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

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Takayuki Mito

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

Differences in K-ras and mitochondrial DNA mutations and microsatellite instability between colorectal cancers of Vietnamese and Japanese patients.

Science.gov (United States)

Miwata, Tomohiro; Hiyama, Toru; Quach, Duc Trong; Le, Huy Minh; Hua, Ha Ngoc Thi; Oka, Shiro; Tanaka, Shinji; Arihiro, Koji; Chayama, Kazuaki

2014-11-30

The incidence of early-onset (under 50 years of age) colorectal cancer (CRC) in the Vietnamese has been reported to be quite higher than that in the Japanese. To clarify the differences in genetic alterations between Vietnamese and Japanese CRCs, we investigated mutations in K-ras and mitochondrial DNA (mtDNA) and high-frequency microsatellite instability (MSI-H) in the CRCs of Vietnamese and Japanese patients. We enrolled 60 Vietnamese and 233 Japanese patients with invasive CRCs. DNA was extracted from formalin-fixed, paraffin-embedded tissue sections. K-ras mutations were examined with PCR-single-strand conformation polymorphism analysis. mtDNA mutations and MSI-H were examined with microsatellite analysis using D310 and BAT-26, respectively. K-ras mutations were examined in 60 Vietnamese and 45 Japanese CRCs. The frequency of the mutations in the Vietnamese CRCs was significantly higher than that in the Japanese CRCs (8 of 24 [33%] vs 5 of 45 [11%], p =0.048). MSI-H was examined in 60 Vietnamese and 130 Japanese CRCs. The frequency of MSI-H in the Vietnamese CRCs was also significantly higher than that in the Japanese CRCs (6 of 27 [22%] vs 10 of 130 [8%], p =0.030). mtDNA mutations were examined in 60 Vietnamese and 138 Japanese CRCs. The frequency of mtDNA mutations in the Vietnamese CRCs was significantly higher than that in the Japanese CRCs (19 of 44 [43%] vs 11 of 133 [9%], p Vietnamese and Japanese patients. These results indicate that the developmental pathways of CRCs in the Vietnamese may differ from those of CRCs in the Japanese.

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

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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.

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.

High frequency of PTEN mutations in nevi and melanomas from xeroderma pigmentosum patients.

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Masaki, Taro; Wang, Yun; DiGiovanna, John J; Khan, Sikandar G; Raffeld, Mark; Beltaifa, Senda; Hornyak, Thomas J; Darling, Thomas N; Lee, Chyi-Chia R; Kraemer, Kenneth H

2014-05-01

We examined nevi and melanomas in 10 xeroderma pigmentosum (XP) patients with defective DNA repair. The lesions had a lentiginous appearance with markedly increased numbers of melanocytes. Using laser capture microdissection, we performed DNA sequencing of 18 benign and atypical nevi and 75 melanomas (melanoma in situ and invasive melanomas). The nevi had a similar high frequency of PTEN mutations as melanomas [61% (11/18) versus 53% (39/73)]. Both had a very high proportion of UV-type mutations (occurring at adjacent pyrimidines) [91% (10/11) versus 92% (36/39)]. In contrast to melanomas in the general population, the frequency of BRAF mutations (11%, 7/61), NRAS mutations (21%, 13/62), and KIT mutations (21%, 6/28) in XP melanomas was lower than for PTEN. Phospho-S6 immunostaining indicated activation of the mTOR pathway in the atypical nevi and melanomas. Thus, the clinical and histological appearances and the molecular pathology of these UV-related XP nevi and melanomas were different from nevi and melanomas in the general population. © 2014 Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

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

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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.

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)

Pre-thymic somatic mutation leads to high mutant frequency at hypoxanthine-guanine phosphoribosyltransferase gene

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Jett, J. [Lawrence Livermore National Lab., CA (United States)

1994-12-01

While characterizing the background mutation spectrum of the Hypoxathine-guanine phosphoribosyltransferase (HPRT) gene in a healthy population, an outlier with a high mutant frequency of thioguanine resistant lymphocytes was found. When studied at the age of 46, this individual had been smoking 60 cigarettes per day for 38 years. His mutant frequency was calculated at 3.6 and 4.2x10{sup {minus}4} for two sampling periods eight months apart. Sequencing analysis of the HPRT gene in his mutant thioguanine resistant T lymphocytes was done to find whether the cells had a high rate of mutation, or if the mutation was due to a single occurrence of mutation and, if so, when in the T lymphocyte development the mutation occurred. By T-cell receptor analysis it has been found that out of 35 thioguanine resistant clones there was no dominant gamma T cell receptor gene rearrangement. During my appointment in the Science & Engineering Research Semester, I found that 34 of those clones have the same base substitution of G{yields}T at cDNA position 197. Due to the consistent mutant frequency from both sampling periods and the varying T cell receptors, the high mutant frequency cannot be due to recent proliferation of a mature mutant T lymphocyte. From the TCR and DNA sequence analysis we conclude that the G{yields}T mutation must have occurred in a T lymphocyte precursor before thymic differentiation so that the thioguanine resistant clones share the same base substitution but not the same gamma T cell receptor gene.

The effects of extremely low frequency magnetic fields on mutation induction in mice

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Wilson, James W. [Department of Genetics, University of Leicester, Leicester LE1 7RH (United Kingdom); Haines, Jackie; Sienkiewicz, Zenon [Centre for Radiation, Chemical and Environmental Hazards, Public Health England, Chilton, Didcot, Oxfordshire OX11 0RQ (United Kingdom); Dubrova, Yuri E., E-mail: yed2@le.ac.uk [Department of Genetics, University of Leicester, Leicester LE1 7RH (United Kingdom)

2015-03-15

Highlights: • The effects of 50 Hz magnetic fields on mutation induction in mice were analyzed. • The frequency of ESTR mutation was established in sperm and blood. • Exposure to 10–300 μT for 2 and 15 h did not result in mutation induction. • Mutagenic effects of 50 Hz magnetic fields are likely to be negligible. - Abstract: The growing human exposure to extremely low frequency (ELF) magnetic fields has raised a considerable concern regarding their genotoxic effects. The aim of this study was to evaluate the in vivo effects of ELF magnetic fields irradiation on mutation induction in the germline and somatic tissues of male mice. Seven week old BALB/c × CBA/Ca F{sub 1} hybrid males were exposed to 10, 100 or 300 μT of 50 Hz magnetic fields for 2 or 15 h. Using single-molecule PCR, the frequency of mutation at the mouse Expanded Simple Tandem Repeat (ESTR) locus Ms6-hm was established in sperm and blood samples of exposed and matched sham-treated males. ESTR mutation frequency was also established in sperm and blood samples taken from male mice exposed to 1 Gy of acute X-rays. The frequency of ESTR mutation in DNA samples extracted from blood of mice exposed to magnetic fields did not significantly differ from that in sham-treated controls. However, there was a marginally significant increase in mutation frequency in sperm but this was not dose-dependent. In contrast, acute exposure X-rays led to significant increases in mutation frequency in sperm and blood of exposed males. The results of our study suggest that, within the range of doses analyzed here, the in vivo mutagenic effects of ELF magnetic fields are likely to be minor if not negligible.

The effects of extremely low frequency magnetic fields on mutation induction in mice

International Nuclear Information System (INIS)

Wilson, James W.; Haines, Jackie; Sienkiewicz, Zenon; Dubrova, Yuri E.

2015-01-01

Highlights: • The effects of 50 Hz magnetic fields on mutation induction in mice were analyzed. • The frequency of ESTR mutation was established in sperm and blood. • Exposure to 10–300 μT for 2 and 15 h did not result in mutation induction. • Mutagenic effects of 50 Hz magnetic fields are likely to be negligible. - Abstract: The growing human exposure to extremely low frequency (ELF) magnetic fields has raised a considerable concern regarding their genotoxic effects. The aim of this study was to evaluate the in vivo effects of ELF magnetic fields irradiation on mutation induction in the germline and somatic tissues of male mice. Seven week old BALB/c × CBA/Ca F 1 hybrid males were exposed to 10, 100 or 300 μT of 50 Hz magnetic fields for 2 or 15 h. Using single-molecule PCR, the frequency of mutation at the mouse Expanded Simple Tandem Repeat (ESTR) locus Ms6-hm was established in sperm and blood samples of exposed and matched sham-treated males. ESTR mutation frequency was also established in sperm and blood samples taken from male mice exposed to 1 Gy of acute X-rays. The frequency of ESTR mutation in DNA samples extracted from blood of mice exposed to magnetic fields did not significantly differ from that in sham-treated controls. However, there was a marginally significant increase in mutation frequency in sperm but this was not dose-dependent. In contrast, acute exposure X-rays led to significant increases in mutation frequency in sperm and blood of exposed males. The results of our study suggest that, within the range of doses analyzed here, the in vivo mutagenic effects of ELF magnetic fields are likely to be minor if not negligible

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

Radiation-induced germ-line mutations detected by a direct comparison of parents and children DNA sequences containing SNPs

International Nuclear Information System (INIS)

Morimyo, M.; Hongo, E.; Higashi, T.; Wu, J.; Matsumoto, I.; Okamoto, M.; Kawano, A.; Tsuji, S.

2003-01-01

Full text: Germ-line mutation is detected in mice but not in humans. To estimate genetic risk of humans, a new approach to extrapolate from animal data to humans or to directly detect radiation-induced mutations in man is expected. We have developed a new method to detect germ-line mutations by directly comparing DNA sequences of parents and children. The nucleotide sequences among mouse strains are almost identical except SNP markers that are detected at 1/1000 frequency. When gamma-irradiated male mice are mated with female mice, heterogeneous nucleotide sequences induced in children DNA are a candidate of mutation, whose assignment can be done by SNP analysis. This system can easily detect all types of mutations such as transition, transversion, frameshift and deletion induced by radiation and can be applied to humans having genetically heterogeneous nucleotide sequences and many SNP markers. C3H male mice of 8 weeks of gestation were irradiated with gamma rays of 3 and 1 Gy and after 3 weeks, they were mated with the same aged C57BL female mice. After 3 weeks breeding, DNA was extracted from parents and children mice. The nucleotide sequences of 150 STS markers containing 300-900 bp and SNPs of parents and children DNA were determined by a direct sequencing; amplification of STS markers by Taq DNA polymerase, purification of PCR products, and DNA sequencing with a dye-terminator method. At each radiation dose, a total amount of 5 Mb DNA sequences were examined to detect radiation-induced mutations. We could find 6 deletions in 3 Gy irradiated mice but not in 1 Gy and control mice. The mutation frequency was about 4.0 x 10 -7 /bp/ Gy or 1.6 x 10 -4 /locus/Gy, and suggested the non-linear increase of mutation rate with dose

The observed human sperm mutation frequency cannot explain the achondroplasia paternal age effect

Science.gov (United States)

Tiemann-Boege, Irene; Navidi, William; Grewal, Raji; Cohn, Dan; Eskenazi, Brenda; Wyrobek, Andrew J.; Arnheim, Norman

2002-01-01

The lifelong spermatogonial stem cell divisions unique to male germ cell production are thought to contribute to a higher mutation frequency in males. The fact that certain de novo human genetic conditions (e.g., achondroplasia) increase in incidence with the age of the father is consistent with this idea. Although it is assumed that the paternal age effect is the result of an increasing frequency of mutant sperm as a man grows older, no direct molecular measurement of the germ-line mutation frequency has been made to confirm this hypothesis. Using sperm DNA from donors of different ages, we determined the frequency of the nucleotide substitution in the fibroblast growth factor receptor 3 (FGFR3) gene that causes achondroplasia. Surprisingly, the magnitude of the increase in mutation frequency with age appears insufficient to explain why older fathers have a greater chance of having a child with this condition. A number of alternatives may explain this discrepancy, including selection for sperm that carry the mutation or an age-dependent increase in premutagenic lesions that remain unrepaired in sperm and are inefficiently detected by the PCR assay. PMID:12397172

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.

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.

Combination of the mutation process with the sensitization and repair processes leading to increased frequencies of mutations in algal populations

International Nuclear Information System (INIS)

Necas, J.

1977-01-01

The possibility of combining the mutation process with the induction of the repair processes was studied to increase the mutation frequencies in algal populations after UV treatment. The repair process induced by visible light was found to be much more effective than the dark repair processes in the chlorococcal algae used. In these algae, visible light possibly does not induce only those repair processes which affect their DNA, but probably also certain recovery processes which affect their damaged structures and physiological functions. A suitable combination of the sensitization of algae cells by a DNA-base analogue before UV treatment and the induction of the light repair and recovery processes resulted in a rather high increase of viable mutations in chlorococcal algae. These findings may be useful in breeding chlorococcal algae, which have no possibility of hybridization other than somatic. (author)

Frequency of factor V Leiden mutation

International Nuclear Information System (INIS)

Nasiruddin; Ali, W.; Rehman, Z.; Anwar, M.; Ayyub, M.; Ali, W.; Ahmed, S.

2005-01-01

Objective: To determine the frequency of factor V Leiden mutation. Design: Observational study. Patients and Methods: Two hundred subjects each of apparently healthy and unrelated Punjabi and Pathan origins were included in the study. Peripheral blood samples were collected in EDTA and DNA extracted by phenol- chloroform extraction method. DNA analysis was done by PCR for restriction fragment length polymorphism. The product was digested overnight with Mn/1 and electrophoresed on acrylamide gel to detect 67 and 153 base pair fragments of factor V Leiden against 37, 67 and 116 base pair fragments of normal factor V. Results: In the 400 subjects studied, only 5 cases of heterozygotes for factor V Leiden were detected. The overall carrier rate was 1.3% (95% CI 0.2-2.2%). The carrier rate in Punjabis and Pathans was 1 % and 1.5% respectively. Conclusion: This study confirms that the prevalence of factor V Leiden is low in Asians and Africans as compared to the European population. (author)

DNA repair of UV photoproducts and mutagenesis in human mitochondrial DNA

International Nuclear Information System (INIS)

Pascucci, B.; Dogliotti, E.; Versteegh, A.; Hoffen, A. van; Zeeland, A.A. van; Mullenders, L.H.F.

1997-01-01

The induction and repair of DNA photolesions and mutations in the mitochondrial (mt) DNA of human cells in culture were analysed after cell exposure to UV-C light. The level of induction of cyclobutane pyrimidine dimers (CPD) in mitochondrial and nuclear DNA was comparable, while a higher frequency of pyrimidine (6-4) pyrimidone photoproducts (6-4 PP) was detected in mitochondrial than in nuclear DNA. Besides the known defect in CPD removal, mitochondria were shown to be deficient also in the excision of 6-4 PP. The effects of repair-defective conditions for the two major UV photolesions on mutagensis was assessed by analysing the frequency and spectrum of spontaneous and UV-induced mutations by restriction site mutation (RSM) method in a restriction endonuclease site, NciI (5'CCCGG3') located within the coding sequence of the mitochondrial gene for tRNA Leu . The spontaneous mutation frequency and spectrum at the NciI site of mitochondrial DNA was very similar to the RSM background mutation frequency (approximately 10 -5 ) and type (predominantly GC > AT transitions at GL 1 ) of the NciI site). Conversely, an approximately tenfold increase over background mutation frequency was recorded after cell exposure to 20 J/m 2 . In this case, the majority of mutations were C > T transitions preferentially located on the non-transcribed DNA strand at C 1 and C 2 of the NciI site. This mutation spectrum is expected by UV mutagenesis. This is the first evidence of induction of mutations in mitochondrial DNA by treatment of human cells with a carcinogen. (author)

Mutation and DNA replication in Escherichia coli treated with low concentrations of N-methyl-N'-nitro-N-nitrosoguanidine

International Nuclear Information System (INIS)

Jimenez-Sanchez, A.; Cerda-Olmedo, E.

