to begin validating dental tissue for potential use in genetic testing, as we achieved excellent DNA yields with minimal effort, even from deciduous teeth shed some years earlier.

  3. 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.

  4. 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.

  5. DNA sequence analysis of methylene chloride-induced HPRT mutations in Chinese hamster ovary cells: comparison with the mutation spectrum obtained for 1,2-dibromoethane and formaldehyde.

    Science.gov (United States)

    Graves, R J; Trueman, P; Jones, S; Green, T

    1996-05-01

    Glutathione-S-transferase-mediated metabolism of methylene chloride (MC) generates S-chloromethylglutathione, which has the potential to react with DNA, and formaldehyde, which is a known mutagen. MC-induced mutations in the HPRT gene of Chinese hamster ovary cells have been sequenced and compared with the mutations induced by 1, 2-dibromoethane (1,2-DEB), which is known to act through a glutathione conjugate, and formaldehyde. All three compounds induced primarily point mutations, with a small number of insertion and deletion events. The most common point mutations induced by MC were GC-->AT transitions (4/8), with two GC-->CG transversions and two AT-->TA transversions. This pattern of mutations showed greater similarity with 1,2-DBE, where the dominant point mutations were GC-->AT transitions (7/9), than formaldehyde, where all mutations were single base transversions and 5/6 occurred from AT base pairs. The mutation sequence results for MC suggest that S-chloromethylglutathione plays a major role in MC mutagenesis, with only a limited contribution from formaldehyde. The involvement of a glutathione (GSH) conjugate in MC mutagenicity would be analogous to the well-characterized pathway of activation of 1,2-DBE.

  6. Conjugation between quinolone-susceptible bacteria can generate mutations in the quinolone resistance-determining region, inducing quinolone resistance.

    Science.gov (United States)

    Pitondo-Silva, André; Martins, Vinicius Vicente; Silva, Carolina Fávero da; Stehling, Eliana Guedes

    2015-02-01

    Quinolones are an important group of antibacterial agents that can inhibit DNA gyrase and topoisomerase IV activity. DNA gyrase is responsible for maintaining bacteria in a negatively supercoiled state, being composed of subunits A and B. Topoisomerase IV is a homologue of DNA gyrase and consists of two subunits codified by the parC and parE genes. Mutations in gyrA and gyrB of DNA gyrase may confer resistance to quinolones, and the majority of resistant strains show mutations between positions 67 and 106 of gyrA, a region denoted the quinolone resistance-determining region (QRDR). The most frequent substitutions occur at positions 83 and 87, but little is known about the mechanisms promoting appearance of mutations in the QRDR. The present study proposes that some mutations in the QRDR could be generated as a result of the natural mechanism of conjugation between bacteria in their natural habitat. This event was observed following conjugation in vitro of two different isolates of quinolone-susceptible Pseudomonas aeruginosa, which transferred plasmids of different molecular weights to a recipient strain of Escherichia coli (HB101), also quinolone-susceptible, generating two different transconjugants that presented mutations in DNA gyrase and acquisition of resistance to all quinolones tested. Copyright © 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

  7. Exome sequencing identifies rare deleterious mutations in DNA repair genes FANCC and BLM as potential breast cancer susceptibility alleles.

    Directory of Open Access Journals (Sweden)

    Ella R Thompson

    2012-09-01

    Full Text Available Despite intensive efforts using linkage and candidate gene approaches, the genetic etiology for the majority of families with a multi-generational breast cancer predisposition is unknown. In this study, we used whole-exome sequencing of thirty-three individuals from 15 breast cancer families to identify potential predisposing genes. Our analysis identified families with heterozygous, deleterious mutations in the DNA repair genes FANCC and BLM, which are responsible for the autosomal recessive disorders Fanconi Anemia and Bloom syndrome. In total, screening of all exons in these genes in 438 breast cancer families identified three with truncating mutations in FANCC and two with truncating mutations in BLM. Additional screening of FANCC mutation hotspot exons identified one pathogenic mutation among an additional 957 breast cancer families. Importantly, none of the deleterious mutations were identified among 464 healthy controls and are not reported in the 1,000 Genomes data. Given the rarity of Fanconi Anemia and Bloom syndrome disorders among Caucasian populations, the finding of multiple deleterious mutations in these critical DNA repair genes among high-risk breast cancer families is intriguing and suggestive of a predisposing role. Our data demonstrate the utility of intra-family exome-sequencing approaches to uncover cancer predisposition genes, but highlight the major challenge of definitively validating candidates where the incidence of sporadic disease is high, germline mutations are not fully penetrant, and individual predisposition genes may only account for a tiny proportion of breast cancer families.

  8. Comparison of DNA Quantification Methods for Next Generation Sequencing.

    Science.gov (United States)

    Robin, Jérôme D; Ludlow, Andrew T; LaRanger, Ryan; Wright, Woodring E; Shay, Jerry W

    2016-04-06

    Next Generation Sequencing (NGS) is a powerful tool that depends on loading a precise amount of DNA onto a flowcell. NGS strategies have expanded our ability to investigate genomic phenomena by referencing mutations in cancer and diseases through large-scale genotyping, developing methods to map rare chromatin interactions (4C; 5C and Hi-C) and identifying chromatin features associated with regulatory elements (ChIP-seq, Bis-Seq, ChiA-PET). While many methods are available for DNA library quantification, there is no unambiguous gold standard. Most techniques use PCR to amplify DNA libraries to obtain sufficient quantities for optical density measurement. However, increased PCR cycles can distort the library's heterogeneity and prevent the detection of rare variants. In this analysis, we compared new digital PCR technologies (droplet digital PCR; ddPCR, ddPCR-Tail) with standard methods for the titration of NGS libraries. DdPCR-Tail is comparable to qPCR and fluorometry (QuBit) and allows sensitive quantification by analysis of barcode repartition after sequencing of multiplexed samples. This study provides a direct comparison between quantification methods throughout a complete sequencing experiment and provides the impetus to use ddPCR-based quantification for improvement of NGS quality.

  9. 21 CFR 864.7280 - Factor V Leiden DNA mutation detection systems.

    Science.gov (United States)

    2010-04-01

    ....7280 Factor V Leiden DNA mutation detection systems. (a) Identification. Factor V Leiden... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Factor V Leiden DNA mutation detection systems. 864.7280 Section 864.7280 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN...

  10. A trade-off between oxidative stress resistance and DNA repair plays a role in the evolution of elevated mutation rates in bacteria

    Science.gov (United States)

    Torres-Barceló, Clara; Cabot, Gabriel; Oliver, Antonio; Buckling, Angus; MacLean, R. Craig

    2013-01-01

    The dominant paradigm for the evolution of mutator alleles in bacterial populations is that they spread by indirect selection for linked beneficial mutations when bacteria are poorly adapted. In this paper, we challenge the ubiquity of this paradigm by demonstrating that a clinically important stressor, hydrogen peroxide, generates direct selection for an elevated mutation rate in the pathogenic bacterium Pseudomonas aeruginosa as a consequence of a trade-off between the fidelity of DNA repair and hydrogen peroxide resistance. We demonstrate that the biochemical mechanism underlying this trade-off in the case of mutS is the elevated secretion of catalase by the mutator strain. Our results provide, to our knowledge, the first experimental evidence that direct selection can favour mutator alleles in bacterial populations, and pave the way for future studies to understand how mutation and DNA repair are linked to stress responses and how this affects the evolution of bacterial mutation rates. PMID:23446530

  11. Circulating tumor DNA outperforms circulating tumor cells for KRAS mutation detection in thoracic malignancies.

    Science.gov (United States)

    Freidin, Maxim B; Freydina, Dasha V; Leung, Maria; Montero Fernandez, Angeles; Nicholson, Andrew G; Lim, Eric

    2015-10-01

    Circulating biomarkers, such as circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), are both considered for blood-based mutation detection, but limited studies have compared them in a head-to-head manner. Using KRAS (Kirsten rat sarcoma viral oncogene homolog), we performed such a comparison in patients who underwent surgery for suspected lung cancer. We recruited 93 patients, including 82 with lung cancer and 11 with benign diseases of the lung. Mutations were detected in codons 12 and 13 of KRAS in DNA extracted from CTCs, plasma, and matched tumors or lung tissues with custom-designed coamplification at lower denaturation temperature (COLD)-PCR assays, high-resolution melt analysis (HRM), and commercial assays (Roche Cobas(®) KRAS mutation test and Qiagen Therascreen(®) pyrosequencing KRAS kit). With the Cobas mutation test, we identified KRAS mutations in 21.3% of tumors. Mutation analysis in matched CTC DNA and ctDNA samples by COLD-PCR/HRM assay revealed mutations in 30.5% (ctDNA) and 23.2% (CTC DNA) of patients with lung cancer. Combined results of different tests revealed KRAS-positive cases for 28% of tumors. The diagnostic sensitivity and specificity of KRAS mutation detection in tumors achieved with ctDNA was 0.96 (95% CI 0.81-1.00) and 0.95 (0.85-0.99), respectively. The diagnostic test performance was lower for CTC DNA, at 0.52 (0.34-0.73) and 0.88 (0.79-0.95). Our results support ctDNA as a preferential specimen type for mutation screening in thoracic malignancies vs CTC DNA, achieving greater mutation detection than either CTCs or limited amounts of tumor tissue alone. © 2015 American Association for Clinical Chemistry.

  12. Presymptomatic DNA testing and prophylactic surgery in families with a BRCA1 or BRCA2 mutation

    NARCIS (Netherlands)

    Meijers-Heijboer, E. J.; Verhoog, L. C.; Brekelmans, C. T.; Seynaeve, C.; Tilanus-Linthorst, M. M.; Wagner, A.; Dukel, L.; Devilee, P.; van den Ouweland, A. M.; van Geel, A. N.; Klijn, J. G.

    2000-01-01

    Germline mutations in the BRCA1 and BRCA2 genes highly predispose to breast and ovarian cancer. In families with BRCA1 or BRCA2 mutations, identification of mutation carriers is clinically relevant in view of the options for surveillance and prevention. We assessed presymptomatic DNA testing and

  13. Simultaneous Profiling of DNA Mutation and Methylation by Melting Analysis Using Magnetoresistive Biosensor Array.

    Science.gov (United States)

    Rizzi, Giovanni; Lee, Jung-Rok; Dahl, Christina; Guldberg, Per; Dufva, Martin; Wang, Shan X; Hansen, Mikkel F

    2017-09-26

    Epigenetic modifications, in particular DNA methylation, are gaining increasing interest as complementary information to DNA mutations for cancer diagnostics and prognostics. We introduce a method to simultaneously profile DNA mutation and methylation events for an array of sites with single site specificity. Genomic (mutation) or bisulphite-treated (methylation) DNA is amplified using nondiscriminatory primers, and the amplicons are then hybridized to a giant magnetoresistive (GMR) biosensor array followed by melting curve measurements. The GMR biosensor platform offers scalable multiplexed detection of DNA hybridization, which is insensitive to temperature variation. The melting curve approach further enhances the assay specificity and tolerance to variations in probe length. We demonstrate the utility of this method by simultaneously profiling five mutation and four methylation sites in human melanoma cell lines. The method correctly identified all mutation and methylation events and further provided quantitative assessment of methylation density validated by bisulphite pyrosequencing.

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

    International Nuclear Information System (INIS)

    Chen Roulei; Song Daojun; Yu Zengliang; Li Yufeng; Liang Yunzhang

    2001-01-01

    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 (S 41 , S 42 , S 45 , S 46 , S 50 , S 52 , S 56 , S 58 ) 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

  15. Highly sensitive detection of mutations in CHO cell recombinant DNA using multi-parallel single molecule real-time DNA sequencing.

    Science.gov (United States)

    Cartwright, Joseph F; Anderson, Karin; Longworth, Joseph; Lobb, Philip; James, David C

    2018-02-10

    High-fidelity replication of biologic-encoding recombinant DNA sequences by engineered mammalian cell cultures is an essential pre-requisite for the development of stable cell lines for the production of biotherapeutics. However, immortalized mammalian cells characteristically exhibit an increased point mutation frequency compared to mammalian cells in vivo, both across their genomes and at specific loci (hotspots). Thus unforeseen mutations in recombinant DNA sequences can arise and be maintained within producer cell populations. These may affect both the stability of recombinant gene expression and give rise to protein sequence variants with variable bioactivity and immunogenicity. Rigorous quantitative assessment of recombinant DNA integrity should therefore form part of the cell line development process and be an essential quality assurance metric for instances where synthetic/multi-component assemblies are utilized to engineer mammalian cells, such as the assessment of recombinant DNA fidelity or the mutability of single-site integration target loci. Based on Pacific Biosciences (Menlo Park, CA) single molecule real-time (SMRT™) circular consensus sequencing (CCS) technology we developed a rDNA sequence analysis tool to process the multi-parallel sequencing of ∼40,000 single recombinant DNA molecules. After statistical filtering of raw sequencing data, we show that this analytical method is capable of detecting single point mutations in rDNA to a minimum single mutation frequency of 0.0042% (sequence. There was no discernable difference between the mutation frequencies of coding and non-coding DNA. The putative ratio of non-synonymous and synonymous changes within the open reading frames (ORFs) in the plasmid sequence indicates that natural selection does not impact upon the prevalence of these mutations. Here we have demonstrated the abundance of mutations that fall outside of the reported range of detection of next generation sequencing (NGS) and second

  16. Risk of colorectal cancer for people with a mutation in both a MUTYH and a DNA mismatch repair gene.

    Science.gov (United States)

    Win, Aung Ko; Reece, Jeanette C; Buchanan, Daniel D; Clendenning, Mark; Young, Joanne P; Cleary, Sean P; Kim, Hyeja; Cotterchio, Michelle; Dowty, James G; MacInnis, Robert J; Tucker, Katherine M; Winship, Ingrid M; Macrae, Finlay A; Burnett, Terrilea; Le Marchand, Loïc; Casey, Graham; Haile, Robert W; Newcomb, Polly A; Thibodeau, Stephen N; Lindor, Noralane M; Hopper, John L; Gallinger, Steven; Jenkins, Mark A

    2015-12-01

    The base excision repair protein, MUTYH, functionally interacts with the DNA mismatch repair (MMR) system. As genetic testing moves from testing one gene at a time, to gene panel and whole exome next generation sequencing approaches, understandin g the risk associated with co-existence of germline mutations in these genes will be important for clinical interpretation and management. From the Colon Cancer Family Registry, we identified 10 carriers who had both a MUTYH mutation (6 with c.1187G>A p.(Gly396Asp), 3 with c.821G>A p.(Arg274Gln), and 1 with c.536A>G p.(Tyr179Cys)) and a MMR gene mutation (3 in MLH1, 6 in MSH2, and 1 in PMS2), 375 carriers of a single (monoallelic) MUTYH mutation alone, and 469 carriers of a MMR gene mutation alone. Of the 10 carriers of both gene mutations, 8 were diagnosed with colorectal cancer. Using a weighted cohort analysis, we estimated that risk of colorectal cancer for carriers of both a MUTYH and a MMR gene mutation was substantially higher than that for carriers of a MUTYH mutation alone [hazard ratio (HR) 21.5, 95% confidence interval (CI) 9.19-50.1; p mutation alone (HR 1.94, 95% CI 0.63-5.99; p = 0.25). Within the limited power of this study, there was no evidence that a monoallelic MUTYH gene mutation confers additional risk of colorectal cancer for carriers of a MMR gene mutation alone. Our finding suggests MUTYH mutation testing in MMR gene mutation carriers is not clinically informative.

  17. Risk of colorectal cancer for people with a mutation in both a MUTYH and a DNA mismatch repair gene

    Science.gov (United States)

    Win, Aung Ko; Reece, Jeanette C.; Buchanan, Daniel D.; Clendenning, Mark; Young, Joanne P.; Cleary, Sean P.; Kim, Hyeja; Cotterchio, Michelle; Dowty, James G.; MacInnis, Robert J.; Tucker, Katherine M.; Winship, Ingrid M.; Macrae, Finlay A.; Burnett, Terrilea; Le Marchand, Loïc; Casey, Graham; Haile, Robert W.; Newcomb, Polly A.; Thibodeau, Stephen N.; Lindor, Noralane M.; Hopper, John L.; Gallinger, Steven; Jenkins, Mark A.

    2015-01-01

    The base excision repair protein, MUTYH, functionally interacts with the DNA mismatch repair (MMR) system. As genetic testing moves from testing one gene at a time, to gene panel and whole exome next generation sequencing approaches, understanding the risk associated with co-existence of germline mutations in these genes will be important for clinical interpretation and management. From the Colon Cancer Family Registry, we identified 10 carriers who had both a MUTYH mutation (6 with c.1187G>A p.(Gly396Asp), 3 with c.821G>A p.(Arg274Gln), and 1 with c.536A>G p.(Tyr179Cys)) and a MMR gene mutation (3 in MLH1, 6 in MSH2, and 1 in PMS2), 375 carriers of a single (monoallelic) MUTYH mutation alone, and 469 carriers of a MMR gene mutation alone. Of the 10 carriers of both gene mutations, 8 were diagnosed with colorectal cancer. Using a weighted cohort analysis, we estimated that risk of colorectal cancer for carriers of both a MUTYH and a MMR gene mutation was substantially higher than that for carriers of a MUTYH mutation alone [hazard ratio (HR) 21.5, 95 % confidence interval (CI) 9.19–50.1; p < 0.001], but not different from that for carriers of a MMR gene mutation alone (HR 1.94, 95 % CI 0.63–5.99; p = 0.25). Within the limited power of this study, there was no evidence that a monoallelic MUTYH gene mutation confers additional risk of colorectal cancer for carriers of a MMR gene mutation alone. Our finding suggests MUTYH mutation testing in MMR gene mutation carriers is not clinically informative. PMID:26202870

  18. Mitochondrial DNA variant discovery and evaluation in human Cardiomyopathies through next-generation sequencing.

    Directory of Open Access Journals (Sweden)

    Michael V Zaragoza

    2010-08-01

    Full Text Available Mutations in mitochondrial DNA (mtDNA may cause maternally-inherited cardiomyopathy and heart failure. In homoplasmy all mtDNA copies contain the mutation. In heteroplasmy there is a mixture of normal and mutant copies of mtDNA. The clinical phenotype of an affected individual depends on the type of genetic defect and the ratios of mutant and normal mtDNA in affected tissues. We aimed at determining the sensitivity of next-generation sequencing compared to Sanger sequencing for mutation detection in patients with mitochondrial cardiomyopathy. We studied 18 patients with mitochondrial cardiomyopathy and two with suspected mitochondrial disease. We "shotgun" sequenced PCR-amplified mtDNA and multiplexed using a single run on Roche's 454 Genome Sequencer. By mapping to the reference sequence, we obtained 1,300x average coverage per case and identified high-confidence variants. By comparing these to >400 mtDNA substitution variants detected by Sanger, we found 98% concordance in variant detection. Simulation studies showed that >95% of the homoplasmic variants were detected at a minimum sequence coverage of 20x while heteroplasmic variants required >200x coverage. Several Sanger "misses" were detected by 454 sequencing. These included the novel heteroplasmic 7501T>C in tRNA serine 1 in a patient with sudden cardiac death. These results support a potential role of next-generation sequencing in the discovery of novel mtDNA variants with heteroplasmy below the level reliably detected with Sanger sequencing. We hope that this will assist in the identification of mtDNA mutations and key genetic determinants for cardiomyopathy and mitochondrial disease.

  19. Genome-wide investigation of DNA methylation marks associated with FV Leiden mutation.

    Science.gov (United States)

    Aïssi, Dylan; Dennis, Jessica; Ladouceur, Martin; Truong, Vinh; Zwingerman, Nora; Rocanin-Arjo, Ares; Germain, Marine; Paton, Tara A; Morange, Pierre-Emmanuel; Gagnon, France; Trégouët, David-Alexandre

    2014-01-01

    In order to investigate whether DNA methylation marks could contribute to the incomplete penetrance of the FV Leiden mutation, a major genetic risk factor for venous thrombosis (VT), we measured genome-wide DNA methylation levels in peripheral blood samples of 98 VT patients carrying the mutation and 251 VT patients without the mutation using the dedicated Illumina HumanMethylation450 array. The genome-wide analysis of 388,120 CpG probes identified three sites mapping to the SLC19A2 locus whose DNA methylation levels differed significantly (pmutation among which 53 were carriers and 161 were non-carriers of the mutation. In both studies, these three CpG sites were also associated (2.33 10-11mutation. A comprehensive linkage disequilibrium (LD) analysis of the whole locus revealed that the original associations were due to LD between the FV Leiden mutation and a block of single nucleotide polymorphisms (SNP) located in SLC19A2. After adjusting for this block of SNPs, the FV Leiden mutation was no longer associated with any CpG site (p>0.05). In conclusion, our work clearly illustrates some promises and pitfalls of DNA methylation investigations on peripheral blood DNA in large epidemiological cohorts. DNA methylation levels at SLC19A2 are influenced by SNPs in LD with FV Leiden, but these DNA methylation marks do not explain the incomplete penetrance of the FV Leiden mutation.

  20. The Application of Next-Generation Sequencing for Mutation Detection in Autosomal-Dominant Hereditary Hearing Impairment.

    Science.gov (United States)

    Gürtler, Nicolas; Röthlisberger, Benno; Ludin, Katja; Schlegel, Christoph; Lalwani, Anil K

    2017-07-01

    Identification of the causative mutation using next-generation sequencing in autosomal-dominant hereditary hearing impairment, as mutation analysis in hereditary hearing impairment by classic genetic methods, is hindered by the high heterogeneity of the disease. Two Swiss families with autosomal-dominant hereditary hearing impairment. Amplified DNA libraries for next-generation sequencing were constructed from extracted genomic DNA, derived from peripheral blood, and enriched by a custom-made sequence capture library. Validated, pooled libraries were sequenced on an Illumina MiSeq instrument, 300 cycles and paired-end sequencing. Technical data analysis was performed with SeqMonk, variant analysis with GeneTalk or VariantStudio. The detection of mutations in genes related to hearing loss by next-generation sequencing was subsequently confirmed using specific polymerase-chain-reaction and Sanger sequencing. Mutation detection in hearing-loss-related genes. The first family harbored the mutation c.5383+5delGTGA in the TECTA-gene. In the second family, a novel mutation c.2614-2625delCATGGCGCCGTG in the WFS1-gene and a second mutation TCOF1-c.1028G>A were identified. Next-generation sequencing successfully identified the causative mutation in families with autosomal-dominant hereditary hearing impairment. The results helped to clarify the pathogenic role of a known mutation and led to the detection of a novel one. NGS represents a feasible approach with great potential future in the diagnostics of hereditary hearing impairment, even in smaller labs.

  1. Structural Learning of Attack Vectors for Generating Mutated XSS Attacks

    Directory of Open Access Journals (Sweden)

    Yi-Hsun Wang

    2010-09-01

    Full Text Available Web applications suffer from cross-site scripting (XSS attacks that resulting from incomplete or incorrect input sanitization. Learning the structure of attack vectors could enrich the variety of manifestations in generated XSS attacks. In this study, we focus on generating more threatening XSS attacks for the state-of-the-art detection approaches that can find potential XSS vulnerabilities in Web applications, and propose a mechanism for structural learning of attack vectors with the aim of generating mutated XSS attacks in a fully automatic way. Mutated XSS attack generation depends on the analysis of attack vectors and the structural learning mechanism. For the kernel of the learning mechanism, we use a Hidden Markov model (HMM as the structure of the attack vector model to capture the implicit manner of the attack vector, and this manner is benefited from the syntax meanings that are labeled by the proposed tokenizing mechanism. Bayes theorem is used to determine the number of hidden states in the model for generalizing the structure model. The paper has the contributions as following: (1 automatically learn the structure of attack vectors from practical data analysis to modeling a structure model of attack vectors, (2 mimic the manners and the elements of attack vectors to extend the ability of testing tool for identifying XSS vulnerabilities, (3 be helpful to verify the flaws of blacklist sanitization procedures of Web applications. We evaluated the proposed mechanism by Burp Intruder with a dataset collected from public XSS archives. The results show that mutated XSS attack generation can identify potential vulnerabilities.

  2. 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.

  3. Noninvasive Test for Mitochondrial DNA A1555G Mutation Associated with Deafness.

    Science.gov (United States)

    Fan, Wenlu; Zhu, Yi; Tang, Xiaowen; Xue, Ling

    2017-01-01

    The homoplasmic mitochondrial DNA (mtDNA) A1555G mutation in the highly conserved decoding site of 12S rRNA has been associated with aminoglycoside-induced and nonsyndromic hearing loss in many families worldwide. The A1555G mutation detection is an important part of hearing screening. At present, blood samples are the most common source of genomic DNA. However, drawing blood is invasive for individuals. The whole genomic DNA of samples carrying mitochondrial DNA A1555G mutation (LX010 and LX044) with aminoglycoside-induced and nonsyndromic deafness as well as wild type were used as templates. The PCR products were analyzed by DNA sequencing, PCR-RFLP, dot blot, and southern blot. The DNA sequencing, PCR-RFLP, dot blot, and southern blot demonstrate that buccal cell DNA can be used for the screening and identification of the A1555G mutation as well as peripheral blood DNA. This study established a convenient, noninvasive and suitable for clinical determination of mtDNA A1555G mutation associated with deafness.

  4. 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

  5. Utility of KRAS mutation detection using circulating cell-free DNA from patients with colorectal cancer.

    Science.gov (United States)

    Yamada, Takeshi; Iwai, Takuma; Takahashi, Goro; Kan, Hayato; Koizumi, Michihiro; Matsuda, Akihisa; Shinji, Seiichi; Yamagishi, Aya; Yokoyama, Yasuyuki; Tatsuguchi, Atsushi; Kawagoe, Tatsuro; Kitano, Shiro; Nakayama, Masato; Matsumoto, Satoshi; Uchida, Eiji

    2016-07-01

    In this study, we evaluated the clinical utility of detecting KRAS mutations in circulating cell-free (ccf)DNA of metastatic colorectal cancer patients. We prospectively recruited 94 metastatic colorectal cancer patients. Circulating cell-free DNA was extracted from plasma samples and analyzed for the presence of seven KRAS point mutations. Using the Invader Plus assay with peptide nucleic acid clamping method and digital PCR, KRAS mutations were detected in the ccfDNA in 35 of 39 patients previously determined to have primary tumors containing KRAS mutations using the Luminex method, and in 5 of 55 patients with tumors containing wild-type KRAS. Curative resection was undertaken in 7 of 34 patients with primary and ccfDNA KRAS mutations, resulting in the disappearance of the mutation from the cell-free DNA in five of seven patients. Three of these patients had tumor recurrence and KRAS mutations in their ccfDNA reappeared. Epidermal growth factor receptor blockade was administered to 24 of the KRAS tumor wild-type patients. Of the 24 patients with wild-type KRAS in their primary tumors, three patients had KRAS mutations in their ccfDNA and did not respond to treatment with epidermal growth factor receptor (EGFR) blockade. We also detected a new KRAS mutation in five patients during chemotherapy with EGFR blockade, before disease progression was detectable with imaging. The detection of KRAS mutations in ccfDNA is an attractive approach for predicting both treatment response and acquired resistance to EGFR blockade, and for detecting disease recurrence. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

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

    Directory of Open Access Journals (Sweden)

    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.

  7. Structural studies of p53 inactivation by DNA-contact mutations and its rescue by suppressor mutations via alternative protein–DNA interactions

    Science.gov (United States)

    Eldar, Amir; Rozenberg, Haim; Diskin-Posner, Yael; Rohs, Remo; Shakked, Zippora

    2013-01-01

    A p53 hot-spot mutation found frequently in human cancer is the replacement of R273 by histidine or cysteine residues resulting in p53 loss of function as a tumor suppressor. These mutants can be reactivated by the incorporation of second-site suppressor mutations. Here, we present high-resolution crystal structures of the p53 core domains of the cancer-related proteins, the rescued proteins and their complexes with DNA. The structures show that inactivation of p53 results from the incapacity of the mutated residues to form stabilizing interactions with the DNA backbone, and that reactivation is achieved through alternative interactions formed by the suppressor mutations. Detailed structural and computational analysis demonstrates that the rescued p53 complexes are not fully restored in terms of DNA structure and its interface with p53. Contrary to our previously studied wild-type (wt) p53-DNA complexes showing non-canonical Hoogsteen A/T base pairs of the DNA helix that lead to local minor-groove narrowing and enhanced electrostatic interactions with p53, the current structures display Watson–Crick base pairs associated with direct or water-mediated hydrogen bonds with p53 at the minor groove. These findings highlight the pivotal role played by R273 residues in supporting the unique geometry of the DNA target and its sequence-specific complex with p53. PMID:23863845

  8. Mutations at position 184 of human immunodeficiency virus type-1 reverse transcriptase affect virus titer and viral DNA synthesis

    International Nuclear Information System (INIS)

    Julias, John G.; Boyer, Paul L.; McWilliams, Mary Jane; Alvord, W. Gregory; Hughes, Stephen H.

    2004-01-01

    Methionine at position 184 of human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) was changed to valine, isoleucine, threonine, or alanine in an HIV-1-based vector. The vectors were analyzed for replication capacity and for resistance to the nucleoside analog 2',3'-dideoxy-3'thiacytidine (3TC) using a single-cycle assay. Viruses containing the valine or isoleucine mutations were highly resistant to 3TC and replicated almost as well as the wild-type virus. The virus containing the threonine mutation was resistant to 3TC, but replicated about 30% as well as the wild-type. The alanine mutation conferred partial resistance to 3TC, but replicated poorly. The amounts of viral DNA synthesized decreased in 3TC-treated cells when the cells were infected with wild-type virus and the M184A mutant. The effect of these mutations on the generation of the ends of the linear viral DNA was determined using the sequence of the 2-LTR circle junctions. The M184T mutation increased the proportion of 2-LTR circle junctions containing a tRNA insertion, suggesting that the mutation affected the RNase H activity of RT

  9. Asymmetric PCR for good quality ssDNA generation towards DNA aptamer production

    Directory of Open Access Journals (Sweden)

    Junji Tominaga4

    2012-04-01

    Full Text Available Aptamers are ssDNA or RNA that binds to wide variety of target molecules with high affinity and specificity producedby systematic evolution of ligands by exponential enrichment (SELEX. Compared to RNA aptamer, DNA aptamer is muchmore stable, favourable to be used in many applications. The most critical step in DNA SELEX experiment is the conversion ofdsDNA to ssDNA. The purpose of this study was to develop an economic and efficient approach of generating ssDNA byusing asymmetric PCR. Our results showed that primer ratio (sense primer:antisense primer of 20:1 and sense primer amountof 10 to 100 pmol, up to 20 PCR cycles using 20 ng of initial template, in combination with polyacrylamide gel electrophoresis,were the optimal conditions for generating good quality and quantity of ssDNA. The generation of ssDNA via this approachcan greatly enhance the success rate of DNA aptamer generation.

  10. Assessment of EGFR mutation status using cell-free DNA from bronchoalveolar lavage fluid.

    Science.gov (United States)

    Park, Sojung; Hur, Jae Young; Lee, Kye Young; Lee, Jae Cheol; Rho, Jin Kyung; Shin, Sun Hwa; Choi, Chang-Min

    2017-08-28

    Much attention has been focused on epidermal growth factor receptor (EGFR) mutation testing since the introduction of EGFR-tyrosine kinase inhibitors have improved survival in EGFR-positive lung cancer patients. Liquid biopsy using circulating tumor cells or cell-free DNA (cfDNA) has enabled less invasive testing, but requires a highly sensitive method. To date, liquid biopsy using bronchoalveolar lavage (BAL) fluid has rarely been used. From 20 patients with lung adenocarcinoma, we isolated cfDNA from 20 samples of cell-free BAL fluid and 19 cell-free bronchial washing samples. cfDNA was examined for EGFR mutations using peptide nucleic acid (PNA)-mediated PCR clamping method. In cases where the results from the tumor biopsy and BAL-derived cfDNA test were not consistent, PANAMutyper™ R EGFR kit was used along with PNA clamping-assisted fluorescence melting curve analysis. We included 17 patients with advanced stage disease and three with non-advanced stage disease. Tumor biopsy detected EGFR mutations in 12 of the patients. One patient had a p.L858R mutation and a de novo p.T790M mutation. The results from PNA-mediated PCR clamping were 75.0% (9/12) concordant with the tumor biopsy results for EGFR mutation status. PANAMutyper with fluorescence melting curve analysis was performed in three cases, which detected EGFR mutations in two more patients (11/12, 91.7%). EGFR mutations were detected in the cfDNA extracted from two bronchial washing samples. cfDNA from BAL fluid could be used for molecular testing of EGFR mutations and identification of p.T790M mutations, with an easily applicable method.

  11. Prevalence of mitochondrial DNA mutations in sporadic patients with nonsyndromic sensorineural hearing loss.

    Science.gov (United States)

    Jiang, Hua; Chen, Jia; Li, Ying; Lin, Peng-Fang; He, Jian-Guo; Yang, Bei-Bei

    2016-01-01

    Several mitochondrial DNA mutations have been reported to be associated with nonsyndromic hearing loss in several families. However, little is known about the prevalence of these mutations in sporadic patients with nonsyndromic sensorineural hearing loss. The purpose of our study was to investigate the incidence of these mitochondrial DNA mutations in such population. A total of 178 sporadic patients with nonsyndromic sensorineural hearing loss were enrolled in this study. Genomic DNA was extracted from the peripheral blood sample. We employed the SNaPshot(®) sequencing method to detect five mitochondrial DNA mutations, including A1555G and A827G in 12S rRNA gene and A7445G, 7472insC, and T7511C in tRNA(Ser(UCN)) gene. Meanwhile, we used polymerase chain reaction and sequenced the products to screen GJB2 gene mutations in patients carrying mitochondrial DNA mutations. We failed to detect the presence of A1555G mutation in 12S rRNA gene, and of A7445G, 7472insC, T7511C mutations in tRNA(Ser(UCN)) gene in our population. However, we found that 6 patients (3.37%) were carriers of a homozygous A827G mutation and one of them also carried homozygous GJB2 235delC mutation. Our findings in the present study indicate that even in sporadic patients with nonsyndromic sensorineural hearing loss, mitochondrial DNA mutations might also contribute to the clinical phenotype. Copyright © 2015 Associação Brasileira de Otorrinolaringologia e Cirurgia Cérvico-Facial. Published by Elsevier Editora Ltda. All rights reserved.

  12. mtDNA mutation C1494T, haplogroup A, and hearing loss in Chinese

    International Nuclear Information System (INIS)

    Wang Chengye; Kong Qingpeng; Yao Yonggang; Zhang Yaping

    2006-01-01

    Mutation C1494T in mitochondrial 12S rRNA gene was recently reported in two large Chinese families with aminoglycoside-induced and nonsyndromic hearing loss (AINHL) and was claimed to be pathogenic. This mutation, however, was first reported in a sample from central China in our previous study that was aimed to reconstruct East Asian mtDNA phylogeny. All these three mtDNAs formed a subclade defined by mutation C1494T in mtDNA haplogroup A. It thus seems that mutation C1494T is a haplogroup A-associated mutation and this matrilineal background may contribute a high risk for the penetrance of mutation C1494T in Chinese with AINHL. To test this hypothesis, we first genotyped mutation C1494T in 553 unrelated individuals from three regional Chinese populations and performed an extensive search for published complete or near-complete mtDNA data sets (>3000 mtDNAs), we then screened the C1494T mutation in 111 mtDNAs with haplogroup A status that were identified from 1823 subjects across China. The search for published mtDNA data sets revealed no other mtDNA besides the above-mentioned three carrying mutation C1494T. None of the 553 randomly selected individuals and the 111 haplogroup A mtDNAs was found to bear this mutation. Therefore, our results suggest that C1494T is a very rare event. The mtDNA haplogroup A background in general is unlikely to play an active role in the penetrance of mutation C1494T in AINHL

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

    International Nuclear Information System (INIS)

    Kukat, Alexandra; Edgar, Daniel; Bratic, Ivana; Maiti, Priyanka; Trifunovic, Aleksandra

    2011-01-01

    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% O 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.

  14. 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.

  15. p53 mutation, allele loss on chromosome 17p, and DNA content in ovarian carcinoma.

    Science.gov (United States)

    McManus, D T; Murphy, M; Arthur, K; Hamilton, P W; Russell, S E; Toner, P G

    1996-06-01

    The aim of this investigation was to explore the relationships between p53 mutation, DNA aneuploidy, 17p deletions, and clinical stage in ovarian cancer. Nuclear suspensions were obtained by tissue disaggregation, stained with propidium iodide, and analysed on a Coulter EPICS Elite flow cytometer. DNA cell cycle analysis was performed using Multicycle software (Phoenix Flow Systems). DNA extracted from paraffin-embedded archival carcinomas/non-tumour tissue was used as template for PCR amplification of the microsatellite dinucleotide repeat polymorphism D17S513, a locus telomeric to p53 on 17p13.1. Allele loss at D17S513 was detected in 64.5 per cent of carcinomas (20 of 31 informative cases). DNA aneuploidy was detected in 20 of 54 (37 per cent) carcinomas. Eight of ten cases previously shown to harbour p53 mutations showed aneuploid DNA content. Although ten other DNA aneuploid cases had shown no p53 mutations, the results show a statistically significant association between p53 mutation and DNA aneuploidy (P p53 mutant cases compared with those showing no p53 mutation (P = 0.02). There was also a significant association between p53 mutations and stage, between ploidy and stage, and between allelic deletions at D17S513 or p53 and stage, but not between these allelic deletions and ploidy. p53 mutations appear to be associated with DNA aneuploidy in ovarian cancer independently of 17p deletions. p53 mutations, DNA aneuploidy, and 17p deletions are associated with late stage.

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

    International Nuclear Information System (INIS)

    Mahmoud, J.; Fossett, N.G.; Arbour-Reily, P.; McDaniel, M.; Tucker, A.; Chang, S.H.; Lee, W.R.

    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

  17. A mutation in the DNA polymerase accessory factor of herpes simplex virus 1 restores viral DNA replication in the presence of raltegravir.

    Science.gov (United States)

    Zhou, Bin; Yang, Kui; Wills, Elizabeth; Tang, Liang; Baines, Joel D

    2014-10-01

    Previous reports showed that raltegravir, a recently approved antiviral compound that targets HIV integrase, can inhibit the nuclease function of human cytomegalovirus (HCMV terminase) in vitro. In this study, subtoxic levels of raltegravir were shown to inhibit the replication of four different herpesviruses, herpes simplex virus 1 (HSV-1), HSV-2, HCMV, and mouse cytomegalovirus, by 30- to 700-fold, depending on the dose and the virus tested. Southern blotting and quantitative PCR revealed that raltegravir inhibits DNA replication of HSV-1 rather than cleavage of viral DNA. A raltegravir-resistant HSV-1 mutant was generated by repeated passage in the presence of 200 μM raltegravir. The genomic sequence of the resistant virus, designated clone 7, contained mutations in 16 open reading frames. Of these, the mutations F198S in unique long region 15 (UL15; encoding the large terminase subunit), A374V in UL32 (required for DNA cleavage and packaging), V296I in UL42 (encoding the DNA polymerase accessory factor), and A224S in UL54 (encoding ICP27, an important transcriptional regulator) were introduced independently into the wild-type HSV-1(F) genome, and the recombinant viruses were tested for raltegravir resistance. Viruses bearing both the UL15 and UL32 mutations inserted within the genome of the UL42 mutant were also tested. While the UL15, UL32, and UL54 mutant viruses were fully susceptible to raltegravir, any virus bearing the UL42 mutation was as resistant to raltegravir as clone 7. Overall, these results suggest that raltegravir may be a valuable therapeutic agent against herpesviruses and the antiviral activity targets the DNA polymerase accessory factor rather than the nuclease activity of the terminase. This paper shows that raltegravir, the antiretrovirus drug targeting integrase, is effective against various herpesviruses. Drug resistance mapped to the herpesvirus DNA polymerase accessory factor, which was an unexpected finding. Copyright © 2014

  18. DNA Damage Is a Potential Marker for TP53 Mutation in Colorectal Carcinogenesis.

    Science.gov (United States)

    Scalise, José Ricardo; Poças, Regina Caeli Guerra; Caneloi, Thamy Pelatieri; Lopes, Camila Oliveira; Kanno, Danilo Toshio; Marques, Mayara Gonçalves; Valdivia, Júlio Cesar Martins; Maximo, Felipe Rodrigues; Pereira, José Aires; Ribeiro, Marcelo Lima; Priolli, Denise Gonçalves

    2016-12-01

    The ability to measure oxidative DNA damage in a tissue allows establishment of the relationship between DNA damage and mutations in normal and neoplastic cells. It is well known that TP53 is a key inhibitor of tumor development and preserves the genome integrity in each cell. The aim of the present study was to investigate the relationship between DNA damage and TP53 mutation in colorectal adenoma and adenocarcinoma, and the value of DNA damage as potential marker of TP53 mutation in non-tumor tissues adjacent to colon malignant lesions. Tissue samples were obtained by colonoscopy from patients with adenoma and/or adenocarcinoma and from healthy volunteers. Diagnosis was defined by histopathology. Immunohistochemistry with computer-assisted image analysis was performed to quantify TP53 mutation. Oxidative DNA damage was determined by comet assay. Statistical analyses were performed with 5 % of significance level. The TP53 level was higher in non-tumor tissues from tumor patients than in normal tissues from healthy volunteers (p = 0.01). Likewise, higher TP53 levels were observed in tumor tissues compared with the non-tumor tissues (p = 0.00). Oxidative DNA damage levels were higher in tumor tissues than in non-tumor tissues (p = 0.00). The amount of TP53 (p = 0.00) and oxidative DNA damage (p = 0.00) in normal and tumor tissue was related. The relationship between oxidative DNA damage and TP53 mutation was demonstrated in all samples (p = 0.00). Oxidative DNA damage is an intervening variable for TP53 mutation in colorectal adenoma-carcinoma. Our data suggests that oxidative DNA damage is a potential marker of TP53 mutation in colorectal carcinogenesis.

  19. Cell-free DNA as biomarker and source for mutation detection in primary colorectal cancer.

    Science.gov (United States)

    Nikolic, Aleksandra; Vlajnic, Marina; Ristanovic, Momcilo; Petrovic, Jelena; Dimitrijevic, Ivan; Krivokapic, Zoran; Radojkovic, Dragica

    2017-01-01

    To analyze if cell-free (cf)DNA levels and the presence of KRAS and BRAF mutations in serum could be used as diagnostic biomarkers in patients with primary colorectal cancer (CRC). This study included 92 individuals who were operated due to primary CRC (N=52;study group) and to hemorrhoids (N=40;control group). Serum cfDNA levels were measured with real-time PCR (RT-PCR) using PicoGreen dsDNA quantitation reagent. Colorectal tissue and related blood and serum samples taken at the time of surgery were subjected to DNA extraction and analysis of KRAS and BRAF mutations based on multiplex SNaPshot assay and DNA sequencing. The average cfDNA concentration was lower in patients of the study group (20±7 ng/μL) in comparison to controls (34±9 ng/μL) and this difference was statistically significant (pmutations in colorectal tumor tissue in 14 cases, but the presence of the mutation was not confirmed in cfDNA extracted from blood samples of these patients. The level of serum cfDNA in CRC is decreased in comparison to patients with hemorrhoids, which questions the usefulness of cfDNA as cancer biomarker. Also, cfDNA does not appear to be suitable as a source for mutation detection in this disease.

  20. Age-associated alterations in the somatic mutation and DNA methylation levels in plants.

    Science.gov (United States)

    Dubrovina, A S; Kiselev, K V

    2016-03-01

    Somatic mutations of the nuclear and mitochondrial DNA and alterations in DNA methylation levels in mammals are well known to play important roles in ageing and various diseases, yet their specific contributions await further investigation. For plants, it has also been proposed that unrepaired DNA damage and DNA polymerase errors accumulate in plant cells and lead to increased somatic mutation rate and alterations in transcription, which eventually contribute to plant ageing. A number of studies also show that DNA methylation levels vary depending on the age of plant tissue and chronological age of a whole plant. Recent studies reveal that prolonged cultivation of plant cells in vitro induces single nucleotide substitutions and increases global DNA methylation level in a time-dependent fashion. Changes in DNA methylation are known to influence DNA repair and can lead to altered mutation rates, and, therefore, it is interesting to investigate both the genetic and epigenetic integrity in relationship to ageing in plants. This review will summarise and discuss the current studies investigating somatic DNA mutation and DNA methylation levels in relation to plant ageing and senescence. The analysis has shown that there still remains a lack of clarity concerning plant biological ageing and the role of the genetic and epigenetic instabilities in this process. © 2015 German Botanical Society and The Royal Botanical Society of the Netherlands.

  1. Role of Mitochondrial DNA Mutations in Cellular Vulnerability to Mitochondria-Specific Environmental Toxins

    National Research Council Canada - National Science Library

    Hirsch, Etienne C

    2005-01-01

    .... To test such a hypothesis in Parkinson's disease we proposed to: 1) develop an animal model with accumulated mtDNA mutations in catecholaminergic neurons by creating a transgenic mouse containing a tyrosine hydroxylase (TH...

  2. Simultaneous Profiling of DNA Mutation and Methylation by Melting Analysis Using Magnetoresistive Biosensor Array

    DEFF Research Database (Denmark)

    Rizzi, Giovanni; Lee, Jung-Rok; Dahl, Christina

    2017-01-01

    specificity. Genomic (mutation) or bisulphite-treated (methylation) DNA is amplified using nondiscriminatory primers, and the amplicons are then hybridized to a giant magnetoresistive (GMR) biosensor array followed by melting curve measurements. The GMR biosensor platform offers scalable multiplexed detection...

  3. Nucleotide selectivity defect and mutator phenotype conferred by a colon cancer-associated DNA polymerase δ mutation in human cells.