1975-01-01

N-Methyl-N'-nitro-N-nitrosoguanidine (nitrosoguanidine) causes an unexpectedly high frequency of closely linked double mutants because of its specificity for chromosome regions in replication. Low nitrosoguanidine concentrations (I μg/ml) in liquid cultures allow replication at the normal rate and are mutagenic. It was expected that mutations would be spread over the chromosome as it replicated, but a high frequency of closely linked double mutants was found. If a thymine auxotroph is grown in the presence of 5-bromodeoxyuridine (BUdR) and nitrosoguanidine and then exposed to 313-nm radiation (which destroys BUdR-substituted DNA), the mutation frequency is much higher among survivors than among non-irradiated cells. It is concluded that nitrosoguanidine inhibits DNA replication in a small fraction of the population and that mutations are induced in that same fraction. Nitrosoguanidine treatment leads to a high frequency of closely linked double mutants under all known conditions

Mitochondrial DNA deletion mutations in adult mouse cardiac side population cells

International Nuclear Information System (INIS)

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

2012-01-01

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

Reduction of spontaneous somatic mutation frequency by a low-dose X irradiation of Drosophila larvae and possible involvement of DNA single-strand damage repair.

Science.gov (United States)

Koana, Takao; Takahashi, Takashi; Tsujimura, Hidenobu

2012-03-01

The third instar larvae of Drosophila were irradiated with X rays, and the somatic mutation frequency in their wings was measured after their eclosion. In the flies with normal DNA repair and apoptosis functions, 0.2 Gy irradiation at 0.05 Gy/min reduced the frequency of the so-called small spot (mutant cell clone with reduced reproductive activity) compared with that in the sham-irradiated flies. When apoptosis was suppressed using the baculovirus p35 gene, the small spot frequency increased four times in the sham-irradiated control group, but the reduction by the 0.2-Gy irradiation was still evident. In a non-homologous end joining-deficient mutant, the small spot frequency was also reduced by 0.2 Gy radiation. In a mutant deficient in single-strand break repair, no reduction in the small spot frequency by 0.2 Gy radiation was observed, and the small spot frequency increased with the radiation dose. Large spot (mutant cell clone with normal reproductive activity) frequency was not affected by suppression of apoptosis and increased monotonically with radiation dose in wild-type larvae and in mutants for single- or double-strand break repair. It is hypothesized that some of the small spots resulted from single-strand damage and, in wild-type larvae, 0.2 Gy radiation activated the normal single-strand break repair gene, which reduced the background somatic mutation frequency.

Tumor‐associated DNA mutation detection in individuals undergoing colonoscopy

OpenAIRE

Fleshner, Phillip; Braunstein, Glenn D.; Ovsepyan, Gayane; Tonozzi, Theresa R.; Kammesheidt, Anja

2017-01-01

Abstract The majority of colorectal cancers (CRC) harbor somatic mutations and epigenetic modifications in the tumor tissue, and some of these mutations can be detected in plasma as circulating tumor DNA (ctDNA). Precancerous colorectal lesions also contain many of these same mutations. This study examined plasma for ctDNA from patients undergoing a screening or diagnostic colonoscopy to determine the sensitivity and specificity of the ctDNA panel for detecting CRC and precancerous lesions. T...

Alkylating agent (MNU)-induced mutation in space environment

Science.gov (United States)

Ohnishi, T.; Takahashi, A.; Ohnishi, K.; Takahashi, S.; Masukawa, M.; Sekikawa, K.; Amano, T.; Nakano, T.; Nagaoka, S.

2001-01-01

In recent years, some contradictory data about the effects of microgravity on radiation-induced biological responses in space experiments have been reported. We prepared a damaged template DNA produced with an alkylating agent (N-methyl-N-nitroso urea; MNU) to measure incorrect base-incorporation during DNA replication in microgravity. We examined whether mutation frequency is affected by microgravity during DNA replication for a DNA template damaged by an alkylating agent. Using an in vitro enzymatic reaction system, DNA synthesis by Taq polymerase or polymerase III was done during a US space shuttle mission (Discovery, STS-91). After the flight, DNA replication and mutation frequencies were measured. We found that there was almost no effect of microgravity on DNA replication and mutation frequency. It is suggested that microgravity might not affect at the stage of substrate incorporation in induced-mutation frequency.

Transfection with extracellularly UV-damaged DNA induces human and rat cells to express a mutator phenotype towards parvovirus H-1

International Nuclear Information System (INIS)

Dinsart, C.; Cornelis, J.J.; Klein, B.; van der Eb, A.J.; Rommelaere, J.

1984-01-01

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

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

Spontaneous mutation by mutagenic repair of spontaneous lesions in DNA

International Nuclear Information System (INIS)

Hastings, P.J.; Quah, S.-K.; Borstel, R.C. von

1976-01-01

It is stated that strains of yeast carrying mutations in many of the steps in pathways repairing radiation-induced damage to DNA have enhanced spontaneous mutation rates. Most strains isolated because they have enhanced spontaneous mutation carry mutations in DNA repair systems. This suggests that much spontaneous mutation arises by mutagenic repair of spontaneous lesions. (author)

Mitochondrial DNA mutations in human tumor cells

OpenAIRE

LI, HUI; HONG, ZE-HUI

2012-01-01

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

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

Science.gov (United States)

2010-04-01

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

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.

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/ ...

Modifying effect of 5-fluoro-2-deoxiuridine on the frequency of x-ray-induced visible mutations in wheat

International Nuclear Information System (INIS)

Azatyan, R.A.; Avakyan, V.A.

1985-01-01

A modifying effect of FUDR on the output of visible mutations induced by X-ray radiation has been studied at soft fall wheat (Tr. aestivum var. turcicum). It is shown that at X-ray radiation of dry seeds with subsequent treatment of FUDR increased is the mutant percent in M 3 which has constituted 0.61% at irradiation in dose of 100 Gy ad 1.03% at irradiation by the same dose and FUDR influence during 10 hours. In analogous variants at irradiation by 150 Gi dose the mutation frequency reached 1.47 and 2.07% respectively. It is shown that FUDR postradiation treatment promotes not only the increase of frequency but consderablly widens the spectrum of induced mutations. A supposition is made that FUDR modyfying effect is caused by DNA synthesis inhibition. A problem is discussed on a possible realization of potential DNA changes caused by irradiation, at additional influence of inhibitor

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.

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

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.

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

EGFR mutation frequency and effectiveness of erlotinib

DEFF Research Database (Denmark)

Weber, Britta; Hager, Henrik; Sorensen, Boe S

2014-01-01

mutation (S768I), and two complex mutations. Seven percent of the patients were never smokers. The differences in median progression-free survival and overall survival between the mutated group and the wild-type group were 8.0 vs. 2.5 months, p...-1 vs. 2-3) and line of treatment (1st vs. 2nd and 3rd) had no influence on outcome in EGFR-mutated patients. CONCLUSION: We found a higher frequency of EGFR mutations than expected in a cohort with less than 10% never smokers. The outcome after treatment with erlotinib was much better in patients......OBJECTIVES: In 2008, we initiated a prospective study to explore the frequency and predictive value of epidermal growth factor receptor (EGFR) mutations in an unselected population of Danish patients with non-small cell lung cancer offered treatment with erlotinib, mainly in second-line. MATERIALS...

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

Science.gov (United States)

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

2018-01-01

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

A Constant Rate of Spontaneous Mutation in DNA-Based Microbes

Science.gov (United States)

Drake, John W.

1991-08-01

In terms of evolution and fitness, the most significant spontaneous mutation rate is likely to be that for the entire genome (or its nonfrivolous fraction). Information is now available to calculate this rate for several DNA-based haploid microbes, including bacteriophages with single- or double-stranded DNA, a bacterium, a yeast, and a filamentous fungus. Their genome sizes vary by ≈6500-fold. Their average mutation rates per base pair vary by ≈16,000-fold, whereas their mutation rates per genome vary by only ≈2.5-fold, apparently randomly, around a mean value of 0.0033 per DNA replication. The average mutation rate per base pair is inversely proportional to genome size. Therefore, a nearly invariant microbial mutation rate appears to have evolved. Because this rate is uniform in such diverse organisms, it is likely to be determined by deep general forces, perhaps by a balance between the usually deleterious effects of mutation and the physiological costs of further reducing mutation rates.

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

Directory of Open Access Journals (Sweden)

Zahra Shajani-Yi

2018-03-01

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

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....

DNA methyltransferase 1 mutations and mitochondrial pathology: is mtDNA methylated?

Directory of Open Access Journals (Sweden)

Alessandra eMaresca

2015-03-01

Full Text Available Autosomal dominant cerebellar ataxia, deafness and narcolepsy (ADCA-DN and Hereditary sensory neuropathy with dementia and hearing loss (HSN1E are two rare, overlapping neurodegenerative syndromes that have been recently linked to allelic dominant pathogenic mutations in the DNMT1 gene, coding for DNA (cytosine-5-methyltransferase 1. DNMT1 is the enzyme responsible for maintaining the nuclear genome methylation patterns during the DNA replication and repair, thus regulating gene expression. The mutations responsible for ADCA-DN and HSN1E affect the replication foci targeting sequence domain, which regulates DNMT1 binding to chromatin. DNMT1 dysfunction is anticipated to lead to a global alteration of the DNA methylation pattern with predictable downstream consequences on gene expression. Interestingly, ADCA-DN and HSN1E phenotypes share some clinical features typical of mitochondrial diseases, such as optic atrophy, peripheral neuropathy and deafness, and some biochemical evidence of mitochondrial dysfunction. The recent discovery of a mitochondrial isoform of DNMT1 and its proposed role in methylating mitochondrial DNA (mtDNA suggests that DNMT1 mutations may directly affect mtDNA and mitochondrial physiology. On the basis of this latter finding the link between DNMT1 abnormal activity and mitochondrial dysfunction in ADCA-DN and HSN1E appears intuitive, however mtDNA methylation remains highly debated. In the last years several groups demonstrated the presence of 5-methylcytosine in mtDNA by different approaches, but, on the other end, the opposite evidence that mtDNA is not methylated has also been published. Since over 1500 mitochondrial proteins are encoded by the nuclear genome, the altered methylation of these genes may well have a critical role in leading to the mitochondrial impairment observed in ADCA-DN and HSN1E. Thus, many open questions still remain unanswered, such as why mtDNA should be methylated, and how this process is

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

Directory of Open Access Journals (Sweden)

Wang Yu-Fen

2006-04-01

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

Extreme assay sensitivity in molecular diagnostics further unveils intratumour heterogeneity in metastatic colorectal cancer as well as artifactual low-frequency mutations in the KRAS gene.

Science.gov (United States)

Mariani, Sara; Bertero, Luca; Osella-Abate, Simona; Di Bello, Cristiana; Francia di Celle, Paola; Coppola, Vittoria; Sapino, Anna; Cassoni, Paola; Marchiò, Caterina

2017-07-25

Gene mutations in the RAS family rule out metastatic colorectal carcinomas (mCRCs) from anti-EGFR therapies. We report a retrospective analysis by Sequenom Massarray and fast COLD-PCR followed by Sanger sequencing on 240 mCRCs. By Sequenom, KRAS and NRAS exons 2-3-4 were mutated in 52.9% (127/240) of tumours, while BRAF codon 600 mutations reached 5% (12/240). Fast COLD-PCR found extra mutations at KRAS exon 2 in 15/166 (9%) of samples, previously diagnosed by Sequenom as wild-type or mutated at RAS (exons 3-4) or BRAF genes. After UDG digestion results were reproduced in 2/12 analysable subclonally mutated samples leading to a frequency of true subclonal KRAS mutations of 1.2% (2.1% of the previous Sequenom wild-type subgroup). In 10 out of 12 samples, the subclonal KRAS mutations disappeared (9 out of 12) or turned to a different sequence variant (1 out of 12). mCRC can harbour coexisting multiple gene mutations. High sensitivity assays allow the detection of a small subset of patients harbouring true subclonal KRAS mutations. However, DNA changes with mutant allele frequencies <3% detected in formalin-fixed paraffin-embedded samples may be artifactual in a non-negligible fraction of cases. UDG pre-treatment of DNA is mandatory to identify true DNA changes in archival samples and avoid misinterpretation due to artifacts.

Modification of mutation frequency in Saccharomyces Cerevisiae

International Nuclear Information System (INIS)

Vashishat, R.K.; Kakar, S.N.

1976-01-01

In a reverse mutation system, using haploid, histidine-requirinq strain of Saccharomyces cerevisiae, the frequency of uv-induced prototrophs increased if the post-irradiation minimal medium was supplemented with limited amounts of histidine. Addition of natural amino acids or RNA bases in the post-irradiation minimal medium, with or without histidine, also increased the uv-induced mutation frequency. Thus, post-irradiation conditions favouring protein and RNA synthesis, are effective in increasing uv-induced mutations in yeast. As compared to uv light, nitrous acid was more effective in inducing reversions in this strain and the frequency increased if the treated cells were plated on minimal medium supplemented with limited amounts of histidine. However, the addition of amino acids or RNA bases decreased the number of revertants. An additional inclusion of histidine reversed the suppressive effect of these metabolites. The mutation induction processes are thus different or differently modifiable in uv and nitrous acid. (author)

Impact of low-frequency hotspot mutation R282Q on the structure of p53 DNA-binding domain as revealed by crystallography at 1.54 Å resolution

Energy Technology Data Exchange (ETDEWEB)

Tu, Chao [Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, MD 21702 (United States); Tan, Yu-Hong [Department of Molecular Biology and Biochemistry, University of California at Irvine, Irvine, CA 92697 (United States); Shaw, Gary [Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, MD 21702 (United States); Zhou, Zheng; Bai, Yawen [Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, Bethesda, MD 20892 (United States); Luo, Ray [Department of Molecular Biology and Biochemistry, University of California at Irvine, Irvine, CA 92697 (United States); Ji, Xinhua, E-mail: jix@ncifcrf.gov [Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, MD 21702 (United States)

2008-05-01

The impact of hotspot mutation R282Q on the structure of human p53 DNA-binding domain has been characterized by X-ray crystallography and molecular-dynamics simulations. Tumor suppressor p53 is a sequence-specific DNA-binding protein and its central DNA-binding domain (DBD) harbors six hotspots (Arg175, Gly245, Arg248, Arg249, Arg273 and Arg282) for human cancers. Here, the crystal structure of a low-frequency hotspot mutant, p53DBD(R282Q), is reported at 1.54 Å resolution together with the results of molecular-dynamics simulations on the basis of the structure. In addition to eliminating a salt bridge, the R282Q mutation has a significant impact on the properties of two DNA-binding loops (L1 and L3). The L1 loop is flexible in the wild type, but it is not flexible in the mutant. The L3 loop of the wild type is not flexible, whereas it assumes two conformations in the mutant. Molecular-dynamics simulations indicated that both conformations of the L3 loop are accessible under biological conditions. It is predicted that the elimination of the salt bridge and the inversion of the flexibility of L1 and L3 are directly or indirectly responsible for deactivating the tumor suppressor p53.