    Science.gov (United States)

    Mertz, T M; Baranovskiy, A G; Wang, J; Tahirov, T H; Shcherbakova, P V

    2017-08-01

    Mutations in the POLD1 and POLE genes encoding DNA polymerases δ (Polδ) and ɛ (Polɛ) cause hereditary colorectal cancer (CRC) and have been found in many sporadic colorectal and endometrial tumors. Much attention has been focused on POLE exonuclease domain mutations, which occur frequently in hypermutated DNA mismatch repair (MMR)-proficient tumors and appear to be responsible for the bulk of genomic instability in these tumors. In contrast, somatic POLD1 mutations are seen less frequently and typically occur in MMR-deficient tumors. Their functional significance is often unclear. Here we demonstrate that expression of the cancer-associated POLD1-R689W allele is strongly mutagenic in human cells. The mutation rate increased synergistically when the POLD1-R689W expression was combined with a MMR defect, indicating that the mutator effect of POLD1-R689W results from a high rate of replication errors. Purified human Polδ-R689W has normal exonuclease activity, but the nucleotide selectivity of the enzyme is severely impaired, providing a mechanistic explanation for the increased mutation rate in the POLD1-R689W-expressing cells. The vast majority of mutations induced by the POLD1-R689W are GC→︀TA transversions and GC→︀AT transitions, with transversions showing a strong strand bias and a remarkable preference for polypurine/polypyrimidine sequences. The mutational specificity of the Polδ variant matches that of the hypermutated CRC cell line, HCT15, in which this variant was first identified. The results provide compelling evidence for the pathogenic role of the POLD1-R689W mutation in the development of the human tumor and emphasize the need to experimentally determine the significance of Polδ variants present in sporadic tumors.

  4. Non-DNA binding, dominant-negative, human PPARγ mutations cause lipodystrophic insulin resistance

    OpenAIRE

    Agostini, Maura; Schoenmakers, Erik; Mitchell, Catherine; Szatmari, Istvan; Savage, David; Smith, Aaron; Rajanayagam, Odelia; Semple, Robert; Luan, Jian'an; Bath, Louise; Zalin, Anthony; Labib, Mourad; Kumar, Sudhesh; Simpson, Helen; Blom, Dirk

    2006-01-01

    PPARgamma is essential for adipogenesis and metabolic homeostasis. We describe mutations in the DNA and ligand binding domains of human PPARgamma in lipodystrophic, severe insulin resistance. These receptor mutants lack DNA binding and transcriptional activity but can translocate to the nucleus, interact with PPARgamma coactivators and inhibit coexpressed wild-type receptor. Expression of PPARgamma target genes is markedly attenuated in mutation-containing versus receptor haploinsufficent pri...

  5. How DNA is damaged by external electric fields: selective mutation vs. random degradation.

    Science.gov (United States)

    Cerón-Carrasco, José Pedro; Cerezo, Javier; Jacquemin, Denis

    2014-05-14

    DNA is constantly exposed to exogenous agents that randomly damage the genetic code. However, external perturbations might also be used to induce malignant cell death if the mutation processes are controlled. The present communication reports a set of parameters allowing DNA mutation through the use of intense external electric fields. This is a step towards the design of pulsed electric field therapy for genetic diseases.

  6. DNA methylation epigenotype and clinical features of NRAS-mutation(+) colorectal cancer.

    Science.gov (United States)

    Takane, Kiyoko; Akagi, Kiwamu; Fukuyo, Masaki; Yagi, Koichi; Takayama, Tadatoshi; Kaneda, Atsushi

    2017-05-01

    Sporadic colorectal cancer (CRC) is classified into several molecular subtypes. We previously established two groups of DNA methylation markers through genome-wide DNA methylation analysis to classify CRC into distinct subgroups: high-, intermediate-, and low-methylation epigenotypes (HME, IME, and LME, respectively). HME CRC, also called CpG island methylator phenotype (CIMP)-high CRC, shows methylation of both Group 1 markers (CIMP markers) and Group 2 markers, while IME/CIMP-low CRC shows methylation of Group 2, but not of Group 1 markers, and LME CRC shows no methylation of either Group 1 or Group 2 markers. While BRAF- and KRAS-mutation(+) CRC strongly correlated with HME and IME, respectively, clinicopathological features of NRAS-mutation(+) CRC, including association with DNA methylation, remain unclear. To characterize NRAS-mutation(+) CRC, the methylation levels of 19 methylation marker genes (6 Group 1 and 13 Group 2) were analyzed in 61 NRAS-mutation(+) and 144 NRAS-mutation(-) CRC cases by pyrosequencing, and their correlation with clinicopathological features was investigated. Different from KRAS-mutation(+) CRC, NRAS-mutation(+) CRC significantly correlated with LME. NRAS-mutation(+) CRC showed significantly better prognosis than KRAS-mutation(+) CRC (P = 3 × 10 -4 ). NRAS-mutation(+) CRC preferentially occurred in elder patients (P = 0.02) and at the distal colon (P = 0.006), showed significantly less lymph vessel invasion (P = 0.002), and correlated with LME (P = 8 × 10 -5 ). DNA methylation significantly accumulated at the proximal colon. NRAS-mutation(+) CRC may constitute a different subgroup from KRAS-mutation(+) CRC, showing significant correlation with LME, older age, distal colon, and relatively better prognosis. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  7. Large Variation in the Ratio of Mitochondrial to Nuclear Mutation Rate across Animals: Implications for Genetic Diversity and the Use of Mitochondrial DNA as a Molecular Marker.

    Science.gov (United States)

    Allio, Remi; Donega, Stefano; Galtier, Nicolas; Nabholz, Benoit

    2017-11-01

    It is commonly assumed that mitochondrial DNA (mtDNA) evolves at a faster rate than nuclear DNA (nuDNA) in animals. This has contributed to the popularity of mtDNA as a molecular marker in evolutionary studies. Analyzing 121 multilocus data sets and four phylogenomic data sets encompassing 4,676 species of animals, we demonstrate that the ratio of mitochondrial over nuclear mutation rate is highly variable among animal taxa. In nonvertebrates, such as insects and arachnids, the ratio of mtDNA over nuDNA mutation rate varies between 2 and 6, whereas it is above 20, on average, in vertebrates such as scaled reptiles and birds. Interestingly, this variation is sufficient to explain the previous report of a similar level of mitochondrial polymorphism, on average, between vertebrates and nonvertebrates, which was originally interpreted as reflecting the effect of pervasive positive selection. Our analysis rather indicates that the among-phyla homogeneity in within-species mtDNA diversity is due to a negative correlation between mtDNA per-generation mutation rate and effective population size, irrespective of the action of natural selection. Finally, we explore the variation in the absolute per-year mutation rate of both mtDNA and nuDNA using a reduced data set for which fossil calibration is available, and discuss the potential determinants of mutation rate variation across genomes and taxa. This study has important implications regarding DNA-based identification methods in predicting that mtDNA barcoding should be less reliable in nonvertebrates than in vertebrates. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  8. Efficiency of EGFR mutation analysis for small microdissected cytological specimens using multitech DNA extraction solution.

    Science.gov (United States)

    Oh, Seo Young; Lee, Hoon Taek

    2015-07-01

    The microdissection method has greatly facilitated the isolation of pure cell populations for accurate analysis of mutations. However, the absence of coverslips in these preparations leads to poor resolution of cellular morphological features. In the current study, the authors developed the MultiTech DNA extraction solution to improve the visualization of cell morphology for microdissection and tested it for the preservation of morphological properties of cells, quality of DNA, and ability to detect mutations. A total of 121 cytological samples, including fine-needle aspirates, sputum, pleural fluid, and bronchial washings, were selected from hospital archives. DNA extracted from microdissected cells was evaluated by epidermal growth factor receptor (EGFR) mutation analysis using pyrosequencing, Sanger sequencing, and peptide nucleic acid (PNA)-mediated real-time polymerase chain reaction clamping. Morphological features of cells as well as DNA quality and quantity were analyzed in several cytological samples to assess the performance of the MultiTech DNA extraction solution. The results were compared with previous EGFR mutation tests. The MultiTech DNA extraction solution improved the morphology of archived stained cells before microdissection and provided a higher DNA yield than the commercial QIAamp DNA Mini Kit in samples containing a minimal number of cells (25-50 cells). The authors were able to detect identical EGFR mutations by using different analysis platforms and consistently identified these mutations in samples comprising as few as 25 microdissected cells. The MultiTech DNA extraction solution is a reliable medium that improves the resolution of cell morphology during microdissection. It was particularly useful in EGFR mutations of samples containing a small number of cells. © 2015 American Cancer Society.

  9. 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

    suppressor mutations function by decreasing the efficiency of replication fork movement in vivo, either by decreasing the dynamic exchange of DNA polymerase subunits in the case of HolC, or by altering the balance between DNA replication and deoxynucleoside triphosphate synthesis in the case of ndk......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...

  10. HIV drug resistance mutations in proviral DNA from a community treatment program.

    Directory of Open Access Journals (Sweden)

    Anne Derache

    Full Text Available Drug resistance mutations archived in resting memory CD4+ cells may persist despite suppression of HIV RNA to <50 copies/ml. We sequenced pol gene from proviral DNA among viremic and suppressed patients to identify drug resistance mutations.The Peninsula AIDS Research Cohort study enrolled and followed over 2 years 120 HIV infected patients from San Mateo and San Francisco Counties. HIV-1 pol genotyping by bulk sequencing was performed on 38 DNA and RNA from viremic patients and DNA only among 82 suppressed patients at baseline. Antiretroviral susceptibility was predicted by HIVDB.stanford.edu.Among 120 subjects, 81% were on antiretroviral therapy and had been treated for a median time of 7 years. Thirty-two viremic patients showed concordant RNA and DNA genotypes (84%; the discordant profiles were mainly observed in patients with low-level viremia. Among suppressed patients, 21 had drug resistance mutations in proviral DNA (26% with potential resistance to one, two or three ARV classes in 16, 4 and 1 samples respectively.The high level of genotype concordance between DNA and RNA in viremic patients suggested that DNA genotyping might be used to assess drug resistance in resource-limited settings, and further investigation of extracted DNA from dried blood spots is needed. Drug resistance mutations in proviral DNA in 26% of subjects with less than 50 copies/ml pose a risk for the transmission of drug resistant virus with virologic failure, treatment interruption or decreased adherence.

  11. Circulating free DNA as biomarker and source for mutation detection in metastatic colorectal cancer.

    Science.gov (United States)

    Spindler, Karen Lise Garm; Pallisgaard, Niels; Andersen, Rikke Fredslund; Brandslund, Ivan; Jakobsen, Anders

    2015-01-01

    Circulating cell-free DNA (cfDNA) in plasma has shown potential as biomarker in various cancers and could become an importance source for tumour mutation detection. The objectives of our study were to establish a normal range of cfDNA in a cohort of healthy individuals and to compare this with four cohorts of metastatic colorectal cancer (mCRC) patients. We also investigated the prognostic value of cfDNA and analysed the tumour-specific KRAS mutations in the plasma. The study was a prospective biomarker evaluation in four consecutive Phase II trials, including 229 patients with chemotherapy refractory mCRC and 100 healthy individuals. Plasma was obtained from an EDTA blood-sample, and the total number of DNA alleles and KRAS mutated alleles were assessed using an in-house ARMS-qPCR as previously described. Median cfDNA levels were higher in mCRC compared to controls (p mutations in plasma and tissue was high (85%). These data confirm the prognostic value of cfDNA measurement in plasma and utility for mutation detection with the method presented.

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

    Indian Academy of Sciences (India)

    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 ...

  13. Effects of TET2 mutations on DNA methylation in chronic myelomonocytic leukemia

    Science.gov (United States)

    TET2 enzymatically converts 5-methyl-cytosine to 5-hydroxymethyl-cytosine, possibly leading to loss of DNA methylation. TET2 mutations are common in myeloid leukemia and were proposed to contribute to leukemogenesis through DNA methylation. To expand on this concept, we studied chronic myelomonocyti...

  14. Mutation of Haemophilus influenzae transforming DNA in vitro with near-ultraviolet radiation: action spectrum

    International Nuclear Information System (INIS)

    Cabrera-Juarez, E.; Setlow, J.K.; Oak Ridge National Lab., Tenn.

    1976-01-01

    Mutations were produced in purified transforming DNA from Haemophilus influenzae by near UV radiation and were assayed as mutants among cells transformed with irradiated DNA. The maximum efficiency of mutation induction was at around 334 nm, and the efficiency dropped off steeply at lower and higher wavelengths. The difference between the action spectrum for mutation and that for the oxygen-independent inactivation of transforming DNA, which had a shoulder at 365 nm, indicates that there are different lesions involved in the inactivating and mutagenic effects of near-UV. The presence of histidine during irradiation enhanced the mutagenic effect at 334 and 365 nm, although it protected against inactivation at 365 nm. The effective near-UV wavelengths for in vitro mutation are to some extent the same as the effective wavelengths for mutation in vivo reported previously. These findings indicate that mutations are produced in vivo by near-UV with DNA as the primary target molecule rather than by a secondary non-photochemical reaction between DNA and some other cell component

  15. 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.

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

    Science.gov (United States)

    Guo, S; Esserlind, A-L; Andersson, Z; Frederiksen, A L; Olesen, J; Vissing, J; Ashina, M

    2016-01-01

    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. In this cross-sectional study, 57 mDNA 3243A>G mutation carriers between May 2012 and October 2014 were included. As a control group, a population-based cohort from our epidemiological studies on migraine in Danes was used. History of headache and migraine was obtained by telephone interview, based on a validated semi-structured questionnaire, performed by trained physicians. The prevalence of migraine is significantly higher in persons carrying the 3243A>G mutation than in controls (58% vs. 18%; P G mutation was found. This finding suggests a clinical association between a monogenetically inherited disorder of mitochondrial dysfunction and susceptibility to migraine. Mitochondrial DNA aberrations may contribute to the pathogenesis of migraine. © 2015 EAN.

  17. 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......) and finally, detection by fluorescence microscopy. The LNA containing probes display high binding affinity and specificity to DNA containing mutations, which allows for the detection of mutation abundance with an intercalating EvaGreen dye. We used a second probe, which increases the overall number of base...... pairs in order to produce a higher fluorescence signal by incorporating more dye molecules. Indeed we show here that using EvaGreen dye and LNA probes, genomic DNA containing BRAF V600E mutation could be detected by fluorescence microscopy at low femtomolar concentrations. Notably, this was at least...

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

    International Nuclear Information System (INIS)

    Li Yufeng; Huang Qunce; Yu Zengliang; Liang Yunzhang

    2003-01-01

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

  19. A single quantum dot-based biosensor for DNA point mutation assay.

    Science.gov (United States)

    Tang, Wei; Zhu, Guichi; Liang, Li; Zhang, Chun-Yang

    2015-09-07

    Sensitive and selective detection of point mutation is essential to molecular biology research and early clinical diagnosis. Here, we demonstrate a single quantum dot (QD)-based biosensor for DNA point mutation assay. In this assay, a mutant target (G/C) remains unchanged after the endonuclease treatment, and the polymerase chain reaction (PCR) may be initiated with the assistance of primers and polymerase, generating a large number of mutant targets. The amplified mutant targets can be captured by biotinylated probes during the process of denaturation and annealing, and Cy5-dGTP may be assembled into the biotinylated probe with the catalysis of polymerase, leading to the formation of Cy5-labeled biotinylated probes. The Cy5-labeled biotinylated probes can be further assembled onto the QD surface to obtain a Cy5-DNA-QD complex, resulting in the generation of fluorescence resonance energy transfer (FRET) between the QD donor and the Cy5 receptor. The mutant targets can be quantitatively evaluated by the measurement of Cy5 counts by total internal reflection fluorescence (TIRF) microscopy. While in the presence of wild-type targets (T/A), no Cy5-dGTP can be assembled into the biotinylated probe due to the presence of a mismatch and consequently no FRET is observed. This single QD-based biosensor exhibits high sensitivity with a detection limit of 5.3 aM (or 32 copies) and can even discriminate as low as 0.01% variant frequency from the mixture of mutant targets and wild-type ones. Importantly, this biosensor can be used for genomic analysis in human lung cancer cells, and may be further applied for an early clinical diagnosis and personalized medicine.

  20. Non-DNA binding, dominant-negative, human PPARγ mutations cause lipodystrophic insulin resistance

    Science.gov (United States)

    Agostini, Maura; Schoenmakers, Erik; Mitchell, Catherine; Szatmari, Istvan; Savage, David; Smith, Aaron; Rajanayagam, Odelia; Semple, Robert; Luan, Jian'an; Bath, Louise; Zalin, Anthony; Labib, Mourad; Kumar, Sudhesh; Simpson, Helen; Blom, Dirk; Marais, David; Schwabe, John; Barroso, Inês; Trembath, Richard; Wareham, Nicholas; Nagy, Laszlo; Gurnell, Mark; O'Rahilly, Stephen; Chatterjee, Krishna

    2006-01-01

    Summary PPARγ is essential for adipogenesis and metabolic homeostasis. We describe mutations in the DNA and ligand binding domains of human PPARγ in lipodystrophic, severe insulin resistance. These receptor mutants lack DNA binding and transcriptional activity but can translocate to the nucleus, interact with PPARγ coactivators and inhibit coexpressed wild-type receptor. Expression of PPARγ target genes is markedly attenuated in mutation-containing versus receptor haploinsufficent primary cells, indicating that such dominant-negative inhibition operates in vivo. Our observations suggest that these mutants restrict wild-type PPARγ action via a non-DNA binding, transcriptional interference mechanism, which may involve sequestration of functionally limiting coactivators. PMID:17011503

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

    Science.gov (United States)

    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

    2015-11-24

    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.

  2. Circulating free DNA as biomarker and source for mutation detection in metastatic colorectal cancer

    DEFF Research Database (Denmark)

    Spindler, Karen Lise Garm; Pallisgaard, Niels; Andersen, Rikke Fredslund

    2015-01-01

    this with four cohorts of metastatic colorectal cancer (mCRC) patients. We also investigated the prognostic value of cfDNA and analysed the tumour-specific KRAS mutations in the plasma. METHODS: The study was a prospective biomarker evaluation in four consecutive Phase II trials, including 229 patients.......6-5.9) months, respectively, HR 1.78, p = 0.0006). Multivariate analysis confirmed an independent prognostic value of cfDNA (HR 1.5 (95% CI 1.3-1.7) for each increase in the cfDNA quartile). The overall concordance of KRAS mutations in plasma and tissue was high (85%). CONCLUSIONS: These data confirm...... the prognostic value of cfDNA measurement in plasma and utility for mutation detection with the method presented....

  3. 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.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    James L Sherwood

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

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

    Science.gov (United States)

    Rangel-López, Angélica; Méndez-Tenorio, Alfonso; Beattie, Kenneth L; Maldonado, Rogelio; Mendoza, Patricia; Vázquez, Guelaguetza; Pérez-Plasencia, Carlos; Sánchez, Martha; Navarro, Guillermo; Salcedo, Mauricio

    2009-01-01

    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 TP53 gene, the results suggest that this microarray system proved to be a rapid, reliable, and effective method for screening all the mutations in TP53 gene. PMID:24198462

  7. 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.

  8. 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.

  9. Biochemical evidence for a mitochondrial genetic modifier in the phenotypic manifestation of Leber's hereditary optic neuropathy-associated mitochondrial DNA mutation.

    Science.gov (United States)

    Jiang, Pingping; Liang, Min; Zhang, Chaofan; Zhao, Xiaoxu; He, Qiufen; Cui, Limei; Liu, Xiaoling; Sun, Yan-Hong; Fu, Qun; Ji, Yanchun; Bai, Yidong; Huang, Taosheng; Guan, Min-Xin

    2016-08-15

    Leber's hereditary optic neuropathy (LHON) is the most common mitochondrial disease. Mitochondrial modifiers are proposed to modify the phenotypic expression of primary LHON-associated mitochondrial DNA (mtDNA) mutations. In this study, we demonstrated that the LHON susceptibility allele (m.14502T > C, p. 58I > V) in the ND6 gene modulated the phenotypic expression of primary LHON-associated m.11778G > A mutation. Twenty-two Han Chinese pedigrees carrying m.14502T > C and m.11778G > A mutations exhibited significantly higher penetrance of optic neuropathy than those carrying only m.11778G > A mutation. We performed functional assays using the cybrid cell models, generated by fusing mtDNA-less ρ o cells with enucleated cells from LHON patients carrying both m.11778G > A and m.14502T > C mutations, only m.14502T > C or m.11778G > A mutation and a control belonging to the same mtDNA haplogroup. These cybrids cell lines bearing m.14502T > C mutation exhibited mild effects on mitochondrial functions compared with those carrying only m.11778G > A mutation. However, more severe mitochondrial dysfunctions were observed in cell lines bearing both m.14502T > C and m.11778G > A mutations than those carrying only m.11778G > A or m.14502T > C mutation. In particular, the m.14502T > C mutation altered assemble of complex I, thereby aggravating the respiratory phenotypes associated with m.11778G > A mutation, resulted in a more defective complex I. Furthermore, more reductions in the levels of mitochondrial ATP and increasing production of reactive oxygen species were also observed in mutant cells bearing both m.14502T > C and m.11778G > A mutation than those carrying only 11778G > A mutation. Our findings provided new insights into the pathophysiology of LHON that were manifested by interaction between primary and secondary mtDNA mutations. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  10. Next generation sequencing of DNA-launched Chikungunya vaccine virus

    Energy Technology Data Exchange (ETDEWEB)

    Hidajat, Rachmat; Nickols, Brian [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States); Forrester, Naomi [Institute for Human Infections and Immunity, Sealy Center for Vaccine Development and Department of Pathology, University of Texas Medical Branch, GNL, 301 University Blvd., Galveston, TX 77555 (United States); Tretyakova, Irina [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States); Weaver, Scott [Institute for Human Infections and Immunity, Sealy Center for Vaccine Development and Department of Pathology, University of Texas Medical Branch, GNL, 301 University Blvd., Galveston, TX 77555 (United States); Pushko, Peter, E-mail: ppushko@medigen-usa.com [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States)

    2016-03-15

    Chikungunya virus (CHIKV) represents a pandemic threat with no approved vaccine available. Recently, we described a novel vaccination strategy based on iDNA® infectious clone designed to launch a live-attenuated CHIKV vaccine from plasmid DNA in vitro or in vivo. As a proof of concept, we prepared iDNA plasmid pCHIKV-7 encoding the full-length cDNA of the 181/25 vaccine. The DNA-launched CHIKV-7 virus was prepared and compared to the 181/25 virus. Illumina HiSeq2000 sequencing revealed that with the exception of the 3′ untranslated region, CHIKV-7 viral RNA consistently showed a lower frequency of single-nucleotide polymorphisms than the 181/25 RNA including at the E2-12 and E2-82 residues previously identified as attenuating mutations. In the CHIKV-7, frequencies of reversions at E2-12 and E2-82 were 0.064% and 0.086%, while in the 181/25, frequencies were 0.179% and 0.133%, respectively. We conclude that the DNA-launched virus has a reduced probability of reversion mutations, thereby enhancing vaccine safety. - Highlights: • Chikungunya virus (CHIKV) is an emerging pandemic threat. • In vivo DNA-launched attenuated CHIKV is a novel vaccine technology. • DNA-launched virus was sequenced using HiSeq2000 and compared to the 181/25 virus. • DNA-launched virus has lower frequency of SNPs at E2-12 and E2-82 attenuation loci.

  11. Next generation sequencing of DNA-launched Chikungunya vaccine virus

    International Nuclear Information System (INIS)

    Hidajat, Rachmat; Nickols, Brian; Forrester, Naomi; Tretyakova, Irina; Weaver, Scott; Pushko, Peter

    2016-01-01

    Chikungunya virus (CHIKV) represents a pandemic threat with no approved vaccine available. Recently, we described a novel vaccination strategy based on iDNA® infectious clone designed to launch a live-attenuated CHIKV vaccine from plasmid DNA in vitro or in vivo. As a proof of concept, we prepared iDNA plasmid pCHIKV-7 encoding the full-length cDNA of the 181/25 vaccine. The DNA-launched CHIKV-7 virus was prepared and compared to the 181/25 virus. Illumina HiSeq2000 sequencing revealed that with the exception of the 3′ untranslated region, CHIKV-7 viral RNA consistently showed a lower frequency of single-nucleotide polymorphisms than the 181/25 RNA including at the E2-12 and E2-82 residues previously identified as attenuating mutations. In the CHIKV-7, frequencies of reversions at E2-12 and E2-82 were 0.064% and 0.086%, while in the 181/25, frequencies were 0.179% and 0.133%, respectively. We conclude that the DNA-launched virus has a reduced probability of reversion mutations, thereby enhancing vaccine safety. - Highlights: • Chikungunya virus (CHIKV) is an emerging pandemic threat. • In vivo DNA-launched attenuated CHIKV is a novel vaccine technology. • DNA-launched virus was sequenced using HiSeq2000 and compared to the 181/25 virus. • DNA-launched virus has lower frequency of SNPs at E2-12 and E2-82 attenuation loci.

  12. HIV-1 Tropism Determines Different Mutation Profiles in Proviral DNA.

    Science.gov (United States)

    Nascimento-Brito, Sieberth; Paulo Zukurov, Jean; Maricato, Juliana T; Volpini, Angela C; Salim, Anna Christina M; Araújo, Flávio M G; Coimbra, Roney S; Oliveira, Guilherme C; Antoneli, Fernando; Janini, Luiz Mário R

    2015-01-01

    In order to establish new infections HIV-1 particles need to attach to receptors expressed on the cellular surface. HIV-1 particles interact with a cell membrane receptor known as CD4 and subsequently with another cell membrane molecule known as a co-receptor. Two major different co-receptors have been identified: C-C chemokine Receptor type 5 (CCR5) and C-X-C chemokine Receptor type 4 (CXCR4) Previous reports have demonstrated cellular modifications upon HIV-1 binding to its co-receptors including gene expression modulations. Here we investigated the effect of viral binding to either CCR5 or CXCR4 co-receptors on viral diversity after a single round of reverse transcription. CCR5 and CXCR4 pseudotyped viruses were used to infect non-stimulated and stimulated PBMCs and purified CD4 positive cells. We adopted the SOLiD methodology to sequence virtually the entire proviral DNA from all experimental infections. Infections with CCR5 and CXCR4 pseudotyped virus resulted in different patterns of genetic diversification. CCR5 virus infections produced extensive proviral diversity while in CXCR4 infections a more localized substitution process was observed. In addition, we present pioneering results of a recently developed method for the analysis of SOLiD generated sequencing data applicable to the study of viral quasi-species. Our findings demonstrate the feasibility of viral quasi-species evaluation by NGS methodologies. We presented for the first time strong evidence for a host cell driving mechanism acting on the HIV-1 genetic variability under the control of co-receptor stimulation. Additional investigations are needed to further clarify this question, which is relevant to viral diversification process and consequent disease progression.

  13. HIV-1 Tropism Determines Different Mutation Profiles in Proviral DNA.

    Directory of Open Access Journals (Sweden)

    Sieberth Nascimento-Brito

    Full Text Available In order to establish new infections HIV-1 particles need to attach to receptors expressed on the cellular surface. HIV-1 particles interact with a cell membrane receptor known as CD4 and subsequently with another cell membrane molecule known as a co-receptor. Two major different co-receptors have been identified: C-C chemokine Receptor type 5 (CCR5 and C-X-C chemokine Receptor type 4 (CXCR4 Previous reports have demonstrated cellular modifications upon HIV-1 binding to its co-receptors including gene expression modulations. Here we investigated the effect of viral binding to either CCR5 or CXCR4 co-receptors on viral diversity after a single round of reverse transcription. CCR5 and CXCR4 pseudotyped viruses were used to infect non-stimulated and stimulated PBMCs and purified CD4 positive cells. We adopted the SOLiD methodology to sequence virtually the entire proviral DNA from all experimental infections. Infections with CCR5 and CXCR4 pseudotyped virus resulted in different patterns of genetic diversification. CCR5 virus infections produced extensive proviral diversity while in CXCR4 infections a more localized substitution process was observed. In addition, we present pioneering results of a recently developed method for the analysis of SOLiD generated sequencing data applicable to the study of viral quasi-species. Our findings demonstrate the feasibility of viral quasi-species evaluation by NGS methodologies. We presented for the first time strong evidence for a host cell driving mechanism acting on the HIV-1 genetic variability under the control of co-receptor stimulation. Additional investigations are needed to further clarify this question, which is relevant to viral diversification process and consequent disease progression.

  14. DNA-based mutation assay GPMA (genome profiling-based mutation assay): reproducibility, parts-per-billion scale sensitivity, and introduction of a mammalian-cell-based approach.

    Science.gov (United States)

    Kumari, Parmila; Gautam, Sunita Ghimire; Baba, Misato; Tsukiashi, Motoki; Matsuoka, Koji; Yasukawa, Kiyoshi; Nishigaki, Koichi

    2017-12-01

    Genome profiling-based mutation assay (GPMA) is, to date, the only DNA sequence-based mutation assay that directly measures DNA alterations induced by mutagens. Here, the all-important congruence of mutagen assignment between DNA-based GPMA and the phenotype-based Ames test (the gold standard of mutagen assays) was confirmed qualitatively and semi-quantitatively by means of 94 chemical species (including previously examined 64). The high sensitivity (on the order of 10 ppb) and reproducibility of GPMA were also corroborated by the match between virtually independent experiments conducted in the distant past (10 years ago) and recently. Meanwhile, a standard experimental framework was established: the conditions of 100 parts per billion (ppb) concentration of a chemical and 15-generation culture of Escherichia coli. Moreover, a mammalian cell line (NIH 3T3) was shown to be suitable as a tester organism for the GPMA approach. Preliminary experimental results suggested that this approach can provide a qualitatively equivalent and quantitatively different mutagen assay results relative to the bacteria-based GPMA (renamed as bGPMA). This finding confirmed the effectiveness of the GPMA approach and indicates that mGPMA is a promising way to detect mammalian-cell mutagens. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  15. A Practical Workshop for Generating Simple DNA Fingerprints of Plants

    Science.gov (United States)

    Rouziere, A.-S.; Redman, J. E.

    2011-01-01

    Gel electrophoresis DNA fingerprints offer a graphical and visually appealing illumination of the similarities and differences between DNA sequences of different species and individuals. A polymerase chain reaction (PCR) and restriction digest protocol was designed to give high-school students the opportunity to generate simple fingerprints of…

  16. 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.

  17. DNA based random key generation and management for OTP encryption.

    Science.gov (United States)

    Zhang, Yunpeng; Liu, Xin; Sun, Manhui

    2017-09-01

    One-time pad (OTP) is a principle of key generation applied to the stream ciphering method which offers total privacy. The OTP encryption scheme has proved to be unbreakable in theory, but difficult to realize in practical applications. Because OTP encryption specially requires the absolute randomness of the key, its development has suffered from dense constraints. DNA cryptography is a new and promising technology in the field of information security. DNA chromosomes storing capabilities can be used as one-time pad structures with pseudo-random number generation and indexing in order to encrypt the plaintext messages. In this paper, we present a feasible solution to the OTP symmetric key generation and transmission problem with DNA at the molecular level. Through recombinant DNA technology, by using only sender-receiver known restriction enzymes to combine the secure key represented by DNA sequence and the T vector, we generate the DNA bio-hiding secure key and then place the recombinant plasmid in implanted bacteria for secure key transmission. The designed bio experiments and simulation results show that the security of the transmission of the key is further improved and the environmental requirements of key transmission are reduced. Analysis has demonstrated that the proposed DNA-based random key generation and management solutions are marked by high security and usability. Published by Elsevier B.V.

  18. Analysis of common mitochondrial DNA mutations by allele-specific oligonucleotide and Southern blot hybridization.

    Science.gov (United States)

    Tang, Sha; Halberg, Michelle C; Floyd, Kristen C; Wang, Jing

    2012-01-01

    Mitochondrial disorders are clinically and genetically heterogeneous. There are a set of recurrent point mutations in the mitochondrial DNA (mtDNA) that are responsible for common mitochondrial diseases, including MELAS (mitochondrial encephalopathy, lactic acidosis, stroke-like episodes), MERRF (myoclonic epilepsy and ragged red fibers), LHON (Leber's hereditary optic neuropathy), NARP (neuropathy, ataxia, retinitis pigmentosa), and Leigh syndrome. Most of the pathogenic mtDNA point mutations are present in the heteroplasmic state, meaning that the wild-type and mutant-containing mtDNA molecules are coexisting. Clinical heterogeneity may be due to the degree of mutant load (heteroplasmy) and distribution of heteroplasmic mutations in affected tissues. Additionally, Kearns-Sayre syndrome and Pearson syndrome are caused by large mtDNA deletions. In this chapter, we describe a multiplex PCR/allele-specific oligonucleotide (ASO) hybridization method for the screening of 13 common point mutations. This method allows the detection of low percentage of mutant heteroplasmy. In addition, a nonradioactive Southern blot hybridization protocol for the analysis of mtDNA large deletions is also described.

  19. Method for detecting point mutations in DNA utilizing fluorescence energy transfer

    Science.gov (United States)

    Parkhurst, Lawrence J.; Parkhurst, Kay M.; Middendorf, Lyle

    2001-01-01

    A method for detecting point mutations in DNA using a fluorescently labeled oligomeric probe and Forster resonance energy transfer (FRET) is disclosed. The selected probe is initially labeled at each end with a fluorescence dye, which act together as a donor/acceptor pair for FRET. The fluorescence emission from the dyes changes dramatically from the duplex stage, wherein the probe is hybridized to the complementary strand of DNA, to the single strand stage, when the probe is melted to become detached from the DNA. The change in fluorescence is caused by the dyes coming into closer proximity after melting occurs and the probe becomes detached from the DNA strand. The change in fluorescence emission as a function of temperature is used to calculate the melting temperature of the complex or T.sub.m. In the case where there is a base mismatch between the probe and the DNA strand, indicating a point mutation, the T.sub.m has been found to be significantly lower than the T.sub.m for a perfectly match probelstand duplex. The present invention allows for the detection of the existence and magnitude of T.sub.m, which allows for the quick and accurate detection of a point mutation in the DNA strand and, in some applications, the determination of the approximate location of the mutation within the sequence.

  20. In utero DNA damage from environmental pollution is associated with somatic gene mutation in newborns

    Energy Technology Data Exchange (ETDEWEB)

    Perera, F.; Hemminki, K.; Jedrychowski, W.; Whyatt, R.; Campbell, U.; Hsu, Y.Z.; Santella, R.; Albertini, R.; O' Neill, J.P. [Columbia University, New York, NY (United States). School of Public Health

    2002-10-01

    Transplacental exposure to carcinogenic air pollutants from the combustion of fossil fuels is a growing health concern, given evidence of the heightened susceptibility of the fetus. These mutagenic/carcinogenic pollutants include aromatic compounds such as polycyclic aromatic hydrocarbons that bind to DNA, forming chemical-DNA adducts. The genotoxic effects of transplacental exposure in humans has been investigated by analyzing aromatic-DNA adducts and the frequency of gene mutations at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in umbilical cord and maternal blood samples. Here the authors show, in a cross-sectional study of 67 mothers and 64 newborns from the Krakow Region of Poland, that aromatic-DNA adducts measured by P-32-postlabeling are positively associated with HPRT mutant frequency in the newborns (beta = 0.56, P = 0.03) after controlling for exposure to tobacco smoke, diet, and socioeconomic status. In contrast to the fetus, HPRT mutations and DNA adducts do not reflect similar exposure periods in the mother, and the maternal biomarkers were not correlated. Adducts were higher in the newborn than the mother, indicating differential susceptibility of the fetus to DNA damage; but HPRT mutation frequency was 4-fold lower, consistent with the long lifetime of the biomarker. These results provide the first demonstration of a molecular link between somatic mutation in the newborn and transplacental exposure to common air pollutants, a finding that is relevant to cancer risk assessment.

  1. Mutation of the little finger domain in human DNA polymerase η alters fidelity when copying undamaged DNA.

    Science.gov (United States)

    Beardslee, Renee A; Suarez, Samuel C; Toffton, Shannon M; McCulloch, Scott D

    2013-10-01

    DNA polymerase η (pol η) synthesizes past cyclobutane pyrimidine dimer and possibly 7,8-dihydro-8-oxoguanine (8-oxoG) lesions during DNA replication. Loss of pol η is associated with an increase in mutation rate, demonstrating its indispensable role in mutation suppression. It has been recently reported that β-strand 12 (amino acids 316-324) of the little finger region correctly positions the template strand with the catalytic core of the enzyme. The authors hypothesized that modification of β-strand 12 residues would disrupt correct enzyme-DNA alignment and alter pol η's activity and fidelity. To investigate this, the authors purified proteins containing the catalytic core of the polymerase, incorporated single amino acid changes to select β-strand 12 residues, and evaluated DNA synthesis activity for each pol η. Lesion bypass efficiencies and replication fidelities when copying DNA-containing cis-syn cyclobutane thymine-thymine dimer and 8-oxoG lesions were determined and compared with the corresponding values for the wild-type polymerase. The results confirm the importance of the β-strand in polymerase function and show that fidelity is most often altered when undamaged DNA is copied. Additionally, it is shown that DNA-protein contacts distal to the active site can significantly affect the fidelity of synthesis. Copyright © 2013 Wiley Periodicals, Inc.

  2. 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......-sectional study, 57 mDNA 3243A>G mutation carriers between May 2012 and October 2014 were included. As a control group, a population-based cohort from our epidemiological studies on migraine in Danes was used. History of headache and migraine was obtained by telephone interview, based on a validated semi......% vs. 6%; P DNA 3243A>G mutation was found. This finding suggests a clinical association between a monogenetically inherited disorder...

  3. A chronocoulometric LNA sensor for amplified detection of K-ras mutation based on site-specific DNA cleavage of restriction endonuclease.

    Science.gov (United States)

    Lin, Liqing; Liu, Ailin; Zhao, Chengfei; Weng, Shaohuang; Lei, Yun; Liu, Qicai; Lin, Xinhua; Chen, Yuanzhong

    2013-04-15

    An amplified chronocoulometric Locked nucleic acid (LNA) sensor (CLS) for selective electrochemical detection of K-ras mutation was developed based on site-specific DNA cleavage of restriction endonuclease EcoRI. Thiolated-hairpin LNA probe with palindrome structure of stem was immobilized on the gold nanoparticles modified gold electrode (NG/AuE). It can be cleaved by EcoRI in the absence of K-ras mutation-type DNA (complementary with the loop part of hairpin probe), but cannot be cleaved in the presence of mutation-type DNA. The difference before and after enzymatic cleavage was then monitored by chronocoulometric biosensor. Electrochemical signals are generated by chronocoulometric interrogation of Hexaammineruthenium (III) chloride (RuHex) that quantitatively binds to surface-confined hairpin LNA probe via electrostatic interactions. The results suggested this CLS had a good specificity to distinguish the K-ras mutation-type, wild-type and non-complementary sequence. There was a good linear relationship between the charge and the logarithmic function of K-ras mutation-type DNA concentration. The detection limit had been estimated as 0.5 fM. It is possible to qualitatively and quantitatively detect K-ras point mutation in pancreatic cancer. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Assessing the contribution of the herpes simplex virus DNA polymerase to spontaneous mutations

    Directory of Open Access Journals (Sweden)

    Leary Jeffry J

    2002-05-01

    Full Text Available Abstract Background The thymidine kinase (tk mutagenesis assay is often utilized to determine the frequency of herpes simplex virus (HSV replication-mediated mutations. Using this assay, clinical and laboratory HSV-2 isolates were shown to have a 10- to 80-fold higher frequency of spontaneous mutations compared to HSV-1. Methods A panel of HSV-1 and HSV-2, along with polymerase-recombinant viruses expressing type 2 polymerase (Pol within a type 1 genome, were evaluated using the tk and non-HSV DNA mutagenesis assays to measure HSV replication-dependent errors and determine whether the higher mutation frequency of HSV-2 is a distinct property of type 2 polymerases. Results Although HSV-2 have mutation frequencies higher than HSV-1 in the tk assay, these errors are assay-specific. In fact, wild type HSV-1 and the antimutator HSV-1 PAAr5 exhibited a 2–4 fold higher frequency than HSV-2 in the non-HSV DNA mutatagenesis assay. Furthermore, regardless of assay, HSV-1 recombinants expressing HSV-2 Pol had error rates similar to HSV-1, whereas the high mutator virus, HSV-2 6757, consistently showed signficant errors. Additionally, plasmid DNA containing the HSV-2 tk gene, but not type 1 tk or LacZ DNA, was shown to form an anisomorphic DNA stucture. Conclusions This study suggests that the Pol is not solely responsible for the virus-type specific differences in mutation frequency. Accordingly, it is possible that (a mutations may be modulated by other viral polypeptides cooperating with Pol, and (b the localized secondary structure of the viral genome may partially account for the apparently enhanced error frequency of HSV-2.

  5. Inflammation-Induced Cell Proliferation Potentiates DNA Damage-Induced Mutations In Vivo

    Science.gov (United States)

    Kiraly, Orsolya; Gong, Guanyu; Olipitz, Werner; Muthupalani, Sureshkumar; Engelward, Bevin P.

    2015-01-01

    Mutations are a critical driver of cancer initiation. While extensive studies have focused on exposure-induced mutations, few studies have explored the importance of tissue physiology as a modulator of mutation susceptibility in vivo. Of particular interest is inflammation, a known cancer risk factor relevant to chronic inflammatory diseases and pathogen-induced inflammation. Here, we used the fluorescent yellow direct repeat (FYDR) mice that harbor a reporter to detect misalignments during homologous recombination (HR), an important class of mutations. FYDR mice were exposed to cerulein, a potent inducer of pancreatic inflammation. We show that inflammation induces DSBs (γH2AX foci) and that several days later there is an increase in cell proliferation. While isolated bouts of inflammation did not induce HR, overlap between inflammation-induced DNA damage and inflammation-induced cell proliferation induced HR significantly. To study exogenously-induced DNA damage, animals were exposed to methylnitrosourea, a model alkylating agent that creates DNA lesions relevant to both environmental exposures and cancer chemotherapy. We found that exposure to alkylation damage induces HR, and importantly, that inflammation-induced cell proliferation and alkylation induce HR in a synergistic fashion. Taken together, these results show that, during an acute bout of inflammation, there is a kinetic barrier separating DNA damage from cell proliferation that protects against mutations, and that inflammation-induced cell proliferation greatly potentiates exposure-induced mutations. These studies demonstrate a fundamental mechanism by which inflammation can act synergistically with DNA damage to induce mutations that drive cancer and cancer recurrence. PMID:25647331

  6. Identification of I1171N resistance mutation in ALK-positive non-small-cell lung cancer tumor sample and circulating tumor DNA.

    Science.gov (United States)

    Johnson, Alison C; Dô, Pascal; Richard, Nicolas; Dubos, Catherine; Michels, Jean Jacques; Bonneau, Jessica; Gervais, Radj

    2016-09-01

    Anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC) is sensitive to ALK inhibitor therapy, but resistance invariably develops and can be mediated by certain secondary mutations. The detection of these mutations is useful to guide treatment decisions, but tumors are not always easily accessible to re-biopsy. We report the case of a patient with ALK-rearranged NSCLC who presented acquired resistance to crizotinib and then alectinib. Sequencing analyses of DNA from a liver metastasis biopsy sample and circulating tumor DNA both found the same I1171N ALK kinase domain mutation, known to confer resistance to certain ALK inhibitors. However, the patient then received ceritinib, a 2nd generation ALK inhibitor, and achieved another partial response. This case underlines how ALK resistance mutation detection in peripheral blood could be a reliable, safer, and less invasive alternative to tissue-based samples in NSCLC. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  7. KRAS mutation screening by chip-based DNA hybridization--a further step towards personalized oncology.

    Science.gov (United States)

    Steinbach, Christine; Steinbrücker, Carolin; Pollok, Sibyll; Walther, Katharina; Clement, Joachim H; Chen, Yuan; Petersen, Iver; Cialla-May, Dana; Weber, Karina; Popp, Jürgen

    2015-04-21

    The use of predictive biomarkers can help to improve therapeutic options for the individual cancer patient. For the treatment of colon cancer patients with anti-EGFR-based drugs, the KRAS mutation status has to be determined to pre-select responders that will benefit from this medication. Amongst others, array-based tests have been established for profiling of the KRAS mutation status. Within this article we describe an on-chip hybridization technique to screen therapeutic relevant KRAS codon 12 mutations. The DNA chip-based platform enables the reliable discrimination of selected mutations by allele-specific hybridization. Here, silver deposits represent robust endpoint signals that allow for a simple naked eye rating. With the here presented assay concept a precise identification of heterozygous and homozygous KRAS mutations, even against a background of up to 95% wild-type DNA, was realizable. The applicability of the test was successfully proven for various cancer cell lines as well as clinical tumour samples. Thus, the chip-based DNA hybridization technique seems to be a promising tool for KRAS mutation analysis to further improve personalized cancer treatment.

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

    International Nuclear Information System (INIS)

    Bragoszewski, Piotr; Kupryjanczyk, Jolanta; Bartnik, Ewa; Rachinger, Andrea; Ostrowski, Jerzy

    2008-01-01

    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. 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. 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. 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

  9. 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.

  10. DNA gyrase and topoisomerase IV mutations in an in vitro fluoroquinolone-resistant Coxiella burnetii strain.

    Science.gov (United States)

    Vranakis, Iosif; Sandalakis, Vassilios; Chochlakis, Dimosthenis; Tselentis, Yannis; Psaroulaki, Anna

    2010-06-01

    The etiological agent of Q fever, Coxiella burnetii, is an obligate intracellular bacterium that multiplies within a vacuole with lysosomal characteristics. Quinolones have been used as an alternative therapy for Q fever. In this study, quinolone-resistance-determining regions of the genes coding for DNA gyrase and topoisomerase IV were analyzed by DNA sequencing from an in vitro fluoroquinolone-resistant C. burnetii strain (Q212). Sequencing and aligning of DNA gyrase encoding genes (gyrA and gyrB) and topoisomerase IV genes (parC and parE) revealed one gyrA mutation leading to the amino acid substitution Asp87Gly (Escherichia coli numbering), two gyrB mutations leading to the amino acid substitutions Ser431Pro and Met518Ile, and three parC mutations leading to the amino acid substitutions Asp69Asn, Thr80Ile, and Gly104Ser. The corresponding alignment of the C. burnetii Q212 reference strain, the in vitro developed fluoroquinolone-resistant C. burnetii Q212 strain, and E. coli resulted in the identification of several other naturally occurring mutations within and outside the quinolone-resistance-determining regions of C. burnetii providing indications of possible natural resistance to fluoroquinolones. The present study adds additional potential mutations in the DNA topoisomerases that may be involved in fluoroquinolone resistance in C. burnetii due to their previous characterization in other bacterial species.