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.

Current study on ionizing radiation-induced mitochondial DNA damage and mutations

International Nuclear Information System (INIS)

Zhou Xin; Wang Zhenhua; Zhang Hong

2012-01-01

Current advance in ionizing radiation-induced mitochondrial DNA damage and mutations is reviewed, in addition with the essential differences between mtDNA and nDNA damage and mutations. To extent the knowledge about radiation induced mitochondrial alterations, the researchers in Institute of Modern Physics, Chinese Academy of Sciences developed some technics such as real-time PCR, long-PCR for accurate quantification of radiation induced damage and mutations, and in-depth investigation about the functional changes of mitochondria based on mtDNA damage and mutations were also carried out. In conclusion, the important role of mitochondrial study in radiation biology is underlined, and further study on mitochondrial study associated with late effect and metabolism changes in radiation biology is pointed out. (authors)

Running on empty: does mitochondrial DNA mutation limit replicative lifespan in yeast?: Mutations that increase the division rate of cells lacking mitochondrial DNA also extend replicative lifespan in Saccharomyces cerevisiae.

Science.gov (United States)

Dunn, Cory D

2011-10-01

Mitochondrial DNA (mtDNA) mutations escalate with increasing age in higher organisms. However, it has so far been difficult to experimentally determine whether mtDNA mutation merely correlates with age or directly limits lifespan. A recent study shows that budding yeast can also lose functional mtDNA late in life. Interestingly, independent studies of replicative lifespan (RLS) and of mtDNA-deficient cells show that the same mutations can increase both RLS and the division rate of yeast lacking the mitochondrial genome. These exciting, parallel findings imply a potential causal relationship between mtDNA mutation and replicative senescence. Furthermore, these results suggest more efficient methods for discovering genes that determine lifespan. Copyright © 2011 WILEY Periodicals, Inc.

DNA replication in necessary for fixing induced mutations to streptomycin-resistance in UV-irradiated Escherichia coli cells

Energy Technology Data Exchange (ETDEWEB)

Dubinin, N P; Filippov, V D

1986-01-01

A suspension of E.coli cells has been subjected to UV radiation, then it has been incubated in the growth medium for 15 min. After that one of the portions was incubated with nalidixic acid (NA), and the other one without it in the presence of an antibiotic. Frequency of mutations depending on or irrespective of photoactivation, has been determined. Dependence of Str mutation fixing, induced by low UV radiation doses, on DNA synthesis is determined. Results indicate that both photoreactivation of mutations and its senstivity to mfd system are simultaneously lost.

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

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

Energy Technology Data Exchange (ETDEWEB)

Mei, Nan [Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, FDA, Jefferson, AR 72079 (United States)]. E-mail: nan.mei@fda.hhs.gov; Arlt, Volker M. [Section of Molecular Carcinogenesis, Institute of Cancer Research, Cotswold Road, Sutton, Surrey SM2 5NG (United Kingdom); Phillips, David H. [Section of Molecular Carcinogenesis, Institute of Cancer Research, Cotswold Road, Sutton, Surrey SM2 5NG (United Kingdom); Heflich, Robert H. [Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, FDA, Jefferson, AR 72079 (United States); Chen, Tao [Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, FDA, Jefferson, AR 72079 (United States)

2006-12-01

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 {sup 32}P-postlabeling and mutant frequency (MF) was determined using the {lambda} Select-cII Mutation Detection System. Three major adducts (7-[deoxyadenosin-N {sup 6}-yl]-aristolactam I, 7-[deoxyadenosin-N {sup 6}-yl]-aristolactam II and 7-[deoxyguanosin-N {sup 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{sup 8} nucleotides in liver and 95-4598 adducts/10{sup 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{sup -6} in liver compared with the MFs of 78-1319 x 10{sup -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 {sup {yields}} T:A transversion was the predominant mutation in AA-treated rats; whereas G:C {sup {yields}} A:T transition was the main type of mutation in control

A comprehensive characterization of mitochondrial DNA mutations in glioblastoma multiforme.

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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.

Mitochondrial DNA mutation screening of male patients with obstructive sleep apnea-hypopnea syndrome.

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Huang, Xiao-Ying; Li, Hong; Xu, Xiao-Mei; Wang, Liang-Xing

2014-08-01

The aim of the present study was to analyze the differences between the genes of the mitochondrial DNA (mtDNA) displacement loop (D-loop) region and the Cambridge Reference sequence, in order to screen the mutation sites and investigate the correlation between mutations, clinical parameters and complications associated with obstructive sleep apnea-hypopnea syndrome (OSAHS). mtDNA was obtained from male patients with OSAHS in the Zhejiang Province. In total, 60 male patients with OSAHS and 102 healthy adults were assessed to determine the levels of fasting blood glucose, total cholesterol, triglyceride (TG) and high-density and low-density lipoproteins (LDL). Furthermore, peripheral mtDNA was extracted and bidirectional sequencing was conducted to enable mutation screening. In the mtDNA D-loop region, 178 mutation sites were identified, of which 115 sites were present in the two groups. The number of non-common sites in the OSAHS group was significantly higher compared with the control group (P0.05). A total of 21 cases in the severe OSAHS group exhibited mutation rates of >10%. In the control group, there were 24 cases where the np73A-G and np263A-G mutations were predominant. The np303-np315 region was identified to be the highly variable region and various mutation forms were observed. Statistically significant differences were observed in the neck perimeter, TG and LDL levels among the OSAHS-no-mutation subgroups (P<0.05) and LDL was shown to be associated with an mtDNA mutation in the OSAHS group. Numerous polymorphic mutation sites were identified in the mtDNA D-loop region of the OSAHS group. Therefore, mtDNA mutation sites may be closely associated with the clinical manifestations and complications of OSAHS.

Application of DNA chips in the analysis of gene mutation in HBV

International Nuclear Information System (INIS)

Wang Yongzhong; Ruan Lihua; Zhou Guoping; Wu Guoxiang; Chen Min

2005-01-01

Objective: To investigate the clinical applicability of DNA chips for analysis of gene mutation in HBV. Methods: Serum HBV DNA from 47 patients with viral hepatitis type B was amplified with PCR. Possible gene mutations were searched for in site 1896 of pre-C section, sites 1762,1764 of BCP section and sites 528, 552 of P section with DNA chip method based upon membrane coloration. Results: In the 32 patients without lamivudine treatment, the results were as follows: (1) 6 specimens with HBsAg + , HBeAg + , HBeAb - , no mutations observed. (2) 13 specimens with HBsAg + , HBeAg - , HBeAb + , mutations at site 1896, pre- C 4 cases, mutations at sites 1762,1764, BCP 11 cases. (3) 13 specimens with HBsAg + , HBeAg + , HBeAb + , mutations at site 1896 pre -C 4 cases, mutations at sites 1762,1764 BCP 13 cases. In the 15 patients after 48 weeks treatment with lamivudine but remained HBV DNA positive, mutations were observed at: site 1896 pre-C, 5 cases, sites 1762,1764 BCP, 6 cases, site 528 P section, 2 cases, site 552 P section, YVDD 4 cases, YIDD 7 cases. Conclusion: Mutations at sites 1896, 1762,1764 were more frequent in patients with HBeAb + and were related to the negative expression of HBeAg, Mutations at 1762,1764 BCP were closely related to the changes of HBeAg/HBeAb. P section mutations were only observed after lamivadine treatment and were related to resistance against the drug. DNA chip method based upon membrane coloration for detection of gene mutation was expedient and specific and worth popularization. (authors)

Frequency and distribution of Notch mutations in tumor cell lines

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

2015-01-01

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

Effect of Ku80 deficiency on mutation frequencies and spectra at a LacZ reporter locus in mouse tissues and cells.

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Rita A Busuttil

Full Text Available Non-homologous end joining (NHEJ is thought to be an important mechanism for preventing the adverse effects of DNA double strand breaks (DSBs and its absence has been associated with premature aging. To investigate the effect of inactivated NHEJ on spontaneous mutation frequencies and spectra in vivo and in cultured cells, we crossed a Ku80-deficient mouse with mice harboring a lacZ-plasmid-based mutation reporter. We analyzed various organs and tissues, as well as cultured embryonic fibroblasts, for mutations at the lacZ locus. When comparing mutant with wild-type mice, we observed a significantly higher number of genome rearrangements in liver and spleen and a significantly lower number of point mutations in liver and brain. The reduced point mutation frequency was not due to a decrease in small deletion mutations thought to be a hallmark of NHEJ, but could be a consequence of increased cellular responses to unrepaired DSBs. Indeed, we found a substantial increase in persistent 53BP1 and gammaH2AX DNA damage foci in Ku80-/- as compared to wild-type liver. Treatment of cultured Ku80-deficient or wild-type embryonic fibroblasts, either proliferating or quiescent, with hydrogen peroxide or bleomycin showed no differences in the number or type of induced genome rearrangements. However, after such treatment, Ku80-deficient cells did show an increased number of persistent DNA damage foci. These results indicate that Ku80-dependent repair of DNA damage is predominantly error-free with the effect of alternative more error-prone pathways creating genome rearrangements only detectable after extended periods of time, i.e., in young adult animals. The observed premature aging likely results from a combination of increased cellular senescence and an increased load of stable, genome rearrangements.

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

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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.

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

Frequency of BRAF V600E Mutation in the Mexican Population of Patients With Metastatic Melanoma

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Erika Ruiz-Garcia

2017-06-01

Full Text Available Purpose: The BRAF V600E mutation has been described in melanomas occurring in the Caucasian, European, and Asian populations. However, in the Mexican population, the status and clinical significance of BRAF mutation has not been researched on a large scale. Methods: Consecutive BRAF-tested Mexican patients with metastatic melanoma (n = 127 were analyzed for mutations in exon 15 of the BRAF gene in genomic DNA by real-time polymerase chain reaction technology for amplification and detection. The results were correlated with the clinical-pathologic features and the prognosis of the patients. Results: The frequency of somatic mutation V600E within the BRAF gene was 54.6% (43 of 127 patients. Nodular melanoma was the most prevalent subtype in our population, with BRAF mutations in 37.2% (16 of 55 patients. In contrast, superficial spread had a frequency of 18.6% BRAF mutation (eight of 24. Other clinicopathologic features were assessed to correlate with the mutation status. Conclusion: This study searched for the most prevalent BRAF V600E mutation type in melanoma in a heterogeneous population from Mexico. Nodular melanoma was found to be the most prevalent in metastatic presentation and the presence of BRAF V600E mutation, perhaps related to the mixed ancestry; in the north, ancestry is predominantly European and in the south, it is predominantly Asian. The outcomes of the mutation correlations were similar to those found in other populations.

Studies of human mutation rates: Progress report

International Nuclear Information System (INIS)

Neel, J.V.

1988-01-01

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

Study of hepatitis B virus gene mutations with enzymatic colorimetry-based DNA microarray.

Science.gov (United States)

Mao, Hailei; Wang, Huimin; Zhang, Donglei; Mao, Hongju; Zhao, Jianlong; Shi, Jian; Cui, Zhichu

2006-01-01

To establish a modified microarray method for detecting HBV gene mutations in the clinic. Site-specific oligonucleotide probes were immobilized to microarray slides and hybridized to biotin-labeled HBV gene fragments amplified from two-step PCR. Hybridized targets were transferred to nitrocellulose membranes, followed by intensity measurement using BCIP/NBT colorimetry. HBV genes from 99 Hepatitis B patients and 40 healthy blood donors were analyzed. Mutation frequencies of HBV pre-core/core and basic core promoter (BCP) regions were found to be significantly higher in the patient group (42%, 40% versus 2.5%, 5%, P colorimetry method exhibited the same level of sensitivity and reproducibility. An enzymatic colorimetry-based DNA microarray assay was successfully established to monitor HBV mutations. Pre-core/core and BCP mutations of HBV genes could be major causes of HBV infection in HBeAg-negative patients and could also be relevant to chronicity and aggravation of hepatitis B.

Assessment of basal-like breast cancer by circulating tumor DNA analysis.

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Wei, Wei; Zhang, Xianyu; Sun, Shanshan; Xia, Bingshu; Liang, Xiaoshuan; Cui, Yan; Gao, Song; Pang, Da

2018-05-01

Standardized methods for the detection and assessment of circulating tumor DNA (ctDNA) in breast cancer are not sufficient. In the present study, the method and the potential application of ctDNA in the diagnosis of breast cancer were explored. DNA was extracted from the tumor tissues, plasma and peripheral blood cells of 11 patients with early-stage invasive breast cancer. Primers were designed against the exons of phosphatidylinositol-4,5-biphosphate 3-kinase catalytic subunit α, p53, epidermal growth factor receptor, Akt and phosphatase and tensin homolog. The amplicon-based method for whole-exon sequencing was performed. The associations between the ctDNA mutant frequency with the tumor DNA mutant frequency, and the ctDNA concentration with clinical data were analyzed. A linear association was identified between the concentration of ctDNA and the tumor volume for the 3 patients with basal-like breast cancer, and not in other subtypes. The mutation frequency differed the least between ctDNA and tissue DNA in basal-like breast cancer. ctDNA retained the constituent ratio of gene mutations identified in the corresponding tumor tissue. The ctDNA detection rate depended to a certain extent on the mutation frequency in tumor tissue; for example, a mutant locus with a mutation frequency of >30% in tissues presented a detection rate of >40% in plasma samples, whereas a locus with a mutation frequency of <10% in tissue was associated with a detection rate of ≤1% in the plasma. Therefore, ctDNA may reflect the mutations observed in cancer. Compared with other subtypes, ctDNA may be a more sensitive biomarker for the assessment of mutation and cancer burden in basal-like breast cancer relative to other subtypes.

Induced mutations in chickpea (Cicer arietinum L.) II. frequency and spectrum of chlorophyll mutations

International Nuclear Information System (INIS)

Kharkwal, M.C.

1998-01-01

A comparative study of frequency and spectrum of chlorophyll mutations induced by two physical (gamma rays, fast neutrons) and two chemical mutagens (NMU, EMS) in relation to the effects in M1 plants and induction of mutations in M2 was made in four chickpea (Cicer arietinum L.) varieties, two desi (G 130 & H 214) one Kabuli (C 104) and one green seeded (L 345). The treatments included three doses each of gamma rays (400, 500 & 600 Gy) and fast neutrons (5, 10 & 15 Gy) and two concentrations with two different durations of two chemical mutagens, NMU [0.01% (20h), & 0.02% (8h)] and EMS [0.1% (20h) & 0.2% (8h)]. The frequencies and spectrum of three different kinds of induced chlorophyll mutations in the order albina (43.5%), chlorina (27.3%) and xantha (24.2%) were recorded. Chemical mutagens were found to be efficient in inducing chlorophyll mutations in chickpea. Highest frequency of mutations was observed in green seeded var. L 345 (83% of M1 families and 19.9/1000 M2 plants). Kabuli var. C 104 was least responsive for chlorophyll mutations

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

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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.

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

International Nuclear Information System (INIS)

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

2014-01-01

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

Radiation-induced mutation at minisatellite loci

International Nuclear Information System (INIS)

Dubrova, Y.E.; Nesterov, V.N.; Krouchinsky, N.G.

1997-01-01

We are studying the radiation-induced increase of mutation rate in minisatellite loci in mice and humans. Minisatellite mutations were scored by multilocus DNA fingerprint analysis in the progeny of γ-irradiated and non-irradiated mice. The frequency of mutation in offspring of irradiated males was 1.7 higher that in the control group. Germline mutation at human minisatellite loci was studied among children born in heavily polluted areas of the Mogilev district of Belarus after the Chernobyl accident and in a control population. The frequency of mutation assayed both by DNA fingerprinting and by eight single locus probes was found to be two times higher in the exposed families than in the control group. Furthermore, mutation rate was correlated with the parental radiation dose for chronic exposure 137 Cs, consistent with radiation-induction of germline mutation. The potential use of minisatellites in monitoring germline mutation in humans will be discussed

The effect of defective DNA double-strand break repair on mutations and chromosome aberrations in the Chinese hamster cell mutant XR-V15B

International Nuclear Information System (INIS)

Helbig, R.; Speit, G.; Zdzienicka, M.Z.

1995-01-01

The radiosensitive Chinese hamster cell line XR-V15B was used to study the effect of decreased rejoining of DNA double-strand breaks (DSBs) on gene mutations and chromosome aberrations. XR-V15B cells are hypersensitive to the cytotoxic effects of neocarzinostatin (NCS) and methyl methanesulfonate (MMS). Both mutagens induced more chromosome aberrations in XR-V15B cells than in the parental cell strain. The clastogenic action of NCS was characterized by the induction of predominantly chromosome-type aberrations in cells of both strains, whereas MMS induced mainly chromatid aberrations. The frequency of induced gene mutations at the hprt locus was not increased compared to the parental V79 cells when considering the same survival level. Molecular analysis by multiplex polymerase chain reaction (PCR) of mutants induced by NCS revealed a high frequency of deletions in cells of both cell lines. Methyl methane-sulfonate induced mainly mutations without visible change in the PCR pattern, which probably represent point mutations. Our findings suggest a link between a defect in DNA DSB repair and increased cytotoxic and clastogenic effects. However, a decreased ability to rejoin DNA DSBs does not seem to influence the incidence and types of gene mutations at the hprt locus induced by NCS and MMS. 28 refs., 4 figs., 3 tabs

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

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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

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

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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.

Optimal control of gene mutation in DNA replication.

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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.

HFE H63D mutation frequency shows an increase in Turkish women with breast cancer

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Guler Emine

2006-02-01

Full Text Available Abstract Background The hereditary hemochromatosis gene HFE plays a pivotal role in iron homeostasis. The association between cancer and HFE hetero- or homozygosity has previously been shown including hepatocellular and nonhepatocellular malignancies. This study was performed to compare frequencies of HFE C282Y and H63D variants in Turkish women with breast cancer and healthy controls. Methods Archived DNA samples of Hacettepe University Oncology Institute were used in this study. The HFE gene was investigated by PCR-RFLP. Results All subjects studied were free from C282Y mutation. Thirty-nine patients had H63D mutation and were all heterozygous. H63D allele frequency was 22.2% (39/176 in the breast cancer patients, and 14% (28/200 in the healthy volunteers. Statistical analysis of cases with HFE H63D phenotype showed significant difference between breast cancer and healthy volunteers (P = 0.02. Conclusion Our results suggest that HFE H63D mutation frequencies were increased in the breast cancer patients in comparison to those in the general population. Also, odds ratios (odds ratio = 2.05 computed in this study suggest that H63D has a positive association with breast cancer.

Mutation induction by ion beams in plants

International Nuclear Information System (INIS)

Tanaka, Atsushi

2001-01-01

The effect of ion beams such as C, He, and Ne ions was investigated on the mutation induction in plants with the expectation that ion beams of high linear energy transfer (LET) can frequently produce large DNA alternation such as inversion, translocation and large deletion rather than point mutation. Mutation frequency was investigated using Arabidopsis visible phenotype loci and was 8 to 33 fold higher for 220 MeV carbon ions than for electrons. Mutation spectrum was investigated on the flower color of chrysanthemum cv to find that flower mutants induced by ion beams show complex and stripe types rather than single color. Polymerase chain reaction analysis was performed to investigate DNA alteration of mutations. In conclusion, the characteristics of ion beams for the mutation induction are 1) high frequency, 2) broad mutation spectrum, and 3) novel mutants. (S. Ohno)

Mutation induction by ion beams in plants

Energy Technology Data Exchange (ETDEWEB)

Tanaka, Atsushi [Japan Atomic Energy Research Inst., Takasaki, Gunma (Japan). Takasaki Radiation Chemistry Research Establishment

2001-03-01

The effect of ion beams such as C, He, and Ne ions was investigated on the mutation induction in plants with the expectation that ion beams of high linear energy transfer (LET) can frequently produce large DNA alternation such as inversion, translocation and large deletion rather than point mutation. Mutation frequency was investigated using Arabidopsis visible phenotype loci and was 8 to 33 fold higher for 220 MeV carbon ions than for electrons. Mutation spectrum was investigated on the flower color of chrysanthemum cv to find that flower mutants induced by ion beams show complex and stripe types rather than single color. Polymerase chain reaction analysis was performed to investigate DNA alteration of mutations. In conclusion, the characteristics of ion beams for the mutation induction are 1) high frequency, 2) broad mutation spectrum, and 3) novel mutants. (S. Ohno)

Mitochondrial DNA Mutation Associated with Leber's Hereditary Optic Neuropathy

Science.gov (United States)

Wallace, Douglas C.; Singh, Gurparkash; Lott, Marie T.; Hodge, Judy A.; Schurr, Theodore G.; Lezza, Angela M. S.; Elsas, Louis J.; Nikoskelainen, Eeva K.

1988-12-01

Leber's hereditary optic neuropathy is a maternally inherited disease resulting in optic nerve degeneration and cardiac dysrhythmia. A mitochondrial DNA replacement mutation was identified that correlated with this disease in multiple families. This mutation converted a highly conserved arginine to a histidine at codon 340 in the NADH dehydrogenase subunit 4 gene and eliminated an Sfa NI site, thus providing a simple diagnostic test. This finding demonstrated that a nucleotide change in a mitochondrial DNA energy production gene can result in a neurological disease.

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

Preimplantation genetic diagnosis for mitochondrial DNA mutations: analysis of one blastomere suffices.

Science.gov (United States)

Sallevelt, Suzanne C E H; Dreesen, Joseph C F M; Coonen, Edith; Paulussen, Aimee D C; Hellebrekers, Debby M E I; de Die-Smulders, Christine E M; Smeets, Hubert J M; Lindsey, Patrick

2017-10-01

Preimplantation genetic diagnosis (PGD) is a reproductive strategy for mitochondrial DNA (mtDNA) mutation carriers, strongly reducing their risk of affected offspring. Embryos either without the mutation or with mutation load below the phenotypic threshold are transferred to the uterus. Because of incidental heteroplasmy deviations in single blastomere and the relatively limited data available, we so far preferred relying on two blastomeres rather than one. Considering the negative effect of a two-blastomere biopsy protocol compared with a single-blastomere biopsy protocol on live birth delivery rate, we re-evaluated the error rate in our current dataset. For the m.3243A>G mutation, sufficient embryos/blastomeres were available for a powerful analysis. The diagnostic error rate, defined as a potential false-negative result, based on a threshold of 15%, was determined in 294 single blastomeres analysed in 73 embryos of 9 female m.3243A>G mutation carriers. Only one out of 294 single blastomeres (0.34%) would have resulted in a false-negative diagnosis. False-positive diagnoses were not detected. Our findings support a single-blastomere biopsy PGD protocol for the m.3243A>G mutation as the diagnostic error rate is very low. As in the early preimplantation embryo no mtDNA replication seems to occur and the mtDNA is divided randomly among the daughter cells, we conclude this result to be independent of the specific mutation and therefore applicable to all mtDNA mutations. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Frequency of Usher syndrome type 1 in deaf children by massively parallel DNA sequencing.

Science.gov (United States)

Yoshimura, Hidekane; Miyagawa, Maiko; Kumakawa, Kozo; Nishio, Shin-Ya; Usami, Shin-Ichi

2016-05-01

Usher syndrome type 1 (USH1) is the most severe of the three USH subtypes due to its profound hearing loss, absent vestibular response and retinitis pigmentosa appearing at a prepubescent age. Six causative genes have been identified for USH1, making early diagnosis and therapy possible through DNA testing. Targeted exon sequencing of selected genes using massively parallel DNA sequencing (MPS) technology enables clinicians to systematically tackle previously intractable monogenic disorders and improve molecular diagnosis. Using MPS along with direct sequence analysis, we screened 227 unrelated non-syndromic deaf children and detected recessive mutations in USH1 causative genes in five patients (2.2%): three patients harbored MYO7A mutations and one each carried CDH23 or PCDH15 mutations. As indicated by an earlier genotype-phenotype correlation study of the CDH23 and PCDH15 genes, we considered the latter two patients to have USH1. Based on clinical findings, it was also highly likely that one patient with MYO7A mutations possessed USH1 due to a late onset age of walking. This first report describing the frequency (1.3-2.2%) of USH1 among non-syndromic deaf children highlights the importance of comprehensive genetic testing for early disease diagnosis.

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

Energy Technology Data Exchange (ETDEWEB)

Cabrera-Juarez, E; Setlow, J K [Escuela Nacional de Ciencias Biologicas, Mexico City. Dept. de Bioquimica; Oak Ridge National Lab., Tenn. (USA). Biology Div.)

1976-05-01

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.

Tyr120Asp mutation alters domain flexibility and dynamics of MeCP2 DNA binding domain leading to impaired DNA interaction: Atomistic characterization of a Rett syndrome causing mutation.

Science.gov (United States)

D'Annessa, Ilda; Gandaglia, Anna; Brivio, Elena; Stefanelli, Gilda; Frasca, Angelisa; Landsberger, Nicoletta; Di Marino, Daniele

2018-05-01

Mutations in the X-linked MECP2 gene represent the main origin of Rett syndrome, causing a profound intellectual disability in females. MeCP2 is an epigenetic transcriptional regulator containing two main functional domains: a methyl-CpG binding domain (MBD) and a transcription repression domain (TRD). Over 600 pathogenic mutations were reported to affect the whole protein; almost half of missense mutations affect the MBD. Understanding the impact of these mutations on the MBD structure and interaction with DNA will foster the comprehension of their pathogenicity and possibly genotype/phenotype correlation studies. Herein, we use molecular dynamics simulations to obtain a detailed view of the dynamics of WT and mutated MBD in the presence and absence of DNA. The pathogenic mutation Y120D is used as paradigm for our studies. Further, since the Y120 residue was previously found to be a phosphorylation site, we characterize the dynamic profile of the MBD also in the presence of Y120 phosphorylation (pY120). We found that addition of a phosphate group to Y120 or mutation in aspartic acid affect domain mobility that samples an alternative conformational space with respect to the WT, leading to impaired ability to interact with DNA. Experimental assays showing a significant reduction in the binding affinity between the mutated MBD and the DNA confirmed our predictions. Copyright © 2018 Elsevier B.V. All rights reserved.

A Phenotype-Driven Approach to Generate Mouse Models with Pathogenic mtDNA Mutations Causing Mitochondrial Disease

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Johanna H.K. Kauppila

2016-09-01

Full Text Available Mutations of mtDNA are an important cause of human disease, but few animal models exist. Because mammalian mitochondria cannot be transfected, the development of mice with pathogenic mtDNA mutations has been challenging, and the main strategy has therefore been to introduce mutations found in cell lines into mouse embryos. Here, we describe a phenotype-driven strategy that is based on detecting clonal expansion of pathogenic mtDNA mutations in colonic crypts of founder mice derived from heterozygous mtDNA mutator mice. As proof of concept, we report the generation of a mouse line transmitting a heteroplasmic pathogenic mutation in the alanine tRNA gene of mtDNA displaying typical characteristics of classic mitochondrial disease. In summary, we describe a straightforward and technically simple strategy based on mouse breeding and histology to generate animal models of mtDNA-mutation disease, which will be of great importance for studies of disease pathophysiology and preclinical treatment trials.

Detection of low frequency FGFR3 mutations in the urine of bladder cancer patients using next-generation deep sequencing

Directory of Open Access Journals (Sweden)

Millholl

2012-06-01

Full Text Available John M Millholland, Shuqiang Li, Cecilia A Fernandez, Anthony P ShuberPredictive Biosciences Inc, Lexington, MA, USAAbstract: Biological fluid-based noninvasive biomarker assays for monitoring and diagnosing disease are clinically powerful. A major technical hurdle for developing these assays is the requirement of high analytical sensitivity so that biomarkers present at very low levels can be consistently detected. In the case of biological fluid-based cancer diagnostic assays, sensitivities similar to those of tissue-based assays are difficult to achieve with DNA markers due to the high abundance of normal DNA background present in the sample. Here we describe a new urine-based assay that uses ultradeep sequencing technology to detect single mutant molecules of fibroblast growth factor receptor 3 (FGFR3 DNA that are indicative of bladder cancer. Detection of FGFR3 mutations in urine would provide clinicians with a noninvasive means of diagnosing early-stage bladder cancer. The single-molecule assay detects FGFR3 mutant DNA when present at as low as 0.02% of total urine DNA and results in 91% concordance with the frequency that FGFR3 mutations are detected in bladder cancer tumors, significantly improving diagnostic performance. To our knowledge, this is the first practical application of next-generation sequencing technology for noninvasive cancer diagnostics.Keywords: FGFR3, mutation, urine, single molecule, sequencing, bladder cancer

[The mutations of the D-loop hypervariable region II and hypervariable region III of mitochondrial DNA in oral squamous cell carcinoma].

Science.gov (United States)

Wang, Yao-Zhong; Jia, Mu-Yun; Yuan, Rong-Tao; Han, Guo-Dong; Bu, Ling-Xue

2010-06-01

To investigate the frequency of mitochondrial DNA (mtDNA) D-loop hypervariable region II (HVR II) and hypervariable region III (HVR III) mutations in oral squamous cell carcinoma (OSCC) and their correlation to provide the new targets for the prevention and treatment of OSCC. The D-loop HVR II and HVR III regions of mtDNA in seven cases with OSCC tissues, matched with paracancerous tissues and normal mucosa tissues from the same case, were amplified by polymerase chain raction (PCR), then were detected by direct sequencing to find the mutantsites after the comparison of all sequencing results with the mtDNA Cambridge sequence in the GenBank database. 82 (56 species) nucleotide changes, with 51(26 species) nucleotide polymorphism, were found after the comparison of all sequencing results with the mtDNA Cambridge sequence in the GenBank database. 31(30 species) mutations, with 21 located within the HVR II and HVR III regions, were found in 3 tumor tissue samples, their paracancerous and normal mucosa tissue were found more polymorphic changes but no mutation. The mtDNA D-loop HVR II and HVR III regions mutation rate was 42.9% (3/7) in OSCC. The mtDNA D-loop HVR II and HVR III regions were highly polymorphic and mutable regions in OSCC. It suggested that the D-loop HVR II and HVR III regions of mtDNA might play a significant role in the tumorigenesis of OSCC. It may become new targets for the gene therapy of OSCC by regulating the above indexes.