  11. rMotifGen: random motif generator for DNA and protein sequences

    Directory of Open Access Journals (Sweden)

    Hardin C Timothy

    2007-08-01

    Full Text Available Abstract Background Detection of short, subtle conserved motif regions within a set of related DNA or amino acid sequences can lead to discoveries about important regulatory domains such as transcription factor and DNA binding sites as well as conserved protein domains. In order to help assess motif detection algorithms on motifs with varying properties and levels of conservation, we have developed a computational tool, rMotifGen, with the sole purpose of generating a number of random DNA or protein sequences containing short sequence motifs. Each motif consensus can be user-defined, randomly generated, or created from a position-specific scoring matrix (PSSM. Insertions and mutations within these motifs are created according to user-defined parameters and substitution matrices. The resulting sequences can be helpful in mutational simulations and in testing the limits of motif detection algorithms. Results Two implementations of rMotifGen have been created, one providing a graphical user interface (GUI for random motif construction, and the other serving as a command line interface. The second implementation has the added advantages of platform independence and being able to be called in a batch mode. rMotifGen was used to construct sample sets of sequences containing DNA motifs and amino acid motifs that were then tested against the Gibbs sampler and MEME packages. Conclusion rMotifGen provides an efficient and convenient method for creating random DNA or amino acid sequences with a variable number of motifs, where the instance of each motif can be incorporated using a position-specific scoring matrix (PSSM or by creating an instance mutated from its corresponding consensus using an evolutionary model based on substitution matrices. rMotifGen is freely available at: http://bioinformatics.louisville.edu/brg/rMotifGen/.

  12. Singlet oxygen-induced mutations in M13 lacZ phage DNA.

    OpenAIRE

    Decuyper-Debergh, D; Piette, J; Van de Vorst, A

    1987-01-01

    The mutagenic consequences of damages to M13 mp19 RF DNA produced by singlet oxygen have been determined in a forward mutational system capable of detecting all classes of mutagenic events. When the damaged M13 mp19 RF DNA is used to transfect competent E. coli JM105 cells, a 16.6-fold increase in mutation frequency is observed at 5% survivors when measured as a loss of alpha-complementation. The enhanced mutagenicity is largely due to single-nucleotide substitutions, frameshift events and do...

  13. 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.

  14. 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.

  15. KRAS-mutated plasma DNA as predictor of outcome from irinotecan monotherapy in metastatic colorectal cancer

    DEFF Research Database (Denmark)

    Spindler, K G; Appelt, A L; Pallisgaard, N

    2013-01-01

    Background:We investigated the clinical implications of KRAS and BRAF mutations detected in both archival tumor tissue and plasma cell-free DNA in metastatic colorectal cancer patients treated with irinotecan monotherapy.Methods:Two hundred and eleven patients receiving second-line irinotecan (350...... mg m(-2) q3w) were included in two independent cohorts. Plasma was obtained from pretreatment EDTA blood-samples. Mutations were detected in archival tumour and plasma with qPCR methods.Results:Mutation status in tumor did not correlate to efficacy in either cohort, whereas none of the patients...... with mutations detectable in plasma responded to therapy. Response rate and disease control rate in plasma KRAS wt patients were 19 and 66% compared with 0 and 37%, in patients with pKRAS mutations, (P=0.04 and 0.01). Tumor KRAS status was not associated with PFS but with OS in the validation cohort. Plasma BRAF...

  16. Reliable Detection of Mismatch Repair Deficiency in Colorectal Cancers Using Mutational Load in Next-Generation Sequencing Panels

    Science.gov (United States)

    Stadler, Zsofia K.; Battaglin, Francesca; Middha, Sumit; Hechtman, Jaclyn F.; Tran, Christina; Cercek, Andrea; Yaeger, Rona; Segal, Neil H.; Varghese, Anna M.; Reidy-Lagunes, Diane L.; Kemeny, Nancy E.; Salo-Mullen, Erin E.; Ashraf, Asad; Weiser, Martin R.; Garcia-Aguilar, Julio; Robson, Mark E.; Offit, Kenneth; Arcila, Maria E.; Berger, Michael F.; Shia, Jinru; Solit, David B.

    2016-01-01

    Purpose Tumor screening for Lynch syndrome is recommended in all or most patients with colorectal cancer (CRC). In metastatic CRC, sequencing of RAS/BRAF is necessary to guide clinical management. We hypothesized that a next-generation sequencing (NGS) panel that identifies RAS/BRAF and other actionable mutations could also reliably identify tumors with DNA mismatch repair protein deficiency (MMR-D) on the basis of increased mutational load. Methods We identified all CRCs that underwent genomic mutation profiling with a custom NGS assay (MSK-IMPACT) between March 2014 and July 2015. Tumor mutational load, with exclusion of copy number changes, was determined for each case and compared with MMR status as determined by routine immunohistochemistry. Results Tumors from 224 patients with unique CRC analyzed for MMR status also underwent MSK-IMPACT. Thirteen percent (n = 28) exhibited MMR-D by immunohistochemistry. Using the 341-gene assay, 100% of the 193 tumors with 150 mutations each. Each of these tumors harbored the P286R hotspot POLE mutation consistent with the ultramutator phenotype. Among MMR-D tumors, the median number of mutations was 50 (range, 20 to 90) compared with six (range, 0 to 17) in MMR-proficient/POLE wild-type tumors (P < .001). With a mutational load cutoff of ≥ 20 and < 150 for MMR-D detection, sensitivity and specificity were both 1.0 (95% CI, 0.93 to 1.0). Conclusion A cutoff for mutational load can be identified via multigene NGS tumor profiling, which provides a highly accurate means of screening for MMR-D in the same assay that is used for tumor genotyping. PMID:27022117

  17. Synergistic Effects of thein cisT251I and P587L Mitochondrial DNA Polymerase γ Disease Mutations.

    Science.gov (United States)

    DeBalsi, Karen L; Longley, Matthew J; Hoff, Kirsten E; Copeland, William C

    2017-03-10

    Human mitochondrial DNA (mtDNA) polymerase γ (Pol γ) is the only polymerase known to replicate the mitochondrial genome. The Pol γ holoenzyme consists of the p140 catalytic subunit (POLG) and the p55 homodimeric accessory subunit (POLG2), which enhances binding of Pol γ to DNA and promotes processivity of the holoenzyme. Mutations within POLG impede maintenance of mtDNA and cause mitochondrial diseases. Two common POLG mutations usually found in cis in patients primarily with progressive external ophthalmoplegia generate T251I and P587L amino acid substitutions. To determine whether T251I or P587L is the primary pathogenic allele or whether both substitutions are required to cause disease, we overproduced and purified WT, T251I, P587L, and T251I + P587L double variant forms of recombinant Pol γ. Biochemical characterization of these variants revealed impaired DNA binding affinity, reduced thermostability, diminished exonuclease activity, defective catalytic activity, and compromised DNA processivity, even in the presence of the p55 accessory subunit. However, physical association with p55 was unperturbed, suggesting intersubunit affinities similar to WT. Notably, although the single mutants were similarly impaired, a dramatic synergistic effect was found for the double mutant across all parameters. In conclusion, our analyses suggest that individually both T251I and P587L substitutions functionally impair Pol γ, with greater pathogenicity predicted for the single P587L variant. Combining T251I and P587L induces extreme thermal lability and leads to synergistic nucleotide and DNA binding defects, which severely impair catalytic activity and correlate with presentation of disease in patients. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Differential strand separation at critical temperature: A minimally disruptive enrichment method for low-abundance unknown DNA mutations

    Science.gov (United States)

    Guha, Minakshi; Castellanos-Rizaldos, Elena; Liu, Pingfang; Mamon, Harvey; Makrigiorgos, G. Mike

    2013-01-01

    Detection of low-level DNA variations in the presence of wild-type DNA is important in several fields of medicine, including cancer, prenatal diagnosis and infectious diseases. PCR-based methods to enrich mutations during amplification have limited multiplexing capability, are mostly restricted to known mutations and are prone to polymerase or mis-priming errors. Here, we present Differential Strand Separation at Critical Temperature (DISSECT), a method that enriches unknown mutations of targeted DNA sequences purely based on thermal denaturation of DNA heteroduplexes without the need for enzymatic reactions. Target DNA is pre-amplified in a multiplex reaction and hybridized onto complementary probes immobilized on magnetic beads that correspond to wild-type DNA sequences. Presence of any mutation on the target DNA forms heteroduplexes that are subsequently denatured from the beads at a critical temperature and selectively separated from wild-type DNA. We demonstrate multiplexed enrichment by 100- to 400-fold for KRAS and TP53 mutations at multiple positions of the targeted sequence using two to four successive cycles of DISSECT. Cancer and plasma-circulating DNA samples containing traces of mutations undergo mutation enrichment allowing detection via Sanger sequencing or high-resolution melting. The simplicity, scalability and reliability of DISSECT make it a powerful method for mutation enrichment that integrates well with existing downstream detection methods. PMID:23258702

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

    International Nuclear Information System (INIS)

    Wessendorf, Petra; Vijg, Jan; Nussenzweig, André; Digweed, Martin

    2014-01-01

    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

  20. Autosomal recessive congenital ichthyosis: CERS3 mutations identified by a next generation sequencing panel targeting ichthyosis genes.

    Science.gov (United States)

    Youssefian, Leila; Vahidnezhad, Hassan; Saeidian, Amir Hossein; Sotoudeh, Soheila; Mahmoudi, Hamidreza; Daneshpazhooh, Maryam; Aghazadeh, Nessa; Adams, Rebecca; Ghanadan, Alireza; Zeinali, Sirous; Fortina, Paolo; Uitto, Jouni

    2017-11-01

    There are at least 38 mutant genes known to be associated with the ichthyosis phenotypes, and autosomal recessive congenital ichthyosis (ARCI) is a specific subgroup caused by mutations in 13 different genes. Mutations in some of these genes, such as CERS3 with only two previous reports, are rare. In this study, we identified mutations in candidate genes in consanguineous families with ARCI with a next generation sequencing (NGS) array that incorporates 38 ichthyosis associated genes. We applied this sequencing array to DNA from 140 ichthyosis families with high prevalence of consanguinity. Among these patients we identified six distinct, previously unreported mutations in CERS3 in six Iranian families. These mutations in each family co-segregated with the ichthyosis phenotype. The patients demonstrated collodion membrane at birth, acrogeria, generalized scaling, and hyperlinearity of the palms and soles. The presence of a significant percentage of CERS3 mutations in our cohort depicts a marked difference between the etiology of ichthyoses in genetically poorly characterized regions and well-characterized western populations. Also, it shows that rare alleles are more prevalent in the gene pool of consanguineous populations and emphasizes the importance of these population studies for better understanding of ichthyosis pathogenesis.

  1. Association of mtDNA mutation with Autism in Iranian patients

    Directory of Open Access Journals (Sweden)

    Massoud Houshmand

    2013-12-01

    Full Text Available The autism spectrum disorders (ASD are amongst the most heritable complex disorders. Although there have been many efforts to locate the genes associated with ASD risk, many has been remained to be disclosed about the genetics of ASD. Scrutiny's have only disclosed a small number of de novo and inherited variants significantly associated with susceptibility to ASD. These only comprise a small number of total genetic risk factors. Some studies confirm the contribution of mitochondrial genome mutations to the pathophysiology of the autism, but some other studies rejected such a contribution. In the current study we tried to scrutinize the association between mitochondrial tRNA genes mutations and the risk of Autism. DNA was extracted from the blood of 24 patients with ASD and 40 age-matched healthy controls from Special Medical Center in Tehran. 22 tRNA genes of mitochondrial genome were PCR amplified using 12 primer pairs and sequenced. Sequencing results were searched for mutations using clustalW Progran and then the association of mutations with the autism risk was assessed by statistical analysis using SPSS version 15. Many of the observed mutations were sporadic mutations without any significant relationship with the risk of autism, and the other mutations including those of high frequency showed no significant relationship with the risk of disease as well (p-value > 0.05 except mutations 16126T>C (p-value=0.01 , 14569G>A(pvalue=0.02 and 1811A>G(p-value=0.04. These three mutations were in the noncoding regions of the mitochondrial genome near tRNA genes. The mutation 16126T>C was in the mtDNA control region.

  2. Predicting the effects of basepair mutations in DNA-protein complexes by thermodynamic integration.

    Science.gov (United States)

    Beierlein, Frank R; Kneale, G Geoff; Clark, Timothy

    2011-09-07

    Thermodynamically rigorous free energy methods in principle allow the exact computation of binding free energies in biological systems. Here, we use thermodynamic integration together with molecular dynamics simulations of a DNA-protein complex to compute relative binding free energies of a series of mutants of a protein-binding DNA operator sequence. A guanine-cytosine basepair that interacts strongly with the DNA-binding protein is mutated into adenine-thymine, cytosine-guanine, and thymine-adenine. It is shown that basepair mutations can be performed using a conservative protocol that gives error estimates of ∼10% of the change in free energy of binding. Despite the high CPU-time requirements, this work opens the exciting opportunity of being able to perform basepair scans to investigate protein-DNA binding specificity in great detail computationally. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  3. Validation of next generation sequencing technologies in comparison to current diagnostic gold standards for BRAF, EGFR and KRAS mutational analysis.

    Science.gov (United States)

    McCourt, Clare M; McArt, Darragh G; Mills, Ken; Catherwood, Mark A; Maxwell, Perry; Waugh, David J; Hamilton, Peter; O'Sullivan, Joe M; Salto-Tellez, Manuel

    2013-01-01

    Next Generation Sequencing (NGS) has the potential of becoming an important tool in clinical diagnosis and therapeutic decision-making in oncology owing to its enhanced sensitivity in DNA mutation detection, fast-turnaround of samples in comparison to current gold standard methods and the potential to sequence a large number of cancer-driving genes at the one time. We aim to test the diagnostic accuracy of current NGS technology in the analysis of mutations that represent current standard-of-care, and its reliability to generate concomitant information on other key genes in human oncogenesis. Thirteen clinical samples (8 lung adenocarcinomas, 3 colon carcinomas and 2 malignant melanomas) already genotyped for EGFR, KRAS and BRAF mutations by current standard-of-care methods (Sanger Sequencing and q-PCR), were analysed for detection of mutations in the same three genes using two NGS platforms and an additional 43 genes with one of these platforms. The results were analysed using closed platform-specific proprietary bioinformatics software as well as open third party applications. Our results indicate that the existing format of the NGS technology performed well in detecting the clinically relevant mutations stated above but may not be reliable for a broader unsupervised analysis of the wider genome in its current design. Our study represents a diagnostically lead validation of the major strengths and weaknesses of this technology before consideration for diagnostic use.

  4. Somatic mitochondrial DNA mutations in cancer escape purifying selection and high pathogenicity mutations lead to the oncocytic phenotype: pathogenicity analysis of reported somatic mtDNA mutations in tumors

    International Nuclear Information System (INIS)

    Pereira, Luísa; Soares, Pedro; Máximo, Valdemar; Samuels, David C

    2012-01-01

    The presence of somatic mitochondrial DNA (mtDNA) mutations in cancer cells has been interpreted in controversial ways, ranging from random neutral accumulation of mutations, to positive selection for high pathogenicity, or conversely to purifying selection against high pathogenicity variants as occurs at the population level. Here we evaluated the predicted pathogenicity of somatic mtDNA mutations described in cancer and compare these to the distribution of variations observed in the global human population and all possible protein variations that could occur in human mtDNA. We focus on oncocytic tumors, which are clearly associated with mitochondrial dysfunction. The protein variant pathogenicity was predicted using two computational methods, MutPred and SNPs&GO. The pathogenicity score of the somatic mtDNA variants were significantly higher in oncocytic tumors compared to non-oncocytic tumors. Variations in subunits of Complex I of the electron transfer chain were significantly more common in tumors with the oncocytic phenotype, while variations in Complex V subunits were significantly more common in non-oncocytic tumors. Our results show that the somatic mtDNA mutations reported over all tumors are indistinguishable from a random selection from the set of all possible amino acid variations, and have therefore escaped the effects of purifying selection that act strongly at the population level. We show that the pathogenicity of somatic mtDNA mutations is a determining factor for the oncocytic phenotype. The opposite associations of the Complex I and Complex V variants with the oncocytic and non-oncocytic tumors implies that low mitochondrial membrane potential may play an important role in determining the oncocytic phenotype

  5. Comprehensive DNA Methylation and Mutation Analyses Reveal a Methylation Signature in Colorectal Sessile Serrated Adenomas.

    Science.gov (United States)

    Patai, Árpád V; Barták, Barbara Kinga; Péterfia, Bálint; Micsik, Tamás; Horváth, Réka; Sumánszki, Csaba; Péter, Zoltán; Patai, Árpád; Valcz, Gábor; Kalmár, Alexandra; Tóth, Kinga; Krenács, Tibor; Tulassay, Zsolt; Molnár, Béla

    2017-07-01

    Colorectal sessile serrated adenomas (SSA) are hypothesized to be precursor lesions of an alternative, serrated pathway of colorectal cancer, abundant in genes with aberrant promoter DNA hypermethylation. In our present pilot study, we explored DNA methylation profiles and examined selected gene mutations in SSA. Biopsy samples from patients undergoing screening colonoscopy were obtained during endoscopic examination. After DNA isolation and quality analysis, SSAs (n = 4) and healthy controls (n = 5) were chosen for further analysis. DNA methylation status of 96 candidate genes was screened by q(RT)PCR using Methyl-Profiler PCR array system. Amplicons for 12 gene mutations were sequenced by GS Junior Instrument using ligated and barcoded adaptors. Analysis of DNA methylation revealed 9 hypermethylated genes in both normal and SSA samples. 12 genes (CALCA, DKK2, GALR2, OPCML, PCDH10, SFRP1, SFRP2, SLIT3, SST, TAC1, VIM, WIF1) were hypermethylated in all SSAs and 2 additional genes (BNC1 and PDLIM4) were hypermethylated in 3 out of 4 SSAs, but in none of the normal samples. 2 SSAs exhibited BRAF mutation and synchronous MLH1 hypermethylation and were microsatellite instable by immunohistochemical analysis. Our combined mutation and DNA methylation analysis revealed that there is a common DNA methylation signature present in pre-neoplastic SSAs. This study advocates for the use of DNA methylation as a potential biomarker for the detection of SSA; however, further investigation is needed to better characterize the molecular background of these newly recognized colorectal lesions.

  6. Does aerobic exercises induce mtDNA mutation in human blood ...

    African Journals Online (AJOL)

    The aim of this study was to determine the effect of eight weeks aerobic training on mitochondrial DNA (mtDNA) mutation in human blood leucocytes. Twenty untrained healthy students (training group: n =10, age = 20.7±1.5 yrs, weight = 67.7±10 kg, BF% = 17.5±7.35 & control group: n =10, age = 21±1.3 yrs, weight ...

  7. Human papillomavirus mutational insertion: specific marker of circulating tumor DNA in cervical cancer patients.

    Directory of Open Access Journals (Sweden)

    Maura Campitelli

    Full Text Available INTRODUCTION: In most cases of cervical cancers, HPV DNA is integrated into the genome of carcinoma cells. This mutational insertion constitutes a highly specific molecular marker of tumor DNA for every patient. Circulating tumor DNA (ctDNA is an emerging marker of tumor dynamics which detection requires specific molecular motif. To determine whether the sequence of the cell-viral junction could be used in clinical practice as a specific marker of ctDNA, we analyzed a series of cervical cancer patient serums. METHODS AND FINDINGS: Serum specimens of 16 patients diagnosed with HPV16/18-associated cervical cancer, and for which the viral integration locus had been previously localized, were analyzed. Sequential serum specimens, taken at different times during the course of the disease, were also available for two of these cases. ctDNA was found in 11 out of 13 patients with tumor size greater than 20 mm at diagnosis, and analysis of sequential serum specimens showed that ctDNA concentration in patients serum was related to tumor dynamics. CONCLUSIONS: We report that HPV mutational insertion constitutes a highly specific molecular marker of ctDNA in HPV-associated tumor patients. Using this original approach, ctDNA was detected in most cervical cancer patients over stage I and ctDNA concentration was found to reflect tumor burden. In addition to its potential prognostic and predictive value, HPV mutation insertion is likely to constitute a new molecular surrogate of minimal residual disease and of subclinical relapse in HPV-associated tumor. This is of major importance in the perspective of specific anti-HPV therapy.

  8. DNA qualification workflow for next generation sequencing of histopathological samples.

    Directory of Open Access Journals (Sweden)

    Michele Simbolo

    Full Text Available Histopathological samples are a treasure-trove of DNA for clinical research. However, the quality of DNA can vary depending on the source or extraction method applied. Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory reproducible results. The qualification process consists of the quantification of double strand DNA (dsDNA and the assessment of its suitability for downstream applications, such as high-throughput next-generation sequencing. We tested the two most frequently used instrumentations to define their role in this process: NanoDrop, based on UV spectroscopy, and Qubit 2.0, which uses fluorochromes specifically binding dsDNA. Quantitative PCR (qPCR was used as the reference technique as it simultaneously assesses DNA concentration and suitability for PCR amplification. We used 17 genomic DNAs from 6 fresh-frozen (FF tissues, 6 formalin-fixed paraffin-embedded (FFPE tissues, 3 cell lines, and 2 commercial preparations. Intra- and inter-operator variability was negligible, and intra-methodology variability was minimal, while consistent inter-methodology divergences were observed. In fact, NanoDrop measured DNA concentrations higher than Qubit and its consistency with dsDNA quantification by qPCR was limited to high molecular weight DNA from FF samples and cell lines, where total DNA and dsDNA quantity virtually coincide. In partially degraded DNA from FFPE samples, only Qubit proved highly reproducible and consistent with qPCR measurements. Multiplex PCR amplifying 191 regions of 46 cancer-related genes was designated the downstream application, using 40 ng dsDNA from FFPE samples calculated by Qubit. All but one sample produced amplicon libraries suitable for next-generation sequencing. NanoDrop UV-spectrum verified contamination of the unsuccessful sample. In conclusion, as qPCR has high costs and is labor intensive, an alternative effective standard

  9. DNA Qualification Workflow for Next Generation Sequencing of Histopathological Samples

    Science.gov (United States)

    Simbolo, Michele; Gottardi, Marisa; Corbo, Vincenzo; Fassan, Matteo; Mafficini, Andrea; Malpeli, Giorgio; Lawlor, Rita T.; Scarpa, Aldo

    2013-01-01

    Histopathological samples are a treasure-trove of DNA for clinical research. However, the quality of DNA can vary depending on the source or extraction method applied. Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory reproducible results. The qualification process consists of the quantification of double strand DNA (dsDNA) and the assessment of its suitability for downstream applications, such as high-throughput next-generation sequencing. We tested the two most frequently used instrumentations to define their role in this process: NanoDrop, based on UV spectroscopy, and Qubit 2.0, which uses fluorochromes specifically binding dsDNA. Quantitative PCR (qPCR) was used as the reference technique as it simultaneously assesses DNA concentration and suitability for PCR amplification. We used 17 genomic DNAs from 6 fresh-frozen (FF) tissues, 6 formalin-fixed paraffin-embedded (FFPE) tissues, 3 cell lines, and 2 commercial preparations. Intra- and inter-operator variability was negligible, and intra-methodology variability was minimal, while consistent inter-methodology divergences were observed. In fact, NanoDrop measured DNA concentrations higher than Qubit and its consistency with dsDNA quantification by qPCR was limited to high molecular weight DNA from FF samples and cell lines, where total DNA and dsDNA quantity virtually coincide. In partially degraded DNA from FFPE samples, only Qubit proved highly reproducible and consistent with qPCR measurements. Multiplex PCR amplifying 191 regions of 46 cancer-related genes was designated the downstream application, using 40 ng dsDNA from FFPE samples calculated by Qubit. All but one sample produced amplicon libraries suitable for next-generation sequencing. NanoDrop UV-spectrum verified contamination of the unsuccessful sample. In conclusion, as qPCR has high costs and is labor intensive, an alternative effective standard workflow for

  10. DNA microarray analysis of fim mutations in Escherichia coli

    DEFF Research Database (Denmark)

    Schembri, Mark; Ussery, David; Workman, Christopher

    2002-01-01

    we have used DNA microarray analysis to examine the molecular events involved in response to fimbrial gene expression in E. coli K-12. Observed differential expression levels of the fim genes were in good agreement with our current knowledge of the stoichiometry of type I fimbriae. Changes in fim...

  11. Compound mitochondrial DNA mutations in a neurological patient ...

    Indian Academy of Sciences (India)

    Research Note Volume 93 Issue 1 April 2014 pp 173-177 ... ataxia; myoclonus; deafness; heteroplasmy; mtDNA; polyneuropathy. ... Korea; Department of Neurology and Brain Research Institute, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea; Department of Neurology, Samsung Medical Center, ...

  12. Compound mitochondrial DNA mutations in a neurological patient ...

    Indian Academy of Sciences (India)

    tials (CMAPs) over the abductor pollicis brevis and adduc- tor digiti quinti, respectively. In the same way, the MNCVs of peroneal and tibial nerves were determined by stimulat- ing the knee and ankle, while recording CMAPs over the. Keywords. ataxia; myoclonus; deafness; heteroplasmy; mtDNA; polyneuropathy. Journal of ...

  13. Compound mitochondrial DNA mutations in a neurological patient ...

    Indian Academy of Sciences (India)

    dPol, polymorphic; AD, Alzeimer's disease; SNHL, sensorineural hearing loss; PEM, progressive encephalopathy; PD, Parkinson's disease;. LHON, Leber hereditary optic neuropathy; MDD, major depressive disorder. Figure 1. Determination of the mtDNA haplogroup. This haplogrouping was performed by the HaploGrep ...

  14. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Yaopromsiri, C. [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Yu, L.D., E-mail: yuld@thep-center.org [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Sarapirom, S. [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Faculty of Science, Maejo University, Bang Khen, Chiang Mai 50290 (Thailand); Thopan, P.; Boonyawan, D. [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand)

    2015-12-15

    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.

  17. Detection of low-level DNA mutation by ARMS-blocker-Tm PCR.

    Science.gov (United States)

    Qu, Shoufang; Liu, Licheng; Gan, Shuzhen; Feng, Huahua; Zhao, Jingyin; Zhao, Jing; Liu, Qi; Gao, Shangxiang; Chen, Weijun; Wang, Mengzhao; Jiang, Yongqiang; Huang, Jie

    2016-02-01

    Low-level DNA mutations play important roles in cancer prognosis and treatment. However, most existing methods for the detection of low-level DNA mutations are insufficient for clinical applications because of the high background of wild-type DNA. In this study, a novel assay based on Tm-dependent inhibition of wild type template amplification was developed. The defining characteristic of this assay is an additional annealing step was introduced into the ARMS-blocker PCR. The temperature of this additional annealing step is equal to the Tm of the blocker. Due to this additional annealing step, the blocker can preferentially and specifically bind the wild-type DNA. Thus, the inhibition of wild type template is realized and the mutant DNA is enriched. The sensitivity of this assay was between 10(-4) and 10(-5), which is approximately 5 to 10 times greater than the sensitivity of the assay without the additional annealing step. To evaluate the performance of this assay in detecting K-ras mutation, we analyzed 100 formalin-fixed paraffin-embedded (FFPE) specimens from colorectal cancer patients using this new assay and Sanger sequencing. Of the clinical samples, 27 samples were positive for K-ras mutation by both methods. Our results indicated that this new assay is a highly selective, convenient, and economical method for detecting rare mutations in the presence of higher concentrations of wild-type DNA. Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  18. DNA strand generation for DNA computing by using a multi-objective differential evolution algorithm.

    Science.gov (United States)

    Chaves-González, José M; Vega-Rodríguez, Miguel A

    2014-02-01

    In this paper, we use an adapted multi-objective version of the differential evolution (DE) metaheuristics for the design and generation of reliable DNA libraries that can be used for computation. DNA sequence design is a very relevant task in many recent research fields, e.g. nanotechnology or DNA computing. Specifically, DNA computing is a new computational model which uses DNA molecules as information storage and their possible biological interactions as processing operators. Therefore, the possible reactions and interactions among molecules must be strictly controlled to prevent incorrect computations. The design of reliable DNA libraries for bio-molecular computing is an NP-hard combinatorial problem which involves many heterogeneous and conflicting design criteria. For this reason, we modelled DNA sequence design as a multiobjective optimization problem and we solved it by using an adapted multi-objective version of DE metaheuristics. Seven different bio-chemical design criteria have been simultaneously considered to obtain high quality DNA sequences which are suitable for molecular computing. Furthermore, we have developed the multiobjective standard fast non-dominated sorting genetic algorithm (NSGA-II) in order to perform a formal comparative study by using multi-objective indicators. Additionally, we have also compared our results with other relevant results published in the literature. We conclude that our proposal is a promising approach which is able to generate reliable real-world DNA sequences that significantly improve other DNA libraries previously published in the literature. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  19. Next-generation sequencing offers new insights into DNA degradation

    DEFF Research Database (Denmark)

    Overballe-Petersen, Søren; Orlando, Ludovic Antoine Alexandre; Willerslev, Eske

    2012-01-01

    The processes underlying DNA degradation are central to various disciplines, including cancer research, forensics and archaeology. The sequencing of ancient DNA molecules on next-generation sequencing platforms provides direct measurements of cytosine deamination, depurination and fragmentation...... rates that previously were obtained only from extrapolations of results from in vitro kinetic experiments performed over short timescales. For example, recent next-generation sequencing of ancient DNA reveals purine bases as one of the main targets of postmortem hydrolytic damage, through base...... elimination and strand breakage. It also shows substantially increased rates of DNA base-loss at guanosine. In this review, we argue that the latter results from an electron resonance structure unique to guanosine rather than adenosine having an extra resonance structure over guanosine as previously suggested....

  20. Prognosis of symptomatic patients with the A3243G mutation of mitochondrial DNA

    Directory of Open Access Journals (Sweden)

    Chi-Hung Liu

    2012-09-01

    Conclusion: Our study found that seizures and status epilepticus are the most important predictive values for a poor outcome in patients with the mtA3243G mutation of mtDNA. Age of onset and visceral organ involvement had no prominent influence on the prognosis. Some medical complications could be well controlled or even reversed after management.

  1. 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

  2. DNA methyltransferase 3B mutations linked to the ICF syndrome cause dysregulation of lymphogenesis genes.

    NARCIS (Netherlands)

    Ehrlich, M.; Buchanan, K.L.; Tsien, F.; Jiang, G.; Sun, B.; Uicker, W.; Weemaes, C.M.R.; Smeets, D.F.C.M.; Sperling, K.; Belohradsky, B.H.; Tommerup, N.; Misek, D.E.; Rouillard, J.M.; Kuick, R.; Hanash, S.M.

    2001-01-01

    ICF (immunodeficiency, centromeric region instability and facial anomalies) is a recessive disease caused by mutations in the DNA methyltransferase 3B gene (DNMT3B). Patients have immunodeficiency, chromosome 1 (Chr1) and Chr16 pericentromeric anomalies in mitogen-stimulated lymphocytes, a small

  3. A Novel Mutation in the DNA Binding Domain of NFKB is Associated with Speckled Leukoplakia.

    Science.gov (United States)

    Govindarajan, Giri Valanthan Veda; Bhanumurthy, Lokesh; Balasubramanian, Anandh; Ramanathan, Arvind

    2016-01-01

    Activation and inactivation of nuclear factor of kappa light chain gene enhancer in B cells (NFKB) is tightly regulated to ensure effective onset and cessation of defensive inflammatory signaling. However, mutations within NFKB, or change in activation and inactivation molecules have been reported in a few cancers. Although oral squamous cell carcinoma is one of the most prevalent forms of cancer in India, with a development associated with malignant transformation of precancerous lesions, the genetic status of NFKB and relative rates of change in oral precancerous lesions remain unknown. Hence in the present study we investigated all twenty four exons of NFKB gene in two precancerous lesions, namely oral submucous fibrosis (OSMF) and oral leukoplakia (OL) to understand its occurrence, incidence and assess its possible contribution to malignant transformation. Chromosomal DNA isolated from twenty five each of OSMF and OL tissue biopsy samples were subjected to PCR amplification with intronic primers flanking twenty four exons of the NFKB gene. The PCR amplicons were subsequently subjected to direct sequencing to elucidate the mutation status. Sequence analysis identified a novel heterozygous mutation, c.419T>A causing substitution of leucine with glutamine at codon 140 (L140Q) in an OL sample. The identification of a substitution mutation L140Q within the DNA binding domain of NFKB in OL suggests that NFKB mutation may be relatively an early event during transformation. To the best of our knowledge, this study is the first to have identified a missense mutation in NFKB in OL.

  4. 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.

  5. Adducts in sperm protamine and DNA (deoxyribonuclease) vs. mutation frequency

    Energy Technology Data Exchange (ETDEWEB)

    Sega, G.A.

    1990-01-01

    In mammals, variability in the genetic sensitivity of different germ-cell stages to mutagens could be the result of how much chemical reaches the different stages, what molecular targets may be affected in the different stages and whether or not repair of lesions occurs. In the mouse, several mutagens have been found that cause their greatest genetic damage in late-spermatid and early-spermatozoa stages and that also bind very strongly to the protamine in these stages. Chemicals which are less genetically damaging to these stages have been found to have much less affinity for protamine. Furthermore, the level of chemical binding to DNA in late-spermatid and early-spermatozoa stages has not been correlated with the level of induced genetic damage, although DNA breakage in these sensitive stages has been shown to increase. This DNA damage is believed to indirectly result from chemical binding to sulfhydryl groups in protamine which prevents normal chromatin condensation within the sperm nucleus. 16 refs., 5 figs.

  6. Synergistic effects of UdgB and Ung in mutation prevention and protection against commonly encountered DNA damaging agents in Mycobacterium smegmatis.

    Science.gov (United States)

    Malshetty, Vidyasagar S; Jain, Ruchi; Srinath, Thiruneelakantan; Kurthkoti, Krishna; Varshney, Umesh

    2010-03-01

    The incorporation of dUMP during replication or the deamination of cytosine in DNA results in the occurrence of uracils in genomes. To maintain genomic integrity, uracil DNA glycosylases (UDGs) excise uracil from DNA and initiate the base-excision repair pathway. Here, we cloned, purified and biochemically characterized a family 5 UDG, UdgB, from Mycobacterium smegmatis to allow us to use it as a model organism to investigate the physiological significance of the novel enzyme. Studies with knockout strains showed that compared with the wild-type parent, the mutation rate of the udgB( -) strain was approximately twofold higher, whereas the mutation rate of a strain deficient in the family 1 UDG (ung(- )) was found to be approximately 8.4-fold higher. Interestingly, the mutation rate of the double-knockout (ung(-)/ udgB(-)) strain was remarkably high, at approximately 19.6-fold. While CG to TA mutations predominated in the ung(-) and ung(-)/udgB(-) strains, AT to GC mutations were enhanced in the udgB(-) strain. The ung(-)/udgB(-) strain was notably more sensitive to acidified nitrite and hydrogen peroxide stresses compared with the single knockouts (ung(-) or udgB(-)). These observations reveal a synergistic effect of UdgB and Ung in DNA repair, and could have implications for the generation of attenuated strains of Mycobacterium tuberculosis.

  7. Clinical phenotype, prognosis and mitochondrial DNA mutation load in mitochondrial encephalomyopathies.

    Science.gov (United States)

    Huang, Chin-Chang; Kuo, Hung-Chou; Chu, Chen-Che; Liou, Chia-Wei; Ma, Yi-Shing; Wei, Yau-Huei

    2002-01-01

    We studied 42 individuals, including 8 patients with either complete or partial syndrome of mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS), 8 patients with either complete or partial syndrome of myoclonic epilepsy with ragged-red fibers (MERRF) and 26 maternal family members who carried either the A3243G or A8344G mutation of mitochondrial DNA (mtDNA). Clinical manifestations and prognosis were followed up in the patients harboring the A3243G or A8344G mutation. The relationship between clinical features and proportions of mutant mtDNAs in muscle biopsies, blood cells and/or hair follicles was studied. In the 8 regularly followed patients with the A3243G mutation, 4 died within 1 month to 7 years due to status epilepticus and/or recurrent stroke-like episodes. Two patients developed marked mental deterioration and 2 remained stationary. All of the patients harboring the A8344G mutation were stable or deteriorated slightly, except for 1 patient who died due to brain herniation after putaminal hemorrhage. The A3243G and A8344G mtDNA mutations were heteroplasmic in the muscle biopsies, blood cells and hair follicles of both the probands and their maternal family members. The mean proportion of A3243G mutant mtDNA in the muscle biopsies of the patients with MELAS syndrome (68.5 +/- 21.3%, range 33-92%) was significantly higher than that of the asymptomatic family members (37.1 +/- 12.6%, range 0-51%). The average proportions of A8344G mutant mtDNA in the muscle biopsies (90.1 +/- 3.9%, range 89-95%) and hair follicles (93.9 +/- 6.4%, range 84-99%) of the patients with MERRF syndrome were also significantly higher than those of the asymptomatic family members (muscle: 40.3 +/- 39.5%, range 1-80%; hair follicles: 51.0 +/- 44.5%, range 0.1-82%). We concluded that measurement of the proportion of mutant mtDNA in muscle biopsies may provide useful information in the identification of symptomatic patients with mitochondrial

  8. Identification and characterization of C106R, a novel mutation in the DNA-binding domain of GCMB, in a family with autosomal-dominant hypoparathyroidism.

    Science.gov (United States)

    Yi, Hyon-Seung; Eom, Young Sil; Park, Ie Byung; Lee, Sangho; Hong, Suntaek; Jüppner, Harald; Mannstadt, Michael; Lee, Sihoon

    2012-05-01

    Glial cells missing B (GCMB) is a transcription factor that is expressed in the parathyroid hormone (PTH)-secreting cells of the parathyroid glands. Several mutations in GCMB have been reported to cause hypoparathyroidism (HP). We identified a family with two individuals in two generations (mother and son), who are affected by autosomal-dominant hypoparathyroidism (AD-HP). A novel heterozygous mutation in exon 2 of GCMB was identified in both affected individuals that changes cysteine at position 106 of the putative DNA-binding domain of GCMB to arginine (C106R). We performed mutational analysis of the genes encoding GCMB, pre-pro PTH, GATA3 and CaSR using polymerase chain reaction (PCR)-amplified genomic DNA. The identified GCMB mutant was characterized by functional studies including nuclear localization, electrophoretic mobility shift assays (EMSA) and luciferase reporter assays, and homology modelling was performed to generate a three-dimensional structural model for the DNA-binding domain of GCMB to predict the structural consequences of the identified mutation. The C106R mutant of GCMB failed to interact with the DNA consensus recognition motif, as determined by EMSA. Furthermore, in comparison with wild-type GCMB, the C106R mutant demonstrated reduced transactivation in luciferase reporter assays; however, the mutant GCMB failed to reduce the activity of the wild-type protein. Consistent with the EMSA findings, homology modelling analysis suggested that replacement of cysteine 106 with arginine would interfere with DNA binding. We have identified a novel GCMB mutation that may explain AD-HP in our family. However, the exact mechanism by which this heterozygous mutation leads to the disease in the described family remains to be elucidated. © 2012 Blackwell Publishing Ltd.

  9. Identification and characterization of C106R, a novel mutation in the DNA-binding domain of GCMB, in a family with autosomal-dominant hypoparathyroidism

    Science.gov (United States)

    Yi, Hyon-Seung; Eom, Young Sil; Park, Ie Byung; Lee, Sangho; Hong, Suntaek; Jüppner, Harald; Mannstadt, Michael; Lee, Sihoon

    2013-01-01

    Summary Overview Glial cells missing B (GCMB) is a transcription factor that is expressed in the parathyroid hormone (PTH)-secreting cells of the parathyroid glands. Several mutations in GCMB have been reported to cause hypoparathyroidism (HP). We identified a family with two individuals in two generations (mother and son), who are affected by autosomal-dominant hypoparathyroidism (AD-HP). A novel heterozygous mutation in exon 2 of GCMB was identified in both affected individuals that changes cysteine at position 106 of the putative DNA-binding domain of GCMB to arginine (C106R). Methods We performed mutational analysis of the genes encoding GCMB, pre-pro PTH, GATA3 and CaSR using polymerase chain reaction (PCR)-amplified genomic DNA. The identified GCMB mutant was characterized by functional studies including nuclear localization, electrophoretic mobility shift assays (EMSA) and luciferase reporter assays, and homology modelling was performed to generate a three-dimensional structural model for the DNA-binding domain of GCMB to predict the structural consequences of the identified mutation. Results The C106R mutant of GCMB failed to interact with the DNA consensus recognition motif, as determined by EMSA. Furthermore, in comparison with wild-type GCMB, the C106R mutant demonstrated reduced transactivation in luciferase reporter assays; however, the mutant GCMB failed to reduce the activity of the wild-type protein. Consistent with the EMSA findings, homology modelling analysis suggested that replacement of cysteine 106 with arginine would interfere with DNA binding. Conclusions We have identified a novel GCMB mutation that may explain AD-HP in our family. However, the exact mechanism by which this heterozygous mutation leads to the disease in the described family remains to be elucidated. PMID:22066718

  10. Mitochondrial DNA triplication and punctual mutations in patients with mitochondrial neuromuscular disorders

    Energy Technology Data Exchange (ETDEWEB)

    Mkaouar-Rebai, Emna, E-mail: emna.mkaouar@gmail.com [Département des Sciences de la Vie, Faculté des Sciences de Sfax, Université de Sfax (Tunisia); Felhi, Rahma; Tabebi, Mouna [Laboratoire de Génétique Moléculaire Humaine, Faculté de Médecine de Sfax, Université de Sfax (Tunisia); Alila-Fersi, Olfa; Chamkha, Imen [Département des Sciences de la Vie, Faculté des Sciences de Sfax, Université de Sfax (Tunisia); Maalej, Marwa; Ammar, Marwa [Laboratoire de Génétique Moléculaire Humaine, Faculté de Médecine de Sfax, Université de Sfax (Tunisia); Kammoun, Fatma [Service de pédiatrie, C.H.U. Hedi Chaker de Sfax (Tunisia); Keskes, Leila [Laboratoire de Génétique Moléculaire Humaine, Faculté de Médecine de Sfax, Université de Sfax (Tunisia); Hachicha, Mongia [Service de pédiatrie, C.H.U. Hedi Chaker de Sfax (Tunisia); Fakhfakh, Faiza, E-mail: faiza.fakhfakh02@gmail.com [Département des Sciences de la Vie, Faculté des Sciences de Sfax, Université de Sfax (Tunisia)

    2016-04-29

    Mitochondrial diseases are a heterogeneous group of disorders caused by the impairment of the mitochondrial oxidative phosphorylation system which have been associated with various mutations of the mitochondrial DNA (mtDNA) and nuclear gene mutations. The clinical phenotypes are very diverse and the spectrum is still expanding. As brain and muscle are highly dependent on OXPHOS, consequently, neurological disorders and myopathy are common features of mtDNA mutations. Mutations in mtDNA can be classified into three categories: large-scale rearrangements, point mutations in tRNA or rRNA genes and point mutations in protein coding genes. In the present report, we screened mitochondrial genes of complex I, III, IV and V in 2 patients with mitochondrial neuromuscular disorders. The results showed the presence the pathogenic heteroplasmic m.9157G>A variation (A211T) in the MT-ATP6 gene in the first patient. We also reported the first case of triplication of 9 bp in the mitochondrial NC7 region in Africa and Tunisia, in association with the novel m.14924T>C in the MT-CYB gene in the second patient with mitochondrial neuromuscular disorder. - Highlights: • We reported 2 patients with mitochondrial neuromuscular disorders. • The heteroplasmic MT-ATP6 9157G>A variation was reported. • A triplication of 9 bp in the mitochondrial NC7 region was detected. • The m.14924T>C transition (S60P) in the MT-CYB gene was found.

  11. Mitochondrial DNA triplication and punctual mutations in patients with mitochondrial neuromuscular disorders

    International Nuclear Information System (INIS)

    Mkaouar-Rebai, Emna; Felhi, Rahma; Tabebi, Mouna; Alila-Fersi, Olfa; Chamkha, Imen; Maalej, Marwa; Ammar, Marwa; Kammoun, Fatma; Keskes, Leila; Hachicha, Mongia; Fakhfakh, Faiza

    2016-01-01

    Mitochondrial diseases are a heterogeneous group of disorders caused by the impairment of the mitochondrial oxidative phosphorylation system which have been associated with various mutations of the mitochondrial DNA (mtDNA) and nuclear gene mutations. The clinical phenotypes are very diverse and the spectrum is still expanding. As brain and muscle are highly dependent on OXPHOS, consequently, neurological disorders and myopathy are common features of mtDNA mutations. Mutations in mtDNA can be classified into three categories: large-scale rearrangements, point mutations in tRNA or rRNA genes and point mutations in protein coding genes. In the present report, we screened mitochondrial genes of complex I, III, IV and V in 2 patients with mitochondrial neuromuscular disorders. The results showed the presence the pathogenic heteroplasmic m.9157G>A variation (A211T) in the MT-ATP6 gene in the first patient. We also reported the first case of triplication of 9 bp in the mitochondrial NC7 region in Africa and Tunisia, in association with the novel m.14924T>C in the MT-CYB gene in the second patient with mitochondrial neuromuscular disorder. - Highlights: • We reported 2 patients with mitochondrial neuromuscular disorders. • The heteroplasmic MT-ATP6 9157G>A variation was reported. • A triplication of 9 bp in the mitochondrial NC7 region was detected. • The m.14924T>C transition (S60P) in the MT-CYB gene was found.