Development and applications of Bacillus subtilis test systems for mutagens, involving DNA-repair deficiency and suppressible auxotrophic mutations

International Nuclear Information System (INIS)

Tanooka, H.

1977-01-01

A mutagen-tester of Bacillus subtilis was constructed and tested with known carcinogens. The parental strain HA101 of Okubo and Yanagida carrying suppressible nonsense mutations in his and met genes was transformed to carry an excision-repair deficiency mutation. The constructed strain TKJ5211 showed a 20-30-fold higher sensitivity for His + reversion than the parental strain when treated with UV and UV-mimetic chemicals but unchanged mutation frequency with X-rays and methyl methanesulfonate. The tester strain was used in a spot test of 30 selected chemicals and also for testing with liver homogenate activation. The results showed an almost equivalent but somewhat broader detection spectrum than the Salmonella typhimurium TA100 system. Another test method used a pair of B. subtilis strains differing in their DNA-repair capacity, i.e. the most UV-sensitive mutant HJ-15 and a wild-type strain, to detect repair-dependent DNA damage produced by chemicals. Spores could be used in either test

TOX3 mutations in breast cancer.

Directory of Open Access Journals (Sweden)

James Owain Jones

Full Text Available TOX3 maps to 16q12, a region commonly lost in breast cancers and recently implicated in the risk of developing breast cancer. However, not much is known of the role of TOX3 itself in breast cancer biology. This is the first study to determine the importance of TOX3 mutations in breast cancers. We screened TOX3 for mutations in 133 breast tumours and identified four mutations (three missense, one in-frame deletion of 30 base pairs in six primary tumours, corresponding to an overall mutation frequency of 4.5%. One potentially deleterious missense mutation in exon 3 (Leu129Phe was identified in one tumour (genomic DNA and cDNA. Whilst copy number changes of 16q12 are common in breast cancer, our data show that mutations of TOX3 are present at low frequency in tumours. Our results support that TOX3 should be further investigated to elucidate its role in breast cancer biology.

Impact of Emergent Circulating Tumor DNA RAS Mutation in Panitumumab-Treated Chemoresistant Metastatic Colorectal Cancer.

Science.gov (United States)

Kim, Tae Won; Peeters, Marc; Thomas, Anne L; Gibbs, Peter; Hool, Kristina; Zhang, Jianqi; Ang, Agnes; Bach, Bruce Allen; Price, Timothy

2018-06-13

The accumulation of emergent RAS mutations during anti-epidermal growth factor receptor (EGFR) therapy is of interest as a mechanism for acquired resistance to anti-EGFR treatment. Plasma analysis of circulating tumor (ct) DNA is a minimally invasive and highly sensitive method to determine RAS mutational status. This biomarker analysis of the global phase III ASPECCT study used next-generation sequencing to detect expanded RAS ctDNA mutations in panitumumab-treated patients. Plasma samples collected at baseline and posttreatment were analyzed categorically for the presence of RAS mutations by the Plasma Select -R™ 64-gene panel at 0.1% sensitivity. Among panitumumab-treated patients with evaluable plasma samples at baseline (n = 238), 188 (79%) were wild-type (WT) RAS, and 50 (21%) were mutant RAS Of the 188 patients with baseline ctDNA WT RAS status, 164 had evaluable posttreatment results with a 32% rate of emergent RAS mutations. The median overall survival (OS) for WT and RAS mutant status by ctDNA at baseline was 13.7 (95% confidence interval: 11.5-15.4) and 7.9 months (6.4-9.6), respectively ( P < 0.0001). Clinical outcomes were not significantly different between patients with and without emergent ctDNA RAS mutations. Although patients with baseline ctDNA RAS mutations had worse outcomes than patients who were WT RAS before initiating treatment, emergent ctDNA RAS mutations were not associated with less favorable patient outcomes in panitumumab-treated patients. Further research is needed to determine a clinically relevant threshold for baseline and emergent ctDNA RAS mutations. Copyright ©2018, American Association for Cancer Research.

UVA activation of N-dialkylnitrosamines releasing nitric oxide, producing strand breaks as well as oxidative damages in DNA, and inducing mutations in the Ames test

International Nuclear Information System (INIS)

Arimoto-Kobayashi, Sakae; Sano, Kayoko; Machida, Masaki; Kaji, Keiko; Yakushi, Keiko

2010-01-01

We investigated the photo-mutagenicity and photo-genotoxicity of N-dialkylnitrosamines and its mechanisms of UVA activation. With simultaneous irradiation of UVA, photo-mutagenicity of seven N-dialkylnitrosamines was observed in Ames bacteria (Salmonella typhimurium TA1535) in the absence of metabolic activation. Mutagenicity of pre-irradiated N-dialkylnitrosamines was also observed with S. typhimurium hisG46, TA100, TA102 and YG7108 in the absence of metabolic activation. UVA-mediated mutation with N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) decreased by adding either the NO or OH radical scavenger. When superhelical DNA was irradiated with N-dialkylnitrosamines, nicked circular DNA appeared. Ten N-dialkylnitrosamines examined produced strand breaks in the treated DNA in the presence of UVA. The level of single-strand breaks in φX174 DNA mediated by N-nitrosomorpholine (NMOR) and UVA decreased by adding either a radical scavenger or superoxide dismutase. When calf thymus DNA was treated with N-dialkylnitrosamines (NDMA, NDEA, NMOR, N-nitrosopyrrolidine (NPYR) and N-nitrosopiperidine (NPIP)) and UVA, the ratio of 8-oxodG/dG in the DNA increased. Action spectra were obtained to determine if nitrosamine acts as a sensitizer of UVA. Both mutation frequency and NO formation were highest at the absorption maximum of nitrosamines, approximately 340 nm. The plots of NO formation and mutation frequency align with the absorption curve of NPYR, NMOR and NDMA. A significant linear correlation between the optical density of N-dialkynitrosamines at 340 nm and NO formation in each irradiated solution was revealed by ANOVA. We would like to propose the hypothesis that the N-nitroso moiety of N-dialkylnitrosamines absorbs UVA photons, UVA-photolysis of N-dialkylnitrosamines brings release of nitric oxide, and subsequent production of alkyl radical cations and active oxygen species follow as secondary events, which cause DNA strand breaks, oxidative and

UVA activation of N-dialkylnitrosamines releasing nitric oxide, producing strand breaks as well as oxidative damages in DNA, and inducing mutations in the Ames test

Energy Technology Data Exchange (ETDEWEB)

Arimoto-Kobayashi, Sakae [Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima, Okayama 700-8530 (Japan); Sano, Kayoko; Machida, Masaki; Kaji, Keiko; Yakushi, Keiko [Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima, Okayama 700-8530 (Japan)

2010-09-10

We investigated the photo-mutagenicity and photo-genotoxicity of N-dialkylnitrosamines and its mechanisms of UVA activation. With simultaneous irradiation of UVA, photo-mutagenicity of seven N-dialkylnitrosamines was observed in Ames bacteria (Salmonella typhimurium TA1535) in the absence of metabolic activation. Mutagenicity of pre-irradiated N-dialkylnitrosamines was also observed with S. typhimurium hisG46, TA100, TA102 and YG7108 in the absence of metabolic activation. UVA-mediated mutation with N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) decreased by adding either the NO or OH radical scavenger. When superhelical DNA was irradiated with N-dialkylnitrosamines, nicked circular DNA appeared. Ten N-dialkylnitrosamines examined produced strand breaks in the treated DNA in the presence of UVA. The level of single-strand breaks in {phi}X174 DNA mediated by N-nitrosomorpholine (NMOR) and UVA decreased by adding either a radical scavenger or superoxide dismutase. When calf thymus DNA was treated with N-dialkylnitrosamines (NDMA, NDEA, NMOR, N-nitrosopyrrolidine (NPYR) and N-nitrosopiperidine (NPIP)) and UVA, the ratio of 8-oxodG/dG in the DNA increased. Action spectra were obtained to determine if nitrosamine acts as a sensitizer of UVA. Both mutation frequency and NO formation were highest at the absorption maximum of nitrosamines, approximately 340 nm. The plots of NO formation and mutation frequency align with the absorption curve of NPYR, NMOR and NDMA. A significant linear correlation between the optical density of N-dialkynitrosamines at 340 nm and NO formation in each irradiated solution was revealed by ANOVA. We would like to propose the hypothesis that the N-nitroso moiety of N-dialkylnitrosamines absorbs UVA photons, UVA-photolysis of N-dialkylnitrosamines brings release of nitric oxide, and subsequent production of alkyl radical cations and active oxygen species follow as secondary events, which cause DNA strand breaks, oxidative and

Clinical differences in patients with mitochondriocytopathies due to nuclear versus mitochondrial DNA mutations.

Science.gov (United States)

Rubio-Gozalbo, M E; Dijkman, K P; van den Heuvel, L P; Sengers, R C; Wendel, U; Smeitink, J A

2000-01-01

Defects in oxidative phosphorylation (OXPHOS) are genetically unique because the different components involved in this process, respiratory chain enzyme complexes (I, III, and IV) and complex V, are encoded by nuclear and mitochondrial genome. The objective of the study was to assess whether there are clinical differences in patients suffering from OXPHOS defects caused by nuclear or mitochondrial DNA (mtDNA) mutations. We studied 16 families with > or = two siblings with a genetically established OXPHOS deficiency, four due to a nuclear gene mutation and 12 due to a mtDNA mutation. Siblings with a nuclear gene mutation showed very similar clinical pictures that became manifest in the first years (ranging from first months to early childhood). There was a severe progressive course. Seven of the eight children died in their first decade. Conversely, siblings with a mtDNA mutation had clinical pictures that varied from almost alike to very distinct. They became symptomatic at an older age (ranging from childhood to adulthood), with the exception of defects associated with Leigh or Leigh-like phenotype. The clinical course was more gradual and relatively less severe; four of the 26 patients died, one in his second year, another in her second decade and two in their sixth decade. There are differences in age at onset, severity of clinical course, outcome, and intrafamilial variability in patients affected of an OXPHOS defect due to nuclear or mtDNA mutations. Patients with nuclear mutations become symptomatic at a young age, and have a severe clinical course. Patients with mtDNA mutations show a wider clinical spectrum of age at onset and severity. These differences may be of importance regarding the choice of which genome to study in affected patients as well as with respect to genetic counseling. Copyright 2000 Wiley-Liss, Inc.

Mutagenic and epigenetic influence of caffeine on the frequencies of UV-induced ouabain-resistant Chinese hamster cells

International Nuclear Information System (INIS)

Chang, Chia-Cheng; Philipps, C.; Trosko, J.E.; Hart, R.W.

1977-01-01

Caffeine, given as a post-treatment to UV-irradiated Chinese hamster cells in vitro, modified the frequency of induced mutations at the ouabain resistance locus. Mutation frequencies were increased when caffeine was added only for the DNA repair and mutation fixation period. When caffeine was added after the DNA repair and mutation fixation period, or immediately after DNA damage and for the entire repair and selection period, mutation frequencies were reduced. A hypothesis, given to explain both results, is that caffeine, by blocking a constitutive 'error-free' postreplication repair process, allows an 'error-prone' DNA repair process to produce many mutations. Moreover, caffeine, possibly by modifying C-AMP metabolism, causes a repression of induced mutations which, in effect, explains its anti-mutagenic and anti-carcinogenic properties

Mutations affecting RNA polymerase I-stimulated exchange and rDNA recombination in yeast

International Nuclear Information System (INIS)

Lin, Y.H.; Keil, R.L.

1991-01-01

HOT1 is a cis-acting recombination-stimulatory sequence isolated from the rDNA repeat unit of yeast. The ability of HOT1 to stimulate mitotic exchange appears to depend on its ability to promote high levels of RNA polymerase I transcription. A qualitative colony color sectoring assay was developed to screen for trans-acting mutations that alter the activity of HOT1. Both hypo-recombination and hyper-recombination mutants were isolated. Genetic analysis of seven HOT1 recombination mutants (hrm) that decrease HOT1 activity shows that they behave as recessive nuclear mutations and belong to five linkage groups. Three of these mutations, hrm1, hrm2, and hrm3, also decrease rDNA exchange but do not alter recombination in the absence of HOT1. Another mutation, hrm4, decreases HOT1-stimulated recombination but does not affect rDNA recombination or exchange in the absence of HOT1. Two new alleles of RAD52 were also isolated using this screen. With regard to HOT1 activity, rad52 is epistatic to all four hrm mutations indicating that the products of the HRM genes and of RAD52 mediate steps in the same recombination pathway. Finding mutations that decrease both the activity of HOT1 and exchange in the rDNA supports the hypothesis that HOT1 plays a role in rDNA recombination

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

Novel Quantitative Real-Time LCR for the Sensitive Detection of SNP Frequencies in Pooled DNA: Method Development, Evaluation and Application

Science.gov (United States)

Psifidi, Androniki; Dovas, Chrysostomos; Banos, Georgios

2011-01-01

Background Single nucleotide polymorphisms (SNP) have proven to be powerful genetic markers for genetic applications in medicine, life science and agriculture. A variety of methods exist for SNP detection but few can quantify SNP frequencies when the mutated DNA molecules correspond to a small fraction of the wild-type DNA. Furthermore, there is no generally accepted gold standard for SNP quantification, and, in general, currently applied methods give inconsistent results in selected cohorts. In the present study we sought to develop a novel method for accurate detection and quantification of SNP in DNA pooled samples. Methods The development and evaluation of a novel Ligase Chain Reaction (LCR) protocol that uses a DNA-specific fluorescent dye to allow quantitative real-time analysis is described. Different reaction components and thermocycling parameters affecting the efficiency and specificity of LCR were examined. Several protocols, including gap-LCR modifications, were evaluated using plasmid standard and genomic DNA pools. A protocol of choice was identified and applied for the quantification of a polymorphism at codon 136 of the ovine PRNP gene that is associated with susceptibility to a transmissible spongiform encephalopathy in sheep. Conclusions The real-time LCR protocol developed in the present study showed high sensitivity, accuracy, reproducibility and a wide dynamic range of SNP quantification in different DNA pools. The limits of detection and quantification of SNP frequencies were 0.085% and 0.35%, respectively. Significance The proposed real-time LCR protocol is applicable when sensitive detection and accurate quantification of low copy number mutations in DNA pools is needed. Examples include oncogenes and tumour suppressor genes, infectious diseases, pathogenic bacteria, fungal species, viral mutants, drug resistance resulting from point mutations, and genetically modified organisms in food. PMID:21283808

Rich Medium Composition Affects Escherichia coli Survival, Glycation, and Mutation Frequency during Long-Term Batch Culture.

Science.gov (United States)

Kram, Karin E; Finkel, Steven E

2015-07-01

Bacteria such as Escherichia coli are frequently grown to high density to produce biomolecules for study in the laboratory. To achieve this, cells can be incubated in extremely rich media that increase overall cell yield. In these various media, bacteria may have different metabolic profiles, leading to changes in the amounts of toxic metabolites produced. We have previously shown that stresses experienced during short-term growth can affect the survival of cells during the long-term stationary phase (LTSP). Here, we incubated cells in LB, 2× yeast extract-tryptone (YT), Terrific Broth, or Super Broth medium and monitored survival during the LTSP, as well as other reporters of genetic and physiological change. We observe differential cell yield and survival in all media studied. We propose that differences in long-term survival are the result of changes in the metabolism of components of the media that may lead to increased levels of protein and/or DNA damage. We also show that culture pH and levels of protein glycation, a covalent modification that causes protein damage, affect long-term survival. Further, we measured mutation frequency after overnight incubation and observed a correlation between high mutation frequencies at the end of the log phase and loss of viability after 4 days of LTSP incubation, indicating that mutation frequency is potentially predictive of long-term survival. Since glycation and mutation can be caused by oxidative stress, we measured expression of the oxyR oxidative stress regulator during log-phase growth and found that higher levels of oxyR expression during the log phase are consistent with high mutation frequency and lower cell density during the LTSP. Since these complex rich media are often used when producing large quantities of biomolecules in the laboratory, the observed increase in damage resulting in glycation or mutation may lead to production of a heterogeneous population of plasmids or proteins, which could affect the

Nuclear DNA but not mtDNA controls tumor phenotypes in mouse cells

International Nuclear Information System (INIS)

Akimoto, Miho; Niikura, Mamoru; Ichikawa, Masami; Yonekawa, Hiromichi; Nakada, Kazuto; Honma, Yoshio; Hayashi, Jun-Ichi

2005-01-01

Recent studies showed high frequencies of homoplasmic mtDNA mutations in various human tumor types, suggesting that the mutated mtDNA haplotypes somehow contribute to expression of tumor phenotypes. We directly addressed this issue by isolating mouse mtDNA-less (ρ 0 ) cells for complete mtDNA replacement between normal cells and their carcinogen-induced transformants, and examined the effect of the mtDNA replacement on expression of tumorigenicity, a phenotype forming tumors in nude mice. The results showed that genome chimera cells carrying nuclear DNA from tumor cells and mtDNA from normal cells expressed tumorigenicity, whereas those carrying nuclear DNA from normal cells and mtDNA from tumor cells did not. These observations provided direct evidence that nuclear DNA, but not mtDNA, is responsible for carcinogen-induced malignant transformation, although it remains possible that mtDNA mutations and resultant respiration defects may influence the degree of malignancy, such as invasive or metastatic properties

Mutation, somatic mutation and diseases of man

International Nuclear Information System (INIS)

Burnet, F.M.

1976-01-01

The relevance of the intrinsic mutagenesis for the evolution process, genetic diseases and the process of aging is exemplified. The fundamental reaction is the function of the DNA and the DNA-enzymes like the DNA-polymerases in replication, repair, and transcription. These defects are responsible for the mutation frequency and the genetic drift in the evolution process. They cause genetic diseases like Xeroderma pigmentosum which is described here in detail. The accumulation of structural and functional mistakes leads to diseases of old age, for example to autoimmune diseases and immune suppression. There is a proportionality between the duration of life and the frequency of mistakes in the enzymatic repair system. No possibility of prophylaxis or therapy is seen. Methods for prognosis could be developed. (AJ) [de

New applications of CRISPR/Cas9 system on mutant DNA detection.

Science.gov (United States)

Jia, Chenqiang; Huai, Cong; Ding, Jiaqi; Hu, Lingna; Su, Bo; Chen, Hongyan; Lu, Daru

2018-01-30

The detection of mutant DNA is critical for precision medicine, but low-frequency DNA mutation is very hard to be determined. CRISPR/Cas9 is a robust tool for in vivo gene editing, and shows the potential for precise in vitro DNA cleavage. Here we developed a DNA mutation detection system based on CRISPR/Cas9 that can detect gene mutation efficiently even in a low-frequency condition. The system of CRISPR/Cas9 cleavage in vitro showed a high accuracy similar to traditional T7 endonuclease I (T7E1) assay in estimating mutant DNA proportion in the condition of normal frequency. The technology was further used for low-frequency mutant DNA detection of EGFR and HBB somatic mutations. To the end, Cas9 was employed to cleave the wild-type (WT) DNA and to enrich the mutant DNA. Using amplified fragment length polymorphism analysis (AFLPA) and Sanger sequencing, we assessed the sensitivity of CRISPR/Cas9 cleavage-based PCR, in which mutations at 1%-10% could be enriched and detected. When combined with blocker PCR, its sensitivity reached up to 0.1%. Our results suggested that this new application of CRISPR/Cas9 system is a robust and potential method for heterogeneous specimens in the clinical diagnosis and treatment management. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterization of the factor VIII defect in 147 patients with sporadic hemophilia A: Family studies indicate a mutation type-dependent sex ratio of mutation frequencies

Energy Technology Data Exchange (ETDEWEB)

Becker, J.; Schmidt, W.; Olek, K. [Univ. of Bonn (Germany)] [and others

1996-04-01

The clinical manifestation of hemophilia A is caused by a wide range of different mutations. In this study the factor VIII genes of 147 severe hemophilia A patients-all exclusively from sporadic families-were screened for mutations by use of the complete panel of modern DNA techniques. The pathogenous defect could be characterized in 126 patients (85.7%). Fifty-five patients (37.4%) showed a F8A-gene inversion, 47 (32.0%) a point mutation, 14 (9.5%) a small deletion, 8 (5.4%) a large deletion, and 2 (1.4%) a small insertion. Further, four (2.7%) mutations were localized but could not be sequenced yet. No mutation could be identified in 17 patients (11.6%). Sixteen (10.9%) of the P identified mutations occurred in the B domain. Four of these were located in an adenosine nucleotide stretch at codon 1192, indicating a mutation hotspot. Somatic mosaicisms were detected in 3 (3.9%) of 76 patients` mothers, comprising 3 of 16 de novo mutations in the patients` mothers. Investigation of family relatives allowed detection of a de novo mutation in 16 of 76 two-generation and 28 of 34 three-generation families. On the basis of these data, the male:female ratio of mutation frequencies (k) was estimated as k = 3.6. By use of the quotients of mutation origin in maternal grandfather to patient`s mother or to maternal grandmother, k was directly estimated as k = 15 and k = 7.5, respectively. Considering each mutation type separately, we revealed a mutation type-specific sex ratio of mutation frequencies. Point mutations showed a 5-to-10-fold-higher and inversions a >10-fold- higher mutation rate in male germ cells, whereas deletions showed a >5-fold-higher mutation rate in female germ cells. Consequently, and in accordance with the data of other diseases like Duchenne muscular dystrophy, our results indicate that at least for X-chromosomal disorders the male:female mutation rate of a disease is determined by its proportion of the different mutation types. 68 refs., 1 fig., 5 tabs.

Enhancement of DNA-transfection frequency by X-rays

Energy Technology Data Exchange (ETDEWEB)

Iwamoto, Ryota; Fushimi, Kazuo; Hiraki, Yoshio; Namba, Masayoshi [Okayama University Medical School (Japan). Institute of Cellular and Molecular Biology

1997-02-01

This study was conducted to evaluate the frequency of DNA transfection into human cells following X-ray irradiation. We transfected plasmid DNA (pSV2neo) into human cells, HeLa and PA-1, by either calcium phosphate precipitation or the lipofection method immediately after irradiating the cells with various doses of X-rays. The transfection frequency was evaluated by counting the number of G418-resistant colonies. When circular plasmid DNA was used, irradiation up to a dose of 2 Gy dose-dependently increased the transfection frequency, which reached a maximum of 5 to 10-fold that of the control unirradiated cells. When linear plasmid DNA was used, the transfection frequency was 2 times higher than that of circular DNA. All five of the clones that were randomly chosen expressed the transfected neo gene. In addition, the pSV2neo gene was randomly integrated into the genomic DNA of each clone. These findings indicate that X-ray treatment can facilitate foreign DNA transfer into human cells and that radiation-induced DNA breaks may promote the insertion of foreign DNA into host DNA. The enhancement of DNA transfection with X-rays may be instrumental in practicing gene therapy. (author)

Enhancement of DNA-transfection frequency by X-rays

International Nuclear Information System (INIS)

Iwamoto, Ryota; Fushimi, Kazuo; Hiraki, Yoshio; Namba, Masayoshi

1997-01-01

This study was conducted to evaluate the frequency of DNA transfection into human cells following X-ray irradiation. We transfected plasmid DNA (pSV2neo) into human cells, HeLa and PA-1, by either calcium phosphate precipitation or the lipofection method immediately after irradiating the cells with various doses of X-rays. The transfection frequency was evaluated by counting the number of G418-resistant colonies. When circular plasmid DNA was used, irradiation up to a dose of 2 Gy dose-dependently increased the transfection frequency, which reached a maximum of 5 to 10-fold that of the control unirradiated cells. When linear plasmid DNA was used, the transfection frequency was 2 times higher than that of circular DNA. All five of the clones that were randomly chosen expressed the transfected neo gene. In addition, the pSV2neo gene was randomly integrated into the genomic DNA of each clone. These findings indicate that X-ray treatment can facilitate foreign DNA transfer into human cells and that radiation-induced DNA breaks may promote the insertion of foreign DNA into host DNA. The enhancement of DNA transfection with X-rays may be instrumental in practicing gene therapy. (author)

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

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.

Characterization of a mutant of Escherichia coli B/R defective in mutation frequency decline

International Nuclear Information System (INIS)

George, D.L.

1974-01-01

A mutant of Escherichia coli B/r designated mfd is very deficient in the ability to exhibit mutation frequency decline (MFD), the characteristic loss of potential suppressor mutations which occurs when protein synthesis is briefly inhibited after irradiation with ultraviolet light (uv). This mutant is known to excise pyrimidine dimers very slowly, although it is as uv-resistant as its mfd + B/r parent strain. We have found that the mfd mutant performs the initial incision step of excision repair normally, but repairs the resulting single-strand breaks much more slowly than the mfd + strain. In spite of the slow dimer excision in the mfd mutant, single-strand DNA breaks do not accumulate during postirradiation incubation, implying that incision and excision are well corrdinated. the prolonged postirradiation lag in cell division and DNA synthesis which accompany slow excision in the mfd strain indicates that resumption of these processes of optimal rates is linked to the timing of excision repair. The normal uv-resistance of the mfd mutant also suggests such coordination and shows that the rate of excision repair is independent of its ultimate efficiency in the removal of potentially lethal uv-induced damage. (U.S.)

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.

Episodic weakness due to mitochondrial DNA MT-ATP6/8 mutations.

Science.gov (United States)

Auré, Karine; Dubourg, Odile; Jardel, Claude; Clarysse, Lucie; Sternberg, Damien; Fournier, Emmanuel; Laforêt, Pascal; Streichenberger, Nathalie; Petiot, Philippe; Gervais-Bernard, Hélène; Vial, Christophe; Bedat-Millet, Anne-Laure; Drouin-Garraud, Valérie; Bouillaud, Frédéric; Vandier, Christophe; Fontaine, Bertrand; Lombès, Anne

2013-11-19

To report that homoplasmic deleterious mutations in the mitochondrial DNA MT-ATP6/8 genes may be responsible for acute episodes of limb weakness mimicking periodic paralysis due to channelopathies and dramatically responding to acetazolamide. Mitochondrial DNA sequencing and restriction PCR, oxidative phosphorylation functional assays, reactive oxygen species metabolism, and patch-clamp technique in cultured skin fibroblasts. Occurrence of a typical MELAS (mitochondrial encephalopathy with lactic acidosis and stroke-like episodes) syndrome in a single member of a large pedigree with episodic weakness associated with a later-onset distal motor neuropathy led to the disclosure of 2 deleterious mitochondrial DNA mutations. The MT-ATP6 m.9185T>C p.Leu220Pro mutation, previously associated with Leigh syndrome, was present in all family members, while the MT-TL1 m.3271T>C mutation, a known cause of MELAS syndrome, was observed in the sole patient with MELAS presentation. Significant defect of complexes V and I as well as oxidative stress were observed in both primary fibroblasts and cybrid cells with 100% m.9185T>C mutation. Permanent plasma membrane depolarization and altered permeability to K(+) in fibroblasts provided a link with the paralysis episodes. Screening of 9 patients, based on their clinical phenotype, identified 4 patients with similar deleterious MT-ATP6 mutations (twice m.9185T>C and once m.9176T>C or m.8893T>C). A fifth patient presented with an original potentially deleterious MT-ATP8 mutation (m.8403T>C). All mutations were associated with almost-normal complex V activity but significant oxidative stress and permanent plasma membrane depolarization. Homoplasmic mutations in the MT-ATP6/8 genes may cause episodic weakness responding to acetazolamide treatment.

Regulation of the DNA Damage Response by DNA-PKcs Inhibitory Phosphorylation of ATM.

Science.gov (United States)

Zhou, Yi; Lee, Ji-Hoon; Jiang, Wenxia; Crowe, Jennie L; Zha, Shan; Paull, Tanya T

2017-01-05

Ataxia-telangiectasia mutated (ATM) regulates the DNA damage response as well as DNA double-strand break repair through homologous recombination. Here we show that ATM is hyperactive when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is chemically inhibited or when the DNA-PKcs gene is deleted in human cells. Pre-incubation of ATM protein with active DNA-PKcs also significantly reduces ATM activity in vitro. We characterize several phosphorylation sites in ATM that are targets of DNA-PKcs and show that phospho-mimetic mutations at these residues significantly inhibit ATM activity and impair ATM signaling upon DNA damage. In contrast, phospho-blocking mutations at one cluster of sites increase the frequency of apoptosis during normal cell growth. DNA-PKcs, which is integral to the non-homologous end joining pathway, thus negatively regulates ATM activity through phosphorylation of ATM. These observations illuminate an important regulatory mechanism for ATM that also controls DNA repair pathway choice. Copyright © 2017 Elsevier Inc. All rights reserved.

Mutational pattern of the nurse shark antigen receptor gene (NAR) is similar to that of mammalian Ig genes and to spontaneous mutations in evolution: the translesion synthesis model of somatic hypermutation.

Science.gov (United States)

Diaz, M; Velez, J; Singh, M; Cerny, J; Flajnik, M F

1999-05-01

The pattern of somatic mutations of shark and frog Ig is distinct from somatic hypermutation of Ig in mammals in that there is a bias to mutate GC base pairs and a low frequency of mutations. Previous analysis of the new antigen receptor gene in nurse sharks (NAR), however, revealed no bias to mutate GC base pairs and the frequency of mutation was comparable to that of mammalian IgG. Here, we analyzed 1023 mutations in NAR and found no targeting of the mechanism to any particular nucleotide but did obtain strong evidence for a transition bias and for strand polarity. As seen for all species studied to date, the serine codon AGC/T in NAR was a mutational hotspot. The NAR mutational pattern is most similar to that of mammalian IgG and furthermore both are strikingly akin to mutations acquired during the neutral evolution of nuclear pseudogenes, suggesting that a similar mechanism is at work for both processes. In yeast, most spontaneous mutations are introduced by the translesion synthesis DNA polymerase zeta (REV3) and in various DNA repair-deficient backgrounds transitions were more often REV3-dependent than were transversions. Therefore, we propose a model of somatic hypermutation where DNA polymerase zeta is recruited to the Ig locus. An excess of DNA glycosylases in germinal center reactions may further enhance the mutation frequency by a REV3-dependent mutagenic process known as imbalanced base excision repair.