  12. [Mutations of DNA gyrase and topoisomerase IV in clinical isolates of fluoroquinolone-resistant Proteus mirabilis].

    Science.gov (United States)

    Saito, Ryoichi; Sato, Kenya; Kumita, Wakako; Inami, Natsuko; Nishiyama, Hiroyuki; Okamura, Noboru; Moriya, Kyoji; Koike, Kazuhiko

    2006-02-01

    The presence of fluoroquinolone resistance-associated mutations within the quinolone resistance-determining region of DNA gyrase and topoisomerase IV was investigated genetically in clinical isolates of Proteus mirabilis recovered from patients with urinay tract infections. Two isolates of fluoroquinolone-resistant P. mirabilis possessed the mutations in GyrA (Ser-83 --> Arg or Ile), GyrB (Ser-464 --> Tyr or Phe) and ParC (Ser-80 --> Ile). A novel mutation with Glu-87 --> Lys in GyrA, where suggested to be responsible for fluoroquinolone resistance, was identified. These results demonstrate that the presence of an additional mutation at Glu-87 in GyrA may contribute to high-level fluoroquinolone resistance, too.

  13. 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.

  14. Roles of DNA polymerase I in leading and lagging-strand replication defined by a high-resolution mutation footprint of ColE1 plasmid replication

    Science.gov (United States)

    Allen, Jennifer M.; Simcha, David M.; Ericson, Nolan G.; Alexander, David L.; Marquette, Jacob T.; Van Biber, Benjamin P.; Troll, Chris J.; Karchin, Rachel; Bielas, Jason H.; Loeb, Lawrence A.; Camps, Manel

    2011-01-01

    DNA polymerase I (pol I) processes RNA primers during lagging-strand synthesis and fills small gaps during DNA repair reactions. However, it is unclear how pol I and pol III work together during replication and repair or how extensive pol I processing of Okazaki fragments is in vivo. Here, we address these questions by analyzing pol I mutations generated through error-prone replication of ColE1 plasmids. The data were obtained by direct sequencing, allowing an accurate determination of the mutation spectrum and distribution. Pol I’s mutational footprint suggests: (i) during leading-strand replication pol I is gradually replaced by pol III over at least 1.3 kb; (ii) pol I processing of Okazaki fragments is limited to ∼20 nt and (iii) the size of Okazaki fragments is short (∼250 nt). While based on ColE1 plasmid replication, our findings are likely relevant to other pol I replicative processes such as chromosomal replication and DNA repair, which differ from ColE1 replication mostly at the recruitment steps. This mutation footprinting approach should help establish the role of other prokaryotic or eukaryotic polymerases in vivo, and provides a tool to investigate how sequence topology, DNA damage, or interactions with protein partners may affect the function of individual DNA polymerases. PMID:21622658

  15. DNA fingerprinting, DNA barcoding, and next generation sequencing technology in plants.

    Science.gov (United States)

    Sucher, Nikolaus J; Hennell, James R; Carles, Maria C

    2012-01-01

    DNA fingerprinting of plants has become an invaluable tool in forensic, scientific, and industrial laboratories all over the world. PCR has become part of virtually every variation of the plethora of approaches used for DNA fingerprinting today. DNA sequencing is increasingly used either in combination with or as a replacement for traditional DNA fingerprinting techniques. A prime example is the use of short, standardized regions of the genome as taxon barcodes for biological identification of plants. Rapid advances in "next generation sequencing" (NGS) technology are driving down the cost of sequencing and bringing large-scale sequencing projects into the reach of individual investigators. We present an overview of recent publications that demonstrate the use of "NGS" technology for DNA fingerprinting and DNA barcoding applications.

  16. DNA microarray analysis of fim mutations in Escherichia coli

    DEFF Research Database (Denmark)

    Schembri, Mark; Ussery, David; Workman, Christopher

    2002-01-01

    Bacterial adhesion is often mediated by complex polymeric surface structures referred to as fimbriae. Type I fimbriae of Escherichia coli represent the archetypical and best characterised fimbrial system. These adhesive organelles mediate binding to D-mannose and are directly associated...... we have used DNA microarray analysis to examine the molecular events involved in response to fimbrial gene expression in E. coli K-12. Observed differential expression levels of the fim genes were in good agreement with our current knowledge of the stoichiometry of type I fimbriae. Changes in fim...

  17. [Exploratory study of circulating tumor DNA detection in early breast cancer: an analysis of 75 next-generation sequencing results].

    Science.gov (United States)

    Zhou, B; Xin, L; Xu, L; Liu, Y H; Zhang, M M; Jing, R L; Liang, X Y; Cao, S B

    2017-11-01

    Objective: To explore the utility of circulating tumor DNA detection in early breast cancer by using next-generation sequencing. Methods: This exploratory study of circulating tumor DNA detection is for early invasive breast cancer patients treated in Breast Disease Center, Peking University First Hospital from December 2015 to July 2016. Plasma samples were collected and were used to isolate plasma cell-free DNA.Exons or hotspots of 247 cancer related genes were sequenced by next-generation sequencing. Mutations and their correlation with clinic-pathological factors were analyzed. The correlation between mutations and clinic-pathological factors was evaluated by χ(2) test or Fisher's exact test. Results: Seventy-five patients were enrolled in this study. All patients were female and aged from 31 to 88 years with median age of 58 years. All patients' clinic-pathological records were complete. Sixty-four mutations in 18 genes (ALK, BCR, ERBB2, ROS1, PDGFRA, EGFR, FGFR2, CYP1B1, CALR, CASP7, BRAF, FGFR1, FGFR3, MET, NRAS, PTEN, KIT, SOD2) were detected in 47 (62.7%) among all 75 patients.Exons were captured in 10 genes, and mutations in 2 of 3 genes analyzed were clustered. Gene mutations were not correlated with menopausal status, histological type, primary tumor (T), regional lymph nodes (N), TNM stage, histological grade, estrogen receptor status, progesterone receptor status, human epidermal growth factor receptor 2 status, Ki-67 and molecular subtype (all P >0.05). Conclusion: Circulating tumor DNA sequencing by next-generation sequencing was useful for detecting breast cancer-related mutations.

  18. Mutations on the DNA binding surface of TBP discriminate between yeast TATA and TATA-less gene transcription.

    Science.gov (United States)

    Kamenova, Ivanka; Warfield, Linda; Hahn, Steven

    2014-08-01

    Most RNA polymerase (Pol) II promoters lack a TATA element, yet nearly all Pol II transcription requires TATA binding protein (TBP). While the TBP-TATA interaction is critical for transcription at TATA-containing promoters, it has been unclear whether TBP sequence-specific DNA contacts are required for transcription at TATA-less genes. Transcription factor IID (TFIID), the TBP-containing coactivator that functions at most TATA-less genes, recognizes short sequence-specific promoter elements in metazoans, but analogous promoter elements have not been identified in Saccharomyces cerevisiae. We generated a set of mutations in the yeast TBP DNA binding surface and found that most support growth of yeast. Both in vivo and in vitro, many of these mutations are specifically defective for transcription of two TATA-containing genes with only minor defects in transcription of two TATA-less, TFIID-dependent genes. TBP binds several TATA-less promoters with apparent high affinity, but our results suggest that this binding is not important for transcription activity. Our results are consistent with the model that sequence-specific TBP-DNA contacts are not important at yeast TATA-less genes and suggest that other general transcription factors or coactivator subunits are responsible for recognition of TATA-less promoters. Our results also explain why yeast TBP derivatives defective for TATA binding appear defective in activated transcription. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  19. Germline mutation rates at tandem repeat loci in DNA-repair deficient mice

    International Nuclear Information System (INIS)

    Barber, Ruth C.; Miccoli, Laurent; Buul, Paul P.W. van; Burr, Karen L.-A.; Duyn-Goedhart, Annemarie van; Angulo, Jaime F.; Dubrova, Yuri E.

    2004-01-01

    Mutation rates at two expanded simple tandem repeat (ESTR) loci were studied in the germline of non-exposed and irradiated severe combined immunodeficient (scid) and poly(ADP-ribose) polymerase (PARP-1 -/- ) deficient male mice. Non-exposed scid and PARP -/- male mice showed considerably elevated ESTR mutation rates, far higher than those in wild-type isogenic mice and other inbred strains. The irradiated scid and PARP-1 -/- male mice did not show any detectable increases in their mutation rate, whereas significant ESTR mutation induction was observed in the irradiated wild-type isogenic males. ESTR mutation spectra in the scid and PARP-1 -/- strains did not differ from those in the isogenic wild-type strains. Considering these data and the results of previous studies, we propose that a delay in repair of DNA damage in scid and PARP-1 -/- mice could result in replication fork pausing which, in turn, may affect ESTR mutation rate in the non-irradiated males. The lack of mutation induction in irradiated scid and PARP-1 -/- can be explained by the high cell killing effects of irradiation on the germline of deficient mice

  20. DNA adduct formation and mutation induction by aristolochic acid in rat kidney and liver

    International Nuclear Information System (INIS)

    Mei, Nan; Arlt, Volker M.; Phillips, David H.; Heflich, Robert H.; Chen, Tao

    2006-01-01

    Aristolochic acid (AA) is a potent nephrotoxin and carcinogen and is the causative factor for Chinese herb nephropathy. AA has been associated with the development of urothelial cancer in humans, and kidney and forestomach tumors in rodents. To investigate the molecular mechanisms responsible for the tumorigenicity of AA, we determined the DNA adduct formation and mutagenicity of AA in the liver (nontarget tissue) and kidney (target tissue) of Big Blue rats. Groups of six male rats were gavaged with 0, 0.1, 1.0 and 10.0 mg AA/kg body weight five times/week for 3 months. The rats were sacrificed 1 day after the final treatment, and the livers and kidneys were isolated. DNA adduct formation was analyzed by 32 P-postlabeling and mutant frequency (MF) was determined using the λ Select-cII Mutation Detection System. Three major adducts (7-[deoxyadenosin-N 6 -yl]-aristolactam I, 7-[deoxyadenosin-N 6 -yl]-aristolactam II and 7-[deoxyguanosin-N 2 -yl]-aristolactam I) were identified. There were strong linear dose-responses for AA-induced DNA adducts in treated rats, ranging from 25 to 1967 adducts/10 8 nucleotides in liver and 95-4598 adducts/10 8 nucleotides in kidney. A similar trend of dose-responses for mutation induction also was found, the MFs ranging from 37 to 666 x 10 -6 in liver compared with the MFs of 78-1319 x 10 -6 that we previously reported for the kidneys of AA-treated rats. Overall, kidneys had at least two-fold higher levels of DNA adducts and MF than livers. Sequence analysis of the cII mutants revealed that there was a statistically significant difference between the mutation spectra in both kidney and liver of AA-treated and control rats, but there was no significant difference between the mutation spectra in AA-treated livers and kidneys. A:T → T:A transversion was the predominant mutation in AA-treated rats; whereas G:C → A:T transition was the main type of mutation in control rats. These results indicate that the AA treatment that eventually

  1. Measurement of oxidatively generated base damage in cellular DNA

    Energy Technology Data Exchange (ETDEWEB)

    Cadet, Jean, E-mail: jean.cadet@cea.fr [Laboratoire ' Lesions des Acides Nucleiques' , SCIB-UMR-E no3 (CEA/UJF), FRE CNRS 3200, Departement de Recherche Fondamentale sur la Matiere Condensee, CEA/Grenoble, F-38054 Grenoble Cedex 9 (France); Douki, Thierry; Ravanat, Jean-Luc [Laboratoire ' Lesions des Acides Nucleiques' , SCIB-UMR-E no3 (CEA/UJF), FRE CNRS 3200, Departement de Recherche Fondamentale sur la Matiere Condensee, CEA/Grenoble, F-38054 Grenoble Cedex 9 (France)

    2011-06-03

    This survey focuses on the critical evaluation of the main methods that are currently available for monitoring single and complex oxidatively generated damage to cellular DNA. Among chromatographic methods, HPLC-ESI-MS/MS and to a lesser extent HPLC-ECD which is restricted to a few electroactive nucleobases and nucleosides are appropriate for measuring the formation of single and clustered DNA lesions. Such methods that require optimized protocols for DNA extraction and digestion are sensitive enough for measuring base lesions formed under conditions of severe oxidative stress including exposure to ionizing radiation, UVA light and high intensity UVC laser pulses. In contrast application of GC-MS and HPLC-MS methods that are subject to major drawbacks have been shown to lead to overestimated values of DNA damage. Enzymatic methods that are based on the use of DNA repair glycosylases in order to convert oxidized bases into strand breaks are suitable, even if they are far less specific than HPLC methods, to deal with low levels of single modifications. Several other methods including immunoassays and {sup 32}P-postlabeling methods that are still used suffer from drawbacks and therefore are not recommended. Another difficult topic is the measurement of oxidatively generated clustered DNA lesions that is currently achieved using enzymatic approaches and that would necessitate further investigations.

  2. Quartz crystal microbalance detection of DNA single-base mutation based on monobase-coded cadmium tellurium nanoprobe.

    Science.gov (United States)

    Zhang, Yuqin; Lin, Fanbo; Zhang, Youyu; Li, Haitao; Zeng, Yue; Tang, Hao; Yao, Shouzhuo

    2011-01-01

    A new method for the detection of point mutation in DNA based on the monobase-coded cadmium tellurium nanoprobes and the quartz crystal microbalance (QCM) technique was reported. A point mutation (single-base, adenine, thymine, cytosine, and guanine, namely, A, T, C and G, mutation in DNA strand, respectively) DNA QCM sensor was fabricated by immobilizing single-base mutation DNA modified magnetic beads onto the electrode surface with an external magnetic field near the electrode. The DNA-modified magnetic beads were obtained from the biotin-avidin affinity reaction of biotinylated DNA and streptavidin-functionalized core/shell Fe(3)O(4)/Au magnetic nanoparticles, followed by a DNA hybridization reaction. Single-base coded CdTe nanoprobes (A-CdTe, T-CdTe, C-CdTe and G-CdTe, respectively) were used as the detection probes. The mutation site in DNA was distinguished by detecting the decreases of the resonance frequency of the piezoelectric quartz crystal when the coded nanoprobe was added to the test system. This proposed detection strategy for point mutation in DNA is proved to be sensitive, simple, repeatable and low-cost, consequently, it has a great potential for single nucleotide polymorphism (SNP) detection. 2011 © The Japan Society for Analytical Chemistry

  3. Correlated Mutation in the Evolution of Catalysis in Uracil DNA Glycosylase Superfamily

    Science.gov (United States)

    Xia, Bo; Liu, Yinling; Guevara, Jose; Li, Jing; Jilich, Celeste; Yang, Ye; Wang, Liangjiang; Dominy, Brian N.; Cao, Weiguo

    2017-04-01

    Enzymes in Uracil DNA glycosylase (UDG) superfamily are essential for the removal of uracil. Family 4 UDGa is a robust uracil DNA glycosylase that only acts on double-stranded and single-stranded uracil-containing DNA. Based on mutational, kinetic and modeling analyses, a catalytic mechanism involving leaving group stabilization by H155 in motif 2 and water coordination by N89 in motif 3 is proposed. Mutual Information analysis identifies a complexed correlated mutation network including a strong correlation in the EG doublet in motif 1 of family 4 UDGa and in the QD doublet in motif 1 of family 1 UNG. Conversion of EG doublet in family 4 Thermus thermophilus UDGa to QD doublet increases the catalytic efficiency by over one hundred-fold and seventeen-fold over the E41Q and G42D single mutation, respectively, rectifying the strong correlation in the doublet. Molecular dynamics simulations suggest that the correlated mutations in the doublet in motif 1 position the catalytic H155 in motif 2 to stabilize the leaving uracilate anion. The integrated approach has important implications in studying enzyme evolution and protein structure and function.

  4. Highly sensitive, non-invasive detection of colorectal cancer mutations using single molecule, third generation sequencing

    Directory of Open Access Journals (Sweden)

    Giancarlo Russo

    2015-12-01

    We present the first study that applies the high read accuracy and depth of single molecule, real time, circular consensus sequencing (SMRT-CCS to the detection of mutations in stool DNA in order to provide a non-invasive, sensitive and accurate test for CRC. In stool DNA isolated from patients diagnosed with adenocarcinoma, we are able to detect mutations at frequencies below 0.5% with no false positives. This approach establishes a foundation for a non-invasive, highly sensitive assay to screen the population for CRC and the early stage adenomas that lead to CRC.

  5. Detection of Hepatitis B Virus M204I Mutation by Quantum Dot-Labeled DNA Probe

    Directory of Open Access Journals (Sweden)

    Cheng Zhang

    2017-04-01

    Full Text Available Quantum dots (QDs are semiconductor nanoparticles with a diameter of less than 10 nm, which have been widely used as fluorescent probes in biochemical analysis and vivo imaging because of their excellent optical properties. Sensitive and convenient detection of hepatitis B virus (HBV gene mutations is important in clinical diagnosis. Therefore, we developed a sensitive, low-cost and convenient QDs-mediated fluorescent method for the detection of HBV gene mutations in real serum samples from chronic hepatitis B (CHB patients who had received lamivudine or telbivudine antiviral therapy. We also evaluated the efficiency of this method for the detection of drug-resistant mutations compared with direct sequencing. In CHB, HBV DNA from the serum samples of patients with poor response or virological breakthrough can be hybridized to probes containing the M204I mutation to visualize fluorescence under fluorescence microscopy, where fluorescence intensity is related to the virus load, in our method. At present, the limits of the method used to detect HBV genetic variations by fluorescence quantum dots is 103 IU/mL. These results show that QDs can be used as fluorescent probes to detect viral HBV DNA polymerase gene variation, and is a simple readout system without complex and expensive instruments, which provides an attractive platform for the detection of HBV M204I mutation.

  6. Familial mild hyperglycemia associated with a novel ABCC8-V84I mutation within three generations

    DEFF Research Database (Denmark)

    Gonsorcikova, Lucie; Vaxillaire, Martine; Pruhova, Stepanka

    2011-01-01

    of the young genes (MODY1-4 and 6), we identified a novel ABCC8 V84I mutation, which segregated with autosomal dominant transmission of mild hyperglycemia within three generations. This mutation that is located in a conserved area of transmembrane domain TMD0 seems to be a rare cause of clinical phenotype...

  7. Systemic oxidatively generated DNA/RNA damage in clinical depression

    DEFF Research Database (Denmark)

    Jorgensen, Anders; Krogh, Jesper; Miskowiak, Kamilla

    2013-01-01

    oxidatively generated DNA and RNA damage, 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo), respectively, were determined in healthy controls (N=28), moderately depressed, non-medicated patients (N=26) and severely depressed patients eligible for electroconvulsive therapy...

  8. Simulations Using Random-Generated DNA and RNA Sequences

    Science.gov (United States)

    Bryce, C. F. A.

    1977-01-01

    Using a very simple computer program written in BASIC, a very large number of random-generated DNA or RNA sequences are obtained. Students use these sequences to predict complementary sequences and translational products, evaluate base compositions, determine frequencies of particular triplet codons, and suggest possible secondary structures.…

  9. DNA-based random number generation in security circuitry.

    Science.gov (United States)

    Gearheart, Christy M; Arazi, Benjamin; Rouchka, Eric C

    2010-06-01

    DNA-based circuit design is an area of research in which traditional silicon-based technologies are replaced by naturally occurring phenomena taken from biochemistry and molecular biology. This research focuses on further developing DNA-based methodologies to mimic digital data manipulation. While exhibiting fundamental principles, this work was done in conjunction with the vision that DNA-based circuitry, when the technology matures, will form the basis for a tamper-proof security module, revolutionizing the meaning and concept of tamper-proofing and possibly preventing it altogether based on accurate scientific observations. A paramount part of such a solution would be self-generation of random numbers. A novel prototype schema employs solid phase synthesis of oligonucleotides for random construction of DNA sequences; temporary storage and retrieval is achieved through plasmid vectors. A discussion of how to evaluate sequence randomness is included, as well as how these techniques are applied to a simulation of the random number generation circuitry. Simulation results show generated sequences successfully pass three selected NIST random number generation tests specified for security applications.

  10. Gamma radiation-induced heritable mutations at repetitive DNA loci in out-bred mice

    International Nuclear Information System (INIS)

    Somers, C.M.; Sharma, R.; Quinn, J.S.; Boreham, D.R.

    2004-01-01

    Recent studies have shown that expanded-simple-tandem-repeat (ESTR) DNA loci are efficient genetic markers for detecting radiation-induced germ line mutations in mice. Dose responses following irradiation, however, have only been characterized in a small number of inbred mouse strains, and no studies have applied Esters to examine potential modifiers of radiation risk, such as adaptive response. We gamma-irradiated groups of male out-bred Swiss-Webster mice with single acute doses of 0.5 and 1.0 Gy, and compared germ line mutation rates at ESTR loci to a sham-irradiated control. To test for evidence of adaptive response we treated a third group with a total dose of 1.1 Gy that was fractionated into a 0.1 Gy adapting dose, followed by a challenge dose of 1.0 Gy 24 h later. Paternal mutation rates were significantly elevated above the control in the 0.5 Gy (2.8-fold) and 1.0 Gy (3.0-fold) groups, but were similar to each other despite the difference in radiation dose. The doubling dose for paternal mutation induction was 0.26 Gy (95% CI = 0.14-0.51 Gy). Males adapted with a 0.1 Gy dose prior to a 1.0 Gy challenge dose had mutation rates that were not significantly elevated above the control, and were 43% reduced compared to those receiving single doses. We conclude that pre-meiotic male germ cells in out-bred Swiss-Webster mice are sensitive to ESTR mutations induced by acute doses of ionizing radiation, but mutation induction may become saturated at a lower dose than in some strains of inbred mice. Reduced mutation rates in the adapted group provide intriguing evidence for suppression of ESTR mutations in the male germline through adaptive response. Repetitive DNA markers may be useful tools for exploration of biological factors affecting the probability of heritable mutations caused by low-dose ionizing radiation exposure. The biological significance of ESTR mutations in terms of radiation risk assessment, however, is still undetermined

  11. Combined mutation and copy-number variation detection by targeted next-generation sequencing in uveal melanoma.

    Science.gov (United States)

    Smit, Kyra N; van Poppelen, Natasha M; Vaarwater, Jolanda; Verdijk, Robert; van Marion, Ronald; Kalirai, Helen; Coupland, Sarah E; Thornton, Sophie; Farquhar, Neil; Dubbink, Hendrikus-Jan; Paridaens, Dion; de Klein, Annelies; Kiliç, Emine

    2018-01-12

    Uveal melanoma is a highly aggressive cancer of the eye, in which nearly 50% of the patients die from metastasis. It is the most common type of primary eye cancer in adults. Chromosome and mutation status have been shown to correlate with the disease-free survival. Loss of chromosome 3 and inactivating mutations in BAP1, which is located on chromosome 3, are strongly associated with 'high-risk' tumors that metastasize early. Other genes often involved in uveal melanoma are SF3B1 and EIF1AX, which are found to be mutated in intermediate- and low-risk tumors, respectively. To obtain genetic information of all genes in one test, we developed a targeted sequencing method that can detect mutations in uveal melanoma genes and chromosomal anomalies in chromosome 1, 3, and 8. With as little as 10 ng DNA, we obtained enough coverage on all genes to detect mutations, such as substitutions, deletions, and insertions. These results were validated with Sanger sequencing in 28 samples. In >90% of the cases, the BAP1 mutation status corresponded to the BAP1 immunohistochemistry. The results obtained in the Ion Torrent single-nucleotide polymorphism assay were confirmed with several other techniques, such as fluorescence in situ hybridization, multiplex ligation-dependent probe amplification, and Illumina SNP array. By validating our assay in 27 formalin-fixed paraffin-embedded and 43 fresh uveal melanomas, we show that mutations and chromosome status can reliably be obtained using targeted next-generation sequencing. Implementing this technique as a diagnostic pathology application for uveal melanoma will allow prediction of the patients' metastatic risk and potentially assess eligibility for new therapies.Modern Pathology advance online publication, 12 January 2018; doi:10.1038/modpathol.2017.187.

  12. Antibiotic resistance in Pseudomonas aeruginosa strains with increased mutation frequency due to inactivation of the DNA oxidative repair system.

    Science.gov (United States)

    Mandsberg, L F; Ciofu, O; Kirkby, N; Christiansen, L E; Poulsen, H E; Høiby, N

    2009-06-01

    The chronic Pseudomonas aeruginosa infection of the lungs of cystic fibrosis (CF) patients is characterized by the biofilm mode of growth and chronic inflammation dominated by polymorphonuclear leukocytes (PMNs). A high percentage of P. aeruginosa strains show high frequencies of mutations (hypermutators [HP]). P. aeruginosa is exposed to oxygen radicals, both those generated by its own metabolism and especially those released by a large number of PMNs in response to the chronic CF lung infection. Our work therefore focused on the role of the DNA oxidative repair system in the development of HP and antibiotic resistance. We have constructed and characterized mutT, mutY, and mutM mutants in P. aeruginosa strain PAO1. The mutT and mutY mutants showed 28- and 7.5-fold increases in mutation frequencies, respectively, over that for PAO1. These mutators had more oxidative DNA damage (higher levels of 7,8-dihydro-8-oxodeoxyguanosine) than PAO1 after exposure to PMNs, and they developed resistance to antibiotics more frequently. The mechanisms of resistance were increased beta-lactamase production and overexpression of the MexCD-OprJ efflux-pump. Mutations in either the mutT or the mutY gene were found in resistant HP clinical isolates from patients with CF, and complementation with wild-type genes reverted the phenotype. In conclusion, oxidative stress might be involved in the development of resistance to antibiotics. We therefore suggest the possible use of antioxidants for CF patients to prevent the development of antibiotic resistance.

  13. Profiling of Somatic Mutations in Phaeochromocytoma and Paraganglioma by Targeted Next Generation Sequencing Analysis

    Directory of Open Access Journals (Sweden)

    Andrea Luchetti

    2015-01-01

    Full Text Available At least 12 genes (FH, HIF2A, MAX, NF1, RET, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, and VHL have been implicated in inherited predisposition to phaeochromocytoma (PCC, paraganglioma (PGL, or head and neck paraganglioma (HNPGL and a germline mutation may be detected in more than 30% of cases. Knowledge of somatic mutations contributing to PCC/PGL/HNPGL pathogenesis has received less attention though mutations in HRAS, HIF2A, NF1, RET, and VHL have been reported. To further elucidate the role of somatic mutation in PCC/PGL/HNPGL tumourigenesis, we employed a next generation sequencing strategy to analyse “mutation hotspots” in 50 human cancer genes. Mutations were identified for HRAS (c.37G>C; p.G13R and c.182A>G; p.Q61R in 7.1% (6/85; for BRAF (c.1799T>A; p.V600E in 1.2% (1/85 of tumours; and for TP53 (c.1010G>A; p.R337H in 2.35% (2/85 of cases. Twenty-one tumours harboured mutations in inherited PCC/PGL/HNPGL genes and no HRAS, BRAF, or TP53 mutations occurred in this group. Combining our data with previous reports of HRAS mutations in PCC/PGL we find that the mean frequency of HRAS/BRAF mutations in sporadic PCC/PGL is 8.9% (24/269 and in PCC/PGL with an inherited gene mutation 0% (0/148 suggesting that HRAS/BRAF mutations and inherited PCC/PGL genes mutations might be mutually exclusive. We report the first evidence for BRAF mutations in the pathogenesis of PCC/PGL/HNPGL.

  14. 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.

  15. Mutation and Methylation Analysis of the Chromodomain-Helicase-DNA Binding 5 Gene in Ovarian Cancer

    Directory of Open Access Journals (Sweden)

    Kylie L. Gorringe

    2008-11-01

    Full Text Available Chromodomain, helicase, DNA binding 5 (CHD5 is a member of a subclass of the chromatin remodeling Swi/Snf proteins and has recently been proposed as a tumor suppressor in a diverse range of human cancers. We analyzed all 41 coding exons of CHD5 for somatic mutations in 123 primary ovarian cancers as well as 60 primary breast cancers using high-resolution melt analysis. We also examined methylation of the CHD5 promoter in 48 ovarian cancer samples by methylation-specific single-stranded conformation polymorphism and bisulfite sequencing. In contrast to previous studies, no mutations were identified in the breast cancers, but somatic heterozygous missense mutations were identified in 3 of 123 ovarian cancers. We identified promoter methylation in 3 of 45 samples with normal CHD5 and in 2 of 3 samples with CHD5 mutation, suggesting these tumors may have biallelic inactivation of CHD5. Hemizygous copy number loss at CHD5 occurred in 6 of 85 samples as assessed by single nucleotide polymorphism array. Tumors with CHD5 mutation or methylation were more likely to have mutation of KRAS or BRAF (P = .04. The aggregate frequency of CHD5 haploinsufficiency or inactivation is 16.2% in ovarian cancer. Thus, CHD5 may play a role as a tumor suppressor gene in ovarian cancer; however, it is likely that there is another target of the frequent copy number neutral loss of heterozygosity observed at 1p36.

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

    Science.gov (United States)

    Tatton-Brown, Katrina; Seal, Sheila; Ruark, Elise; Harmer, Jenny; Ramsay, Emma; del Vecchio Duarte, Silvana; Zachariou, Anna; Hanks, Sandra; O’Brien, Eleanor; Aksglaede, Lise; Baralle, Diana; Dabir, Tabib; Gener, Blanca; Goudie, David; Homfray, Tessa; Kumar, Ajith; Pilz, Daniela T; Selicorni, Angelo; Temple, I Karen; Van Maldergem, Lionel; Yachelevich, Naomi; van Montfort, Robert; Rahman, Nazneen

    2014-01-01

    Overgrowth disorders are a heterogeneous group of conditions characterised by increased growth parameters and variable other clinical features, such as intellectual disability and facial dysmorphism1. To identify novel causes of human overgrowth we performed exome sequencing in 10 proband-parent trios and detected two de novo DNMT3A mutations. We identified 11 additional de novo mutations through DNMT3A sequencing of a further 142 individuals with overgrowth. The mutations were all located in functional DNMT3A domains and protein modelling suggests they interfere with domain-domain interactions and histone binding. No similar mutations were present in 1000 UK population controls (13/152 vs 0/1000; P<0.0001). Mutation carriers had a distinctive facial appearance, intellectual disability and increased height. DNMT3A encodes a key methyltransferase essential for establishing the methylation imprint in embryogenesis and is commonly somatically mutated in acute myeloid leukaemia2-4. Thus DNMT3A joins an emerging group of epigenetic DNA and histone modifying genes associated with both developmental growth disorders and haematological malignancies5. PMID:24614070

  17. 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.

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

    International Nuclear Information System (INIS)

    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

  19. A straightforward assay to evaluate DNA integrity and optimize next-generation sequencing for clinical diagnosis in oncology.

    Science.gov (United States)

    Bettoni, Fabiana; Koyama, Fernanda Christtanini; de Avelar Carpinetti, Paola; Galante, Pedro Alexandre Favoretto; Camargo, Anamaria Aranha; Asprino, Paula Fontes

    2017-12-01

    Next generation sequencing (NGS) has become an informative tool to guide cancer treatment and conduce a personalized approach in oncology. The biopsy collected for pathologic analysis is usually stored as formalin-fixed paraffin-embedded (FFPE) blocks and then availed for molecular diagnostic, resulting in DNA molecules that are invariably fragmented and chemically modified. In an attempt to improve NGS based diagnostics in oncology we developed a straightforward DNA integrity assessment assay based on qPCR, defining clear parameters to whether NGS sequencing results is accurate or when it should be analyzed with caution. We performed DNA extraction from 12 tumor samples from diverse tissues and accessed DNA integrity by straightforward qPCR assays. In order to perform a cancer panel NGS sequencing, DNA library preparation was performed using RNA capture baits. Reads were aligned to the reference human genome and mutation calls were further validated by Sanger sequencing. Results obtained by the DNA integrity assays correlated to the efficiency of the pre-capture library preparation in up to 0.94 (Pearson's test). Moreover, sequencing results showed that poor integrity DNA leads to high rates of false positive mutation calls, specially C:G>T:A and C:G>A:T. Poor quality FFPE DNA samples are prone to generating false positive mutation calls. These are especially perilous in cases in which subclonal populations are expected, such as in advance disease, since it could lead clinicians to erroneous conclusions and equivocated conduct. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  20. Effect of transforming DNA on growth and frequency of mutation of Streptococcus pneumoniae.

    Science.gov (United States)

    Grist, R W; Butler, L O

    1983-01-01

    We studied the effect of the presence of homologous transforming DNA on the growth of several transformable strains of Streptococcus pneumoniae and on the frequency of mutation of these strains to various antibiotic resistances. We observed no effect on growth until the strains became competent, when growth was depressed. At the end of the competence period, some strains showed recovery to varying degrees, whereas others showed evidence of cell death. Growth was also depressed by the presence of DNA from Escherichia coli, indicating that recombination was not likely to be the cause of the observed effect. Furthermore, cell death was not caused by the induction of a prophage. Several of the strains showed increased mutation frequencies during the competence period, although treatment with E. coli DNA gave no such effect, indicating that the mutagenesis was due to recombination. We observed no mutagenesis due to UV irradiation of the strains. The possibility that integration of the transforming DNA may produce lesions which induce error-prone repair is discussed. Furthermore, a strain that showed no mutability by transforming DNA, indicating the presence of a more efficient repair system, gave evidence of producing higher amounts of the hex system when competent, and the possible relationship between these properties is discussed.

  1. Determination of mutated genes in the presence of wild-type DNA by using molecular beacons as probe

    Science.gov (United States)

    Zhang, Yonghua; Ai, Junjie; Gu, Qiaorong; Gao, Qiang; Qi, Honglan; Zhang, Chengxiao

    2017-03-01

    Low-abundance mutations in the presence of wild-type DNA can be determined using molecular beacon (MB) as probe. A MB is generally used as DNA probe because it can distinguish single-base mismatched target DNA from fully matched target DNA. However, the probe can not determine low-abundance mutations in the presence of wild-type DNA. In this study, this limitation is addressed by enhancing the stability of unpaired base-containing dsDNA with a hydrogen-bonding ligand, which was added after hybridization of the MB to the target DNA. The ligand formed hydrogen bonds with unpaired bases and stabilized the unpaired base-containing dsDNA if target DNA is mutated one. As a result, more MBs were opened by the mutant genes in the presence of the ligand and a further increase in the fluorescence intensity was obtained. By contrast, fluorescence intensity did not change if target DNA is wild-type one. Consequent increase in the fluorescence intensity of the MB was regarded as a signal derived from mutant genes. The proposed method was applied in synthetic template systems to determine point mutation in DNA obtained from PCR analysis. The method also allows rapid and simple discrimination of a signal if it is originated in the presence of mutant gene or alternatively by a lower concentration of wild gene.

  2. Identification of unique repeated patterns, location of mutation in DNA finger printing using artificial intelligence technique.

    Science.gov (United States)

    Mukunthan, B; Nagaveni, N

    2014-01-01

    In genetic engineering, conventional techniques and algorithms employed by forensic scientists to assist in identification of individuals on the basis of their respective DNA profiles involves more complex computational steps and mathematical formulae, also the identification of location of mutation in a genomic sequence in laboratories is still an exigent task. This novel approach provides ability to solve the problems that do not have an algorithmic solution and the available solutions are also too complex to be found. The perfect blend made of bioinformatics and neural networks technique results in efficient DNA pattern analysis algorithm with utmost prediction accuracy.

  3. 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

  4. 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

  5. Detection of coding microsatellite frameshift mutations in DNA mismatch repair-deficient mouse intestinal tumors.

    Science.gov (United States)

    Woerner, Stefan M; Tosti, Elena; Yuan, Yan P; Kloor, Matthias; Bork, Peer; Edelmann, Winfried; Gebert, Johannes

    2015-11-01

    Different DNA mismatch repair (MMR)-deficient mouse strains have been developed as models for the inherited cancer predisposing Lynch syndrome. It is completely unresolved, whether coding mononucleotide repeat (cMNR) gene mutations in these mice can contribute to intestinal tumorigenesis and whether MMR-deficient mice are a suitable molecular model of human microsatellite instability (MSI)-associated intestinal tumorigenesis. A proof-of-principle study was performed to identify mouse cMNR-harboring genes affected by insertion/deletion mutations in MSI murine intestinal tumors. Bioinformatic algorithms were developed to establish a database of mouse cMNR-harboring genes. A panel of five mouse noncoding mononucleotide markers was used for MSI classification of intestinal matched normal/tumor tissues from MMR-deficient (Mlh1(-/-) , Msh2(-/-) , Msh2(LoxP/LoxP) ) mice. cMNR frameshift mutations of candidate genes were determined by DNA fragment analysis. Murine MSI intestinal tumors but not normal tissues from MMR-deficient mice showed cMNR frameshift mutations in six candidate genes (Elavl3, Tmem107, Glis2, Sdccag1, Senp6, Rfc3). cMNRs of mouse Rfc3 and Elavl3 are conserved in type and length in their human orthologs that are known to be mutated in human MSI colorectal, endometrial and gastric cancer. We provide evidence for the utility of a mononucleotide marker panel for detection of MSI in murine tumors, the existence of cMNR instability in MSI murine tumors, the utility of mouse subspecies DNA for identification of polymorphic repeats, and repeat conservation among some orthologous human/mouse genes, two of them showing instability in human and mouse MSI intestinal tumors. MMR-deficient mice hence are a useful molecular model system for analyzing MSI intestinal carcinogenesis. © 2014 Wiley Periodicals, Inc.

  6. 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...

  7. DNA stabilization by the upregulation of estrogen signaling in BRCA gene mutation carriers

    Directory of Open Access Journals (Sweden)

    Suba Z

    2015-05-01

    Full Text Available Zsuzsanna Suba Surgical and Molecular Tumor Pathology Centre, National Institute of Oncology, Budapest, Hungary Abstract: Currently available scientific evidence erroneously suggests that mutagenic weakness or loss of the BRCA1/2 genes may liberate the proliferative effects of estrogen signaling, which provokes DNA damage and genomic instability. Conversely, BRCA mutation seems to be an imbalanced defect, crudely inhibiting the upregulation of estrogen receptor expression and liganded transcriptional activity, whereas estrogen receptor-repressor functions become predominant. In BRCA-proficient cases, estrogen signaling orchestrates the activity of cell proliferation and differentiation with high safety, while upregulating the expression and DNA-stabilizing impact of BRCA genes. In turn, BRCA proteins promote estrogen signaling by proper estrogen synthesis via CYP19 gene regulation and by induction of the appropriate expression and transcriptional activity of estrogen receptors. In this exquisitely organized regulatory system, the dysfunction of each player may jeopardize genome stability and lead to severe chronic diseases, such as cancer development. Female organs, such as breast, endometrium, and ovary, exhibiting regular cyclic proliferative activity are particularly vulnerable in case of disturbances in either estrogen signaling or BRCA-mediated DNA repair. BRCA mutation carrier women may apparently be healthy or exhibit clinical signs of deficient estrogen signaling in spite of hyperestrogenism. Even women who enjoy sufficient compensatory DNA-defending activities are at risk of tumor development because many endogenous and environmental factors may jeopardize the mechanisms of extreme compensatory processes. Natural estrogens have numerous benefits in tumor prevention and therapy even in BRCA mutation carriers. There are no toxic effects even in sky-high doses and all physiologic cellular functions are strongly upregulated, while malignant

  8. Variability in Chromatin Architecture and Associated DNA Repair at Genomic Positions Containing Somatic Mutations.

    Science.gov (United States)

    Lim, Byungho; Mun, Jihyeob; Kim, Yong Sung; Kim, Seon-Young

    2017-06-01

    Dynamic chromatin structures result in differential chemical reactivity to mutational processes throughout the genome. To identify chromatin features responsible for mutagenesis, we compared chromatin architecture around single-nucleotide variants (SNV), insertion/deletions (indels), and their context-matched, nonmutated positions. We found epigenetic differences between genomic regions containing missense SNVs and those containing frameshift indels across multiple cancer types. Levels of active histone marks were higher around frameshift indels than around missense SNV, whereas repressive histone marks exhibited the reverse trend. Accumulation of repressive histone marks and nucleosomes distinguished mutated positions (both SNV and indels) from the context-matched, nonmutated positions, whereas active marks were associated with substitution- and cancer type-specific mutagenesis. We also explained mutagenesis based on genome maintenance mechanisms, including nucleotide excision repair (NER), mismatch repair (MMR), and DNA polymerase epsilon (POLE). Regional NER variation correlated strongly with chromatin features; NER machineries exhibited shifted or depleted binding around SNV, resulting in decreased NER at mutation positions, especially at sites of recurrent mutations. MMR-deficient tumors selectively acquired SNV in regions with high active histone marks, especially H3K36me3, whereas POLE-deficient tumors selectively acquired indels and SNV in regions with low active histone marks. These findings demonstrate the importance of fine-scaled chromatin structures and associated DNA repair mechanisms in mutagenesis. Cancer Res; 77(11); 2822-33. ©2017 AACR . ©2017 American Association for Cancer Research.

  9. cDNA library generation from ribonucleoprotein particles.

    Science.gov (United States)

    Rederstorff, Mathieu; Hüttenhofer, Alexander

    2011-02-01

    Most, if not all, known noncoding RNAs (ncRNAs) are associated with RNA binding proteins, thus forming ribonucleoprotein particles or RNPs. Here we describe a protocol for the generation of a specialized cDNA library from RNPs, thereby increasing the proportion of functional ncRNA species in the library. To that end, cellular extracts are fractionated on 10-30% glycerol gradients. Subsequently, RNP-derived ncRNAs are isolated and 3'-tailed by cytidine triphosphate and poly(A) polymerase; this is followed by 5' adapter ligation by T4 RNA ligase. Reverse transcription of ncRNAs into cDNAs is carried out with an oligo-d(G) anchor primer. The generated cDNA libraries are subsequently submitted to high-throughput sequencing. This RNP selection procedure increases the probability of the presence of biologically relevant ncRNA species in the library compared with libraries generation methods that use size-selected, protein-devoid ncRNAs. The protocol enables the generation of deep-sequencing-compatible cDNA libraries that code for functional ncRNAs within 1 week.

  10. Structure and Function of p53-DNA Complexes with Inactivation and Rescue Mutations: A Molecular Dynamics Simulation Study.

    Directory of Open Access Journals (Sweden)

    Balu Kamaraj

    Full Text Available The tumor suppressor protein p53 can lose its function upon DNA-contact mutations (R273C and R273H in the core DNA-binding domain. The activity can be restored by second-site suppressor or rescue mutations (R273C_T284R, R273H_T284R, and R273H_S240R. In this paper, we elucidate the structural and functional consequence of p53 proteins upon DNA-contact mutations and rescue mutations and the underlying mechanisms at the atomic level by means of molecular dynamics simulations. Furthermore, we also apply the docking approach to investigate the binding phenomena between the p53 protein and DNA upon DNA-contact mutations and rescue mutations. This study clearly illustrates that, due to DNA-contact mutants, the p53 structure loses its stability and becomes more rigid than the native protein. This structural loss might affect the p53-DNA interaction and leads to inhibition of the cancer suppression. Rescue mutants (R273C_T284R, R273H_T284R and R273H_S240R can restore the functional activity of the p53 protein upon DNA-contact mutations and show a good interaction between the p53 protein and a DNA molecule, which may lead to reactivate the cancer suppression function. Understanding the effects of p53 cancer and rescue mutations at the molecular level will be helpful for designing drugs for p53 associated cancer diseases. These drugs should be designed so that they can help to inhibit the abnormal function of the p53 protein and to reactivate the p53 function (cell apoptosis to treat human cancer.

  11. Oxidatively generated DNA/RNA damage in psychological stress states

    DEFF Research Database (Denmark)

    Jørgensen, Anders

    2013-01-01

    Both non-pathological psychological stress states and mental disorders are associated with molecular, cellular and epidemiological signs of accelerated aging. Oxidative stress on nucleic acids is a critical component of cellular and organismal aging, and a suggested pathogenic mechanism in several...... age-related somatic disorders. The overall aim of the PhD project was to investigate the relation between psychopathology, psychological stress, stress hormone secretion and oxidatively generated DNA and RNA damage, as measured by the urinary excretion of markers of whole-body DNA/RNA oxidation (8......-oxodG and 8-oxoGuo, respectively). The main hypothesis was that psychological stress states are associated with increased DNA/RNA damage from oxidation. In a study of 40 schizophrenia patients and 40 healthy controls matched for age and gender, we found that 8-oxodG/8-oxoGuo excretion was increased...

  12. DNA hypermethylation, Her-2/neu overexpression and p53 mutations in ovarian carcinoma.

    Science.gov (United States)

    Feng, Qinghua; Deftereos, Georgios; Hawes, Stephen E; Stern, Joshua E; Willner, Julia B; Swisher, Elizabeth M; Xi, Longfu; Drescher, Charles; Urban, Nicole; Kiviat, Nancy

    2008-11-01

    To define patterns of aberrant DNA methylation, p53 mutation and Her-2/neu overexpression in tissues from benign (n=29), malignant (n=100), and border line malignant ovaries (n=10), as compared to normal (n=68) ovarian tissues. Further, to explore the relationship between the presence of genetic and epigenetic abnormalities in ovarian cancers, and assess the association between epigenetic changes and clinical stage of malignancy at presentation and response to therapy. The methylation status of 23 genes that were previously reported associated with various epithelial malignancies was assessed in normal and abnormal ovarian tissues by methylation-specific PCR. The presence of p53 mutation (n=82 cases) and Her-2/neu overexpression (n=51 cases) were assessed by DNA sequencing and immunohistochemistry, respectively. Methylation of four genes (MINT31, HIC1, RASSF1, and CABIN1) was significantly associated with ovarian cancer but not other ovarian pathology. Her-2/neu overexpression was associated with aberrant methylation of three genes (MINT31, RASSF1, and CDH13), although aberrant methylation was not associated with p53 mutations. Methylation of RASSF1 and HIC1 was more frequent in early compared to late stage ovarian cancer, while methylation of CABIN1 and RASSF1 was associated with response to chemotherapy. DNA methylation of tumor suppressor genes is a frequent event in ovarian cancer, and in some cases is associated with Her-2/neu overexpression. Methylation of CABIN1 and RASSF1 may have the utility to predict response to therapy.

  13. Thermodynamic framework to assess low abundance DNA mutation detection by hybridization

    Science.gov (United States)

    Willems, Hanny; Jacobs, An; Hadiwikarta, Wahyu Wijaya; Venken, Tom; Valkenborg, Dirk; Van Roy, Nadine; Vandesompele, Jo; Hooyberghs, Jef

    2017-01-01

    The knowledge of genomic DNA variations in patient samples has a high and increasing value for human diagnostics in its broadest sense. Although many methods and sensors to detect or quantify these variations are available or under development, the number of underlying physico-chemical detection principles is limited. One of these principles is the hybridization of sample target DNA versus nucleic acid probes. We introduce a novel thermodynamics approach and develop a framework to exploit the specific detection capabilities of nucleic acid hybridization, using generic principles applicable to any platform. As a case study, we detect point mutations in the KRAS oncogene on a microarray platform. For the given platform and hybridization conditions, we demonstrate the multiplex detection capability of hybridization and assess the detection limit using thermodynamic considerations; DNA containing point mutations in a background of wild type sequences can be identified down to at least 1% relative concentration. In order to show the clinical relevance, the detection capabilities are confirmed on challenging formalin-fixed paraffin-embedded clinical tumor samples. This enzyme-free detection framework contains the accuracy and efficiency to screen for hundreds of mutations in a single run with many potential applications in molecular diagnostics and the field of personalised medicine. PMID:28542229

  14. Thermodynamic framework to assess low abundance DNA mutation detection by hybridization.

    Directory of Open Access Journals (Sweden)

    Hanny Willems

    Full Text Available The knowledge of genomic DNA variations in patient samples has a high and increasing value for human diagnostics in its broadest sense. Although many methods and sensors to detect or quantify these variations are available or under development, the number of underlying physico-chemical detection principles is limited. One of these principles is the hybridization of sample target DNA versus nucleic acid probes. We introduce a novel thermodynamics approach and develop a framework to exploit the specific detection capabilities of nucleic acid hybridization, using generic principles applicable to any platform. As a case study, we detect point mutations in the KRAS oncogene on a microarray platform. For the given platform and hybridization conditions, we demonstrate the multiplex detection capability of hybridization and assess the detection limit using thermodynamic considerations; DNA containing point mutations in a background of wild type sequences can be identified down to at least 1% relative concentration. In order to show the clinical relevance, the detection capabilities are confirmed on challenging formalin-fixed paraffin-embedded clinical tumor samples. This enzyme-free detection framework contains the accuracy and efficiency to screen for hundreds of mutations in a single run with many potential applications in molecular diagnostics and the field of personalised medicine.

  15. 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.

  16. 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.

  17. 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

  18. DNA Repair Domain Modeling Can Predict Cell Death and Mutation Frequency for Wide Range Spectrum of Radiation

    Science.gov (United States)

    Viger, Louise; Ponomarev, Artem L.; Plante, Ianik; Evain, Trevor; Penninckx, Sebastien; Blattnig, Steve R.; Costes, Sylvain V.

    2017-01-01

    Exploration missions to Mars and other destinations raise many questions about the health of astronauts. The continuous exposure of astronauts to galactic cosmic rays is one of the main concerns for long-term missions. Cosmic ionizing radiations are composed of different ions of various charges and energies notably, highly charged energy (HZE) particles. The HZE particles have been shown to be more carcinogenic than low-LET radiation, suggesting the severity of chromosomal aberrations induced by HZE particles is one possible explanation. However, most mathematical models predicting cell death and mutation frequency are based on directly fitting various HZE dose response and are in essence empirical approaches. In this work, we assume a simple biological mechanism to model DNA repair and use it to simultaneously explain the low- and high-LET response using the exact same fitting parameters. Our work shows that the geometrical position of DNA repair along tracks of heavy ions are sufficient to explain why high-LET particles can induce more death and mutations. Our model is based on assuming DNA double strand breaks (DSBs) are repaired within repair domain, and that any DSBs located within the same repair domain cluster into one repair unit, facilitating chromosomal rearrangements and increasing the probability of cell death. We introduced this model in 2014 using simplified microdosimetry profiles to predict cell death. In this work, we collaborated with NASA Johnson Space Center to generate more accurate microdosimetry profiles derived by Monte Carlo techniques, taking into account track structure of HZE particles and simulating DSBs in realistic cell geometry. We simulated 224 data points (D, A, Z, E) with the BDSTRACKS model, leading to a large coverage of LET from 10 to 2,400 keV/µm. This model was used to generate theoretical RBE for various particles and energies for both cell death and mutation frequencies. The RBE LET dependence is in agreement with

  19. 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.

  20. Substrate rescue of DNA polymerase β containing a catastrophic L22P mutation.

    Science.gov (United States)

    Kirby, Thomas W; Derose, Eugene F; Beard, William A; Shock, David D; Wilson, Samuel H; London, Robert E

    2014-04-15

    DNA polymerase (pol) β is a multidomain enzyme with two enzymatic activities that plays a central role in the overlapping base excision repair and single-strand break repair pathways. The high frequency of pol β variants identified in tumor-derived tissues suggests a possible role in the progression of cancer, making the determination of the functional consequences of these variants of interest. Pol β containing a proline substitution for leucine 22 in the lyase domain (LD), identified in gastric tumors, has been reported to exhibit severe impairment of both lyase and polymerase activities. Nuclear magnetic resonance (NMR) spectroscopic evaluations of both pol β and the isolated LD containing the L22P mutation demonstrate destabilization sufficient to result in LD-selective unfolding with minimal structural perturbations to the polymerase domain. Unexpectedly, addition of single-stranded or hairpin DNA resulted in partial refolding of the mutated lyase domain, both in isolation and for the full-length enzyme. Further, formation of an abortive ternary complex using Ca(2+) and a complementary dNTP indicates that the fraction of pol β(L22P) containing the folded LD undergoes conformational activation similar to that of the wild-type enzyme. Kinetic characterization of the polymerase activity of L22P pol β indicates that the L22P mutation compromises DNA binding, but nearly wild-type catalytic rates can be observed at elevated substrate concentrations. The organic osmolyte trimethylamine N-oxide (TMAO) is similarly able to induce folding and kinetic activation of both polymerase and lyase activities of the mutant. Kinetic data indicate synergy between the TMAO cosolvent and substrate binding. NMR data indicate that the effect of the DNA results primarily from interaction with the folded LD(L22P), while the effect of the TMAO results primarily from destabilization of the unfolded LD(L22P). These studies illustrate that substrate-induced catalytic activation of pol

  1. Molecular targets, DNA breakage, DNA repair: Their roles in mutation induction in mammalian germ cells

    Energy Technology Data Exchange (ETDEWEB)

    Sega, G.A.

    1989-01-01

    Variability in genetic sensitivity among different germ-cell stages in the mammal to various mutagens could be the result of how much chemical reaches the different stages, what molecular targets may be affected in the different stages and whether or not repair of lesions occurs. Several chemicals have been found to bind very strongly to protamine in late-spermatid and early-spermatozoa stages in the mouse. The chemicals also produce their greatest genetic damage in these same germ-cell stages. While chemical binding to DNA has not been correlated with the level of induced genetic damage, DNA breakage in the sensitive stages has been shown to increase. This DNA breakage is believed to indirectly result from chemical binding to sulfhydryl groups in protamine which prevents normal chromatin condensation within the sperm nucleus. 22 refs., 5 figs.

  2. Association of EGFR L858R Mutation in Circulating Free DNA With Survival in the EURTAC Trial.

    Science.gov (United States)

    Karachaliou, Niki; Mayo-de las Casas, Clara; Queralt, Cristina; de Aguirre, Itziar; Melloni, Boris; Cardenal, Felipe; Garcia-Gomez, Ramon; Massuti, Bartomeu; Sánchez, José Miguel; Porta, Ruth; Ponce-Aix, Santiago; Moran, Teresa; Carcereny, Enric; Felip, Enriqueta; Bover, Isabel; Insa, Amelia; Reguart, Noemí; Isla, Dolores; Vergnenegre, Alain; de Marinis, Filippo; Gervais, Radj; Corre, Romain; Paz-Ares, Luis; Morales-Espinosa, Daniela; Viteri, Santiago; Drozdowskyj, Ana; Jordana-Ariza, Núria; Ramirez-Serrano, Jose Luis; Molina-Vila, Miguel Angel; Rosell, Rafael

    2015-05-01

    The EURTAC trial demonstrated the greater efficacy of erlotinib compared with chemotherapy for the first-line treatment of European patients with advanced non-small-cell lung cancer (NSCLC) harboring oncogenic epidermal growth factor receptor (EGFR) mutations (exon 19 deletion or L858R mutation in exon 21) in tumor tissue. To assess the feasibility of using circulating free DNA (cfDNA) from blood samples as a surrogate for tumor biopsy for determining EGFR mutation status and to correlate EGFR mutations in cfDNA with outcome. This prespecified analysis was a secondary objective of the EURTAC trial using patients included in the EURTAC trial from 2007 to 2011 with available baseline serum or plasma samples. Patients had advanced NSCLC, oncogenic EGFR mutations in the tumor, and no prior chemotherapy for metastatic disease and were treated with erlotinib or chemotherapy. EGFR mutations were examined in cfDNA isolated from 97 baseline blood samples by our novel peptide nucleic acid-mediated 5´ nuclease real-time polymerase chain reaction (TaqMan) assay. Overall survival (OS), progression-free survival (PFS), and response to therapy were correlated with type of EGFR mutations in cfDNA. In samples from 76 of 97 (78%) patients with usable blood samples, EGFR mutations in cfDNA were detected. Median OS was shorter in patients with the L858R mutation in cfDNA than in those with the exon 19 deletion (13.7 [95% CI, 7.1-17.7] vs 30.0 [95% CI, 19.3-37.7] months; P < .001). Univariate analyses of patients with EGFR mutations in cfDNA identified the L858R mutation in tumor tissue or in cfDNA as a marker of shorter OS (hazard ratio [HR], 2.70 [95% CI, 1.60-4.56]; P < .001) and PFS (HR, 2.04 [95% CI, 1.20-3.48]; P = .008). For patients with the L858R mutation in tissue, median OS was 13.7 (95% CI, 7.1-17.7) months for patients with the L858R mutation in cfDNA and 27.7 (95% CI, 16.1-46.2) months for those in whom the mutation was not detected in cfDNA (HR, 2.22 [95% CI

  3. A novel mutation in homeobox DNA binding domain of HOXC13 gene underlies pure hair and nail ectodermal dysplasia (ECTD9) in a Pakistani family.

    Science.gov (United States)

    Khan, Anwar Kamal; Muhammad, Noor; Aziz, Abdul; Khan, Sher Alam; Shah, Khadim; Nasir, Abdul; Khan, Muzammil Ahmad; Khan, Saadullah

    2017-04-12

    Pure hair and nail ectodermal dysplasia (PHNED) is a congenital disorder of hair abnormalities and nail dysplasia. Both autosomal recessive and dominant inheritance fashion of PHNED occurs. In literature, to date, five different forms of PHNED have been reported at molecular level, having three genes known and two loci with no gene yet. In this study, a four generations consanguineous family of Pakistani origin with autosomal recessive PHNED was investigated. Affected members exhibited PHNED phenotypes with involvement of complete hair loss and nail dysplasia. To screen for mutation in the genes (HOXC13, KRT74, KRT85), its coding exons and exons-intron boundaries were sequenced. The 3D models of normal and mutated HOXC13 were predicted by using homology modeling. Through investigating the family to known loci, the family was mapped to ectodermal dysplasia 9 (ECTD9) loci with genetic address of 12q13.13. Mutation screening revealed a novel missense mutation (c.929A > C; p.Asn310Thr) in homeobox DNA binding domain of HOXC13 gene in affected members of the family. Due to mutation, loss of hydrogen bonding and difference in potential energy occurs, which may resulting in alteration of protein function. This is the first mutation reported in homeodomain, while 5 th mutation reported in HOXC13 gene causing PHNED.

  4. 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...

  5. 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

    . 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...

  6. MLPA and cDNA analysis improves COL4A5 mutation detection in X-linked Alport syndrome

    DEFF Research Database (Denmark)

    Hertz, JM; Juncker, I; Marcussen, N

    2008-01-01

    for 10-15% of mutations. We have established a method for mutation analysis of COL4A5 based on reverse transcriptase-polymerase chain reaction analysis of mRNA from cultured skin fibroblasts and multiplex ligation-dependent probe amplification (MLPA) on genomic DNA. One advantage of using skin biopsies...

  7. DNA and cancer biology: role in radiation and drug sensitivity, carcinogenesis and mutations

    International Nuclear Information System (INIS)

    Yielding, K.L.

    1974-01-01

    The DNA excision repair mechanism is an important factor in the resistance exhibited by tumor cells toward both x rays and alkylating agents as demonstrated by the fact that the chemical alterations to cellular DNA caused by these agents are substrates for the repair enzymes. Furthermore, experiments performed in our laboratory demonstrate that: (a) tumor sensitivity to alkylating agents and x-ray can be increased by inhibition of the repair process, and (b) there is a suggestion that this sensitization can be achieved with some degree of selectivity, thereby improving the balance of sensitivites between tumor and normal tissue. Other work from this laboratory has shown that cocarcinogens probably act by preventing repair of carcinogenic damage to the DNA genome. The possibility has also been raised that mistakes made during repair synthesis might be responsible for some genetic diversity and for the mutations which arise in resting cells. (U.S.)

  8. Human Prolactin Point Mutations and Their Projected Effect on Vasoinhibin Generation and Vasoinhibin-Related Diseases

    Directory of Open Access Journals (Sweden)

    Jakob Triebel

    2017-11-01

    Full Text Available BackgroundA dysregulation of the generation of vasoinhibin hormones by proteolytic cleavage of prolactin (PRL has been brought into context with diabetic retinopathy, retinopathy of prematurity, preeclampsia, pregnancy-induced hypertension, and peripartum cardiomyopathy. Factors governing vasoinhibin generation are incompletely characterized, and the composition of vasoinhibin isoforms in human tissues or compartments, such as the circulation, is unknown. The aim of this study was to determine the possible contribution of PRL point mutations to the generation of vasoinhibins as well as to project their role in vasoinhibin-related diseases.MethodsProlactin sequences, point mutations, and substrate specificity information about the PRL cleaving enzymes cathepsin D, matrix metalloproteinases 8 and 13, and bone-morphogenetic protein 1 were retrieved from public databases. The consequences of point mutations in regard to their possible effect on vasoinhibin levels were projected on the basis of a score indicating the suitability of a particular sequence for enzymatic cleavage that result in vasoinhibin generation. The relative abundance and type of vasoinhibin isoforms were estimated by comparing the relative cleavage efficiency of vasoinhibin-generating enzymes.ResultsSix point mutations leading to amino acid substitutions in vasoinhibin-generating cleavage sites were found and projected to either facilitate or inhibit vasoinhibin generation. Four mutations affecting vasoinhibin generation in cancer tissues were found. The most likely composition of the relative abundance of vasoinhibin isoforms is projected to be 15 > 17.2 > 16.8 > 17.7 > 18 kDa vasoinhibin.ConclusionProlactin point mutations are likely to influence vasoinhibin levels by affecting the proteolysis efficiency of vasoinhibin-generating enzymes and should be monitored in patients with vasoinhibin-related diseases. Attempts to characterize vasoinhibin-related diseases

  9. 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.

  10. Accurate mitochondrial DNA sequencing using off-target reads provides a single test to identify pathogenic point mutations.

    Science.gov (United States)

    Griffin, Helen R; Pyle, Angela; Blakely, Emma L; Alston, Charlotte L; Duff, Jennifer; Hudson, Gavin; Horvath, Rita; Wilson, Ian J; Santibanez-Koref, Mauro; Taylor, Robert W; Chinnery, Patrick F

    2014-12-01

    Mitochondrial disorders are a common cause of inherited metabolic disease and can be due to mutations affecting mitochondrial DNA or nuclear DNA. The current diagnostic approach involves the targeted resequencing of mitochondrial DNA and candidate nuclear genes, usually proceeds step by step, and is time consuming and costly. Recent evidence suggests that variations in mitochondrial DNA sequence can be obtained from whole-exome sequence data, raising the possibility of a comprehensive single diagnostic test to detect pathogenic point mutations. We compared the mitochondrial DNA sequence derived from off-target exome reads with conventional mitochondrial DNA Sanger sequencing in 46 subjects. Mitochondrial DNA sequences can be reliably obtained using three different whole-exome sequence capture kits. Coverage correlates with the relative amount of mitochondrial DNA in the original genomic DNA sample, heteroplasmy levels can be determined using variant and total read depths, and-providing there is a minimum read depth of 20-fold-rare sequencing errors occur at a rate similar to that observed with conventional Sanger sequencing. This offers the prospect of using whole-exome sequence in a diagnostic setting to screen not only all protein coding nuclear genes but also all mitochondrial DNA genes for pathogenic mutations. Off-target mitochondrial DNA reads can also be used to assess quality control and maternal ancestry, inform on ethnic origin, and allow genetic disease association studies not previously anticipated with existing whole-exome data sets.

  11. A Rapid and Sensitive Method for Detection of the T790M Mutation of EGFR in Plasma DNA.

    Science.gov (United States)

    Kimura, Hideharu; Nishikawa, Shingo; Koba, Hayato; Yoneda, Taro; Sone, Takashi; Kasahara, Kazuo

    2016-01-01

    Epidermal growth factor receptor (EGFR) T790M mutation is associated with resistance to EGFR tyrosine kinase inhibitors' (EGFR-TKIs) in non-small cell lung cancer (NSCLC). The aims of this study are to develop a blood-based, non-invasive approach to detecting the EGFR T790M mutation in advanced NSCLC patients, using PointMan™ EGFR DNA Enrichment Kit which is a novel method for selective amplification of genotype specific sequences.Pairs of blood samples and tumor tissues were collected from NSCLC patients with an EGFR activating mutation and who were resistant to EGFR-TKI treatment. EGFR T790M mutation in plasma DNA were detected using the PointMan™ EGFR DNA Enrichment Kit. The concentrations of plasma DNA were determined using quantitative real-time PCR.Of the 52 patients enrolled in this study, 41 of the patients' plasma samples were collected at post EGFR-TKIs. Nineteen (46.3 %) of the 41 patients had an EGFR T790M mutation in their plasma DNA as detected using the PointMan™ EGFR DNA Enrichment Kit after disease progression to EFGR-TKI. Of 11 cases with a detected T790M mutation from tumor tissues, 10 (90.9 %) also had a detectable T790M mutation in the plasma DNA. There was no difference in the progression-free survival between patients with T790M and those without T790M.The PointMan™ proved to be a useful method for determining plasma EGFR T790M mutation status.

  12. Peptide Synthesis on a Next-Generation DNA Sequencing Platform.

    Science.gov (United States)

    Svensen, Nina; Peersen, Olve B; Jaffrey, Samie R

    2016-09-02

    Methods for displaying large numbers of peptides on solid surfaces are essential for high-throughput characterization of peptide function and binding properties. Here we describe a method for converting the >10(7) flow cell-bound clusters of identical DNA strands generated by the Illumina DNA sequencing technology into clusters of complementary RNA, and subsequently peptide clusters. We modified the flow-cell-bound primers with ribonucleotides thus enabling them to be used by poliovirus polymerase 3D(pol) . The primers hybridize to the clustered DNA thus leading to RNA clusters. The RNAs fold into functional protein- or small molecule-binding aptamers. We used the mRNA-display approach to synthesize flow-cell-tethered peptides from these RNA clusters. The peptides showed selective binding to cognate antibodies. The methods described here provide an approach for using DNA clusters to template peptide synthesis on an Illumina flow cell, thus providing new opportunities for massively parallel peptide-based assays. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. DNA-Mutation Inventory to Refine and Enhance Cancer Treatment (DIRECT): a catalog of clinically relevant cancer mutations to enable genome-directed anticancer therapy.

    Science.gov (United States)

    Yeh, Paul; Chen, Heidi; Andrews, Jenny; Naser, Riyad; Pao, William; Horn, Leora

    2013-04-01

    Tumor gene mutation status is becoming increasingly important in the treatment of patients with cancer. A comprehensive catalog of tumor gene-response outcomes from individual patients is needed, especially for actionable mutations and rare variants. We created a proof-of-principle database [DNA-mutation Inventory to Refine and Enhance Cancer Treatment (DIRECT)], starting with lung cancer-associated EGF receptor (EGFR) mutations, to provide a resource for clinicians to prioritize treatment decisions based on a patient's tumor mutations at the point of care. A systematic search of literature published between June 2005 and May 2011 was conducted through PubMed to identify patient-level, mutation-drug response in patients with non-small cell lung cancer (NSCLC) with EGFR mutant tumors. Minimum inclusion criteria included patient's EGFR mutation, corresponding treatment, and an associated radiographic outcome. A total of 1,021 patients with 1,070 separate EGFR tyrosine kinase inhibitor therapy responses from 116 different publications were included. About 188 unique EGFR mutations occurring in 207 different combinations were identified: 149 different mutation combinations were associated with disease control and 42 were associated with disease progression. Four secondary mutations, in 16 different combinations, were associated with acquired resistance. As tumor sequencing becomes more common in oncology, this comprehensive electronic catalog can enable genome-directed anticancer therapy. DIRECT will eventually encompass all tumor mutations associated with clinical outcomes on targeted therapies. Users can make specific queries at http://www.mycancergenome.org/about/direct to obtain clinically relevant data associated with various mutations. ©2013 AACR.

  14. Generation of mutation hotspots in ageing bacterial colonies

    DEFF Research Database (Denmark)

    Sekowska, Agnieszka; Wendel, Sofie; Nørholm, Morten

    How do ageing bacterial colonies generate adaptive mutants? Over a period of two months, we isolated on ageing colonies outgrowing mutants able to use a new carbon source, and sequenced their genomes. This allowed us to uncover exquisite details on the molecular mechanism behind their adaptation:...

  15. Generation and characterization of the Western Regional Research Center Brachypodium T-DNA insertional mutant collection.

    Directory of Open Access Journals (Sweden)

    Jennifer N Bragg

    Full Text Available The model grass Brachypodium distachyon (Brachypodium is an excellent system for studying the basic biology underlying traits relevant to the use of grasses as food, forage and energy crops. To add to the growing collection of Brachypodium resources available to plant scientists, we further optimized our Agrobacterium tumefaciens-mediated high-efficiency transformation method and generated 8,491 Brachypodium T-DNA lines. We used inverse PCR to sequence the DNA flanking the insertion sites in the mutants. Using these flanking sequence tags (FSTs we were able to assign 7,389 FSTs from 4,402 T-DNA mutants to 5,285 specific insertion sites (ISs in the Brachypodium genome. More than 29% of the assigned ISs are supported by multiple FSTs. T-DNA insertions span the entire genome with an average of 19.3 insertions/Mb. The distribution of T-DNA insertions is non-uniform with a larger number of insertions at the distal ends compared to the centromeric regions of the chromosomes. Insertions are correlated with genic regions, but are biased toward UTRs and non-coding regions within 1 kb of genes over exons and intron regions. More than 1,300 unique genes have been tagged in this population. Information about the Western Regional Research Center Brachypodium insertional mutant population is available on a searchable website (http://brachypodium.pw.usda.gov designed to provide researchers with a means to order T-DNA lines with mutations in genes of interest.

  16. 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....... RESULTS: The study included 246 patients receiving a minimum of 1 treatment cycle, and all but four were evaluable for response according to RECIST. Forty-three patients (17.5%) presented with a KRAS mutation. OS was 8.9 months and PFS by intention to treat 5.4 months. Patients with a detectable plasma......-KRAS mutation had a significantly shorter OS and PFS compared to the wild type (WT) patients (median OS 4.8 months versus 9.5 months, HR 1.87, 95% CI 1.23-2.84, p=0.0002 and median PFS 3.0 months versus 5.6 months, HR 1.60, 95% CI 1.09-2.37, p=0.0043). A multivariate Cox regression analysis confirmed...

  17. 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.

  18. Nanopore-based fourth-generation DNA sequencing technology.

    Science.gov (United States)

    Feng, Yanxiao; Zhang, Yuechuan; Ying, Cuifeng; Wang, Deqiang; Du, Chunlei

    2015-02-01

    Nanopore-based sequencers, as the fourth-generation DNA sequencing technology, have the potential to quickly and reliably sequence the entire human genome for less than $1000, and possibly for even less than $100. The single-molecule techniques used by this technology allow us to further study the interaction between DNA and protein, as well as between protein and protein. Nanopore analysis opens a new door to molecular biology investigation at the single-molecule scale. In this article, we have reviewed academic achievements in nanopore technology from the past as well as the latest advances, including both biological and solid-state nanopores, and discussed their recent and potential applications. Copyright © 2015 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.

  19. Nanopore-based Fourth-generation DNA Sequencing Technology

    Directory of Open Access Journals (Sweden)

    Yanxiao Feng

    2015-02-01

    Full Text Available Nanopore-based sequencers, as the fourth-generation DNA sequencing technology, have the potential to quickly and reliably sequence the entire human genome for less than $1000, and possibly for even less than $100. The single-molecule techniques used by this technology allow us to further study the interaction between DNA and protein, as well as between protein and protein. Nanopore analysis opens a new door to molecular biology investigation at the single-molecule scale. In this article, we have reviewed academic achievements in nanopore technology from the past as well as the latest advances, including both biological and solid-state nanopores, and discussed their recent and potential applications.

  20. 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.

  1. 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.

  2. Frequency of Mitochondrial DNA D-Loop Somatic Mutations in Patients with HTLV-I

    Directory of Open Access Journals (Sweden)

    Toktam Zolfaghari

    2017-08-01

    Full Text Available Human T-cell Lymphotropic virus type-1 (HTLV-1 is endemic in Northeast of Iran. Still, it is unclear that genetic background has role in infection by HTLV-1. Methods: We ascertained the frequency of mitochondrial DNA (mtDNA D-loop region nucleotide changes in 45 HTLV-1 infected individuals and 463 healthy control subjects using Sanger sequencing method. Results: Out of totally 164 identified single nucleotide polymorphisms (SNPs among HTLV-1 patients, 89 SNPs found statistically significant in comparison to the control group (P<0.05. In this study, no deletion was identified in mtDNA D-loop region. But, for the first time a high frequency of point mutations was observed in HTLV-1 patients. Conclusion: Such nucleotide changes in HTLV-1 patients propose that these mutations may result in impaired mitochondria function directly and/or indirectly. Moreover, these variations may act as a predisposing factor along with the environmental factors, and might play an important role in pathogenesis of HTLV-1.

  3. Senataxin Mutation Reveals How R-Loops Promote Transcription by Blocking DNA Methylation at Gene Promoters.

    Science.gov (United States)

    Grunseich, Christopher; Wang, Isabel X; Watts, Jason A; Burdick, Joshua T; Guber, Robert D; Zhu, Zhengwei; Bruzel, Alan; Lanman, Tyler; Chen, Kelian; Schindler, Alice B; Edwards, Nancy; Ray-Chaudhury, Abhik; Yao, Jianhua; Lehky, Tanya; Piszczek, Grzegorz; Crain, Barbara; Fischbeck, Kenneth H; Cheung, Vivian G

    2018-02-01

    R-loops are three-stranded nucleic acid structures found abundantly and yet often viewed as by-products of transcription. Studying cells from patients with a motor neuron disease (amyotrophic lateral sclerosis 4 [ALS4]) caused by a mutation in senataxin, we uncovered how R-loops promote transcription. In ALS4 patients, the senataxin mutation depletes R-loops with a consequent effect on gene expression. With fewer R-loops in ALS4 cells, the expression of BAMBI, a negative regulator of transforming growth factor β (TGF-β), is reduced; that then leads to the activation of the TGF-β pathway. We uncovered that genome-wide R-loops influence promoter methylation of over 1,200 human genes. DNA methyl-transferase 1 favors binding to double-stranded DNA over R-loops. Thus, in forming R-loops, nascent RNA blocks DNA methylation and promotes further transcription. Hence, our results show that nucleic acid structures, in addition to sequences, influence the binding and activity of regulatory proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. 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).

  5. 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...... in only one family, where a homozygous 2 bp deletion was associated with fatal infantile lactic acidosis. We here report a patient with a novel homozygous missense mutation in SUCLG1, whose phenotype is similar to that of patients with SUCLA2 mutations....

  6. Next generation sequencing improves the accuracy of KRAS mutation analysis in endoscopic ultrasound fine needle aspiration pancreatic lesions.

    Directory of Open Access Journals (Sweden)

    Dario de Biase

    Full Text Available The use of endoscopic ultrasonography has allowed for improved detection and pathologic analysis of fine needle aspirate material for pancreatic lesion diagnosis. The molecular analysis of KRAS has further improved the clinical sensitivity of preoperative analysis. For this reason, the use of highly analytical sensitive and specific molecular tests in the analysis of material from fine needle aspirate specimens has become of great importance. In the present study, 60 specimens from endoscopic ultrasonography fine needle aspirate were analyzed for KRAS exon 2 and exon 3 mutations, using three different techniques: Sanger sequencing, allele specific locked nucleic acid PCR and Next Generation sequencing (454 GS-Junior, Roche. Moreover, KRAS was also tested in wild-type samples, starting from DNA obtained from cytological smears after pathological evaluation. Sanger sequencing showed a clinical sensitivity for the detection of the KRAS mutation of 42.1%, allele specific locked nucleic acid of 52.8% and Next Generation of 73.7%. In two wild-type cases the re-sequencing starting from selected material allowed to detect a KRAS mutation, increasing the clinical sensitivity of next generation sequencing to 78.95%. The present study demonstrated that the performance of molecular analysis could be improved by using highly analytical sensitive techniques. The Next Generation Sequencing allowed to increase the clinical sensitivity of the test without decreasing the specificity of the analysis. Moreover we observed that it could be useful to repeat the analysis starting from selectable material, such as cytological smears to avoid false negative results.

  7. PGD for the m.14487 T>C mitochondrial DNA mutation resulted in the birth of a healthy boy.

    Science.gov (United States)

    Sallevelt, Suzanne C E H; Dreesen, Joseph C F M; Drüsedau, Marion; Hellebrekers, Debby M E I; Paulussen, Aimee D C; Coonen, Edith; van Golde, Ronald J T; Geraedts, Joep P M; Gianaroli, Luca; Magli, Maria C; Zeviani, Massimo; Smeets, Hubert J M; de Die-Smulders, Christine E M

    2017-03-01

    We report on the first PGD performed for the m.14487 T>C mitochondrial DNA (mtDNA) mutation in the MT-ND6 gene, associated with Leigh syndrome. The female carrier gave birth to a healthy baby boy at age 42. This case adds to the successes of PGD for mtDNA mutations. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  8. Clinical significance of disease-specific MYD88 mutations in circulating DNA in primary central nervous system lymphoma.

    Science.gov (United States)

    Hattori, Keiichiro; Sakata-Yanagimoto, Mamiko; Suehara, Yasuhito; Yokoyama, Yasuhisa; Kato, Takayasu; Kurita, Naoki; Nishikii, Hidekazu; Obara, Naoshi; Takano, Shingo; Ishikawa, Eiichi; Matsumura, Akira; Hasegawa, Yuichi; Chiba, Shigeru

    2018-01-01

    Recent sequencing studies demonstrated the MYD88 L265P mutation in more than 70% of primary central nervous system lymphomas (PCNSL), and the clinical significance of this mutation has been proposed as diagnostic and prognostic markers in PCNSL. In contrast, mutational analyses using cell-free DNAs have been reported in a variety of systemic lymphomas. To investigate how sensitively the MYD88 L265P mutation can be identified in cell-free DNA from PCNSL patients, we carried out droplet digital PCR (ddPCR) and targeted deep sequencing (TDS) in 14 consecutive PCNSL patients from whom paired tumor-derived DNA and cell-free DNA was available at diagnosis. The MYD88 L265P mutation was found in tumor-derived DNA from all 14 patients (14/14, 100%). In contrast, among 14 cell-free DNAs evaluated by ddPCR (14/14) and TDS (13/14), the MYD88 L265P mutation was detected in eight out of 14 (ddPCR) and in 0 out of 13 (TDS) samples, implying dependence on the detection method. After chemotherapy, the MYD88 L265P mutation in cell-free DNAs was traced in five patients; unexpectedly, the mutations disappeared after chemotherapy was given, and they remained undetectable in all patients. These observations suggest that ddPCR can sensitively detect the MYD88 L265P mutation in cell-free DNA and could be used as non-invasive diagnostics, but may not be applicable for monitoring minimal residual diseases in PCNSL. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  9. Prognostic Significance of Circulating RET M918T Mutated Tumor DNA in Patients With Advanced Medullary Thyroid Carcinoma.

    Science.gov (United States)

    Cote, Gilbert J; Evers, Caitlin; Hu, Mimi I; Grubbs, Elizabeth G; Williams, Michelle D; Hai, Tao; Duose, Dzifa Y; Houston, Michal R; Bui, Jacquelin H; Mehrotra, Meenakshi; Waguespack, Steven G; Busaidy, Naifa L; Cabanillas, Maria E; Habra, Mouhammed Amir; Luthra, Rajyalakshmi; Sherman, Steven I

    2017-09-01

    Interpretation of calcitonin measurement to predict the prognosis of medullary thyroid carcinoma (MTC) requires multiple measurements over an extended time period, making it an imperfect biomarker for evaluating prognosis or disease behavior. Single circulating cell-free DNA (cfDNA) values have been shown to be a valuable prognostic marker for several solid tumors. We tested the hypothesis that cfDNA containing the RET M918T mutation could be detected in the blood of patients with advanced MTC whose tumor harbored an M918T mutation and would be able to predict overall survival more reliably than calcitonin. The level of cfDNA containing RET M918T mutation was measured in the plasma of patients with MTC via droplet digital polymerase chain reaction. Patients had a confirmed sporadic MTC diagnosis, a serum calcitonin measurement >100 pg/mL, and tumor tissue biopsy results providing RET M918T mutation status. There were 75 patients included in this study, 50 of whom harbored an RET M918T mutation by tissue biopsy. RET M918T cfDNA was detected in 16 of 50 patients (32%) with a positive tissue biopsy. The detection of RET M918T cfDNA strongly correlated with worse overall survival and more accurately predicted a worse outcome than calcitonin doubling time. Liquid biopsy is able to detect RET M918T mutations in patient plasma with high specificity but low sensitivity. In patients with established somatic RET M918T mutations, the allelic fraction of circulating tumor DNA is prognostic for overall survival and may play a role in monitoring response to treatment. Copyright © 2017 Endocrine Society

  10. Epidermal growth factor receptor mutation status in cell-free DNA supernatant of bronchial washings and brushings.

    Science.gov (United States)

    Kawahara, Akihiko; Fukumitsu, Chihiro; Taira, Tomoki; Abe, Hideyuki; Takase, Yorihiko; Murata, Kazuya; Yamaguchi, Tomohiko; Azuma, Koichi; Ishii, Hidenobu; Takamori, Shinzo; Akiba, Jun; Hoshino, Tomoaki; Kage, Masayoshi

    2015-10-01

    The aim of the current study was to examine whether it would be possible to detect epidermal growth factor receptor (EGFR) mutations in cytology cell-free DNA (ccfDNA) from the supernatant fluids of bronchial cytology samples. This study investigated cell damage via immunostaining with a cleaved caspase 3 antibody and the quantity of cell-free DNA in supernatant fluid from 2 cancer cell lines, and the EGFR mutation status was evaluated via polymerase chain reaction (PCR) analysis. EGFR mutations were also evaluated via PCR analysis in 74 clinical samples of ccfDNA from bronchial washing samples with physiological saline or from bronchial brushing liquid-based cytology samples with CytoRich Red. The quantity and fragmentation of cell-free DNA in the supernatant fluid and the cell damage and cleaved caspase 3 expression in the sediment gradually increased in a time-dependent manner in the cell lines. In the 74 clinical samples, the quantity of ccfDNA extracted from the supernatant was adequate to perform the PCR assay, whereas the quality of ccfDNA in physiological saline was often decreased. The detection of EGFR mutations with ccfDNA showed a sensitivity of 88.0%, a specificity of 100%, a positive predictive value of 100%, a negative predictive value of 89.7%, and an accuracy of 94.1% in samples with malignant or atypical cells. These results suggest that activating EGFR mutations can be detected with ccfDNA extracted from the supernatant fluid of liquid-based samples via a PCR assay. This could be a rapid and sensitive method for achieving a parallel diagnosis by both morphology and DNA analysis in non-small cell lung cancer patients. © 2015 American Cancer Society.

  11. Mutation Detection in Patients with Retinal Dystrophies Using Targeted Next Generation Sequencing.

    Directory of Open Access Journals (Sweden)

    Nicole Weisschuh

    Full Text Available Retinal dystrophies (RD constitute a group of blinding diseases that are characterized by clinical variability and pronounced genetic heterogeneity. The different nonsyndromic and syndromic forms of RD can be attributed to mutations in more than 200 genes. Consequently, next generation sequencing (NGS technologies are among the most promising approaches to identify mutations in RD. We screened a large cohort of patients comprising 89 independent cases and families with various subforms of RD applying different NGS platforms. While mutation screening in 50 cases was performed using a RD gene capture panel, 47 cases were analyzed using whole exome sequencing. One family was analyzed using whole genome sequencing. A detection rate of 61% was achieved including mutations in 34 known and two novel RD genes. A total of 69 distinct mutations were identified, including 39 novel mutations. Notably, genetic findings in several families were not consistent with the initial clinical diagnosis. Clinical reassessment resulted in refinement of the clinical diagnosis in some of these families and confirmed the broad clinical spectrum associated with mutations in RD genes.

  12. DNA polymerase eta is a limiting factor for A:T mutations in Ig genes and contributes to antibody affinity maturation.

    Science.gov (United States)

    Masuda, Keiji; Ouchida, Rika; Yokoi, Masayuki; Hanaoka, Fumio; Azuma, Takachika; Wang, Ji-Yang

    2008-10-01

    DNA polymerase eta (POLH) is required for the generation of A:T mutations during the somatic hypermutation of Ig genes in germinal center B cells. It remains unclear, however, whether POLH is a limiting factor for A:T mutations and how the absence of POLH might affect antibody affinity maturation. We found that the heterozygous Polh+/- mice exhibited a significant reduction in the frequency of A:T mutations in Ig genes, with each type of base substitutions at a level intermediate between the Polh+/+ and Polh(-/-) mice. These observations suggest that Polh is haplo-insufficient for the induction of A:T mutations in Ig genes. Intriguingly, there was also a reduction of C to T and G to A transitions in Polh+/- mice as compared with WT mice. Polh(-/-) mice produced decreased serum titers of high-affinity antibodies against a T-dependent antigen, which was associated with a significant reduction in the number of plasma cells secreting high-affinity antibodies. Analysis of the V region revealed that aa substitutions caused by A:T mutations were greatly reduced in Polh(-/-) mice. These results demonstrate that POLH is a limiting factor for A:T mutations and contributes to the efficient diversification of Ig genes and affinity maturation of antibodies.

  13. Determining the Location of DNA Modification and Mutation Caused by UVB Light in Skin Cancer

    Science.gov (United States)

    2015-09-01

    were being administered. To address this we obtained an LED bulb from Qphotonics that emits light at 315nm ± 10 nm (10) and incorporated it into a...Award Number: W81XWH-12-1-0333 TITLE: Determining the Location of DNA Modification and Mutation Caused by UVB Light in Skin Cancer PRINCIPAL...Caused by UVB Light in Skin Cancer 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Monica Ransom, PhD Betty Diamond 5d. PROJECT NUMBER

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

    Directory of Open Access Journals (Sweden)

    Lucy F Stead

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

  15. 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

  16. 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

  17. A hotspot in the glucocorticoid receptor DNA-binding domain susceptible to loss of function mutation.

    Science.gov (United States)

    Banuelos, Jesus; Shin, Soon Cheon; Lu, Nick Z

    2015-04-01

    Glucocorticoids (GCs) are used to treat a variety of inflammatory disorders and certain cancers. However, GC resistance occurs in subsets of patients. We found that EL4 cells, a GC-resistant mouse thymoma cell line, harbored a point mutation in their GC receptor (GR) gene, resulting in the substitution of arginine 493 by a cysteine in the second zinc finger of the DNA-binding domain. Allelic discrimination analyses revealed that the R493C mutation occurred on both alleles. In the absence of GCs, the GR in EL4 cells localized predominantly in the cytoplasm and upon dexamethasone treatment underwent nuclear translocation, suggesting that the ligand binding ability of the GR in EL4 cells was intact. In transient transfection assays, the R493C mutant could not transactivate the MMTV-luciferase reporter. Site-directed mutagenesis to revert the R493C mutation restored the transactivation activity. Cotransfection experiments showed that the R493C mutant did not inhibit the transcriptional activities of the wild-type GR. In addition, the R493C mutant did not repress either the AP-1 or NF-κB reporters as effectively as WT GR. Furthermore, stable expression of the WT GR in the EL4 cells enabled GC-mediated gene regulation, specifically upregulation of IκBα and downregulation of interferon γ and interleukin 17A. Arginine 493 is conserved among multiple species and all human nuclear receptors and its mutation has also been found in the human GR, androgen receptor, and mineralocorticoid receptor. Thus, R493 is necessary for the transcriptional activity of the GR and a hotspot for mutations that result in GC resistance. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Correlation of BRAF Mutation Status in Circulating-Free DNA and Tumor and Association with Clinical Outcome across Four BRAFi and MEKi Clinical Trials.

    Science.gov (United States)

    Santiago-Walker, Ademi; Gagnon, Robert; Mazumdar, Jolly; Casey, Michelle; Long, Georgina V; Schadendorf, Dirk; Flaherty, Keith; Kefford, Richard; Hauschild, Axel; Hwu, Patrick; Haney, Patricia; O'Hagan, Anne; Carver, Jennifer; Goodman, Vicki; Legos, Jeffrey; Martin, Anne-Marie

    2016-02-01

    Tumor-derived circulating cell-free DNA (cfDNA) is a potential alternative source from which to derive tumor mutation status. cfDNA data from four clinical studies of the BRAF inhibitor (BRAFi) dabrafenib or the MEK inhibitor (MEKi) trametinib were analyzed to determine the association between BRAF mutation status in cfDNA and tumor tissue, and the association of BRAF cfDNA mutation status with baseline factors and clinical outcome. Patients with BRAF V600 mutation-positive melanoma were enrolled in each study after central confirmation of BRAF status in tumor using a PCR-based assay. BRAF mutation status in cfDNA from patient plasma collected at baseline, 732 of 836 (88%) enrolled patients in total, was determined. BRAF mutations were detectable in cfDNA in 76% and 81% of patients with BRAF V600E/V600K-positive tumors, respectively. Patients negative for BRAF mutations in cfDNA had longer progression-free survival (PFS) and overall survival in each of the four studies, compared with patients with detectable cfDNA BRAF mutations. The presence of BRAF-mutant cfDNA was an independent prognostic factor for PFS after multivariate adjustment for baseline factors in three of four studies. Patients negative for BRAF mutation-positive cfDNA in plasma had higher response rates to dabrafenib and trametinib. BRAF mutations in cfDNA are detectable in >75% of late-stage melanoma patients with BRAF mutation-positive tumors. The lack of circulating, BRAF mutation-positive cfDNA is clinically significant for metastatic melanoma patients, and may be a prognostic marker for better disease outcome. ©2015 American Association for Cancer Research.

  19. Generation of a luciferase-based reporter for CHH and CG DNA methylation in Arabidopsis thaliana.

    Science.gov (United States)

    Dinh, Thanh Theresa; O'Leary, Michael; Won, So Youn; Li, Shengben; Arroyo, Lorena; Liu, Xigang; Defries, Andrew; Zheng, Binglian; Cutler, Sean R; Chen, Xuemei

    2013-04-05

    DNA methylation ensures genome integrity and regulates gene expression in diverse eukaryotes. In Arabidopsis, methylation occurs in three sequence contexts: CG, CHG and CHH. The initial establishment of DNA methylation at all three sequence contexts occurs through a process known as RNA-directed DNA methylation (RdDM), in which small RNAs bound by Argonaute4 (AGO4) guide DNA methylation at homologous loci through the de novo methyltransferase DRM2. Once established, DNA methylation at each of the three sequence contexts is maintained through different mechanisms. Although some players involved in RdDM and maintenance methylation have been identified, the underlying molecular mechanisms are not fully understood. To aid the comprehensive identification of players in DNA methylation, we generated a transgenic reporter system that permits genetic and chemical genetic screens in Arabidopsis. A dual 35S promoter (d35S) driven luciferase (LUC) reporter was introduced into Arabidopsis and LUCL, a line with a low basal level of luciferase activity, was obtained. LUCL was found to be a multi-copy, single-insertion transgene that contains methylated cytosines in CG, CHG and CHH contexts, with the highest methylation in the CG context. Methylation was present throughout the promoter and LUC coding region. Treatment with an inhibitor of cytosine methylation de-repressed luciferase activity. A mutation in MET1, which encodes the CG maintenance methyltransferase, drastically reduced CG methylation and de-repressed LUC expression. Mutations in AGO4 and DRM2 also de-repressed LUC expression, albeit to a smaller extent than loss of MET1. Using LUCL as a reporter line, we performed a chemical screen for compounds that de-repress LUC expression, and identified a chemical, methotrexate, known to be involved in biogenesis of the methyl donor. We developed a luciferase-based reporter system, LUCL, which reports both RdDM and CG maintenance methylation in Arabidopsis. The low basal level

  20. 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

    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......, colon, or urine. Thus, lard intake at the expense of other nutrients and a large increase in the fat energy consumption affects the redox state locally in the liver cytosol, but does not induce DNA-damage, systemic oxidative stress or a dose-dependent increase in mutation frequency in rat colon or liver.......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...