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

Frequency of common CFTR gene mutations in Venezuelan patients with cystic fibrosis

OpenAIRE

Sánchez, Karen; Arcia, Orlando; Matute, Xiorama; Mindiola, Luz; Chaustre, Ismenia; Takiff, Howard

2014-01-01

Mutations in the CFTR gene in Cystic Fibrosis (CF) patients have geographic differences and there is scant data on their prevalence in Venezuelan patients. This study determined the frequency of common CFTR gene mutations in these patients. We amplified and sequenced exons 7, 10, 11, 19, 20 and 21, which contain the most common CFTR mutations, from 105 Venezuelan patients in the National CF Program. Eleven different mutations were identified, four with frequencies greater than 1%: p.Phe508del...

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

Mutant DNA quantification by digital PCR can be confounded by heating during DNA fragmentation.

Science.gov (United States)

Kang, Qing; Parkin, Brian; Giraldez, Maria D; Tewari, Muneesh

2016-04-01

Digital PCR (dPCR) is gaining popularity as a DNA mutation quantification method for clinical specimens. Fragmentation prior to dPCR is required for non-fragmented genomic DNA samples; however, the effect of fragmentation on DNA analysis has not been well-studied. Here we evaluated three fragmentation methods for their effects on dPCR point mutation assay performance. Wild-type (WT) human genomic DNA was fragmented by heating, restriction digestion, or acoustic shearing using a Covaris focused-ultrasonicator. dPCR was then used to determine the limit of blank (LoB) by quantifying observed WT and mutant allele counts of the proto-oncogenes KRAS and BRAF in the WT DNA sample. DNA fragmentation by heating to 95°C, while the simplest and least expensive method, produced a high background mutation frequency for certain KRAS mutations relative to the other methods. This was due to heat-induced mutations, specifically affecting dPCR assays designed to interrogate guanine to adenine (G>A) mutations. Moreover, heat-induced fragmentation overestimated gene copy number, potentially due to denaturation and partition of single-stranded DNA into different droplets. Covaris acoustic shearing and restriction enzyme digestion showed similar LoBs and gene copy number estimates to one another. It should be noted that moderate heating, commonly used in genomic DNA extraction protocols, did not significantly increase observed KRAS mutation counts.

Myopathic mtDNA Depletion Syndrome Due to Mutation in TK2 Gene.

Science.gov (United States)

Martín-Hernández, Elena; García-Silva, María Teresa; Quijada-Fraile, Pilar; Rodríguez-García, María Elena; Rivera, Henry; Hernández-Laín, Aurelio; Coca-Robinot, David; Fernández-Toral, Joaquín; Arenas, Joaquín; Martín, Miguel A; Martínez-Azorín, Francisco

2017-01-01

Whole-exome sequencing was used to identify the disease gene(s) in a Spanish girl with failure to thrive, muscle weakness, mild facial weakness, elevated creatine kinase, deficiency of mitochondrial complex III and depletion of mtDNA. With whole-exome sequencing data, it was possible to get the whole mtDNA sequencing and discard any pathogenic variant in this genome. The analysis of whole exome uncovered a homozygous pathogenic mutation in thymidine kinase 2 gene ( TK2; NM_004614.4:c.323 C>T, p.T108M). TK2 mutations have been identified mainly in patients with the myopathic form of mtDNA depletion syndromes. This patient presents an atypical TK2-related myopathic form of mtDNA depletion syndromes, because despite having a very low content of mtDNA (TK2 gene in mtDNA depletion syndromes and expanded the phenotypic spectrum.

Mutations of mtDNA polymerase-γ and hyperlactataemia in the HIV ...

African Journals Online (AJOL)

Mutations of mtDNA polymerase-γ and hyperlactataemia in the HIV-infected Zulu population of South Africa. ... D B A Ojwach, C Aldous, P Kocheleff, B Sartorius ... of their capacity to impede human mitochondrial DNA polymerase-γ (POLG), ...

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

Frequent occurrence of mitochondrial DNA mutations in Barrett's metaplasia without the presence of dysplasia.

Directory of Open Access Journals (Sweden)

Soong Lee

Full Text Available BACKGROUND: Barrett's esophagus (BE is one of the most common premalignant lesions and can progress to esophageal adenocarcinoma (EA. The numerous molecular events may play a role in the neoplastic transformation of Barrett's mucosa such as the change of DNA ploidy, p53 mutation and alteration of adhesion molecules. However, the molecular mechanism of the progression of BE to EA remains unclear and most studies of mitochondrial DNA (mtDNA mutations in BE have performed on BE with the presence of dysplasia. METHODS/FINDINGS: Thus, the current study is to investigate new molecular events (Barrett's esophageal tissue-specific-mtDNA alterations/instabilities in mitochondrial genome and causative factors for their alterations using the corresponding adjacent normal mucosal tissue (NT and tissue (BT from 34 patients having Barrett's metaplasia without the presence of dysplasia. Eighteen patients (53% exhibited mtDNA mutations which were not found in adjacent NT. mtDNA copy number was about 3 times higher in BT than in adjacent NT. The activity of the mitochondrial respiratory chain enzyme complexes in tissues from Barrett's metaplasia without the presence of dysplasia was impaired. Reactive oxygen species (ROS level in BT was significantly higher than those in corresponding samples. CONCLUSION/SIGNIFICANCE: High ROS level in BT may contribute to the development of mtDNA mutations, which may play a crucial role in disease progression and tumorigenesis in BE.

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

Mitochondrial DNA Mutations in Epithelial Ovarian Tumor Progression

Science.gov (United States)

2007-12-01

Panici PL, Fazio VM: Mutations of D310 mitochondrial mononu- cleotide repeat in primary tumors and cytological speci- mens . Cancer Lett 2003, 190:73...BR: Detection of LOH and mitochondrial DNA alter- ations in ductal lavage and nipple aspirate fluids from high- risk patients. Breast Cancer Res

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

2018-01-01

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.

Quantitative evaluation of DNA damage and mutation rate by atmospheric and room-temperature plasma (ARTP) and conventional mutagenesis.

Science.gov (United States)

Zhang, Xue; Zhang, Chong; Zhou, Qian-Qian; Zhang, Xiao-Fei; Wang, Li-Yan; Chang, Hai-Bo; Li, He-Ping; Oda, Yoshimitsu; Xing, Xin-Hui

2015-07-01

DNA damage is the dominant source of mutation, which is the driving force of evolution. Therefore, it is important to quantitatively analyze the DNA damage caused by different mutagenesis methods, the subsequent mutation rates, and their relationship. Atmospheric and room temperature plasma (ARTP) mutagenesis has been used for the mutation breeding of more than 40 microorganisms. However, ARTP mutagenesis has not been quantitatively compared with conventional mutation methods. In this study, the umu test using a flow-cytometric analysis was developed to quantify the DNA damage in individual viable cells using Salmonella typhimurium NM2009 as the model strain and to determine the mutation rate. The newly developed method was used to evaluate four different mutagenesis systems: a new ARTP tool, ultraviolet radiation, 4-nitroquinoline-1-oxide (4-NQO), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis. The mutation rate was proportional to the corresponding SOS response induced by DNA damage. ARTP caused greater DNA damage to individual living cells than the other conventional mutagenesis methods, and the mutation rate was also higher. By quantitatively comparing the DNA damage and consequent mutation rate after different types of mutagenesis, we have shown that ARTP is a potentially powerful mutagenesis tool with which to improve the characteristics of microbial cell factories.

General misincorporation frequency: Re-evaluation of the fidelity of DNA polymerases.

Science.gov (United States)

Yang, Jie; Li, Bianbian; Liu, Xiaoying; Tang, Hong; Zhuang, Xiyao; Yang, Mingqi; Xu, Ying; Zhang, Huidong; Yang, Chun

2018-02-19

DNA replication in cells is performed in the presence of four dNTPs and four rNTPs. In this study, we re-evaluated the fidelity of DNA polymerases using the general misincorporation frequency consisting of three incorrect dNTPs and four rNTPs but not using the traditional special misincorporation frequency with only the three incorrect dNTPs. We analyzed both the general and special misincorporation frequencies of nucleotide incorporation opposite dG, rG, or 8-oxoG by Pseudomonas aeruginosa phage 1 (PaP1) DNA polymerase Gp90 or Sulfolobus solfataricus DNA polymerase Dpo4. Both misincorporation frequencies of other DNA polymerases published were also summarized and analyzed. The general misincorporation frequency is obviously higher than the special misincorporation frequency for many DNA polymerases, indicating the real fidelity of a DNA polymerase should be evaluated using the general misincorporation frequency. Copyright © 2018 Elsevier Inc. All rights reserved.

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.

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

% vs. 6%; P persons with the mDNA 3243A>G mutation was found. This finding suggests a clinical association between a monogenetically inherited disorder......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...

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.

Chloroplast mutations induced by 9-aminoacridine hydrochloride are independent of the plastome mutator in Oenothera.

Science.gov (United States)

GuhaMajumdar, M; Baldwin, S; Sears, B B

2004-02-01

Oenothera plants homozygous for the recessive plastome mutator allele ( pm) show chloroplast DNA (cpDNA) mutation frequencies that are about 1,000-fold higher than spontaneous levels. The pm-encoded gene product has been hypothesized to have a function in cpDNA replication, repair and/or mutation avoidance. Previous chemical mutagenesis experiments with the alkylating agent nitroso-methyl urea (NMU) showed a synergistic effect of NMU on the induction of mutations in the pm line, suggesting an interaction between the pm-encoded gene product and one of the repair systems that corrects alkylation damage. The goal of the experiments described here was to examine whether the pm activity extends to the repair of damage caused by non-alkylating mutagens. To this end, the intercalating mutagen, 9-aminoacridine hydrochloride (9AA) was tested for synergism with the plastome mutator. A statistical analysis of the data reported here indicates that the pm-encoded gene product is not involved in the repair of the 9AA-induced mutations. However, the recovery of chlorotic sectors in plants derived from the mutagenized seeds shows that 9AA can act as a mutagen of the chloroplast genome.

Survey on the frequency of somatic mutations in A-bomb survivors

International Nuclear Information System (INIS)

Akiyama, Mitoshi

1992-01-01

Several methods have recently been established for quantitatively detecting somatic cell mutations on a specific locus using human blood cells. These methods have enabled the biological estimation of A-bomb radiation doses in surveys on somatic cell mutations. This paper outlines HPRT, GPA, and TCR assays used to measure somatic cell mutations, focusing on the outcome in A-bomb survivors. HPRT assay is based on colony formation with interleukin-2. The frequency of HPRT mutant cells was significantly increased with advancing age in A-bomb survivors and was positively correlated with the frequency of chromosomal aberrations in lymphocytes. There was also a significantly positive correlation between HPRT mutant cell frequencies and DS86 estimated doses, although the slope was slow. In GPA assay, flow cytometric measurements of fluorescence-labeled erythrocytes are used to detect somatic cell mutations. There was a positive correlation between GPA mutant cell frequencies and age in A-bomb survivors. The GPA mutant cell frequencies showed much more positive correlation with lymphocyte chromosomal aberration frequencies than the HPRT mutant cell frequencies. When anti-CD3 antibody and anti-CD4 antibody are labeled with different fluorescences and are analyzed by using flow cytometry, TCR mutant cells having CD3 - 4 + can be detected. When the frequency of TCR mutant cells was examined in 342 A-bomb survivors, it did not correlate with radiation doses. This implies that TCR assay may be unadequate for biological estimation of A-bomb radiation doses throughout a lifetime of A-bomb survivors, because TCR mutant cells seems to be unable to live for a long time due to national selection. (N.K.)

Translesion DNA synthesis and mutation induced in a plasmid with a single adduct of the environmental contaminant 3-nitrobenzanthrone in SOS-induced Escherichia coli

International Nuclear Information System (INIS)

Kawanishi, M.; Kanno, T.; Yagi, T.; Enya-Takamura, T.; Fuchs, R.P.

2003-01-01

Full text: 3-Nitrobenzanthrone (NBA) is a powerfully mutagenic nitrated aromatic hydrocarbon found in diesel exhaust and in airborne particulate matters. NBA forms an unusual DNA adduct in vitro that has a C-C bond between the C-8 position of deoxyguanosine and the C-2 position of NBA. We previously found that this adduct is also present in the human cells treated with NBA, and induces mutations in supF shuttle vector system. In this study, we analyzed translesion DNA synthesis (TLS) over a single adduct in lacZ' gene in a plasmid in uvrAmutS Escherichia coli. The result showed that the adduct blocked DNA replication and an observed TLS frequency was 5.4% in non-SOS-induced E. coli. All progenies after the TLS had no mutation. On the other hand, TLS increased to 11.3%, and 4.8% of them had mostly G to T mutations in SOS-induced E. coli. These results suggest that this unusual adduct would be one of causes of lung cancer that is increasing in the urban areas polluted with diesel exhaust. It must be interesting to reveal which DNA polymerase is involved in this TLS

POLE mutations in families predisposed to cutaneous melanoma

DEFF Research Database (Denmark)

Aoude, Lauren G; Heitzer, Ellen; Johansson, Peter

2015-01-01

Germline mutations in the exonuclease domain of POLE have been shown to predispose to colorectal cancers and adenomas. POLE is an enzyme involved in DNA repair and chromosomal DNA replication. In order to assess whether such mutations might also predispose to cutaneous melanoma, we interrogated...... variants in the exonuclease domain of POLE. Although this frequency is not significantly higher than that in unselected Caucasian controls, we observed multiple cancer types in the melanoma families, suggesting that some germline POLE mutations may predispose to a broad spectrum of cancers, including...

Founder Mutations in Xeroderma Pigmentosum

Science.gov (United States)

Tamura, Deborah; DiGiovanna, John J.; Kraemer, Kenneth H.

2012-01-01

In this issue, Soufir et al. report a founder mutation in the XPC DNA repair gene in 74% of families with xeroderma pigmentosum (XP) in the Maghreb region (Algeria, Morocco, and Tunisia) of northern Africa. These patients have a high frequency of skin cancer. The presence of this founder mutation provides an opportunity for genetic counseling and early diagnosis of XP. PMID:20463673

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.

Mitochondrial DNA depletion syndrome due to mutations in the RRM2B gene.

Science.gov (United States)

Bornstein, Belén; Area, Estela; Flanigan, Kevin M; Ganesh, Jaya; Jayakar, Parul; Swoboda, Kathryn J; Coku, Jorida; Naini, Ali; Shanske, Sara; Tanji, Kurenai; Hirano, Michio; DiMauro, Salvatore

2008-06-01

Mitochondrial DNA depletion syndrome (MDS) is characterized by a reduction in mtDNA copy number and has been associated with mutations in eight nuclear genes, including enzymes involved in mitochondrial nucleotide metabolism (POLG, TK2, DGUOK, SUCLA2, SUCLG1, PEO1) and MPV17. Recently, mutations in the RRM2B gene, encoding the p53-controlled ribonucleotide reductase subunit, have been described in seven infants from four families, who presented with various combinations of hypotonia, tubulopathy, seizures, respiratory distress, diarrhea, and lactic acidosis. All children died before 4 months of age. We sequenced the RRM2B gene in three unrelated cases with unexplained severe mtDNA depletion. The first patient developed intractable diarrhea, profound weakness, respiratory distress, and died at 3 months. The other two unrelated patients had a much milder phenotype and are still alive at ages 27 and 36 months. All three patients had lactic acidosis and severe depletion of mtDNA in muscle. Muscle histochemistry showed RRF and COX deficiency. Sequencing the RRM2B gene revealed three missense mutations and two single nucleotide deletions in exons 6, 8, and 9, confirming that RRM2B mutations are important causes of MDS and that the clinical phenotype is heterogeneous and not invariably fatal in infancy.