  1. Contribution of Topoisomerase IV and DNA Gyrase Mutations in Streptococcus pneumoniae to Resistance to Novel Fluoroquinolones

    Science.gov (United States)

    Pestova, Ekaterina; Beyer, Rebecca; Cianciotto, Nicholas P.; Noskin, Gary A.; Peterson, Lance R.

    1999-01-01

    In this study, we assessed the activity of ciprofloxacin, levofloxacin, sparfloxacin, and trovafloxacin against clinical isolates of Streptococcus pneumoniae that were resistant to the less-recently developed fluoroquinolones by using defined amino acid substitutions in DNA gyrase and topoisomerase IV. The molecular basis for resistance was assessed by using mutants selected with trovafloxacin, ciprofloxacin, and levofloxacin in vitro. This demonstrated that the primary target of trovafloxacin in S. pneumoniae is the ParC subunit of DNA topoisomerase IV, similar to most other fluoroquinolones. However, first-step mutants bearing the Ser79→Phe/Tyr substitution in topoisomerase IV subunit ParC were susceptible to trovafloxacin with a minimum inhibitory concentration of 0.25 μg/ml, and mutations in the structural genes for both topoisomerase IV subunit ParC (parC) and the DNA gyrase subunit (gyrA) were required to achieve levels of resistance above the breakpoint. The data also suggest that enhanced activity of trovafloxacin against pneumococci is due to a combination of factors that may include reduced efflux of this agent and an enhanced activity against both DNA gyrase and topoisomerase IV. PMID:10428926

  2. Ethidium Bromide Modifies The Agarose Electrophoretic Mobility of CAG•CTG Alternative DNA Structures Generated by PCR

    Directory of Open Access Journals (Sweden)

    Mário Gomes-Pereira

    2017-05-01

    Full Text Available The abnormal expansion of unstable simple sequence DNA repeats can cause human disease through a variety of mechanisms, including gene loss-of-function, toxic gain-of-function of the encoded protein and toxicity of the repeat-containing RNA transcript. Disease-associated unstable DNA repeats display unusual biophysical properties, including the ability to adopt non-B-DNA structures. CAG•CTG trinucleotide sequences, in particular, have been most extensively studied and they can fold into slipped-stranded DNA structures, which have been proposed as mutation intermediates in repeat size expansion. Here, we describe a simple assay to detect unusual DNA structures generated by PCR amplification, based on their slow electrophoretic migration in agarose and on the effects of ethidium bromide on the mobility of structural isoforms through agarose gels. Notably, the inclusion of ethidium bromide in agarose gels and running buffer eliminates the detection of additional slow-migrating DNA species, which are detected in the absence of the intercalating dye and may be incorrectly classified as mutant alleles with larger than actual expansion sizes. Denaturing and re-annealing experiments confirmed the slipped-stranded nature of the additional DNA species observed in agarose gels. Thus, we have shown that genuine non-B-DNA conformations are generated during standard PCR amplification of CAG•CTG sequences and detected by agarose gel electrophoresis. In contrast, ethidium bromide does not change the multi-band electrophoretic profiles of repeat-containing PCR products through native polyacrylamide gels. These data have implications for the analysis of trinucleotide repeat DNA and possibly other types of unstable repetitive DNA sequences by standard agarose gel electrophoresis in diagnostic and research protocols. We suggest that proper sizing of CAG•CTG PCR products in agarose gels should be performed in the presence of ethidium bromide.

  3. Analysis of mutation/rearrangement frequencies and methylation patterns at a given DNA locus using restriction fragment length polymorphism.

    Science.gov (United States)

    Boyko, Alex; Kovalchuk, Igor

    2010-01-01

    Restriction fragment length polymorphism (RFLP) is a difference in DNA sequences of organisms belonging to the same species. RFLPs are typically detected as DNA fragments of different lengths after digestion with various restriction endonucleases. The comparison of RFLPs allows investigators to analyze the frequency of occurrence of mutations, such as point mutations, deletions, insertions, and gross chromosomal rearrangements, in the progeny of stressed plants. The assay involves restriction enzyme digestion of DNA followed by hybridization of digested DNA using a radioactively or enzymatically labeled probe. Since DNA can be digested with methylation sensitive enzymes, the assay can also be used to analyze a methylation pattern of a particular locus. Here, we describe RFLP analysis using methylation-insensitive and methylation-sensitive enzymes.

  4. The de novo cytosine methyltransferase DRM2 requires intact UBA domains and a catalytically mutated paralog DRM3 during RNA-directed DNA methylation in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Ian R Henderson

    2010-10-01

    Full Text Available Eukaryotic DNA cytosine methylation can be used to transcriptionally silence repetitive sequences, including transposons and retroviruses. This silencing is stable between cell generations as cytosine methylation is maintained epigenetically through DNA replication. The Arabidopsis thaliana Dnmt3 cytosine methyltransferase ortholog DOMAINS rearranged methyltransferase2 (DRM2 is required for establishment of small interfering RNA (siRNA directed DNA methylation. In mammals PIWI proteins and piRNA act in a convergently evolved RNA-directed DNA methylation system that is required to repress transposon expression in the germ line. De novo methylation may also be independent of RNA interference and small RNAs, as in Neurospora crassa. Here we identify a clade of catalytically mutated DRM2 paralogs in flowering plant genomes, which in A.thaliana we term domains rearranged methyltransferase3 (DRM3. Despite being catalytically mutated, DRM3 is required for normal maintenance of non-CG DNA methylation, establishment of RNA-directed DNA methylation triggered by repeat sequences and accumulation of repeat-associated small RNAs. Although the mammalian catalytically inactive Dnmt3L paralogs act in an analogous manner, phylogenetic analysis indicates that the DRM and Dnmt3 protein families diverged independently in plants and animals. We also show by site-directed mutagenesis that both the DRM2 N-terminal UBA domains and C-terminal methyltransferase domain are required for normal RNA-directed DNA methylation, supporting an essential targeting function for the UBA domains. These results suggest that plant and mammalian RNA-directed DNA methylation systems consist of a combination of ancestral and convergent features.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1988-03-01

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

  6. Mutation Detection in Patients with Retinal Dystrophies Using Targeted Next Generation Sequencing

    DEFF Research Database (Denmark)

    Weisschuh, Nicole; Mayer, Anja K; Strom, Tim M

    2016-01-01

    Retinal dystrophies (RD) constitute a group of blinding diseases that are characterized by clinical variability and pronounced genetic heterogeneity. The different nonsyndromic and syndromic forms of RD can be attributed to mutations in more than 200 genes. Consequently, next generation sequencin...

  7. Comparative study of IDH1 mutations in gliomas by high resolution melting analysis, immunohistochemistry and direct DNA sequencing.

    Science.gov (United States)

    Li, Juan; Zhang, Haiyan; Wang, Li; Yang, Chuanhong; Lai, Huangwen; Zhang, Wei; Chen, Xiaodong; Wang, Jie

    2015-09-01

    Patients with glioblastomas with a specific mutation in the isocitrate dehydrogenase 1 (IDH1) gene have a better prognosis than those with gliomas with wild‑type IDH1. IDH1 analysis has become part of the standard diagnostic procedure and a promising tool used for stratification in clinical trials. The present study aimed to compare high resolution melting (HRM) analysis, immunohistochemistry (IHC) and direct DNA sequencing for the detection of IDH mutations in gliomas. Fifty‑one formalin‑fixed paraffin‑embedded tumor samples were selected. For the HRM analysis and direct DNA sequencing, DNA was extracted from the tissues. For IHC, sections were stained with an anti‑IDH1‑R132H specific antibody. The HRM analysis method identified 33 cases of IDH1 gene mutations, and all mutations occurred at the R132H site. There were 33 cases of IDH1 gene mutations found by IHC, which was consistent with that identified using the HRM analysis method. However, only 30 IDH1 samples were confirmed by sequencing, in which mutations occurred at the IDH1 exon 4 R132H site. No mutation was detected in the other three of these 33 cases (two grade II oligodendroglioma and one grade II diffuse astrocytoma) by sequencing, while IHC was positive for IDH1‑R132H. The results showed that the mutation detection rate was not identified to be significantly different (P=0.250) when determined by the HRM analysis method or by direct DNA sequencing, as the concordant rate between the two methods was high (κ=0.866). The HRM analysis method in glioma IDH1 gene mutation detection has advantages of high sensitivity, good repeatability, simple operation and accurate results. It provides a novel method for detecting mutations of the IDH1 gene in paraffin embedded tissue samples of clinical glioma. Related to a small amount of sample, there was no evidence showing that HRM analysis method is superior to IHC. Direct DNA sequencing, HRM analysis and IHC results were consistent; however, HRM and

  8. Complex mutations & subpopulations of deletions at exon 19 of EGFR in NSCLC revealed by next generation sequencing: potential clinical implications.

    Directory of Open Access Journals (Sweden)

    Antonio Marchetti

    Full Text Available Microdeletions at exon 19 are the most frequent genetic alterations affecting the Epidermal Growth Factor Receptor (EGFR gene in non-small cell lung cancer (NSCLC and they are strongly associated with response to treatment with tyrosine kinase inhibitors. A series of 116 NSCLC DNA samples investigated by Sanger Sequencing (SS, including 106 samples carrying exon 19 EGFR deletions and 10 without deletions (control samples, were subjected to deep next generation sequencing (NGS. All samples with deletions at SS showed deletions with NGS. No deletions were seen in control cases. In 93 (88% cases, deletions detected by NGS were exactly corresponding to those identified by SS. In 13 cases (12% NGS resolved deletions not accurately characterized by SS. In 21 (20% cases the NGS showed presence of complex (double/multiple frameshift deletions producing a net in-frame change. In 5 of these cases the SS could not define the exact sequence of mutant alleles, in the other 16 cases the results obtained by SS were conventionally considered as deletions plus insertions. Different interpretative hypotheses for complex mutations are discussed. In 46 (43% tumors deep NGS showed, for the first time to our knowledge, subpopulations of DNA molecules carrying EGFR deletions different from the main one. Each of these subpopulations accounted for 0.1% to 17% of the genomic DNA in the different tumors investigated. Our findings suggest that a region in exon 19 is highly unstable in a large proportion of patients carrying EGFR deletions. As a corollary to this study, NGS data were compared with those obtained by immunohistochemistry using the 6B6 anti-mutant EGFR antibody. The immunoreaction was E746-A750del specific. In conclusion, NGS analysis of EGFR exon 19 in NSCLCs allowed us to formulate a new interpretative hypothesis for complex mutations and revealed the presence of subpopulations of deletions with potential pathogenetic and clinical impact.

  9. Novel compound heterozygous DNA ligase IV mutations in an adolescent with a slowly-progressing radiosensitive-severe combined immunodeficiency.

    Science.gov (United States)

    Tamura, Shinobu; Higuchi, Kohei; Tamaki, Masaharu; Inoue, Chizuko; Awazawa, Ryoko; Mitsuki, Noriko; Nakazawa, Yuka; Mishima, Hiroyuki; Takahashi, Kenzo; Kondo, Osamu; Imai, Kohsuke; Morio, Tomohiro; Ohara, Osamu; Ogi, Tomoo; Furukawa, Fukumi; Inoue, Masami; Yoshiura, Koh-ichiro; Kanazawa, Nobuo

    2015-10-01

    We herein describe a case of a 17-year-old boy with intractable common warts, short stature, microcephaly and slowly-progressing pancytopenia. Simultaneous quantification of T-cell receptor recombination excision circles (TREC) and immunoglobulin κ-deleting recombination excision circles (KREC) suggested very poor generation of both T-cells and B-cells. By whole exome sequencing, novel compound heterozygous mutations were identified in the patient's DNA ligase IV (LIG4) gene. The diagnosis of LIG4 syndrome was confirmed by delayed DNA double-strand break repair kinetics in γ-irradiated fibroblasts from the patient and their restoration by an introduction of wild-type LIG4. Although the patient received allogeneic hematopoietic stem cell transplantation from his haploidentical mother, he unfortunately expired due to an insufficiently reconstructed immune system. An earlier definitive diagnosis using TREC/KREC quantification and whole exome sequencing would thereby allow earlier intervention, which would be essential for improving long-term survival in similar cases with slowly-progressing LIG4 syndrome masked in adolescents. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Detection of cystic fibrosis delta F508 mutation by anti-double-stranded DNA antibody.

    Science.gov (United States)

    Hopfer, S M; Makowski, G S; Davis, E L; Aslanzadeh, J

    1995-01-01

    This study evaluated an enzyme immunoassay (EIA) as a screening tool for detection of the most common mutation (delta F508) in cystic fibrosis (CF). Guthrie card bloodspots were extracted to remove whole blood polymerase chain reaction (PCR) inhibitors. The washed filter paper was directly amplified under standard (98 bp amplicons) or modified PCR conditions (491 bp amplicons) for the delta F508 mutation. Monoclonal anti-double stranded deoxyribonucleic acid antibody was used to detect competent hybrid formation between PCR products and normal (N) and mutant (M) cDNA (deoxyribonucleic acid) probes coated to microtiter plate wells. Under standard conditions, mean relative color production (N/M) via an enzyme-linked horseradish peroxidase secondary antibody was 8.8, 0.6 and 0.04 for individuals normal, heterozygous and homozygous for the CF delta F508 mutation, respectively (n = 27). Comparison of EIA results to those obtained by tris-borate-EDTA/8 percent polyacrylamide gel electrophoresis (TBE-PAGE) yielded excellent correlation (100 percent) for all three genotypes (n = 27). In comparison to TBE-PAGE, the EIA was 5 to 10 fold more sensitive when serially diluted PCR samples were evaluated. Larger PCR products (491 bp amplicons) for the CF delta F508 mutation obtained under modified conditions were not resolved by TBE-PAGE. The EIA, however, demonstrated equal sensitivity to the 98 bp and 491 bp amplicons. Performance time for TBE-PAGE analysis was substantially shorter (25 percent) than the EIA (3.5 to 4 h and 4.5 to 5 h, respectively) when small batches of samples (n = 5) were analyzed. The TBE-PAGE was not, however, convenient for screening large numbers of PCR-amplified samples (n > 15).

  11. 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.

  12. Colon cancer-associated mutator DNA polymerase δ variant causes expansion of dNTP pools increasing its own infidelity.

    Science.gov (United States)

    Mertz, Tony M; Sharma, Sushma; Chabes, Andrei; Shcherbakova, Polina V

    2015-05-12

    Defects in DNA polymerases δ (Polδ) and ε (Polε) cause hereditary colorectal cancer and have been implicated in the etiology of some sporadic colorectal and endometrial tumors. We previously reported that the yeast pol3-R696W allele mimicking a human cancer-associated variant, POLD1-R689W, causes a catastrophic increase in spontaneous mutagenesis. Here, we describe the mechanism of this extraordinary mutator effect. We found that the mutation rate increased synergistically when the R696W mutation was combined with defects in Polδ proofreading or mismatch repair, indicating that pathways correcting DNA replication errors are not compromised in pol3-R696W mutants. DNA synthesis by purified Polδ-R696W was error-prone, but not to the extent that could account for the unprecedented mutator phenotype of pol3-R696W strains. In a search for cellular factors that augment the mutagenic potential of Polδ-R696W, we discovered that pol3-R696W causes S-phase checkpoint-dependent elevation of dNTP pools. Abrogating this elevation by strategic mutations in dNTP metabolism genes eliminated the mutator effect of pol3-R696W, whereas restoration of high intracellular dNTP levels restored the mutator phenotype. Further, the use of dNTP concentrations present in pol3-R696W cells for in vitro DNA synthesis greatly decreased the fidelity of Polδ-R696W and produced a mutation spectrum strikingly similar to the spectrum observed in vivo. The results support a model in which (i) faulty synthesis by Polδ-R696W leads to a checkpoint-dependent increase in dNTP levels and (ii) this increase mediates the hypermutator effect of Polδ-R696W by facilitating the extension of mismatched primer termini it creates and by promoting further errors that continue to fuel the mutagenic pathway.

  13. Targeted DNA methylation analysis by next-generation sequencing.

    Science.gov (United States)

    Masser, Dustin R; Stanford, David R; Freeman, Willard M

    2015-02-24

    The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

  14. 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.

  15. 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.

  16. 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.

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

    International Nuclear Information System (INIS)

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

    2003-01-01

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

  18. Effects of the ssb-1 and ssb-113 mutations on survival and DNA repair in UV-irradiated delta uvrB strains of Escherichia coli K-12.

    OpenAIRE

    Wang, T C; Smith, K C

    1982-01-01

    The molecular defect in DNA repair caused by ssb mutations (single-strand binding protein) was studied by analyzing DNA synthesis and DNA double-strand break production in UV-irradiated Escherichia coli delta uvrB strains. The presence of the ssb-113 mutation produced a large inhibition of DNA synthesis and led to the formation of double-strand breaks, whereas the ssb-1 mutation produced much less inhibition of DNA synthesis and fewer double-strand breaks. We suggest that the single-strand bi...

  19. 454 next generation-sequencing outperforms allele-specific PCR, Sanger sequencing, and pyrosequencing for routine KRAS mutation analysis of formalin-fixed, paraffin-embedded samples

    Directory of Open Access Journals (Sweden)

    Altimari A

    2013-08-01

    Full Text Available Annalisa Altimari,1,* Dario de Biase,2,* Giovanna De Maglio,3 Elisa Gruppioni,1 Elisa Capizzi,1 Alessio Degiovanni,1 Antonia D'Errico,1 Annalisa Pession,2 Stefano Pizzolitto,3 Michelangelo Fiorentino,1,# Giovanni Tallini2,#1Laboratory of Molecular Oncologic and Transplantation Pathology, S. Orsola-Malpighi Hospital, Bologna, 2Laboratory of Molecular Pathology, Anatomic Pathology, Bellaria Hospital, Bologna, 3Department of Pathology, S. Maria della Misericordia Hospital, Udine, Italy*These authors contributed equally to this work #These authors share senior authorshipAbstract: Detection of KRAS mutations in archival pathology samples is critical for therapeutic appropriateness of anti-EGFR monoclonal antibodies in colorectal cancer. We compared the sensitivity, specificity, and accuracy of Sanger sequencing, ARMS-Scorpion (TheraScreen® real-time polymerase chain reaction (PCR, pyrosequencing, chip array hybridization, and 454 next-generation sequencing to assess KRAS codon 12 and 13 mutations in 60 nonconsecutive selected cases of colorectal cancer. Twenty of the 60 cases were detected as wild-type KRAS by all methods with 100% specificity. Among the 40 mutated cases, 13 were discrepant with at least one method. The sensitivity was 85%, 90%, 93%, and 92%, and the accuracy was 90%, 93%, 95%, and 95% for Sanger sequencing, TheraScreen real-time PCR, pyrosequencing, and chip array hybridization, respectively. The main limitation of Sanger sequencing was its low analytical sensitivity, whereas TheraScreen real-time PCR, pyrosequencing, and chip array hybridization showed higher sensitivity but suffered from the limitations of predesigned assays. Concordance between the methods was k = 0.79 for Sanger sequencing and k > 0.85 for the other techniques. Tumor cell enrichment correlated significantly with the abundance of KRAS-mutated deoxyribonucleic acid (DNA, evaluated as ΔCt for TheraScreen real-time PCR (P = 0.03, percentage of mutation for

  20. The Decrease in Mitochondrial DNA Mutation Load Parallels Visual Recovery in a Leber Hereditary Optic Neuropathy Patient

    Directory of Open Access Journals (Sweden)

    Sonia Emperador

    2018-02-01

    Full Text Available The onset of Leber hereditary optic neuropathy is relatively rare in childhood and, interestingly, the rate of spontaneous visual recovery is very high in this group of patients. Here, we report a child harboring a rare pathological mitochondrial DNA mutation, present in heteroplasmy, associated with the disease. A patient follow-up showed a rapid recovery of the vision accompanied by a decrease of the percentage of mutated mtDNA. A retrospective study on the age of recovery of all childhood-onset Leber hereditary optic neuropathy patients reported in the literature suggested that this process was probably related with pubertal changes.

  1. Next Generation Sequencing approach to molecular diagnosis of Duchenne muscular dystrophy; identification of a novel mutation.

    Science.gov (United States)

    Ebrahimzadeh-Vesal, Reza; Teymoori, Atieh; Azimi-Nezhad, Mohsen; Hosseini, Forough Sadat

    2018-02-20

    Duchenne Muscular Dystrophy (DMD; MIM 310200) is one of the most common and severe type of hereditary muscular dystrophies. The disease is caused by mutations in the dystrophin gene. The dystrophin gene is associated with X-linked recessive Duchenne and Becker muscular dystrophy. This disease occurs almost exclusively in males. The clinical symptoms of muscle weakness usually begin at childhood. The main symptoms of this disorder are gradually muscular weakness. The affected patients have inability to standing up and walking. Death is usually due to respiratory infection or cardiomyopathy. In this article, we have reported the discovery of a new nonsense mutation that creates abnormal stop codon in the dystrophin gene. This mutation was detected using Next Generation Sequencing (NGS) technique. The subject was a 17-year-old male with muscular dystrophy that who was suspected of having DMD. He was referred to Hakim medical genetics center of Neyshabur, IRAN. Copyright © 2017. Published by Elsevier B.V.

  2. Preliminary studies on DNA retardation by MutS applied to the detection of point mutations in clinical samples

    Energy Technology Data Exchange (ETDEWEB)

    Stanislawska-Sachadyn, Anna [Gdansk University of Technology, Microbiology Department, Narutowicza 11/12, 80952 Gdansk (Poland)]. E-mail: annast@altis.chem.pg.gda.pl; Paszko, Zygmunt [Maria Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology, Endocrinology Department, 5 W.K. Roentgen, 02-781 Warsaw (Poland); Kluska, Anna [Maria Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology, Endocrinology Department, 5 W.K. Roentgen, 02-781 Warsaw (Poland); Skasko, Elzibieta [Maria Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology, Endocrinology Department, 5 W.K. Roentgen, 02-781 Warsaw (Poland); Sromek, Maria [Maria Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology, Endocrinology Department, 5 W.K. Roentgen, 02-781 Warsaw (Poland); Balabas, Aneta [Maria Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology, Endocrinology Department, 5 W.K. Roentgen, 02-781 Warsaw (Poland); Janiec-Jankowska, Aneta [Maria Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology, Endocrinology Department, 5 W.K. Roentgen, 02-781 Warsaw (Poland); Wisniewska, Alicja [Maria Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology, Endocrinology Department, 5 W.K. Roentgen, 02-781 Warsaw (Poland); Kur, Jozef [Gdansk University of Technology, Microbiology Department, Narutowicza 11/12, 80952 Gdansk (Poland); Sachadyn, Pawel [Gdansk University of Technology, Microbiology Department, Narutowicza 11/12, 80952 Gdansk (Poland)

    2005-02-15

    MutS ability to bind DNA mismatches was applied to the detection of point mutations in PCR products. MutS recognized mismatches from single up to five nucleotides and retarded the electrophoretic migration of mismatched DNA. The electrophoretic detection of insertions/deletions above three nucleotides is also possible without MutS, thanks to the DNA mobility shift caused by the presence of large insertion/deletion loops in the heteroduplex DNA. Thus, the method enables the search for a broad range of mutations: from single up to several nucleotides. The mobility shift assays were carried out in polyacrylamide gels stained with SYBR-Gold. One assay required 50-200 ng of PCR product and 1-3 {mu}g of Thermus thermophilus his{sub 6}-MutS protein. The advantages of this approach are: the small amounts of DNA required for the examination, simple and fast staining, no demand for PCR product purification, no labelling and radioisotopes required. The method was tested in the detection of cancer predisposing mutations in RET, hMSH2, hMLH1, BRCA1, BRCA2 and NBS1 genes. The approach appears to be promising in screening for unknown point mutations.

  3. Methanol-based fixation is superior to buffered formalin for next-generation sequencing of DNA from clinical cancer samples.

    Science.gov (United States)

    Piskorz, A M; Ennis, D; Macintyre, G; Goranova, T E; Eldridge, M; Segui-Gracia, N; Valganon, M; Hoyle, A; Orange, C; Moore, L; Jimenez-Linan, M; Millan, D; McNeish, I A; Brenton, J D

    2016-03-01

    Next-generation sequencing (NGS) of tumour samples is a critical component of personalised cancer treatment, but it requires high-quality DNA samples. Routine neutral-buffered formalin (NBF) fixation has detrimental effects on nucleic acids, causing low yields, as well as fragmentation and DNA base changes, leading to significant artefacts. We have carried out a detailed comparison of DNA quality from matched samples isolated from high-grade serous ovarian cancers from 16 patients fixed in methanol and NBF. These experiments use tumour fragments and mock biopsies to simulate routine practice, ensuring that results are applicable to standard clinical biopsies. Using matched snap-frozen tissue as gold standard comparator, we show that methanol-based fixation has significant benefits over NBF, with greater DNA yield, longer fragment size and more accurate copy-number calling using shallow whole-genome sequencing (WGS). These data also provide a new approach to understand and quantify artefactual effects of fixation using non-negative matrix factorisation to analyse mutational spectra from targeted and WGS data. We strongly recommend the adoption of methanol fixation for sample collection strategies in new clinical trials. This approach is immediately available, is logistically simple and can offer cheaper and more reliable mutation calling than traditional NBF fixation. © The Author 2015. Published by Oxford University Press on behalf of the European Society for Medical Oncology.

  4. Mutation spectrum resulting in M13mp2 phage DNA exposed to N-nitrosoproline with UVA irradiation.

    Science.gov (United States)

    Horai, Yumi; Ando, Yoshiko; Kimura, Sachiko; Arimoto-Kobayashi, Sakae

    2017-09-01

    N-nitrosoproline (NPRO) is endogenously formed from proline and nitrite. In an effort to delineate the mechanism of NPRO-induced photomutagenicity, we investigated the mutagenic spectrum of NPRO on M13mp2 DNA with UVA irradiation. Following exposure to NPRO and UVA, the mutation frequency increased significantly in an NPRO and UVA dose-dependent manner. The sequence data derived from seventy of the mutants indicated that mutagenesis resulted mainly from an increase in single-base substitutions, the most frequent being GC to CG transversions. Non-clustering of the GC to CG mutations suggests that NPRO+UVA damage to DNA is random. These transversions may be caused by guanine adducts in DNA or in part by oxidatively modified guanine in DNA exposed to NPRO and UVA. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. 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

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

    Science.gov (United States)

    Bennett, Richard R; Schneider, Hal E; Estrella, Elicia; Burgess, Stephanie; Cheng, Andrew S; Barrett, Caitlin; Lip, Va; Lai, Poh San; Shen, Yiping; Wu, Bai-Lin; Darras, Basil T; Beggs, Alan H; Kunkel, Louis M

    2009-10-18

    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. 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 patients). Methods and assay sequences are reported in

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

    International Nuclear Information System (INIS)

    Swayne, Breanne G.; Kawata, Alice; Behan, Nathalie A.; Williams, Andrew; Wade, Mike G.; MacFarlane, Amanda J.; Yauk, Carole L.

    2012-01-01

    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.

  8. 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.

  9. Macrolide Resistance in Treponema pallidum Correlates With 23S rDNA Mutations in Recently Isolated Clinical Strains.

    Science.gov (United States)

    Molini, Barbara J; Tantalo, Lauren C; Sahi, Sharon K; Rodriguez, Veronica I; Brandt, Stephanie L; Fernandez, Mark C; Godornes, Charmie B; Marra, Christina M; Lukehart, Sheila A

    2016-09-01

    High rates of 23S rDNA mutations implicated in macrolide resistance have been identified in Treponema pallidum samples from syphilis patients in many countries. Nonetheless, some clinicians have been reluctant to abandon azithromycin as a treatment for syphilis, citing the lack of a causal association between these mutations and clinical evidence of drug resistance. Although azithromycin resistance has been demonstrated in vivo for the historical Street 14 strain, no recent T. pallidum isolates have been tested. We used the well-established rabbit model of syphilis to determine the in vivo efficacy of azithromycin against 23S rDNA mutant strains collected in 2004 to 2005 from patients with syphilis in Seattle, Wash. Groups of 9 rabbits were each infected with a strain containing 23S rDNA mutation A2058G (strains UW074B, UW189B, UW391B) or A2059G (strains UW228B, UW254B, and UW330B), or with 1 wild type strain (Chicago, Bal 3, and Mexico A). After documentation of infection, 3 animals per strain were treated with azithromycin, 3 were treated with benzathine penicillin G, and 3 served as untreated control groups. Treatment efficacy was documented by darkfield microscopic evidence of T. pallidum, serological response, and rabbit infectivity test. Azithromycin uniformly failed to cure rabbits infected with strains harboring either 23S rDNA mutation, although benzathine penicillin G was effective. Infections caused by wild type strains were successfully treated by either azithromycin or benzathine penicillin G. A macrolide resistant phenotype was demonstrated for all strains harboring a 23S rDNA mutation, demonstrating that either A2058G or A2059G mutation confers in vivo drug resistance.

  10. DNA biosensor based on hybridization refractory mutation system approach for single mismatch detection.

    Science.gov (United States)

    Joda, Hamdi; Beni, Valerio; Katakis, Ioanis; O'Sullivan, Ciara K

    2015-04-01

    We report on a simple approach to enhance solid-phase hybridization-based single base mismatch discrimination at high ionic strength based on the deliberate insertion of a natural DNA base mismatch in the surface-tethered probe. A large drop in hybridization signal of single base mismatched alleles using the designed probe as compared with the conventional probe, from 80% to less than 25% of the signal obtained with the fully complementary, non-mutation-containing sequence, when using colorimetric detection was further improved to 20% when using electrochemical detection, attributable to a difference of spacing of immobilized probes. Finally, the designed probe was used for the electrochemical detection of the DQA1*05:05 allele amplified from real human blood samples. Copyright © 2014 Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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.

  12. Comparison of the quantification of KRAS mutations by digital PCR and E-ice-COLD-PCR in circulating-cell-free DNA from metastatic colorectal cancer patients.

    Science.gov (United States)

    Sefrioui, David; Mauger, Florence; Leclere, Laurence; Beaussire, Ludivine; Di Fiore, Frédéric; Deleuze, Jean-François; Sarafan-Vasseur, Nasrin; Tost, Jörg

    2017-02-01

    Circulating cell-free DNA (ccfDNA) bears great promise as biomarker for personalized medicine, but ccfDNA is present only at low levels in the plasma or serum of cancer patients. E-ice-COLD-PCR is a recently developed enrichment method to detect and identify mutations present at low-abundance in clinical samples. However, recent studies have shown the importance to accurately quantify low-abundance mutations as clinically important decisions will depend on certain mutation thresholds. The possibility for an enrichment method to accurately quantify the mutation levels remains a point of concern and might limit its clinical applicability. In the present study, we compared the quantification of KRAS mutations in ccfDNA from metastatic colorectal cancer patients by E-ice-COLD-PCR with two digital PCR approaches. For the quantification of mutations by E-ice-COLD-PCR, cell lines with known mutations diluted into WT genomic DNA were used for calibration. E-ice-COLD-PCR and the two digital PCR approaches showed the same range of the mutation level and were concordant for mutation levels below the clinical relevant threshold. E-ice-COLD-PCR can accurately detect and quantify low-abundant mutations in ccfDNA and has a shorter time to results making it compatible with the requirements of analyses in a clinical setting without the loss of quantitative accuracy. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  13. Towards next-generation biodiversity assessment using DNA metabarcoding

    DEFF Research Database (Denmark)

    Taberlet, Pierre; Coissac, Eric; Pompanon, Francois

    2012-01-01

    Virtually all empirical ecological studies require species identification during data collection. DNA metabarcoding refers to the automated identification of multiple species from a single bulk sample containing entire organisms or from a single environmental sample containing degraded DNA (soil,...

  14. Linking the generation of DNA adducts to lung cancer.

    Science.gov (United States)

    Ceppi, Marcello; Munnia, Armelle; Cellai, Filippo; Bruzzone, Marco; Peluso, Marco E M

    2017-09-01

    Worldwide, lung cancer is the leading cause of cancer death. DNA adducts are considered a reliable biomarker that reflects carcinogen exposure to tobacco smoke, but the central question is what is the relationship of DNA adducts and cancer? Therefore, we investigated this relationship by a meta-analysis of twenty-two studies with bronchial adducts for a total of 1091 subjects, 887 lung cancer cases and 204 apparently healthy individuals with no evidence of lung cancer. Our study shows that these adducts are significantly associated to increase lung cancer risk. The value of Mean Ratio lung-cancer (MR) of bronchial adducts resulting from the random effects model was 2.64, 95% C.I. 2.00-3.50, in overall lung cancer cases as compared to controls. The significant difference, with lung cancer patients having significant higher levels of bronchial adducts than controls, persisted after stratification for smoking habits. The MR lung-cancer value between lung cancer patients and controls for smokers was 2.03, 95% C.I. 1.42-2.91, for ex-smokers 3.27, 95% C.I. 1.49-7.18, and for non-smokers was 3.81, 95% C.I. 1.85-7.85. Next, we found that the generation of bronchial adducts is significantly related to inhalation exposure to tobacco smoke carcinogens confirming its association with volatile carcinogens. The MR smoking estimate of bronchial adducts resulting from meta-regression was 2.28, 95% Confidence Interval (C.I.) 1.10-4.73, in overall smokers in respect to non-smokers. The present work provides strengthening of the hypothesis that bronchial adducts are not simply relate to exposure, but are a cause of chemical-induced lung cancer. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. A novel cell line generated using the CRISPR/Cas9 technology as universal quality control material for KRAS G12V mutation testing.

    Science.gov (United States)

    Jia, Shiyu; Zhang, Rui; Lin, Guigao; Peng, Rongxue; Gao, Peng; Han, Yanxi; Fu, Yu; Ding, Jiansheng; Wu, Qisheng; Zhang, Kuo; Xie, Jiehong; Li, Jinming

    2018-01-30

    KRAS mutations are the key indicator for EGFR monoclonal antibody-targeted therapy and acquired drug resistance, and their accurate detection is critical to the clinical decision-making of colorectal cancer. However, no proper quality control material is available for the current detection methods, particularly next-generation sequencing (NGS). The ideal quality control material for NGS needs to provide both the tumor mutation gene and the matched background genomic DNA, which is uncataloged in public databases, to accurately distinguish germline polymorphisms and somatic mutations. We developed a novel KRAS G12V mutant cell line using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technique to make up for the deficiencies in existing quality control material and further validated the feasibility of the cell line as quality control material by amplification refractory mutation system (ARMS), Sanger sequencing, digital PCR (dPCR), and NGS. We verified that the edited cell line specifically had the G12V mutation, and the validation results presented a high consistency among the four methods of detection. The three cell lines screened contained the G12V mutation and the mutation allele fractions of G12V-1, G12V-2, and G12V-3 were 52.01%, 82.06%, and 17.29%, respectively. The novel KRAS G12V cell line generated using the CRISPR/Cas9 gene editing system is suitable as a quality control material for all current detection methods and provides a new direction in the development of quality control material. © 2018 Wiley Periodicals, Inc.

  16. A single aspartate mutation in the conserved catalytic site of Rev3L generates a hypomorphic phenotype in vivo and in vitro.

    Science.gov (United States)

    Fritzen, Rémi; Delbos, Frédéric; De Smet, Annie; Palancade, Benoît; Canman, Christine E; Aoufouchi, Said; Weill, Jean-Claude; Reynaud, Claude-Agnès; Storck, Sébastien

    2016-10-01

    Rev3, the catalytic subunit of yeast DNA polymerase ζ, is required for UV resistance and UV-induced mutagenesis, while its mammalian ortholog, REV3L, plays further vital roles in cell proliferation and embryonic development. To assess the contribution of REV3L catalytic activity to its in vivo function, we generated mutant mouse strains in which one or two Ala residues were substituted to the Asp of the invariant catalytic YGDTDS motif. The simultaneous mutation of both Asp (ATA) phenocopies the Rev3l knockout, which proves that the catalytic activity is mandatory for the vital functions of Rev3L, as reported recently. Surprisingly, although the mutation of the first Asp severely impairs the enzymatic activity of other B-family DNA polymerases, the corresponding mutation of Rev3 (ATD) is hypomorphic in yeast and mouse, as it does not affect viability and proliferation and moderately impacts UVC-induced cell death and mutagenesis. Interestingly, Rev3l hypomorphic mutant mice display a distinct, albeit modest, alteration of the immunoglobulin gene mutation spectrum at G-C base pairs, further documenting its role in this process. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2017-03-27

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

  18. Escherichia coli DNA polymerase III is responsible for the high level of spontaneous mutations in mutT strains.

    Science.gov (United States)

    Yamada, Masami; Shimizu, Masatomi; Katafuchi, Atsushi; Grúz, Petr; Fujii, Shingo; Usui, Yukio; Fuchs, Robert P; Nohmi, Takehiko

    2012-12-01

    Reactive oxygen species induce oxidative damage in DNA precursors, i.e. dNTPs, leading to point mutations upon incorporation. Escherichia coli mutT strains, deficient in the activity hydrolysing 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP), display more than a 100-fold higher spontaneous mutation frequency over the wild-type strain. 8-oxo-dGTP induces A to C transversions when misincorporated opposite template A. Here, we report that DNA pol III incorporates 8-oxo-dGTP ≈ 20 times more efficiently opposite template A compared with template C. Single, double or triple deletions of pol I, pol II, pol IV or pol V had modest effects on the mutT mutator phenotype. Only the deletion of all four polymerases led to a 70% reduction of the mutator phenotype. While pol III may account for nearly all 8-oxo-dGTP incorporation opposite template A, it only extends ≈ 30% of them, the remaining 70% being extended by the combined action of pol I, pol II, pol IV or pol V. The unique property of pol III, a C-family DNA polymerase present only in eubacteria, to preferentially incorporate 8-oxo-dGTP opposite template A during replication might explain the high spontaneous mutation frequency in E. coli mutT compared with the mammalian counterparts lacking the 8-oxo-dGTP hydrolysing activities. © 2012 Blackwell Publishing Ltd.

  19. DNA mismatch repair gene mutations in 55 kindreds with verified or putative hereditary non-polyposis colorectal cancer

    NARCIS (Netherlands)

    NystromLahti, M; Wu, Y; Moisio, AL; Hofstra, RMW; Osinga, J; MEcklin, JP; Jarvinen, HJ; Leisti, J; Buys, CHCM; delaChapelle, A; Peltomaki, P

    The DNA mismatch repair genes MSH2 and MLH1 have been shown to account for a major share of hereditary non-polyposis colorectal cancer (HNPCC). We searched for germline mutations in these genes in 35 HNPCC kindreds fulfilling the Amsterdam diagnostic criteria and in a further 20 kindreds with an

  20. No evidence of association between optic neuritis and secondary LHON mtDNA mutations in patients with multiple sclerosis

    DEFF Research Database (Denmark)

    Andalib, Sasan; Talebi, Mahnaz; Sakhinia, Ebrahim

    2017-01-01

    primers and restriction endonucleases for seven secondary LHON mutations. Products were visualized using 3% agarose gel electrophoresis with the aid of DNA safe stain in a UV transilluminator. Accuracy of the genotyping procedure was confirmed by sequencing. Data was analyzed using chi square and Fisher...

  1. Infantile Encephalopathy and Defective Mitochondrial DNA Translation in Patients with Mutations of Mitochondrial Elongation Factors EFG1 and EFTu

    Science.gov (United States)

    Valente, Lucia; Tiranti, Valeria; Marsano, René Massimiliano; Malfatti, Edoardo; Fernandez-Vizarra, Erika; Donnini, Claudia; Mereghetti, Paolo; De Gioia, Luca; Burlina, Alberto; Castellan, Claudio; Comi, Giacomo P.; Savasta, Salvatore; Ferrero, Iliana; Zeviani, Massimo

    2007-01-01

    Mitochondrial protein translation is a complex process performed within mitochondria by an apparatus composed of mitochondrial DNA (mtDNA)–encoded RNAs and nuclear DNA–encoded proteins. Although the latter by far outnumber the former, the vast majority of mitochondrial translation defects in humans have been associated with mutations in RNA-encoding mtDNA genes, whereas mutations in protein-encoding nuclear genes have been identified in a handful of cases. Genetic investigation involving patients with defective mitochondrial translation led us to the discovery of novel mutations in the mitochondrial elongation factor G1 (EFG1) in one affected baby and, for the first time, in the mitochondrial elongation factor Tu (EFTu) in another one. Both patients were affected by severe lactic acidosis and rapidly progressive, fatal encephalopathy. The EFG1-mutant patient had early-onset Leigh syndrome, whereas the EFTu-mutant patient had severe infantile macrocystic leukodystrophy with micropolygyria. Structural modeling enabled us to make predictions about the effects of the mutations at the molecular level. Yeast and mammalian cell systems proved the pathogenic role of the mutant alleles by functional complementation in vivo. Nuclear-gene abnormalities causing mitochondrial translation defects represent a new, potentially broad field of mitochondrial medicine. Investigation of these defects is important to expand the molecular characterization of mitochondrial disorders and also may contribute to the elucidation of the complex control mechanisms, which regulate this fundamental pathway of mtDNA homeostasis. PMID:17160893

  2. Systematic biochemical analysis of somatic missense mutations in DNA polymerase β found in prostate cancer reveal alteration of enzymatic function.

    Science.gov (United States)

    An, Chang Long; Chen, Desheng; Makridakis, Nick M

    2011-04-01

    DNA polymerase β is essential for short-patch base excision repair. We have previously identified 20 somatic pol β mutations in prostate tumors, many of them missense. In the current article we describe the effect of all of these somatic missense pol β mutations (p.K27N, p.E123K, p.E232K, p.P242R, p.E216K, p.M236L, and the triple mutant p.P261L/T292A/I298T) on the biochemical properties of the polymerase in vitro, following bacterial expression and purification of the respective enzymatic variants. We report that all missense somatic pol β mutations significantly affect enzyme function. Two of the pol β variants reduce catalytic efficiency, while the remaining five missense mutations alter the fidelity of DNA synthesis. Thus, we conclude that a significant proportion (9 out of 26; 35%) of prostate cancer patients have functionally important somatic mutations of pol β. Many of these missense mutations are clonal in the tumors, and/or are associated with loss of heterozygosity and microsatellite instability. These results suggest that interfering with normal polymerase β function may be a frequent mechanism of prostate tumor progression. Furthermore, the availability of detailed structural information for pol β allows understanding of the potential mechanistic effects of these mutants on polymerase function. © 2011 Wiley-Liss, Inc.

  3. 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.

  4. Germline mutations in DNA repair genes predispose asbestos-exposed patients to malignant pleural mesothelioma.

    Science.gov (United States)

    Betti, Marta; Casalone, Elisabetta; Ferrante, Daniela; Aspesi, Anna; Morleo, Giulia; Biasi, Alessandra; Sculco, Marika; Mancuso, Giuseppe; Guarrera, Simonetta; Righi, Luisella; Grosso, Federica; Libener, Roberta; Pavesi, Mansueto; Mariani, Narciso; Casadio, Caterina; Boldorini, Renzo; Mirabelli, Dario; Pasini, Barbara; Magnani, Corrado; Matullo, Giuseppe; Dianzani, Irma

    2017-10-01

    Malignant pleural mesothelioma (MPM) is a rare, aggressive cancer caused by asbestos exposure. An inherited predisposition has been suggested to explain multiple cases in the same family and the observation that not all individuals highly exposed to asbestos develop the tumor. Germline mutations in BAP1 are responsible for a rare cancer predisposition syndrome that includes predisposition to mesothelioma. We hypothesized that other genes involved in hereditary cancer syndromes could be responsible for the inherited mesothelioma predisposition. We investigated the prevalence of germline variants in 94 cancer-predisposing genes in 93 MPM patients with a quantified asbestos exposure. Ten pathogenic truncating variants (PTVs) were identified in PALB2, BRCA1, FANCI, ATM, SLX4, BRCA2, FANCC, FANCF, PMS1 and XPC. All these genes are involved in DNA repair pathways, mostly in homologous recombination repair. Patients carrying PTVs represented 9.7% of the panel and showed lower asbestos exposure than did all the other patients (p = 0.0015). This suggests that they did not efficiently repair the DNA damage induced by asbestos and leading to carcinogenesis. This study shows that germline variants in several genes may increase MPM susceptibility in the presence of asbestos exposure and may be important for specific treatment. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  5. Screening for germline mutations in mismatch repair genes in patients with Lynch syndrome by next generation sequencing.

    Science.gov (United States)

    Soares, Barbara Luísa; Brant, Ayslan Castro; Gomes, Renan; Pastor, Tatiane; Schneider, Naye Balzan; Ribeiro-Dos-Santos, Ândrea; de Assumpção, Paulo Pimentel; Achatz, Maria Isabel W; Ashton-Prolla, Patrícia; Moreira, Miguel Angelo Martins

    2017-09-20

    Lynch syndrome (LS) is an autosomal dominant disorder, with high penetrance that affects approximately 3% of the cases of colorectal cancer. Affected individuals inherit germline mutations in genes responsible for DNA mismatch repair, mainly at MSH2, MLH1, MSH6 and PMS2. The molecular screening of these individuals is frequently costly and time consuming due to the large size of these genes. In addition, PMS2 mutation detection is often a challenge because there are 16 different pseudogenes identified until now. In the present work we evaluate a molecular screening strategy based in next generation sequencing (NGS) in order to optimize the mutation detection in LS patients. We established 16 multiplex PCRs for MSH2, MSH6 and MLH1 and 5 Long-Range PCRs for PMS2, coupled with NGS. The strategy was validated by screening 66 patients who filled Bethesda and Amsterdam criteria for LS from health institutions of Brazil. The mean depth of coverage for MSH2, MSH6, MLH1 and PMS2 genes was 7.988, 36.313, 11.899 and 4.772 times, respectively. Ninety-four variants were found in exons and flanking intron/exon regions for the four MMR genes. Twenty-five were pathogenic or VUS and found in 32 patients (7 in MSH2, 5 in MSH6, 12 in MLH1 e 1 in PMS2). All variants were confirmed by Sanger sequencing. The strategy was efficient to reduce time consuming and costs to identify genetic changes at these MMR genes, reducing in three times the number of PCR reactions performed per patient and was efficient in identifying variants at PMS2 gene.

  6. Profiling cancer gene mutations in clinical formalin-fixed, paraffin-embedded colorectal tumor specimens using targeted next-generation sequencing.

    Science.gov (United States)

    Zhang, Liangxuan; Chen, Liangjing; Sah, Sachin; Latham, Gary J; Patel, Rajesh; Song, Qinghua; Koeppen, Hartmut; Tam, Rachel; Schleifman, Erica; Mashhedi, Haider; Chalasani, Sreedevi; Fu, Ling; Sumiyoshi, Teiko; Raja, Rajiv; Forrest, William; Hampton, Garret M; Lackner, Mark R; Hegde, Priti; Jia, Shidong

    2014-04-01

    The success of precision oncology relies on accurate and sensitive molecular profiling. The Ion AmpliSeq Cancer Panel, a targeted enrichment method for next-generation sequencing (NGS) using the Ion Torrent platform, provides a fast, easy, and cost-effective sequencing workflow for detecting genomic "hotspot" regions that are frequently mutated in human cancer genes. Most recently, the U.K. has launched the AmpliSeq sequencing test in its National Health Service. This study aimed to evaluate the clinical application of the AmpliSeq methodology. We used 10 ng of genomic DNA from formalin-fixed, paraffin-embedded human colorectal cancer (CRC) tumor specimens to sequence 46 cancer genes using the AmpliSeq platform. In a validation study, we developed an orthogonal NGS-based resequencing approach (SimpliSeq) to assess the AmpliSeq variant calls. Validated mutational analyses revealed that AmpliSeq was effective in profiling gene mutations, and that the method correctly pinpointed "true-positive" gene mutations with variant frequency >5% and demonstrated high-level molecular heterogeneity in CRC. However, AmpliSeq enrichment and NGS also produced several recurrent "false-positive" calls in clinically druggable oncogenes such as PIK3CA. AmpliSeq provided highly sensitive and quantitative mutation detection for most of the genes on its cancer panel using limited DNA quantities from formalin-fixed, paraffin-embedded samples. For those genes with recurrent "false-positive" variant calls, caution should be used in data interpretation, and orthogonal verification of mutations is recommended for clinical decision making.

  7. Enzymatic recognition of DNA damage induced by UVB-photosensitized titanium dioxide and biological consequences in Saccharomyces cerevisiae: Evidence for oxidatively DNA damage generation

    International Nuclear Information System (INIS)

    Pinto, A. Viviana; Deodato, Elder L.; Cardoso, Janine S.; Oliveira, Eliza F.; Machado, Sergio L.; Toma, Helena K.; Leitao, Alvaro C.; Padula, Marcelo de

    2010-01-01

    Although titanium dioxide (TiO 2 ) has been considered to be biologically inert, finding use in cosmetics, paints and food colorants, recent reports have demonstrated that when TiO 2 is attained by UVA radiation oxidative genotoxic and cytotoxic effects are observed in living cells. However, data concerning TiO 2 -UVB association is poor, even if UVB radiation represents a major environmental carcinogen. Herein, we investigated DNA damage, repair and mutagenesis induced by TiO 2 associated with UVB irradiation in vitro and in vivo using Saccharomyces cerevisiae model. It was found that TiO 2 plus UVB treatment in plasmid pUC18 generated, in addition to cyclobutane pyrimidine dimers (CPDs), specific damage to guanine residues, such as 8-oxo-7,8-dihydroguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG), which are characteristic oxidatively generated lesions. In vivo experiments showed that, although the presence of TiO 2 protects yeast cells from UVB cytotoxicity, high mutation frequencies are observed in the wild-type (WT) and in an ogg1 strain (deficient in 8-oxoG and FapyG repair). Indeed, after TiO 2 plus UVB treatment, induced mutagenesis was drastically enhanced in ogg1 cells, indicating that mutagenic DNA lesions are repaired by the Ogg1 protein. This effect could be attenuated by the presence of metallic ion chelators: neocuproine or dipyridyl, which partially block oxidatively generated damage occurring via Fenton reactions. Altogether, the results indicate that TiO 2 plus UVB potentates UVB oxidatively generated damage to DNA, possibly via Fenton reactions involving the production of DNA base damage, such as 8-oxo-7,8-dihydroguanine.

  8. Enzymatic recognition of DNA damage induced by UVB-photosensitized titanium dioxide and biological consequences in Saccharomyces cerevisiae: Evidence for oxidatively DNA damage generation

    Energy Technology Data Exchange (ETDEWEB)

    Pinto, A. Viviana, E-mail: alicia.pinto@incqs.fiocruz.br [Laboratorio de Diagnostico Molecular e Hematologia, Faculdade de Farmacia, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21941-540, Rio de Janeiro (Brazil); Laboratorio de Radiobiologia Molecular, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21949-900, Rio de Janeiro (Brazil); Deodato, Elder L. [Laboratorio de Diagnostico Molecular e Hematologia, Faculdade de Farmacia, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21941-540, Rio de Janeiro (Brazil); Laboratorio de Radiobiologia Molecular, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21949-900, Rio de Janeiro (Brazil); Cardoso, Janine S. [Laboratorio de Radiobiologia Molecular, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21949-900, Rio de Janeiro (Brazil); Oliveira, Eliza F.; Machado, Sergio L.; Toma, Helena K. [Laboratorio de Diagnostico Molecular e Hematologia, Faculdade de Farmacia, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21941-540, Rio de Janeiro (Brazil); Leitao, Alvaro C. [Laboratorio de Radiobiologia Molecular, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21949-900, Rio de Janeiro (Brazil); Padula, Marcelo de [Laboratorio de Diagnostico Molecular e Hematologia, Faculdade de Farmacia, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21941-540, Rio de Janeiro (Brazil)

    2010-06-01

    Although titanium dioxide (TiO{sub 2}) has been considered to be biologically inert, finding use in cosmetics, paints and food colorants, recent reports have demonstrated that when TiO{sub 2} is attained by UVA radiation oxidative genotoxic and cytotoxic effects are observed in living cells. However, data concerning TiO{sub 2}-UVB association is poor, even if UVB radiation represents a major environmental carcinogen. Herein, we investigated DNA damage, repair and mutagenesis induced by TiO{sub 2} associated with UVB irradiation in vitro and in vivo using Saccharomyces cerevisiae model. It was found that TiO{sub 2} plus UVB treatment in plasmid pUC18 generated, in addition to cyclobutane pyrimidine dimers (CPDs), specific damage to guanine residues, such as 8-oxo-7,8-dihydroguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG), which are characteristic oxidatively generated lesions. In vivo experiments showed that, although the presence of TiO{sub 2} protects yeast cells from UVB cytotoxicity, high mutation frequencies are observed in the wild-type (WT) and in an ogg1 strain (deficient in 8-oxoG and FapyG repair). Indeed, after TiO{sub 2} plus UVB treatment, induced mutagenesis was drastically enhanced in ogg1 cells, indicating that mutagenic DNA lesions are repaired by the Ogg1 protein. This effect could be attenuated by the presence of metallic ion chelators: neocuproine or dipyridyl, which partially block oxidatively generated damage occurring via Fenton reactions. Altogether, the results indicate that TiO{sub 2} plus UVB potentates UVB oxidatively generated damage to DNA, possibly via Fenton reactions involving the production of DNA base damage, such as 8-oxo-7,8-dihydroguanine.

  9. Targeted next generation sequencing identifies clinically actionable mutations in patients with melanoma.

    Science.gov (United States)

    Jeck, William R; Parker, Joel; Carson, Craig C; Shields, Janiel M; Sambade, Maria J; Peters, Eldon C; Burd, Christin E; Thomas, Nancy E; Chiang, Derek Y; Liu, Wenjin; Eberhard, David A; Ollila, David; Grilley-Olson, Juneko; Moschos, Stergios; Neil Hayes, D; Sharpless, Norman E

    2014-07-01

    Somatic sequencing of cancers has produced new insight into tumorigenesis, tumor heterogeneity, and disease progression, but the vast majority of genetic events identified are of indeterminate clinical significance. Here, we describe a NextGen sequencing approach to fully analyzing 248 genes, including all those of known clinical significance in melanoma. This strategy features solution capture of DNA followed by multiplexed, high-throughput sequencing and was evaluated in 31 melanoma cell lines and 18 tumor tissues from patients with metastatic melanoma. Mutations in melanoma cell lines correlated with their sensitivity to corresponding small molecule inhibitors, confirming, for example, lapatinib sensitivity in ERBB4 mutant lines and identifying a novel activating mutation of BRAF. The latter event would not have been identified by clinical sequencing and was associated with responsiveness to a BRAF kinase inhibitor. This approach identified focal copy number changes of PTEN not found by standard methods, such as comparative genomic hybridization (CGH). Actionable mutations were found in 89% of the tumor tissues analyzed, 56% of which would not be identified by standard-of-care approaches. This work shows that targeted sequencing is an attractive approach for clinical use in melanoma. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  10. Catalytic effects of mutations of distant protein residues in human DNA polymerase β: theory and experiment.

    Science.gov (United States)

    Klvaňa, Martin; Murphy, Drew L; Jeřábek, Petr; Goodman, Myron F; Warshel, Arieh; Sweasy, Joann B; Florián, Jan

    2012-11-06

    We carried out free-energy calculations and transient kinetic experiments for the insertion of the right (dC) and wrong (dA) nucleotides by wild-type (WT) and six mutant variants of human DNA polymerase β (Pol β). Since the mutated residues in the point mutants, I174S, I260Q, M282L, H285D, E288K, and K289M, were not located in the Pol β catalytic site, we assumed that the WT and its point mutants share the same dianionic phosphorane transition-state structure of the triphosphate moiety of deoxyribonucleotide 5'-triphosphate (dNTP) substrate. On the basis of this assumption, we have formulated a thermodynamic cycle for calculating relative dNTP insertion efficiencies, Ω = (k(pol)/K(D))(mut)/(k(pol)/K(D))(WT) using free-energy perturbation (FEP) and linear interaction energy (LIE) methods. Kinetic studies on five of the mutants have been published previously using different experimental conditions, e.g., primer-template sequences. We have performed a presteady kinetic analysis for the six mutants for comparison with wild-type Pol β using the same conditions, including the same primer/template DNA sequence proximal to the dNTP insertion site used for X-ray crystallographic studies. This consistent set of kinetic and structural data allowed us to eliminate the DNA sequence from the list of factors that can adversely affect calculated Ω values. The calculations using the FEP free energies scaled by 0.5 yielded 0.9 and 1.1 standard deviations from the experimental log Ω values for the insertion of the right and wrong dNTP, respectively. We examined a hybrid FEP/LIE method in which the FEP van der Waals term for the interaction of the mutated amino acid residue with its surrounding environment was replaced by the corresponding van der Waals term calculated using the LIE method, resulting in improved 0.4 and 1.0 standard deviations from the experimental log Ω values. These scaled FEP and FEP/LIE methods were also used to predict log Ω for R283A and R283L Pol

  11. Case Report Identification of a novel SLC45A2 mutation in albinism by targeted next-generation sequencing.

    Science.gov (United States)

    Xue, J J; Xue, J F; Xue, H Q; Guo, Y Y; Liu, Y; Ouyang, N

    2016-09-19

    Albinism is a diverse group of hypopigmentary disorders caused by multiple-genetic defects. The genetic diagnosis of patients affected with albinism by Sanger sequencing is often complex, expensive, and time-consuming. In this study, we performed targeted next-generation sequencing to screen for 16 genes in a patient with albinism, and identified 21 genetic variants, including 19 known single nucleotide polymorphisms, one novel missense mutation (c.1456 G>A), and one disease-causing mutation (c.478 G>C). The novel mutation was not observed in 100 controls, and was predicted to be a damaging mutation by SIFT and Polyphen. Thus, we identified a novel mutation in SLC45A2 in a Chinese family, expanding the mutational spectrum of albinism. Our results also demonstrate that targeted next-generation sequencing is an effective genetic test for albinism.

  12. A conservative isoleucine to leucine mutation causes major rearrangements and cold sensitivity in KlenTaq1 DNA polymerase.

    Science.gov (United States)

    Wu, Eugene Y; Walsh, Amanda R; Materne, Emma C; Hiltner, Emily P; Zielinski, Bryan; Miller, Bill R; Mawby, Lily; Modeste, Erica; Parish, Carol A; Barnes, Wayne M; Kermekchiev, Milko B

    2015-01-27

    Assembly of polymerase chain reactions at room temperature can sometimes lead to low yields or unintentional products due to mispriming. Mutation of isoleucine 707 to leucine in DNA polymerase I from Thermus aquaticus substantially decreases its activity at room temperature without compromising its ability to amplify DNA. To understand why a conservative change to the enzyme over 20 Å from the active site can have a large impact on its activity at low temperature, we solved the X-ray crystal structure of the large (5'-to-3' exonuclease-deleted) fragment of Taq DNA polymerase containing the cold-sensitive mutation in the ternary (E-DNA-ddNTP) and binary (E-DNA) complexes. The I707L KlenTaq1 ternary complex was identical to the wild-type in the closed conformation except for the mutation and a rotamer change in nearby phenylalanine 749, suggesting that the enzyme should remain active. However, soaking out of the nucleotide substrate at low temperature results in an altered binary complex made possible by the rotamer change at F749 near the tip of the polymerase O-helix. Surprisingly, two adenosines in the 5'-template overhang fill the vacated active site by stacking with the primer strand, thereby blocking the active site at low temperature. Replacement of the two overhanging adenosines with pyrimidines substantially increased activity at room temperature by keeping the template overhang out of the active site, confirming the importance of base stacking. These results explain the cold-sensitive phenotype of the I707L mutation in KlenTaq1 and serve as an example of a large conformational change affected by a conservative mutation.

  13. Mutation in cytochrome b gene of mitochondrial DNA in a family with fibromyalgia is associated with NLRP3-inflammasome activation.

    Science.gov (United States)

    Cordero, Mario D; Alcocer-Gómez, Elísabet; Marín-Aguilar, Fabiola; Rybkina, Tatyana; Cotán, David; Pérez-Pulido, Antonio; Alvarez-Suarez, José Miguel; Battino, Maurizio; Sánchez-Alcazar, José Antonio; Carrión, Angel M; Culic, Ognjen; Navarro-Pando, José M; Bullón, Pedro

    2016-02-01

    Fibromyalgia (FM) is a worldwide diffuse musculoskeletal chronic pain condition that affects up to 5% of the general population. Many symptoms associated with mitochondrial diseases are reported in patients with FM such as exercise intolerance, fatigue, myopathy and mitochondrial dysfunction. In this study, we report a mutation in cytochrome b gene of mitochondrial DNA (mtDNA) in a family with FM with inflammasome complex activation. mtDNA from blood cells of five patients with FM were sequenced. We clinically and genetically characterised a patient with FM and family with a new mutation in mtCYB. Mitochondrial mutation phenotypes were determined in skin fibroblasts and transmitochondrial cybrids. After mtDNA sequence in patients with FM, we found a mitochondrial homoplasmic mutation m.15804T>C in the mtCYB gene in a patient and family, which was maternally transmitted. Mutation was observed in several tissues and skin fibroblasts showed a very significant mitochondrial dysfunction and oxidative stress. Increased NLRP3-inflammasome complex activation was observed in blood cells from patient and family. We propose further studies on mtDNA sequence analysis in patients with FM with evidences for maternal inheritance. The presence of similar symptoms in mitochondrial myopathies could unmask mitochondrial diseases among patients with FM. On the other hand, the inflammasome complex activation by mitochondrial dysfunction could be implicated in the pathophysiology of mitochondrial diseases. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  14. Mutation rate, spectrum, topology, and context-dependency in the DNA mismatch repair-deficient Pseudomonas fluorescens ATCC948.

    Science.gov (United States)

    Long, Hongan; Sung, Way; Miller, Samuel F; Ackerman, Matthew S; Doak, Thomas G; Lynch, Michael

    2014-12-23

    High levels of genetic diversity exist among natural isolates of the bacterium Pseudomonas fluorescens, and are especially elevated around the replication terminus of the genome, where strain-specific genes are found. In an effort to understand the role of genetic variation in the evolution of Pseudomonas, we analyzed 31,106 base substitutions from 45 mutation accumulation lines of P. fluorescens ATCC948, naturally deficient for mismatch repair, yielding a base-substitution mutation rate of 2.34 × 10(-8) per site per generation (SE: 0.01 × 10(-8)) and a small-insertion-deletion mutation rate of 1.65 × 10(-9) per site per generation (SE: 0.03 × 10(-9)). We find that the spectrum of mutations in prophage regions, which often contain virulence factors and antibiotic resistance, is highly similar to that in the intergenic regions of the host genome. Our results show that the mutation rate varies around the chromosome, with the lowest mutation rate found near the origin of replication. Consistent with observations from other studies, we find that site-specific mutation rates are heavily influenced by the immediately flanking nucleotides, indicating that mutations are context dependent. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  15. Current status and future potential of somatic mutation testing from circulating free DNA in patients with solid tumours.

    Science.gov (United States)

    Aung, K L; Board, R E; Ellison, G; Donald, E; Ward, T; Clack, G; Ranson, M; Hughes, A; Newman, W; Dive, C

    2010-12-01

    Genetic alterations can determine the natural history of cancer and its treatment response. With further advances in DNA sequencing technology, multiple novel genetic alterations will be discovered which could be exploited as prognostic, predictive and pharmacodynamic biomarkers in the development and use of cancer therapeutics. As such, the importance in clinical practice of efficient and robust somatic mutation testing in solid tumours cannot be overemphasized in the current era of personalized medicine. However, significant challenges remain regarding the testing of genetic biomarkers in clinical practice. Reliance on archived formalin fixed, paraffin embedded tumour, obtained from diagnostic biopsies, for testing somatic genetic alterations could restrict the scientific community in asking relevant questions about a patient's cancer biology. Problems inherent with using formalin fixed, archival tissue are well recognized and difficult to resolve. It could be argued that to achieve rapid and efficient incorporation of genetic biomarkers into clinical practice, somatic mutation testing in cancer patients should be simpler, less invasive using a readily available clinical sample, whilst maintaining robustness and reproducibility. In this regard, use of circulating free DNA (cfDNA) from plasma or serum as an alternative and/or additional source of DNA to test cancer specific genetic alterations is an attractive proposition. In light of encouraging results from recent studies, this mini review will discuss the current role and future potential of somatic mutation testing from circulating or cell free DNA derived from the blood of patients with solid tumours.

  16. Bone metastasis in prostate cancer: Recurring mitochondrial DNA mutation reveals selective pressure exerted by the bone microenvironment.

    Science.gov (United States)

    Arnold, Rebecca S; Fedewa, Stacey A; Goodman, Michael; Osunkoya, Adeboye O; Kissick, Haydn T; Morrissey, Colm; True, Lawrence D; Petros, John A

    2015-09-01

    Cancer progression and metastasis occur such that cells with acquired mutations enhancing growth and survival (or inhibiting cell death) increase in number, a concept that has been recognized as analogous to Darwinian evolution of species since Peter C. Nowell's description in 1976. Selective forces include those intrinsic to the host (including metastatic site) as well as those resulting from anti-cancer therapies. By examining the mutational status of multiple tumor sites within an individual patient some insight may be gained into those genetic variants that enhance site-specific metastasis. By comparing these data across multiple individuals, recurrent patterns may identify alterations that are fundamental to successful site-specific metastasis. We sequenced the mitochondrial genome in 10 prostate cancer patients with bone metastases enrolled in a rapid autopsy program. Patients had late stage disease and received androgen ablation and frequently other systemic therapies. For each of 9 patients, 4 separate tissues were sequenced: the primary prostate cancer, a soft tissue metastasis, a bone metastasis and an uninvolved normal tissue that served as the non-cancerous control. An additional (10th) patient had no primary prostate available for sequencing but had both metastatic sites (and control DNA) sequenced. We then examined the number and location of somatically acquired mitochondrial DNA (mtDNA) mutations in the primary tumor and two metastatic sites in each individual patient. Finally, we compared patients with each other to determine any common patterns of somatic mutation. Somatic mutations were significantly more numerous in the bone compared to either the primary tumor or soft tissue metastases. A missense mutation at nucleotide position (n.p.) 10398 (A10398G; Thr114Ala) in the respiratory complex I gene ND3 was the most common (7 of 10 patients) and was detected only in the bone. Other notable somatic mutations that occurred in more than one patient

  17. Bias-corrected targeted next-generation sequencing for rapid, multiplexed detection of actionable alterations in cell-free DNA from advanced lung cancer patients

    Science.gov (United States)

    Paweletz, Cloud P.; Sacher, Adrian G.; Raymond, Chris K.; Alden, Ryan S.; O'Connell, Allison; Mach, Stacy L.; Kuang, Yanan; Gandhi, Leena; Kirschmeier, Paul; English, Jessie M.; Lim, Lee P.; Jänne, Pasi A.; Oxnard, Geoffrey R.

    2015-01-01

    Purpose Tumor genotyping is a powerful tool for guiding non-small cell lung cancer (NSCLC) care, however comprehensive tumor genotyping can be logistically cumbersome. To facilitate genotyping, we developed a next-generation sequencing (NGS) assay using a desktop sequencer to detect actionable mutations and rearrangements in cell-free plasma DNA (cfDNA). Experimental Design An NGS panel was developed targeting 11 driver oncogenes found in NSCLC. Targeted NGS was performed using a novel methodology that maximizes on-target reads, and minimizes artifact, and was validated on DNA dilutions derived from cell lines. Plasma NGS was then blindly performed on 48 patients with advanced, progressive NSCLC and a known tumor genotype, and explored in two patients with incomplete tumor genotyping. Results NGS could identify mutations present in DNA dilutions at ≥0.4% allelic frequency with 100% sensitivity/specificity. Plasma NGS detected a broad range of driver and resistance mutations, including ALK, ROS1, and RET rearrangements, HER2 insertions, and MET amplification, with 100% specificity. Sensitivity was 77% across 62 known driver and resistance mutations from the 48 cases; in 29 cases with common EGFR and KRAS mutations, sensitivity was similar to droplet digital PCR. In two cases with incomplete tumor genotyping, plasma NGS rapidly identified a novel EGFR exon 19 deletion and a missed case of MET amplification. Conclusion Blinded to tumor genotype, this plasma NGS approach detected a broad range of targetable genomic alterations in NSCLC with no false positives including complex mutations like rearrangements and unexpected resistance mutations such as EGFR C797S. Through use of widely available vacutainers and a desktop sequencing platform, this assay has the potential to be implemented broadly for patient care and translational research. PMID:26459174

  18. Concordance of mutation detection in circulating tumor DNA in early clinical trials using different blood collection protocols

    DEFF Research Database (Denmark)

    Ahlborn, Lise B.; Madsen, Mette; Jonson, Lars

    2017-01-01

    in a clinical setting. Here we investigate the concordance between standard blood collection for molecular analysis using immediate separation of plasma, compared to the use of collection tubes allowing for delayed processing. Methods: In this study, we measured the fractional abundance of tumor specific...... patients with advanced solid cancers enrolled in early clinical trials. Results: Concordance in the fractional abundance of mutations in ctDNA isolated from blood collected in either K3EDTA or BCT tubes from patients with different solid cancers was observed. Conclusions: This study indicates that BCT...... mutations (BRAF p.V600E and PIK3CA p.H1047R) in ctDNA isolated from blood samples collected in either cell-stabilizing Cell-Free DNA BCT tubes (delayed processing within 72 hours) or standard K3EDTA tubes (immediate processing within 15 minutes). Twenty-five blood sample pairs (EDTA/BCT) were collected from...

  19. MELAS syndrome associated with both A3243G-tRNALeu mutation and multiple mitochondrial DNA deletions.

    Science.gov (United States)

    Aharoni, Sharon; Traves, Teres A; Melamed, Eldad; Cohen, Sarit; Silver, Esther Leshinsky

    2010-09-15

    The syndrome of mitochondrial encephalopathy, lactic acidosis, and stroke-like episode (MELAS) is characterized clinically by recurrent focal neurological deficits, epilepsy, and short stature. The phenotypic spectrum is extremely diverse, with multisystemic organ involvement leading to isolated diabetes, deafness, renal tubulopathy, hypertrophic cardiomyopathy, and retinitis pigmentosa. In 80% of cases, the syndrome is associated with an AG transmission mutation (A3243G) in the tRNALeu gene of the mitochondrial DNA (mtDNA). We describe a woman with a unique combination of the MELAS A3243G mutation and multiple mtDNA deletions with normal POLG sequence. The patient presented with diabetes mellitus, sensorineural deafness, short stature, and mental disorientation. All her three children died in early adolescence. 2010 Elsevier B.V. All rights reserved.

  20. 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; Daneshvar, Bahram; Autrup, Herman

    2003-01-01

    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......, colon, or urine. Thus, lard intake at the expense of other nutrients and a large increase in the fat energy consumption affects the redox state locally in the liver cytosol, but does not induce DNA-damage, systemic oxidative stress or a dose-dependent increase in mutation frequency in rat colon or liver........ The DNA-adduct level measured by 32P-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...

  1. 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

    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......, colon, or urine. Thus, lard intake at the expense of other nutrients and a large increase in the fat energy consumption affects the redox state locally in the liver cytosol, but does not induce DNA-damage, systemic oxidative stress or a dose-dependent increase in mutation frequency in rat 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...

  2. 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)

    Qu Jia; Li Ronghua; Tong Yi; Hu Yongwu; Zhou Xiangtian; Qian Yaping; Lu Fan; Guan Minxin

    2005-01-01

    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

  3. Gene editing by co-transformation of TALEN and chimeric RNA/DNA oligonucleotides on the rice OsEPSPS gene and the inheritance of mutations.

    Directory of Open Access Journals (Sweden)

    Mugui Wang

    Full Text Available Although several site-specific nucleases (SSNs, such as zinc-finger nucleases (ZFNs, transcription activator-like effector nucleases (TALENs, and the clustered regularly interspaced short palindromic repeat (CRISPR/Cas, have emerged as powerful tools for targeted gene editing in many organisms, to date, gene targeting (GT in plants remains a formidable challenge. In the present study, we attempted to substitute a single base in situ on the rice OsEPSPS gene by co-transformation of TALEN with chimeric RNA/DNA oligonucleotides (COs, including different strand composition such as RNA/DNA (C1 or DNA/RNA (C2 but contained the same target base to be substituted. In contrast to zero GT event obtained by the co-transformation of TALEN with homologous recombination plasmid (HRP, we obtained one mutant showing target base substitution although accompanied by undesired deletion of 12 bases downstream the target site from the co-transformation of TALEN and C1. In addition to this typical event, we also obtained 16 mutants with different length of base deletions around the target site among 105 calli lines derived from transformation of TALEN alone (4/19 as well as co-transformation of TELAN with either HRP (5/30 or C1 (2/25 or C2 (5/31. Further analysis demonstrated that the homozygous gene-edited mutants without foreign gene insertion could be obtained in one generation. The induced mutations in transgenic generation were also capable to pass to the next generation stably. However, the genotypes of mutants did not segregate normally in T1 population, probably due to lethal mutations. Phenotypic assessments in T1 generation showed that the heterozygous plants with either one or three bases deletion on target sequence, called d1 and d3, were more sensitive to glyphosate and the heterozygous d1 plants had significantly lower seed-setting rate than wild-type.

  4. Do mutations in DNMT3A/3B affect global DNA hypomethylation among benzene-exposed workers in Southeast China?: Effects of mutations in DNMT3A/3B on global DNA hypomethylation.

    Science.gov (United States)

    Zhang, Guang-Hui; Lu, Ye; Ji, Bu-Qiang; Ren, Jing-Chao; Sun, Pin; Ding, Shibin; Liao, Xiaoling; Liao, Kaiju; Liu, Jinyi; Cao, Jia; Lan, Qing; Rothman, Nathaniel; Xia, Zhao-Lin

    2017-12-01

    Global DNA hypomethylation is commonly observed in benzene-exposed workers, but the underlying mechanisms remain unclear. We sought to discover the relationships among reduced white blood cell (WBC) counts, micronuclear (MN) frequency, and global DNA methylation to determine whether there were associations with mutations in DNMT3A/3B. Therefore, we recruited 410 shoe factory workers and 102 controls from Wenzhou in Zhenjiang Province. A Methylated DNA Quantification Kit was used to quantify global DNA methylation, and single nucleotide polymorphisms (SNPs) in DNMT3A (rs36012910, rs1550117, and R882) and DNMT3B (rs1569686, rs2424909, and rs2424913) were identified using the restriction fragment length polymorphism method. A multilinear regression analysis demonstrated that the benzene-exposed workers experienced significant global DNA hypomethylation compared with the controls (β = -0.51, 95% CI: -0.69 to -0.32, P benzene exposure and that mutations in DNMTs are significantly associated with global DNA methylation, which might have influenced the induction of MN following exposure to benzene. Environ. Mol. Mutagen. 58:678-687, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  5. Mixed phenotype acute leukemia contains heterogeneous genetic mutations by next-generation sequencing

    Science.gov (United States)

    Quesada, Andrés E.; Hu, Zhihong; Routbort, Mark J.; Patel, Keyur P.; Luthra, Rajyalakshmi; Loghavi, Sanam; Zuo, Zhuang; Yin, C. Cameron; Kanagal-Shamanna, Rashmi; Wang, Sa A.; Jorgensen, Jeffrey L.; Medeiros, L. Jeffrey; Ok, Chi Young

    2018-01-01

    Mixed phenotype acute leukemia (MPAL) is an uncommon manifestation of acute leukemia. The aim of this study is to further characterize the genetic landscape of de novo cases of MPAL that fulfill the 2016 World Health Organization (WHO) classification criteria for this entity. We identified 14 cases examined by next generation sequencing (NGS) using 28 (n = 10), 53 (n = 3) or 81 (n = 1) gene panels: 7 cases with a B-cell/myeloid (B/My) immunophenotype, 6 T-cell/myeloid (T/My) immunophenotype, and 1 B-cell/T-cell (B/T) immunophenotype. A total of 25 distinct mutations were identified in 15 different genes in 9/14 (64%) patients. FLT3-ITD was the only recurrent mutation in 2 patients. B/My MPAL cases less commonly harbored mutations compared with T/My MPAL cases (43% vs. 100%, p = 0.07). In contrast, B/My MPALs more commonly showed a complex karyotype compared to T/My MPALs (71% vs. 17%, p = 0.1). With NGS and karyotype combined, most (93%) MPAL cases had mutations or cytogenetic abnormalities. With a median follow-up of 12.5 months, there were no significant differences in median overall survival (OS) between patients with B/My or T/My MPAL (17.8 and 6.5 months, respectively, p = 0.81) or between patients with MPAL with versus without gene mutations (6.5 and 13.3 months, respectively, p = 0.86). Our data suggest that the distinguishing cases of MPAL according to immunophenotype has value because the underlying mechanisms of leukemogenesis might differ between B/My and T/My MPAL. PMID:29492206

  6. Generating Exome Enriched Sequencing Libraries from Formalin-Fixed, Paraffin-Embedded Tissue DNA for Next Generation Sequencing

    Science.gov (United States)

    Marosy, Beth A.; Craig, Brian D.; Hetrick, Kurt N.; Witmer, P. Dane; Ling, Hua; Griffith, Sean M.; Myers, Ben; Ostrander, Elaine A.; Stanford, Janet L.; Brody, Lawrence C.; Doheny, Kimberly F.

    2016-01-01

    This unit describes a protocol for generating exome enriched sequencing libraries using DNA extracted from Formalin Fixed Paraffin Embedded (FFPE) samples. Utilizing commercially available kits, we present a low input FFPE workflow starting with 50ng of DNA. This procedure includes a repair step to address damage caused by FFPE preservation that improves sequence quality. Subsequently, libraries undergo an in-solution targeted selection for exons, followed by sequencing using the Illumina next generation short read sequencing platform. PMID:28075488

  7. N-acetoxy-N-2-acetylaminofluorene induced frameshift mutations: a comparison between the DNA modification spectrum and the mutation spectrum

    International Nuclear Information System (INIS)

    Fuchs, R.P.P.; Koffel-Schwartz, N.; Daune, M.

    1983-01-01

    We describe the analysis of forward mutations induced in the tetracycline resistance gene of the plasmid pBR322 by directing the reaction of the carcinogen N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF) to a small restriction fragment (BamHI, SalI) that is located in the proximal part of the antibiotic-resistance gene. Mutant plasmids obtained both in wild type and excision repair deficient (uvrA) bacterial cells are compared. Preliminary data showing the distribution of the -AAF adducts along this restriction fragments are discussed in relation to the observed spectrum of mutations. 20 references, 4 figures

  8. [Genotyping of Vaginal Candida glabrata Isolates Using Microsatellite Marker Analysis and DNA Sequencing to Identify Mutations Associated with Antifungal Resistance].

    Science.gov (United States)

    Döğen, Aylin; Durukan, Hüseyin; Güzel, Ahmet Barış; Oksüz, Zehra; Kaplan, Engin; Serin, Mehmet Sami; Serin, Ayşe; Emekdaş, Gürol; Aslan, Gönül; Tezcan, Seda; Kalkancı, Ayşe; Ilkit, Macit

    2013-01-01

    Vulvovaginal candidosis is the second most common cause of vaginitis (17-39%) after bacterial vaginosis (22-50%). Since the diagnosis of vulvovaginal candidosis mainly depends on clinical findings without mycologic confirmatory tests and treated empirically, the actual incidence rate of vulvovaginal candidosis is unknown. Approximately 70-90% of vulvovaginal candidosis cases are caused by Candida albicans, however the increasing incidence of C.glabrata infections and its reduced susceptibility to azole drug therapy have generated increasing attention. The epidemiology and population structure of vulvovaginal candidosis due to C.glabrata are poorly characterized. This study was aimed to genotype the C.glabrata strains isolated from vaginal samples in Cukurova region, Turkey by microsatellite markers, to investigate the antifungal susceptibility profiles of the strains and to determine the molecular mechanisms leading to phenotypical azole resistance. A total of 34 unrelated vaginal C.glabrata strains isolated from patients with acute (n= 11) and recurrent (n= 14) vulvovaginal candidosis, control group (n= 9) without vaginitis symptoms, and a reference strain of C.glabrata CBS 138 (ATCC 2001) were included in the study. These isolates were genotyped using multiple-locus variable number tandem repeat analysis of three microsatellite markers (RPM2, MTI, and Cg6). Analysis of microsatellite markers was performed by fragment size determination of RPM2, MTI, and Cg6 PCR products through capillary electrophoresis. For each of the evaluated strains, DNA sequence analysis was performed for one gene (CgERG11) and four loci (CgPDR1, NTM1, TRP1, and URA3) to detect mutations possibly associated with antifungal resistance in each strain. In vitro susceptibility profiles of the strains to 13 antifungals and boric acid were determined according to CLSI document M27-A3 to investigate possible relationships between detected mutations and phenotypic resistance. C.glabrata CBS 138

  9. Noninvasive detection through REMS-PCR technique of K-ras mutations in stool DNA of patients with colorectal cancer.

    Science.gov (United States)

    Mixich, Francisc; Ioana, Mihai; Voinea, Florea; Săftoiu, Adrian; Ciurea, Tudorel

    2007-03-01

    Tumor exfoliated cells that shed into stool are attractive targets for molecular screening and early detection of colon malignancies. Many studies have suggested that the detection of activated ras may have diagnostic or prognostic importance. The aim of this study was to establish the suitability for use in diagnostic laboratories of the noninvasive screening test of K-ras mutation determination in the stool and its routine prognostic value in colorectal cancer. Paired stool and tissue specimens obtained after polypectomy and colorectal biopsy from 28 patients diagnosed solely by histopathological findings with primary colorectal carcinoma, were prospectively studied for K-ras codon 12 mutations by restriction endonuclease-mediated selective (REMS)-PCR. DNA was obtained in 28 of tissue samples (100%) and 26 of stool samples (92.8%). K-ras codon 12 mutation was seen in 14 (50.0%) paired stool and tissue samples. Mutation detection was possible in 1000-fold excess of wild-type sequence. These results may be important in the design of genetic screening programs, determination of prognosis, early detection and treatment for patients with colon malignancy. The sensitivity and specificity of K-ras determination on stool-derived DNA in patients with colorectal carcinoma, support the opportunity of a large-scale trial to validate its use as a screening test. REMS- PCR is not labor intensive, but a sensitive, rapid, and robust assay for the detection of point mutations, and was introduced by us in a routine diagnostic laboratory.

  10. Importance of a stable topoisomerase IB clamping for an efficient DNA processing: Effect of the Lys(369)Glu mutation.

    Science.gov (United States)

    Vieira, Sara; Castelli, Silvia; Desideri, Alessandro

    2015-11-01

    The role of lysine 369 of human topoisomerase IB in recognizing, clamping and processing its DNA substrate was experimentally investigated. Lys(369) is located in one of the two lips that interact to each other allowing the protein to embrace and firmly bind the DNA substrate. The lysine was mutated to a glutamate residue and the catalytic activity of the mutant enzyme was assayed. The mutant shows a distributive behavior, has a reduced binding to the substrate and a lower cleavage extent when compared to the wild type enzyme. The mutant displays reduced sensitivity to CPT both "in vitro" and in an "in vivo" yeast model, likely because of the low amount of cleaved DNA, however it displays cleavage and religation rates comparable to the wild type. These results demonstrate that the mutation causes a destabilization of the lips clamping around the DNA, impairing the protein-DNA interaction, emphasizing the importance of the ionic pair in tuning the stability of the protein-DNA complex. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Rapid infectious disease identification by next-generation DNA sequencing.

    Science.gov (United States)

    Ellis, Jeremy E; Missan, Dara S; Shabilla, Matthew; Martinez, Delyn; Fry, Stephen E

    2017-07-01

    Currently, there is a critical need to rapidly identify infectious organisms in clinical samples. Next-Generation Sequencing (NGS) could surmount the deficiencies of culture-based methods; however, there are no standardized, automated programs to process NGS data. To address this deficiency, we developed the Rapid Infectious Disease Identification (RIDI™) system. The system requires minimal guidance, which reduces operator errors. The system is compatible with the three major NGS platforms. It automatically interfaces with the sequencing system, detects their data format, configures the analysis type, applies appropriate quality control, and analyzes the results. Sequence information is characterized using both the NCBI database and RIDI™ specific databases. RIDI™ was designed to identify high probability sequence matches and more divergent matches that could represent different or novel species. We challenged the system using defined American Type Culture Collection (ATCC) reference standards of 27 species, both individually and in varying combinations. The system was able to rapidly detect known organisms in DNA sequence reads at the genus-level and 75.3% at the species-level in reference standards. It has a limit of detection of 146cells/ml in simulated clinical samples, and is also able to identify the components of polymicrobial samples with 16.9% discrepancy at the genus-level and 31.2% at the species-level. Thus, the system's effectiveness may exceed current methods, especially in situations where culture methods could produce false negatives or where rapid results would influence patient outcomes. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Ligation bias in Illumina next-generation DNA libraries

    DEFF Research Database (Denmark)

    Seguin-Orlando, Andaine; Schubert, Mikkel; Clary, Joel

    2013-01-01

    that library preparation based on adapter ligation at AT-overhangs are biased against DNA templates starting with thymine residues, contrarily to blunt-end adapter ligation. We observe the same bias on fresh DNA extracts sheared on Bioruptor, Covaris and nebulizers. This contradicts previous reports suggesting...... that this bias could originate from the methods used for shearing DNA. This also suggests that AT-overhang adapter ligation efficiency is affected in a sequence-dependent manner and results in an uneven representation of different genomic contexts. We then show how this bias could affect the base composition...... of ancient DNA libraries prepared following AT-overhang ligation, mainly by limiting the ability to ligate DNA templates starting with thymines and therefore deaminated cytosines. This results in particular nucleotide misincorporation damage patterns, deviating from the signature generally expected...

  13. Damages induced in lambda phage DNA by enzyme-generated triplet acetone

    International Nuclear Information System (INIS)

    Menck, C.F.; Cabral Neto, J.B.; Gomes, R.A.; Faljoni-Alario, A.

    1985-01-01

    Exposure of lambda phage to triplet acetone, generated during the aerobic oxidation of isobutanal by peroxidase, leads to genome lesions. The majority of these lesions are detected as DNA single-strand breaks only in alkaline conditions, so true breaks were not observed. Also, no sites sensitive to UV-endonuclease from Micrococcus luteus were found in DNA from treated phage. The participation of triplet acetone in the generation of such DNA damage is discussed. (Author) [pt

  14. A Conservative Isoleucine to Leucine Mutation Causes Major Rearrangements and Cold Sensitivity in KlenTaq1 DNA Polymerase

    Science.gov (United States)

    2015-01-01

    Assembly of polymerase chain reactions at room temperature can sometimes lead to low yields or unintentional products due to mispriming. Mutation of isoleucine 707 to leucine in DNA polymerase I from Thermus aquaticus substantially decreases its activity at room temperature without compromising its ability to amplify DNA. To understand why a conservative change to the enzyme over 20 Å from the active site can have a large impact on its activity at low temperature, we solved the X-ray crystal structure of the large (5′-to-3′ exonuclease-deleted) fragment of Taq DNA polymerase containing the cold-sensitive mutation in the ternary (E–DNA–ddNTP) and binary (E–DNA) complexes. The I707L KlenTaq1 ternary complex was identical to the wild-type in the closed conformation except for the mutation and a rotamer change in nearby phenylalanine 749, suggesting that the enzyme should remain active. However, soaking out of the nucleotide substrate at low temperature results in an altered binary complex made possible by the rotamer change at F749 near the tip of the polymerase O-helix. Surprisingly, two adenosines in the 5′-template overhang fill the vacated active site by stacking with the primer strand, thereby blocking the active site at low temperature. Replacement of the two overhanging adenosines with pyrimidines substantially increased activity at room temperature by keeping the template overhang out of the active site, confirming the importance of base stacking. These results explain the cold-sensitive phenotype of the I707L mutation in KlenTaq1 and serve as an example of a large conformational change affected by a conservative mutation. PMID:25537790

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

    International Nuclear Information System (INIS)

    Narahari, P.

    1978-01-01

    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 3 H-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 M 1 plants, M 1 spikes and M 2 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)

  16. 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 various...... combinations. Consequently, it is difficult to predict the "phenotypic risk profile" of 3243A>G mutation-positive subjects. The 3243A>G mutation coexists in cells with wild-type mtDNA, a phenomenon called heteroplasmy. The marked variability in mutation loads in different tissues is the main explanation...... for the different phenotypes associated with this mutation. AIM: The aim of the study was to screen asymptomatic and oligosymptomatic 3243A>G mtDNA carriers for diabetes and myopathy. METHODS: The study is a case-control study. Nineteen adult 3243A>G carriers presumed to be normoglycemic and matched healthy...

  17. Using high-sensitivity sequencing for the detection of mutations in BTK and PLCγ2 genes in cellular and cell-free DNA and correlation with progression in patients treated with BTK inhibitors.

    Science.gov (United States)

    Albitar, Adam; Ma, Wanlong; DeDios, Ivan; Estella, Jeffrey; Ahn, Inhye; Farooqui, Mohammed; Wiestner, Adrian; Albitar, Maher

    2017-03-14

    Patients with chronic lymphocytic leukemia (CLL) that develop resistance to Bruton tyrosine kinase (BTK) inhibitors are typically positive for mutations in BTK or phospholipase c gamma 2 (PLCγ2). We developed a high sensitivity (HS) assay utilizing wild-type blocking polymerase chain reaction achieved via bridged and locked nucleic acids. We used this high sensitivity assay in combination with Sanger sequencing and next generation sequencing (NGS) and tested cellular DNA and cell-free DNA (cfDNA) from patients with CLL treated with the BTK inhibitor, ibrutinib. We also tested ibrutinib-naïve patients with CLL. HS testing achieved 100x greater sensitivity than Sanger. HS Sanger sequencing was capable of detecting sequencing, plasma cfDNA is more reliable than cellular DNA in detecting mutations. Our studies indicate that wild-type blocking and HS sequencing is necessary for proper and early detection of BTK or PLCγ2 mutations in monitoring patients treated with BTK inhibitors. Furthermore, cfDNA from plasma is very reliable sample-type for testing.

  18. 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.

  19. mtDNA-Server: next-generation sequencing data analysis of human mitochondrial DNA in the cloud.

    Science.gov (United States)

    Weissensteiner, Hansi; Forer, Lukas; Fuchsberger, Christian; Schöpf, Bernd; Kloss-Brandstätter, Anita; Specht, Günther; Kronenberg, Florian; Schönherr, Sebastian

    2016-07-08

    Next generation sequencing (NGS) allows investigating mitochondrial DNA (mtDNA) characteristics such as heteroplasmy (i.e. intra-individual sequence variation) to a higher level of detail. While several pipelines for analyzing heteroplasmies exist, issues in usability, accuracy of results and interpreting final data limit their usage. Here we present mtDNA-Server, a scalable web server for the analysis of mtDNA studies of any size with a special focus on usability as well as reliable identification and quantification of heteroplasmic variants. The mtDNA-Server workflow includes parallel read alignment, heteroplasmy detection, artefact or contamination identification, variant annotation as well as several quality control metrics, often neglected in current mtDNA NGS studies. All computational steps are parallelized with Hadoop MapReduce and executed graphically with Cloudgene. We validated the underlying heteroplasmy and contamination detection model by generating four artificial sample mix-ups on two different NGS devices. Our evaluation data shows that mtDNA-Server detects heteroplasmies and artificial recombinations down to the 1% level with perfect specificity and outperforms existing approaches regarding sensitivity. mtDNA-Server is currently able to analyze the 1000G Phase 3 data (n = 2,504) in less than 5 h and is freely accessible at https://mtdna-server.uibk.ac.at. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. Colon cancer-associated mutator DNA polymerase δ variant causes expansion of dNTP pools increasing its own infidelity

    Science.gov (United States)

    Mertz, Tony M.; Sharma, Sushma; Chabes, Andrei; Shcherbakova, Polina V.

    2015-01-01

    Defects in DNA polymerases δ (Polδ) and ε (Polε) cause hereditary colorectal cancer and have been implicated in the etiology of some sporadic colorectal and endometrial tumors. We previously reported that the yeast pol3-R696W allele mimicking a human cancer-associated variant, POLD1-R689W, causes a catastrophic increase in spontaneous mutagenesis. Here, we describe the mechanism of this extraordinary mutator effect. We found that the mutation rate increased synergistically when the R696W mutation was combined with defects in Polδ proofreading or mismatch repair, indicating that pathways correcting DNA replication errors are not compromised in pol3-R696W mutants. DNA synthesis by purified Polδ-R696W was error-prone, but not to the extent that could account for the unprecedented mutator phenotype of pol3-R696W strains. In a search for cellular factors that augment the mutagenic potential of Polδ-R696W, we discovered that pol3-R696W causes S-phase checkpoint-dependent elevation of dNTP pools. Abrogating this elevation by strategic mutations in dNTP metabolism genes eliminated the mutator effect of pol3-R696W, whereas restoration of high intracellular dNTP levels restored the mutator phenotype. Further, the use of dNTP concentrations present in pol3-R696W cells for in vitro DNA synthesis greatly decreased the fidelity of Polδ-R696W and produced a mutation spectrum strikingly similar to the spectrum observed in vivo. The results support a model in which (i) faulty synthesis by Polδ-R696W leads to a checkpoint-dependent increase in dNTP levels and (ii) this increase mediates the hypermutator effect of Polδ-R696W by facilitating the extension of mismatched primer termini it creates and by promoting further errors that continue to fuel the mutagenic pathway. PMID:25827231

  1. Human Induced Pluripotent Stem Cells Harbor Homoplasmic and Heteroplasmic Mitochondrial DNA Mutations While Maintaining Human Embryonic Stem Cell-like Metabolic Reprogramming

    NARCIS (Netherlands)

    Prigione, A.; Lichtner, B.; Kuhl, H.; Struys, E.A.; Wamelink, M.M.C.; Lehrach, H.; Ralser, M.; Timmermann, B.; Adjaye, J.

    2011-01-01

    Human induced pluripotent stem cells (iPSCs) have been recently found to harbor genomic alterations. However, the integrity of mitochondrial DNA (mtDNA) within reprogrammed cells has yet to be investigated. mtDNA mutations occur at a high rate and contribute to the pathology of a number of human

  2. Clinical and molecular analysis of a four-generation Chinese family with aminoglycoside-induced and nonsyndromic hearing loss associated with the mitochondrial 12S rRNA C1494T mutation

    International Nuclear Information System (INIS)

    Wang Qiuju; Li Qingzhong; Han Dongyi; Zhao Yali; Zhao Lidong; Qian Yaping; Yuan Hu; Li Ronghua; Zhai Suoqiang; Young Wieyen; Guan Minxin

    2006-01-01

    We report here the clinical, genetic, and molecular characterization of a four-generation Chinese family with aminoglycoside-induced and nonsyndromic hearing loss. Five of nine matrilineal relatives had aminoglycoside-induced hearing loss. These matrilineal relatives exhibited variable severity and audiometric configuration of hearing impairment, despite sharing some common features: being bilateral and having sensorineural hearing impairment. Sequence analysis of mitochondrial DNA (mtDNA) in the pedigree identified 16 variants and the homoplasmic 12S rRNA C1494T mutation, which was associated with hearing loss in the other large Chinese family. In fact, the occurrence of the C1494T mutation in these genetically unrelated pedigrees affected by hearing impairment strongly indicated that this mutation is involved in the pathogenesis of aminoglycoside-induced and nonsyndromic hearing loss. However, incomplete penetrance of hearing loss indicated that the C1494T mutation itself is not sufficient to produce a clinical phenotype but requires the involvement of modifier factors for the phenotypic expression. Those mtDNA variants, showing no evolutional conservation, may not have a potential modifying role in the pathogenesis of the C1494T mutation. However, nuclear background seems to contribute to the phenotypic variability of matrilineal relatives in this family. Furthermore, aminoglycosides modulate the expressivity and penetrance of deafness associated with the C1494T mutation in this family

  3. Generation of Oxtr cDNA(HA)-Ires-Cre Mice for Gene Expression in an Oxytocin Receptor Specific Manner.

    Science.gov (United States)

    Hidema, Shizu; Fukuda, Tomokazu; Hiraoka, Yuichi; Mizukami, Hiroaki; Hayashi, Ryotaro; Otsuka, Ayano; Suzuki, Shingo; Miyazaki, Shinji; Nishimori, Katsuhiko

    2016-05-01

    The neurohypophysial hormone oxytocin (OXT) and its receptor (OXTR) have critical roles in the regulation of pro-social behaviors, including social recognition, pair bonding, parental behavior, and stress-related responses. Supporting this hypothesis, a portion of patients suffering from autism spectrum disorder have mutations, such as single nucleotide polymorphisms, or epigenetic modifications in their OXTR gene. We previously reported that OXTR-deficient mice exhibit pervasive social deficits, indicating the critical role of OXTR in social behaviors. In the present study, we generated Oxtr cDNA(HA)-Ires-Cre knock-in mice, expressing both OXTR and Cre recombinase under the control of the endogenous Oxtr promoter. Knock-in cassette of Oxtr cDNA(HA)-Ires-Cre consisted of Oxtr cDNA tagged with the hemagglutinin epitope at the 3' end (Oxtr cDNA(HA)), internal ribosomal entry site (Ires), and Cre. Cre was expressed in the uterus, mammary gland, kidney, and brain of Oxtr cDNA(HA)-Ires-Cre knock-in mice. Furthermore, the distribution of Cre in the brain was similar to that observed in Oxtr-Venus fluorescent protein expressing mice (Oxtr-Venus), another animal model previously generated by our group. Social behavior of Oxtr cDNA(HA)-Ires-Cre knock-in mice was similar to that of wild-type animals. We demonstrated that this construct is expressed in OXTR-expressing neurons specifically after an infection with the recombinant adeno-associated virus carrying the flip-excision switch vector. Using this system, we showed the transport of the wheat-germ agglutinin tracing molecule from the OXTR-expressing neurons to the innervated neurons in knock-in mice. This study might contribute to the monosynaptic analysis of neuronal circuits and to the optogenetic analysis of neurons expressing OXTR. © 2015 Wiley Periodicals, Inc.

  4. FOXL2 402C>G Mutation Can Be Identified in the Circulating Tumor DNA of Patients with Adult-Type Granulosa Cell Tumor.

    Science.gov (United States)

    Färkkilä, Anniina; McConechy, Melissa K; Yang, Winnie; Talhouk, Aline; Ng, Ying; Lum, Amy; Morin, Ryan D; Bushell, Kevin; Riska, Annika; McAlpine, Jessica N; Gilks, C Blake; Unkila-Kallio, Leila; Anttonen, Mikko; Huntsman, David G

    2017-01-01

    Adult granulosa cell tumors (AGCTs) of the ovary are molecularly characterized by the pathognomonic FOXL2 402C>G (C134W) mutation. To improve diagnostics and follow-up, we developed a specific digital droplet PCR (ddPCR) assay to detect the FOXL2 mutation in the circulating tumor DNA (ctDNA) of AGCT patients. Optimization of the ddPCR assay was performed using a TaqMan primer/probe with the RainDance RainDrop digital PCR system. The ddPCR assay was performed on circulating cell-free DNA extracted from 120 serial plasma samples collected prospectively from 35 AGCT patients. The ddPCR assay included a preamplification step that is sensitive and specific for detecting the FOXL2-mutated ctDNA at levels as low as 0.05%. FOXL2 ctDNA mutations were detected in the plasma of 12 of 33 AGCT patients (36%), with both primary (6 of 17, 35%) and recurrent (6 of 31, 19%) tumors. The median tumor size was significantly larger in ctDNA mutation-positive compared with mutation-negative samples (13.5 cm versus 7.5 cm; P = 0.003). The ctDNA FOXL2 mutation was detected in four patients without clinical disease, of which one relapsed during follow-up. As proof of concept, we established that specific molecular diagnosis of AGCT and detection of AGCT recurrence can be achieved noninvasively using ctDNA FOXL2 mutation testing. Further studies are needed to determine the clinical value of ctDNA mutation testing. Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  5. Generation of heritable germline mutations in the jewel wasp Nasonia vitripennis using CRISPR/Cas9.

    Science.gov (United States)

    Li, Ming; Au, Lauren Yun Cook; Douglah, Deema; Chong, Abigail; White, Bradley J; Ferree, Patrick M; Akbari, Omar S

    2017-04-19

    The revolutionary RNA-guided endonuclease CRISPR/Cas9 system has proven to be a powerful tool for gene editing in a plethora of organisms. Here, utilizing this system we developed an efficient protocol for the generation of heritable germline mutations in the parasitoid jewel wasp, Nasonia vitripennis, a rising insect model organism for the study of evolution, development of axis pattern formation, venom production, haplo-diploid sex determination, and host-symbiont interactions. To establish CRISPR-directed gene editing in N. vitripennis, we targeted a conserved eye pigmentation gene cinnabar, generating several independent heritable germline mutations in this gene. Briefly, to generate these mutants, we developed a protocol to efficiently collect N. vitripennis eggs from a parasitized flesh fly pupa, Sarcophaga bullata, inject these eggs with Cas9/guide RNA mixtures, and transfer injected eggs back into the host to continue development. We also describe a flow for screening mutants and establishing stable mutant strains through genetic crosses. Overall, our results demonstrate that the CRISPR/Cas9 system is a powerful tool for genome manipulation in N. vitripennis, with strong potential for expansion to target critical genes, thus allowing for the investigation of several important biological phenomena in this organism.

  6. 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...... and recent investigations have shown TET2 mutations in diffuse large B-cell lymphoma. However, the detection rates and the types of TET2 mutations vary, and the relation to global methylation patterns has not been investigated. Here, we show TET2 mutations in 12 of 100 diffuse large B-cell lymphomas with 7...

  7. 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.

  8. [Correlation analysis between abundance of K-ras mutation in plasma free DNA and its correlation with clinical outcome and prognosis in patients with metastatic colorectal cancer].

    Science.gov (United States)

    Bai, Yan-qing; Liu, Xiao-jing; Wang, Yan; Ge, Fei-jiao; Zhao, Chuan-hua; Fu, Ya-li; Lin, Li; Xu, Jian-ming

    2013-09-01

    To detect K-ras gene mutations in plasma free DNA by peptide nucleic acid clamp PCR assay (PNA-PCR) and nested primer PCR, and to analyze the correlation between K-ras mutations and prognosis in patients with metastatic colorectal cancer (mCRC). Peripheral blood was collected and free DNA was extracted from plasma in 106 patients with mCRC. Nested primer PCR and PNA-PCR were used to detect K-ras gene mutation in the plasma free DNA. The patients were divided into three groups by K-ras status: wild-type group (wild-type determined by both methods), low mutation group (mutation by PNA-PCR method, wild-type by nested primer PCR method) and high mutation group (mutation by two methods). The correlation between K-ras mutations and prognosis was analyzed. The mutation rate of K-ras in tumor tissues of the 106 patients was 40.6%. The Mutation rate of K-ras in plasma free DNA detected by PNA-PCR was 31.1%, significantly higher than that of 15.1% detected by nested primer PCR (P = 0.006). The consistent rate of the K-ras status in plasma free DNA detected by PNA-PCR and that in tumor tissue detected by traditional method was up to 83.0%. The median overall survival (OS) of patients of the wild type, low mutation and high mutation groups was 23.5 months, 17.3 months and 13.9 months, respectively (P = 0.002). The median progression-free survival (PFS) of the K-ras wild-type, low mutation and high mutation groups with first-line chemotherapy was 6.8 months, 6.1 months and 3.2 months, respectively (P = 0.002), and the median OS of them were 23.0 months, 15.5 months and 13.9 months, respectively (P = 0.036). The overall response rate (ORR) was improved in the K-ras wide-type patients who received cetuximab combined with chemotherapy as first-line therapy (75.0% vs. 23.4%, P = 0.058). Cetuximab combined with in second-line therapy chemotherapy led to a significant improvement in disease control rate (DCR) ( 100% vs. 35.7%, P mutation in plasma free DNA can be used to substitute

  9. Mutation detection with next-generation resequencing through a mediator genome.

    Directory of Open Access Journals (Sweden)

    Omri Wurtzel

    Full Text Available The affordability of next generation sequencing (NGS is transforming the field of mutation analysis in bacteria. The genetic basis for phenotype alteration can be identified directly by sequencing the entire genome of the mutant and comparing it to the wild-type (WT genome, thus identifying acquired mutations. A major limitation for this approach is the need for an a-priori sequenced reference genome for the WT organism, as the short reads of most current NGS approaches usually prohibit de-novo genome assembly. To overcome this limitation we propose a general framework that utilizes the genome of relative organisms as mediators for comparing WT and mutant bacteria. Under this framework, both mutant and WT genomes are sequenced with NGS, and the short sequencing reads are mapped to the mediator genome. Variations between the mutant and the mediator that recur in the WT are ignored, thus pinpointing the differences between the mutant and the WT. To validate this approach we sequenced the genome of Bdellovibrio bacteriovorus 109J, an obligatory bacterial predator, and its prey-independent mutant, and compared both to the mediator species Bdellovibrio bacteriovorus HD100. Although the mutant and the mediator sequences differed in more than 28,000 nucleotide positions, our approach enabled pinpointing the single causative mutation. Experimental validation in 53 additional mutants further established the implicated gene. Our approach extends the applicability of NGS-based mutant analyses beyond the domain of available reference genomes.

  10. Mutation Detection with Next-Generation Resequencing through a Mediator Genome

    Energy Technology Data Exchange (ETDEWEB)

    Wurtzel, Omri; Dori-Bachash, Mally; Pietrokovski, Shmuel; Jurkevitch, Edouard; Sorek, Rotem; Ben-Jacob, Eshel

    2010-12-31

    The affordability of next generation sequencing (NGS) is transforming the field of mutation analysis in bacteria. The genetic basis for phenotype alteration can be identified directly by sequencing the entire genome of the mutant and comparing it to the wild-type (WT) genome, thus identifying acquired mutations. A major limitation for this approach is the need for an a-priori sequenced reference genome for the WT organism, as the short reads of most current NGS approaches usually prohibit de-novo genome assembly. To overcome this limitation we propose a general framework that utilizes the genome of relative organisms as mediators for comparing WT and mutant bacteria. Under this framework, both mutant and WT genomes are sequenced with NGS, and the short sequencing reads are mapped to the mediator genome. Variations between the mutant and the mediator that recur in the WT are ignored, thus pinpointing the differences between the mutant and the WT. To validate this approach we sequenced the genome of Bdellovibrio bacteriovorus 109J, an obligatory bacterial predator, and its prey-independent mutant, and compared both to the mediator species Bdellovibrio bacteriovorus HD100. Although the mutant and the mediator sequences differed in more than 28,000 nucleotide positions, our approach enabled pinpointing the single causative mutation. Experimental validation in 53 additional mutants further established the implicated gene. Our approach extends the applicability of NGS-based mutant analyses beyond the domain of available reference genomes.

  11. 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

    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...

  12. Osimertinib benefit in EGFR-mutant NSCLC patients with T790M-mutation detected by circulating tumour DNA.

    Science.gov (United States)

    Remon, J; Caramella, C; Jovelet, C; Lacroix, L; Lawson, A; Smalley, S; Howarth, K; Gale, D; Green, E; Plagnol, V; Rosenfeld, N; Planchard, D; Bluthgen, M V; Gazzah, A; Pannet, C; Nicotra, C; Auclin, E; Soria, J C; Besse, B

    2017-04-01

    Approximately 50% of epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC) patients treated with EGFR tyrosine kinase inhibitors (TKIs) will acquire resistance by the T790M mutation. Osimertinib is the standard of care in this situation. The present study assesses the efficacy of osimertinib when T790M status is determined in circulating cell-free tumour DNA (ctDNA) from blood samples in progressing advanced EGFR-mutant NSCLC patients. ctDNA T790M mutational status was assessed by Inivata InVision™ (eTAm-Seq™) assay in 48 EGFR-mutant advanced NSCLC patients with acquired resistance to EGFR TKIs without a tissue biopsy between April 2015 and April 2016. Progressing T790M-positive NSCLC patients received osimertinib (80 mg daily). The objectives were to assess the response rate to osimertinib according to Response Evaluation Criteria in Solid Tumours (RECIST) 1.1, the progression-free survival (PFS) on osimertinib, and the percentage of T790M positive in ctDNA. The ctDNA T790M mutation was detected in 50% of NSCLC patients. Among assessable patients, osimertinib gave a partial response rate of 62.5% and a stable disease rate of 37.5%. All responses were confirmed responses. After median follow up of 8 months, median PFS by RECIST criteria was not achieved (95% CI: 4-NA), with 6- and 12-months PFS of 66.7% and 52%, respectively. ctDNA from liquid biopsy can be used as a surrogate marker for T790M in tumour tissue. © The Author 2017. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  13. Targeted next-generation sequencing identification of mutations in disease resistance gene anologs (RGAs) in wild and cultivated beets

    Science.gov (United States)

    Resistance gene analogs (RGAs) were searched bioinformatically in the sugar beet (Beta vulgaris L.) genome as potential candidates for improving resistance against different diseases. In the present study, Ion Torrent sequencing technology was used to identify mutations in 21 RGAs. The DNA samples o...

  14. Spontaneous mutations in the flhD operon generate motility heterogeneity in Escherichia coli biofilm.

    Science.gov (United States)

    Horne, Shelley M; Sayler, Joseph; Scarberry, Nicholas; Schroeder, Meredith; Lynnes, Ty; Prüß, Birgit M

    2016-11-08

    :1, and 1:10. After 3 weeks, biofilm of the mixed cultures contained up to five times more biomass than biofilm of each of the individual strains. Mutations in the flhD operon can exert positive or negative effects on motility, depending on the site of the mutation. We believe that this is a mechanism to generate motility heterogeneity within E. coli biofilm, which may help to maintain biofilm biomass over extended periods of time.

  15. 9,10-Phenanthrenequinone induces DNA deletions and forward mutations via oxidative mechanisms in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Rodriguez, Chester E; Sobol, Zhanna; Schiestl, Robert H

    2008-03-01

    The estimated cancer risk from diesel exhaust particles (DEP) in the air is approximately 70% of the cancer risk from all air pollutants. DEP is comprised of a complex mixture of chemicals whose carcinogenic potential has not been adequately assessed. The polycyclic aromatic hydrocarbon quinone 9,10-phenanthrenequinone (9,10 PQ) is a major component of DEP and a suspect genotoxic agent for DEP induced DNA damage. 9,10 PQ undergoes redox cycling to produce reactive oxygen species that can lead to oxidative DNA damage. We used two systems in the yeast Saccharomyces cerevisiae to examine possible differential genotoxicity of 9,10 PQ. The DEL assay measures intra-chromosomal homologous recombination leading to DNA deletions and the CAN assay measures forward mutations leading to canavanine resistance. Cells were exposed to 9,10 PQ aerobically and anaerobically followed by DNA damage assessment. The results indicate that 9,10 PQ induces DNA deletions and point mutations in the presence of oxygen while exhibiting negligible effects anaerobically. In contrast to the cytotoxicity observed aerobically, the anaerobic effects of 9,10 PQ seem to be cytostatic in nature, reducing growth without affecting cell viability. Thus, 9,10 PQ requires oxygen for genotoxicity while different toxicities exhibited aerobically and anaerobically suggest multiple mechanisms of action.

  16. Somatic mutation detection using various targeted detection assays in paired samples of circulating tumor DNA, primary tumor and metastases from patients undergoing resection of colorectal liver metastases.

    Science.gov (United States)

    Beije, Nick; Helmijr, Jean C; Weerts, Marjolein J A; Beaufort, Corine M; Wiggin, Matthew; Marziali, Andre; Verhoef, Cornelis; Sleijfer, Stefan; Jansen, Maurice P H M; Martens, John W M

    2016-12-01

    Assessing circulating tumor DNA (ctDNA) is a promising method to evaluate somatic mutations from solid tumors in a minimally-invasive way. In a group of twelve metastatic colorectal cancer (mCRC) patients undergoing liver metastasectomy, from each patient DNA from cell-free DNA (cfDNA), the primary tumor, metastatic liver tissue, normal tumor-adjacent colon or liver tissue, and whole blood were obtained. Investigated was the feasibility of a targeted NGS approach to identify somatic mutations in ctDNA. This targeted NGS approach was also compared with NGS preceded by mutant allele enrichment using synchronous coefficient of drag alteration technology embodied in the OnTarget assay, and for selected mutations with digital PCR (dPCR). All tissue and cfDNA samples underwent IonPGM sequencing for a CRC-specific 21-gene panel, which was analyzed using a standard and a modified calling pipeline. In addition, cfDNA, whole blood and normal tissue DNA were analyzed with the OnTarget assay and with dPCR for specific mutations in cfDNA as detected in the corresponding primary and/or metastatic tumor tissue. NGS with modified calling was superior to standard calling and detected ctDNA in the cfDNA of 10 patients harboring mutations in APC, ATM, CREBBP, FBXW7, KRAS, KMT2D, PIK3CA and TP53. Using this approach, variant allele frequencies in plasma ranged predominantly from 1 to 10%, resulting in limited concordance between ctDNA and the primary tumor (39%) and the metastases (55%). Concordance between ctDNA and tissue markedly improved when ctDNA was evaluated for KRAS, PIK3CA and TP53 mutations by the OnTarget assay (80%) and digital PCR (93%). Additionally, using these techniques mutations were observed in tumor-adjacent tissue with normal morphology in the majority of patients, which were not observed in whole blood. In conclusion, in these mCRC patients with oligometastatic disease NGS on cfDNA was feasible, but had limited sensitivity to detect all somatic mutations present

  17. Variables that influence BRAF mutation probability: A next-generation sequencing, non-interventional investigation of BRAFV600 mutation status in melanoma.

    Directory of Open Access Journals (Sweden)

    Maria Rita Gaiser

    Full Text Available The incidence of melanoma, particularly in older patients, has steadily increased over the past few decades. Activating mutations of BRAF, the majority occurring in BRAFV600, are frequently detected in melanoma; however, the prognostic significance remains unclear. This study aimed to define the probability and distribution of BRAFV600 mutations, and the clinico-pathological factors that may affect BRAF mutation status, in patients with advanced melanoma using next-generation sequencing.This was a non-interventional, retrospective study of BRAF mutation testing at two German centers, in Heidelberg and Tübingen. Archival tumor samples from patients with histologically confirmed melanoma (stage IIIB, IIIC, IV were analyzed using PCR amplification and deep sequencing. Clinical, histological, and mutation data were collected. The statistical influence of patient- and tumor-related characteristics on BRAFV600 mutation status was assessed using multiple logistic regression (MLR and a prediction profiler.BRAFV600 mutation status was assessed in 453 samples. Mutations were detected in 57.6% of patients (n = 261, with 48.1% (n = 102 at the Heidelberg site and 66.0% (n = 159 at the Tübingen site. The decreasing influence of increasing age on mutation probability was quantified. A main effects MLR model identified age (p = 0.0001, center (p = 0.0004, and melanoma subtype (p = 0.014 as significantly influencing BRAFV600 mutation probability; ultraviolet (UV exposure showed a statistical trend (p = 0.1419. An interaction model of age versus other variables showed that center (p<0.0001 and melanoma subtype (p = 0.0038 significantly influenced BRAF mutation probability; age had a statistically significant effect only as part of an interaction with both UV exposure (p = 0.0110 and melanoma subtype (p = 0.0134.This exploratory study highlights that testing center, melanoma subtype, and age in combination with UV exposure and melanoma subtype significantly

  18. On the Sequence-Directed Nature of Human Gene Mutation: The Role of Genomic Architecture and the Local DNA Sequence Environment in Mediating Gene Mutations Underlying Human Inherited Disease

    Science.gov (United States)

    Cooper, David N.; Bacolla, Albino; Férec, Claude; Vasquez, Karen M.; Kehrer-Sawatzki, Hildegard; Chen, Jian-Min

    2011-01-01

    Different types of human gene mutation may vary in size, from structural variants (SVs) to single base-pair substitutions, but what they all have in common is that their nature, size and location are often determined either by specific characteristics of the local DNA sequence environment or by higher-order features of the genomic architecture. The human genome is now recognized to contain ‘pervasive architectural flaws’ in that certain DNA sequences are inherently mutation-prone by virtue of their base composition, sequence repetitivity and/or epigenetic modification. Here we explore how the nature, location and frequency of different types of mutation causing inherited disease are shaped in large part, and often in remarkably predictable ways, by the local DNA sequence environment. The mutability of a given gene or genomic region may also be influenced indirectly by a variety of non-canonical (non-B) secondary structures whose formation is facilitated by the underlying DNA sequence. Since these non-B DNA structures can interfere with subsequent DNA replication and repair, and may serve to increase mutation frequencies in generalized fashion (i.e. both in the context of subtle mutations and SVs), they have the potential to serve as a unifying concept in studies of mutational mechanisms underlying human inherited disease. PMID:21853507

  19. Monitoring the effect of first line treatment in RAS/RAF mutated metastatic colorectal cancer by serial analysis of tumor specific DNA in plasma

    DEFF Research Database (Denmark)

    Brenner Thomsen, Caroline Emilie; Hansen, T F; Andersen, R F

    2018-01-01

    BACKGROUND: Precision medicine calls for an early indicator of treatment efficiency. Circulating tumor DNA (ctDNA) is a promising marker in this setting. Our prospective study explored the association between disease development and change of ctDNA during first line chemotherapy in patients...... with RAS/RAF mutated metastatic colorectal cancer (mCRC). METHODS: The study included 138 patients with mCRC receiving standard first line treatment. In patients with RAS/RAF mutated tumor DNA the same mutation was quantified in the plasma using droplet digital PCR. The fractional abundance of ct...... on continuous treatment. The first increase in ctDNA level occurred at a median of 51 days before radiologically confirmed progression. CONCLUSIONS: The results indicate that the ctDNA level holds potential as a clinically valuable marker in first line treatment of mCRC. A rapid decrease was associated...

  20. mtDNA Mutations and Their Role in Aging, Diseases and Forensic Sciences

    OpenAIRE

    Zapico, Sara C.; Ubelaker, Douglas H.

    2013-01-01

    Mitochondria are independent organelles with their own DNA. As a primary function, mitochondria produce the energy for the cell through Oxidative Phosphorylation (OXPHOS) in the Electron Transport Chain (ETC). One of the toxic products of this process is Reactive Oxygen Species (ROS), which can induce oxidative damage in macromolecules like lipids, proteins and DNA. Mitochondrial DNA (mtDNA) is less protected and has fewer reparation mechanisms than nuclear DNA (nDNA), and as such is more exp...

  1. 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

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

    International Nuclear Information System (INIS)

    Hassim, Farzanah; Papadopoulos, Andrea O.; Kana, Bavesh D.; Gordhan, Bhavna G.

    2015-01-01

    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 ( 1 0 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 homologues

  3. The erratic mitochondrial clock: variations of mutation rate, not population size, affect mtDNA diversity across birds and mammals

    Directory of Open Access Journals (Sweden)

    Galtier Nicolas

    2009-03-01

    Full Text Available Abstract Background During the last ten years, major advances have been made in characterizing and understanding the evolution of mitochondrial DNA, the most popular marker of molecular biodiversity. Several important results were recently reported using mammals as model organisms, including (i the absence of relationship between mitochondrial DNA diversity and life-history or ecological variables, (ii the absence of prominent adaptive selection, contrary to what was found in invertebrates, and (iii the unexpectedly large variation in neutral substitution rate among lineages, revealing a possible link with species maximal longevity. We propose to challenge these results thanks to the bird/mammal comparison. Direct estimates of population size are available in birds, and this group presents striking life-history trait differences with mammals (higher mass-specific metabolic rate and longevity. These properties make birds the ideal model to directly test for population size effects, and to discriminate between competing hypotheses about the causes of substitution rate variation. Results A phylogenetic analysis of cytochrome b third-codon position confirms that the mitochondrial DNA mutation rate is quite variable in birds, passerines being the fastest evolving order. On average, mitochondrial DNA evolves slower in birds than in mammals of similar body size. This result is in agreement with the longevity hypothesis, and contradicts the hypothesis of a metabolic rate-dependent mutation rate. Birds show no footprint of adaptive selection on cytochrome b evolutionary patterns, but no link between direct estimates of population size and cytochrome b diversity. The mutation rate is the best predictor we have of within-species mitochondrial diversity in birds. It partly explains the differences in mitochondrial DNA diversity patterns observed between mammals and birds, previously interpreted as reflecting Hill-Robertson interferences with the W

  4. The reliable assurance of detecting somatic mutations in cancer-related genes by next-generation sequencing: the results of external quality assessment in China.

    Science.gov (United States)

    Zhang, Rui; Ding, Jiansheng; Han, Yanxi; Yi, Lang; Xie, Jiehong; Yang, Xin; Fan, Gaowei; Wang, Guojing; Hao, Mingju; Zhang, Dong; Zhang, Kuo; Lin, Guigao; Li, Jinming

    2016-09-06

    To evaluate the proficiencies of laboratories utilizing next-generation sequencing (NGS) to detect somatic mutations in cancer-related genes, an external quality assessment (EQA) was implemented by the National Center for Clinical Laboratories of China in 2015. We prepared a panel of samples that comprised eight samples made by mixing synthetic mutated DNA fragments with normal human genomic DNA and one reference sample containing only genomic DNA. We validated our sample panel, and then distributed it to laboratories across China. We received complete results from 64 laboratories. The performances of 51.6 % (33/64) respondent labs were acceptable and 26.6 % (17/64) of the labs returned perfect results. In total, 449 mistakes were reported, including 201 false-negatives (201/449, 44.8 %) and 222 false-positives (222/449, 49.4 %) and 26 slightly discordant results (26/449, 5.8 %). We believe these unsatisfactory results and varied performances are mainly due to the enrichment methods used, the diverse sequencing chemistries of the different NGS platforms, and other errors within the sequencing process. The results indicate that our sample panel is suitable for use in EQA studies, and that further laboratory training in targeted NGS testing is urgently required. To address this, we propose a targeted NGS workflow with details on quality assurance procedures according to the current guidelines.

  5. Personalizing cancer treatment in the age of global genomic analyses: PALB2 gene mutations and the response to DNA damaging agents in pancreatic cancer.

    Science.gov (United States)

    Villarroel, Maria C; Rajeshkumar, N V; Garrido-Laguna, Ignacio; De Jesus-Acosta, Ana; Jones, Siân; Maitra, Anirban; Hruban, Ralph H; Eshleman, James R; Klein, Alison; Laheru, Daniel; Donehower, Ross; Hidalgo, Manuel

    2011-01-01

    Metastasis and drug resistance are the major causes of mortality in patients with pancreatic cancer. Once developed, the progression of pancreatic cancer metastasis is virtually unstoppable with current therapies. Here, we report the remarkable clinical outcome of a patient with advanced, gemcitabine-resistant, pancreatic cancer who was later treated with DNA damaging agents, on the basis of the observation of significant activity of this class of drugs against a personalized xenograft generated from the patient's surgically resected tumor. Mitomycin C treatment, selected on the basis of its robust preclinical activity in a personalized xenograft generated from the patient's tumor, resulted in long-lasting (36+ months) tumor response. Global genomic sequencing revealed biallelic inactivation of the gene encoding PalB2 protein in this patient's cancer; the mutation is predicted to disrupt BRCA1 and BRCA2 interactions critical to DNA double-strand break repair. This work suggests that inactivation of the PALB2 gene is a determinant of response to DNA damage in pancreatic cancer and a new target for personalizing cancer treatment. Integrating personalized xenografts with unbiased exomic sequencing led to customized therapy, tailored to the genetic environment of the patient's tumor, and identification of a new biomarker of drug response in a lethal cancer. ©2010 AACR.

  6. Mutational profiling of acute myeloid leukemia with normal cytogenetics in Brazilian patients: the value of next-generation sequencing for genomic classification.

    Science.gov (United States)

    de Noronha, Thiago Rodrigo; Mitne-Neto, Miguel; Chauffaille, Maria de Lourdes

    2017-12-01

    Karyotype (KT) aberrations are important prognostic factors for acute myeloid leukemia (AML); however, around 50% of cases present normal results. Single nucleotide polymorphism array can detect chromosomal gains, losses or uniparental disomy that are invisible to KT, thus improving patients' risk assessment. However, when both tests are normal, important driver mutations can be detected by the use of next-generation sequencing (NGS). Fourteen adult patients with AML with normal cytogenetics were investigated by NGS for 19 AML-related genes. Every patient presented at least one mutation: DNMT3A in nine patients; IDH2 in six; IDH1 in three; NRAS and NPM1 in two; and TET2 , ASXL1 , PTPN11 , and RUNX1 in one patient. No mutations were found in FLT3 , KIT , JAK2 , CEBPA , GATA2 , TP53 , BRAF , CBL , KRAS, and WT1 genes. Twelve patients (86%) had at least one mutation in genes related with DNA methylation ( DNMT3A , IDH1 , IDH2, and TET2 ), which is involved in regulation of gene expression and genomic stability. All patients could be reclassified based on genomic status and nine had their prognosis modified. In summary, NGS offers insights into the molecular pathogenesis and biology of cytogenetically normal AML in Brazilian patients, indicating that the prognosis could be further stratified by different mutation combinations. This study shows a different frequency of mutations in Brazilian population that should be confirmed. © American Federation for Medical Research (unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  7. Research on mutation generation in higher plants with heavy ions at NIRS-HIMAC

    International Nuclear Information System (INIS)

    Okamura, M.; Watanabe, S.; Watanabe, M.; Toguri, T.; Furusawa, Y.

    2006-01-01

    Plants are closely related to medical treatment in medicine, foods, herbs and medical care by gardening. Ion beams have much higher linear energy transfer (LET) and relative biological effectiveness than those of gamma rays and X-rays. Ion beams are supposed to be useful as new mutagen to obtain novel mutants with superior characteristics in higher plants. In this study, the influence of heavy ions irradiation on bud growth was examined in carnation and the mutation generation was inspected in babies' breath. The growth of carnation buds began to decrease at 10 Gy and the median growth dose was estimated at 35 Gy for 290 Mev/u carbon ion beams. Mutants with petaloid leaves were observed in babies' breath by the irradiation of 290 Mev/u carbon ion beams at 20Gy. We will examine the mutation rates and spectrum for 290 MeV/u carbon, 400 MeV/u neon and 500 MeV/u argon ion beams to find optimum use of the beams in plant breeding. The efficient system to generate useful mutants using heavy ions at NIRS-HIMAC will be developed in higher plants. (author)

  8. Mutational specificity of alkylating agents and the influence of DNA repair

    Energy Technology Data Exchange (ETDEWEB)

    Horsfall, M.J.; Gordon, A.J.; Burns, P.A.; Zielenska, M.; van der Vliet, G.M.; Glickman, B.W. (York Univ., Toronto, Ontario (Canada))

    1990-01-01

    Alkylating treatments predominantly induce G:C = greater than A:T transitions, consistent with the predicted significance of the miscoding potential of the O6-alG lesion. However, the frequency and distribution of these events induced by any one compound may be diagnostic. SN1 agents that act via an alkyldiazonium cation, such as the N-nitroso compounds, preferentially generate G:C = greater than A:T transitions at 5'-RG-3' sites, while the more SN2 alkylsulfates and alkylalkane-sulfonates do not. The precise nature of this site bias and the possibility of strand bias are target dependent. The extent of this site bias and the contribution of other base substitutions are substituent size dependent. A similar 5'-RT-3' effect is seen for A:T = greater than G:C transitions, presumably directed by O4-alT lesions. The 5'-RG-3' effect, at least, likely reflects a deposition specificity arising from some aspect of helix geometry, although it may be further exaggerated by alkylation-specific repair. Excision repair appears to preferentially reduce the occurrence of ethylation-induced G:C = greater than A:T and A:T = greater than G:C transitions at sites flanked by A:T base pairs. This may be due to an enhancement of the helical distortion imposed by damage at such positions. A similar effect is not seen for methylation-induced mutations and in the case of propyl adducts, the influence of excision repair on the ultimate distribution of mutation cannot be as easily defined with respect to neighbouring sequence. 199 references.

  9. A Dominant Mutation in Human RAD51 Reveals Its Function in DNA Interstrand Crosslink Repair Independent of Homologous Recombination.

    Science.gov (United States)

    Wang, Anderson T; Kim, Taeho; Wagner, John E; Conti, Brooke A; Lach, Francis P; Huang, Athena L; Molina, Henrik; Sanborn, Erica M; Zierhut, Heather; Cornes, Belinda K; Abhyankar, Avinash; Sougnez, Carrie; Gabriel, Stacey B; Auerbach, Arleen D; Kowalczykowski, Stephen C; Smogorzewska, Agata

    2015-08-06

    Repair of DNA interstrand crosslinks requires action of multiple DNA repair pathways, including homologous recombination. Here, we report a de novo heterozygous T131P mutation in RAD51/FANCR, the key recombinase essential for homologous recombination, in a patient with Fanconi anemia-like phenotype. In vitro, RAD51-T131P displays DNA-independent ATPase activity, no DNA pairing capacity, and a co-dominant-negative effect on RAD51 recombinase function. However, the patient cells are homologous recombination proficient due to the low ratio of mutant to wild-type RAD51 in cells. Instead, patient cells are sensitive to crosslinking agents and display hyperphosphorylation of Replication Protein A due to increased activity of DNA2 and WRN at the DNA interstrand crosslinks. Thus, proper RAD51 function is important during DNA interstrand crosslink repair outside of homologous recombination. Our study provides a molecular basis for how RAD51 and its associated factors may operate in a homologous recombination-independent manner to maintain genomic integrity. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Machine learning classifier for identification of damaging missense mutations exclusive to human mitochondrial DNA-encoded polypeptides.

    Science.gov (United States)

    Martín-Navarro, Antonio; Gaudioso-Simón, Andrés; Álvarez-Jarreta, Jorge; Montoya, Julio; Mayordomo, Elvira; Ruiz-Pesini, Eduardo

    2017-03-07

    Several methods have been developed to predict the pathogenicity of missense mutations but none has been specifically designed for classification of variants in mtDNA-encoded polypeptides. Moreover, there is not available curated dataset of neutral and damaging mtDNA missense variants to test the accuracy of predictors. Because mtDNA sequencing of patients suffering mitochondrial diseases is revealing many missense mutations, it is needed to prioritize candidate substitutions for further confirmation. Predictors can be useful as screening tools but their performance must be improved. We have developed a SVM classifier (Mitoclass.1) specific for mtDNA missense variants. Training and validation of the model was executed with 2,835 mtDNA damaging and neutral amino acid substitutions, previously curated by a set of rigorous pathogenicity criteria with high specificity. Each instance is described by a set of three attributes based on evolutionary conservation in Eukaryota of wildtype and mutant amino acids as well as coevolution and a novel evolutionary analysis of specific substitutions belonging to the same domain of mitochondrial polypeptides. Our classifier has performed better than other web-available tested predictors. We checked performance of three broadly used predictors with the total mutations of our curated dataset. PolyPhen-2 showed the best results for a screening proposal with a good sensitivity. Nevertheless, the number of false positive predictions was too high. Our method has an improved sensitivity and better specificity in relation to PolyPhen-2. We also publish predictions for the complete set of 24,201 possible missense variants in the 13 human mtDNA-encoded polypeptides. Mitoclass.1 allows a better selection of candidate damaging missense variants from mtDNA. A careful search of discriminatory attributes and a training step based on a curated dataset of amino acid substitutions belonging exclusively to human mtDNA genes allows an improved

  11. Evaluation of yield in Gamma Radiated Lines Selected from Mutated Generations of Mungbean (Vigna radiata)

    International Nuclear Information System (INIS)

    Aye Thandar; Phyu Hnin Htike; Myo Myint

    2010-12-01

    The induced mutation through different gamma radiation frequencies 50, 100, 150, 200, 250, 300, 350, 400, 450 and 500Gy in mungbean was studied for yield components in M3 generation. A Randomized Complete Block Design (RCBD) was employed with three replications in this experiment. The data collected from M3 generation were subjected to statistical analysis with the help of Excel (Microsoft office 2007) and for pairs wise comparison of groups was by SPSS program.In primary yield components, there were no significant difference in M3 generation of pods per plant, pod length and seeds per pod except 100 seeds weight. The plant treated with 250 Gy and 400Gy exploited the maximum value of one hundred seeds weight and yield per plant , respectively. Although there was no significant difference in secondary yield components; 50% flowering days, 50% maturity days and plant height in this generation, highest plant height at 200Gy and early flowering and maturity at 300Gy were obserded. The selection of individual plants in the M3 generation was carried out for high yield. In mutant selection, 250Gy and 400Gy revealed relatively more number of plants having good characters such as more number of pods per plant and longer pod length but not in other treatments and control.

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

    Science.gov (United States)

    Yin, Qian Qian; Li, Zhen Peng; Zhao, Hai; Pan, Dong; Wang, Yan; Xu, Wei Si; Xing, Hui; Feng, Yi; Jiang, Shi Bo; Shao, Yi Ming; Ma, Li Ying

    2016-04-01

    To 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. Forty-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. Compared 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, PDNA, and 5 of these samples with different DRMs between proviral DNA and paired viral RNA showed a higher level of drug resistance to the first-line drugs. Considering 'minority resistant variants', 22 samples (28.57%) were associated with a higher level of drug resistance to the tested RTIs for proviral DNA when compared with paired viral RNA. Compared with viral RNA, the distinctive information of DRMs and 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. Copyright © 2016 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  13. Ultraviolet light induction of diphtheria toxin-resistant mutations in normal and DNA repair-deficient human and Chinese hamster fibroblasts

    International Nuclear Information System (INIS)

    Trosko, J.E.; Schultz, R.S.; Chang, C.C.; Glover, T.

    1980-01-01

    The role on unrepaired DNA lesions in the production of mutations is suspected of contributing to the initiation phase of carcinogenesis. Since the molecular basis of mutagenesis is not understood in eukaryotic cells, development of new genetic markers for quantitative in vitro measurement of mutations for mammalian cells is needed. Furthermore, mammalian cells, genetically deficient for various DNA repair enzymes, will be needed to study the role of unrepaired DNA lesions in mutagenesis. The results in this report relate to preliminary attempts to characterize the diphtheria toxin resistance marker as a useful quantitative genetic marker in human cells and to isolate and characterize various DNA repair-deficient Chinese hamster cells

  14. Does DNA methylation pattern mark generative development in winter rape?

    Czech Academy of Sciences Publication Activity Database

    Filek, M.; Janiak, A.; Szarejko, I.; Grabczynska, J.; Macháčková, Ivana; Krekule, Jan

    2006-01-01

    Roč. 61, 5-6 (2006), s. 387-396 ISSN 0939-5075 R&D Projects: GA AV ČR IAA600040612 Institutional research plan: CEZ:AV0Z50380511 Keywords : DNA methylation * rape * vernalization Subject RIV: EF - Botanics Impact factor: 0.720, year: 2006

  15. A Microbiome DNA Enrichment Method for Next-Generation Sequencing Sample Preparation.

    Science.gov (United States)

    Yigit, Erbay; Feehery, George R; Langhorst, Bradley W; Stewart, Fiona J; Dimalanta, Eileen T; Pradhan, Sriharsa; Slatko, Barton; Gardner, Andrew F; McFarland, James; Sumner, Christine; Davis, Theodore B

    2016-07-01

    "Microbiome" is used to describe the communities of microorganisms and their genes in a particular environment, including communities in association with a eukaryotic host or part of a host. One challenge in microbiome analysis concerns the presence of host DNA in samples. Removal of host DNA before sequencing results in greater sequence depth of the intended microbiome target population. This unit describes a novel method of microbial DNA enrichment in which methylated host DNA such as human genomic DNA is selectively bound and separated from microbial DNA before next-generation sequencing (NGS) library construction. This microbiome enrichment technique yields a higher fraction of microbial sequencing reads and improved read quality resulting in a reduced cost of downstream data generation and analysis. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  16. 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.

  17. 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

    2014-10-07

    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.

  18. 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

    controls were subjected to an oral glucose tolerance test. Twenty-six adult 3243A>G carriers with unknown myopathy status and 17 healthy controls had a maximal cycle test and a muscle biopsy performed. The mutation loads were quantified in blood and muscle biopsies and correlated to the clinical......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...... combinations. Consequently, it is difficult to predict the "phenotypic risk profile" of 3243A>G mutation-positive subjects. The 3243A>G mutation coexists in cells with wild-type mtDNA, a phenomenon called heteroplasmy. The marked variability in mutation loads in different tissues is the main explanation...

  19. Reduction in oxidatively generated DNA damage following smoking cessation

    Directory of Open Access Journals (Sweden)

    Freund Harold G

    2011-05-01

    Full Text Available Abstract Background Cigarette smoking is a known cause of cancer, and cancer may be in part due to effects of oxidative stress. However, whether smoking cessation reverses oxidatively induced DNA damage unclear. The current study sought to examine the extent to which three DNA lesions showed significant reductions after participants quit smoking. Methods Participants (n = 19 in this study were recruited from an ongoing 16-week smoking cessation clinical trial and provided blood samples from which leukocyte DNA was extracted and assessed for 3 DNA lesions (thymine glycol modification [d(TgpA]; formamide breakdown of pyrimidine bases [d(TgpA]; 8-oxo-7,8-dihydroguanine [d(Gh] via liquid chromatography tandem mass spectrometry (LC-MS/MS. Change in lesions over time was assessed using generalized estimating equations, controlling for gender, age, and treatment condition. Results Overall time effects for the d(TgpA (χ2(3 = 8.068, p fpA (χ2(3 = 8.477, p h (χ2(3 = 37.599, p gpA and d(PfpA lesions show