The spectrum of HNF1A gene mutations in Greek patients with MODY3: relative frequency and identification of seven novel germline mutations.

Science.gov (United States)

Tatsi, Christina; Kanaka-Gantenbein, Christina; Vazeou-Gerassimidi, Adriani; Chrysis, Dionysios; Delis, Dimitrios; Tentolouris, Nikolaos; Dacou-Voutetakis, Catherine; Chrousos, George P; Sertedaki, Amalia

2013-11-01

Maturity-Onset Diabetes of the Young (MODY) is the most common type of monogenic diabetes accounting for 1-2% of the population with diabetes. The relative incidence of HNF1A-MODY (MODY3) is high in European countries; however, data are not available for the Greek population. The aims of this study were to determine the relative frequency of MODY3 in Greece, the type of the mutations observed, and their relation to the phenotype of the patients. Three hundred ninety-five patients were referred to our center because of suspected MODY during a period of 15 yr. The use of Denaturing Gradient Gel Electrophoresis of polymerase chain reaction amplified DNA revealed 72 patients carrying Glucokinase gene mutations (MODY2) and 8 patients carrying HNF1A gene mutations (MODY3). After using strict criteria, 54 patients were selected to be further evaluated by direct sequencing or by multiplex ligation probe amplification (MLPA) for the presence of HNF1A gene mutations. In 16 unrelated patients and 13 of their relatives, 15 mutations were identified in the HNF1A gene. Eight of these mutations were previously reported, whereas seven were novel. Clinical features, such as age of diabetes at diagnosis or severity of hyperglycemia, were not related to the mutation type or location. In our cohort of patients fulfilling strict clinical criteria for MODY, 12% carried an HNF1A gene mutation, suggesting that defects of this gene are responsible for a significant proportion of monogenic diabetes in the Greek population. No clear phenotype-genotype correlations were identified. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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

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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.

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

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Christina L. Zheng

2014-11-01

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

Evaluation of the metabolic fate of munitions material (TNT & RDX) in plant systems. Initial assessment of plant DNA mutation spectra as a biomarker

Energy Technology Data Exchange (ETDEWEB)

Leung, F.; Cataldo, D.A.; Fellows, R.J.; Jarrell, A.E.; Harvey, S.D.

1995-09-01

Munitions material can enter the environment as a result of manufacturing activities and field usage. Predictor methodologies, or biomarkers would enhance evaluation of environmental impacts. The goal of this exploratory study deoxyribonucleic acid (DNA) mutation frequency as a biomarker for munitions exposure. The approach e resolution of an effective repetitive sequence probe for the identification of characteristic mutations, and (2) the development of a testing media [a clonal cell line of carrot (Daucus carota) spension cells]. Commercially available probes demonstrated marginal resolution therefore a low-C{sub o}t library was then constructed. Three colonies from the low-C{sub o}t DNA library were screened and the DNA isolates sequenced. A suspension culture of carrot (Daucus carota) was developed. A mutation spectra experiment was initiated at a 10-mg TNT/L exposure concentration with the attempt to clone over 1500 single TNT-exposed cells. Over the following six months greater than 98% of the initially isolated cells were unable to survive and produce micro calluses. The remaining calli were too few to be statistically significant and the experiment was terminated. The biomarker concept itself remains to be disproved, but the need for large numbers of uniform clones to differentiate true mutations suggest that more direct techniques using whole tissues need to be developed.

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

International Nuclear Information System (INIS)

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

1978-01-01

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

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 ...

MELAS syndrome, cardiomyopathy, rhabdomyolysis, and autism associated with the A3260G mitochondrial DNA mutation.

Science.gov (United States)

Connolly, Barbara S; Feigenbaum, Annette S J; Robinson, Brian H; Dipchand, Anne I; Simon, David K; Tarnopolsky, Mark A

2010-11-12

The A to G transition mutation at position 3260 of the mitochondrial genome is usually associated with cardiomyopathy and myopathy. One Japanese kindred reported the phenotype of mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS syndrome) in association with the A3260G mtDNA mutation. We describe the first Caucasian cases of MELAS syndrome associated with the A3260G mutation. Furthermore, this mutation was associated with exercise-induced rhabdomyolysis, hearing loss, seizures, cardiomyopathy, and autism in the large kindred. We conclude that the A3260G mtDNA mutation is associated with wide phenotypic heterogeneity with MELAS and other "classical" mitochondrial phenotypes being manifestations. Copyright © 2010 Elsevier Inc. All rights reserved.

Influence of inhibitors of poly(ADP-ribose) polymerase on DNA repair, chromosomal alterations, and mutations

Energy Technology Data Exchange (ETDEWEB)

Natarajan, A.T.; van Zeeland, A.A.; Zwanenburg, T.S.

1983-01-01

The influence of inhibitors of poly(ADP-ribose) polymerase such as 3-aminobenzamide (3AB) and benzamide (B) on the spontaneously occurring as well as mutagen induced chromosomal aberrations, sister chromatid exchanges (SCEs) and point mutations has been studied. In addition, the influence of 3AB on DNA repair was measured following treatment with physical and chemical mutagens. Post treatment of X-irradiated mammalian cells with 3AB increases the frequencies of induced chromosomal aberrations by a factor of 2 to 3. 3AB, when present in the medium containing bromodeoxyuridine(BrdUrd) during two cell cycles, increases the frequencies of SCEs in Chinese hamster ovary cells (CHO) in a concentration dependent manner leading to about a 10-fold increase at 10 mM concentration. The extent of increase in the frequencies of SCEs due to 1 mM 3AB in several human cell lines has been studied, including those derived from patients suffering from genetic diseases such as ataxia telangiectasia (A-T), Fanconi's anemia (FA), and Huntington's chorea. None of these syndromes showed any increased response when compared to normal cells. 3AB, however, increased the frequencies of spontaneously occurring chromosomal aberrations in A-T and FA cells. 3AB does not influence the frequencies of SCEs induced by UV or mitomycin C (MMC) in CHO cells. However, it increases the frequencies of SCEs induced by ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS). Under the conditions in which 3AB increases the frequencies of spontaneously occurring as well as induced SCEs, it does not increase the frequencies of point mutations in hypoxanthine-guanine phosphoribosyltransferase (HGPRT) locus. 3AB does not influence the amount of repair replication following dimethylsulphate (DMS) treatment of human fibroblasts, or UV irradiated human lymphocytes.

Mutation spectrum produced on PBR322 by 8-Methoxypsoralen plus UV-A light

International Nuclear Information System (INIS)

Bauluz, C.; Vidania, R.

1990-01-01

The mutagenic effect of 8-MOP+UVA (PUVA treatment) on pBR322 has been analysed by determining the frequency of mutation in the tet gene and identifying the type and position of the mutations produced inside a 276 pb-fragment (Bam-H1-SalI) of the same gene. pBR322 DNA was irradiated with UVA light in the presence of increasing concentrations of 8-Methoxypsoralen (8-MOP). The number of psoralen adducts formed in pBR322 upon that treatment ranged from 0 to 10.7 adducts per plasmid molecule. Modified DNA samples were used to transform several strains of E. Coli (differing in their repair capacities), both in constitutive conditions and after sos pre-induction by 254 nm-irradiation of cells. Mutation frequencies in the tet gene showed to increase in the wild type and uvrA strains along with the number of psoralen adducts per plasmid molecule; higher mutation frequencies were found in cells that had been previously irradiated to induce the SOS expression. Mutant plasmids were isolated from ApRTcS colonies and sequenced by the method of Maxam and Gilbert. Mutations appeared to be unique in most of the cases and were always punctual, i.e. affecting only to one base pair. The relative positions of the mutations showed a high frequency of coincidence among the sequenced fragments, indicating the existence of several DNA regions with high probability to mutated ('hot spots'). (author)

The occurrence and frequency of genomic mutations that mediate ...

African Journals Online (AJOL)

The occurrence and frequency of genomic mutations that mediate Isoniazid and Rifampicin resistance in Mycobacterium tuberculosis isolates from untreated pulmonary Tuberculosis cases in urban Blantyre, Malawi.

TRAIP promotes DNA damage response during genome replication and is mutated in primordial dwarfism.

Science.gov (United States)

Harley, Margaret E; Murina, Olga; Leitch, Andrea; Higgs, Martin R; Bicknell, Louise S; Yigit, Gökhan; Blackford, Andrew N; Zlatanou, Anastasia; Mackenzie, Karen J; Reddy, Kaalak; Halachev, Mihail; McGlasson, Sarah; Reijns, Martin A M; Fluteau, Adeline; Martin, Carol-Anne; Sabbioneda, Simone; Elcioglu, Nursel H; Altmüller, Janine; Thiele, Holger; Greenhalgh, Lynn; Chessa, Luciana; Maghnie, Mohamad; Salim, Mahmoud; Bober, Michael B; Nürnberg, Peter; Jackson, Stephen P; Hurles, Matthew E; Wollnik, Bernd; Stewart, Grant S; Jackson, Andrew P

2016-01-01

DNA lesions encountered by replicative polymerases threaten genome stability and cell cycle progression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism. We establish that TRAIP relocalizes to sites of DNA damage, where it is required for optimal phosphorylation of H2AX and RPA2 during S-phase in response to ultraviolet (UV) irradiation, as well as fork progression through UV-induced DNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes. Human genetics thus identifies TRAIP as a component of the DNA damage response to replication-blocking DNA lesions.

Abiotic stress leads to somatic and heritable changes in homologous recombination frequency, point mutation frequency and microsatellite stability in Arabidopsis plants

International Nuclear Information System (INIS)

Yao Youli; Kovalchuk, Igor

2011-01-01

In earlier studies, we showed that abiotic stresses, such as ionizing radiation, heavy metals, temperature and water, trigger an increase in homologous recombination frequency (HRF). We also demonstrated that many of these stresses led to inheritance of high-frequency homologous recombination, HRF. Although an increase in recombination frequency is an important indicator of genome rearrangements, it only represents a minor portion of possible stress-induced mutations. Here, we analyzed the influence of heat, cold, drought, flood and UVC abiotic stresses on two major types of mutations in the genome, point mutations and small deletions/insertions. We used two transgenic lines of Arabidopsis thaliana, one allowing an analysis of reversions in a stop codon-containing inactivated β-glucuronidase transgene and another one allowing an analysis of repeat stability in a microsatellite-interrupted β-glucuronidase transgene. The transgenic Arabidopsis line carrying the β-glucuronidase-based homologous recombination substrate was used as a positive control. We showed that the majority of stresses increased the frequency of point mutations, homologous recombination and microsatellite instability in somatic cells, with the frequency of homologous recombination being affected the most. The analysis of transgenerational changes showed an increase in HRF to be the most prominent effect observed in progeny. Significant changes in recombination frequency were observed upon exposure to all types of stress except drought, whereas changes in microsatellite instability were observed upon exposure to UVC, heat and cold. The frequency of point mutations in the progeny of stress-exposed plants was the least affected; an increase in mutation frequency was observed only in the progeny of plants exposed to UVC. We thus conclude that transgenerational changes in genome stability in response to stress primarily involve an increase in recombination frequency.

A Comparative Study for Detection of EGFR Mutations in Plasma Cell-Free DNA in Korean Clinical Diagnostic Laboratories

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Yoonjung Kim

2018-01-01

Full Text Available Liquid biopsies to genotype the epidermal growth factor receptor (EGFR for targeted therapy have been implemented in clinical decision-making in the field of lung cancer, but harmonization of detection methods is still scarce among clinical laboratories. We performed a pilot external quality assurance (EQA scheme to harmonize circulating tumor DNA testing among laboratories. For EQA, we created materials containing different levels of spiked cell-free DNA (cfDNA in normal plasma. The limit of detection (LOD of the cobas® EGFR Mutation Test v2 (Roche Molecular Systems was also evaluated. From November 2016 to June 2017, seven clinical diagnostic laboratories participated in the EQA program. The majority (98.94% of results obtained using the cobas assay and next-generation sequencing (NGS were acceptable. Quantitative results from the cobas assay were positively correlated with allele frequencies derived from digital droplet PCR measurements and showed good reproducibility among laboratories. The LOD of the cobas assay was 5~27 copies/mL for p.E746_A750del (exon 19 deletion, 35~70 copies/mL for p.L858R, 18~36 copies/mL for p.T790M, and 15~31 copies/mL for p.A767_V769dup (exon 20 insertion. Deep sequencing of materials (>100,000X depth of coverage resulted in detection of low-level targets present at frequencies of 0.06~0.13%. Our results indicate that the cobas assay is a reliable and rapid method for detecting EGFR mutations in plasma cfDNA. Careful interpretation is particularly important for p.T790M detection in the setting of relapse. Individual laboratories should optimize NGS performance to maximize clinical utility.

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.

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...

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

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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.

Effects of TET2 mutations on DNA methylation in chronic myelomonocytic leukemia

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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...

Inherited and environmentally induced differences in mutation frequencies between wild strains of Sordaria fimicola from "Evolution Canyon".

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Lamb, B C; Saleem, M; Scott, W; Thapa, N; Nevo, E

1998-05-01

We have studied whether there is natural genetic variation for mutation frequencies, and whether any such variation is environment-related. Mutation frequencies differed significantly between wild strains of the fungus Sordaria fimicola isolated from a harsher or a milder microscale environment in "Evolution Canyon," Israel. Strains from the harsher, drier, south-facing slope had higher frequencies of new spontaneous mutations and of accumulated mutations than strains from the milder, lusher, north-facing slope. Collective total mutation frequencies over many loci for ascospore pigmentation were 2.3, 3.5 and 4.4% for three strains from the south-facing slope, and 0.9, 1.1, 1.2, 1.3 and 1.3% for five strains from the north-facing slope. Some of this between-slope difference was inherited through two generations of selfing, with average spontaneous mutation frequencies of 1.9% for south-facing slope strains and 0.8% for north-facing slope strains. The remainder was caused by different frequencies of mutations arising in the original environments. There was also significant heritable genetic variation in mutation frequencies within slopes. Similar between-slope differences were found for ascospore germination-resistance to acriflavine, with much higher frequencies in strains from the south-facing slope. Such inherited variation provides a basis for natural selection for optimum mutation rates in each environment.

ASSOCIATION OF HFE GENE MUTATION IN THALASSEMIA MAJOR PATIENTS

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Amit Kumar Tiwari

2016-11-01

Full Text Available BACKGROUND Thalassemia major patients are dependent on frequent blood transfusion and consequently develop iron overload. HFE gene mutations (C282Y, H63D and S65C in hereditary haemochromatosis has been shown to be associated with iron overload. The study aims at finding the association of HFE gene mutations in β-thalassemia major patients. MATERIALS AND METHODS A descriptive observational pilot study was conducted including fifty diagnosed -thalassemia major cases. DNA analysis by PCR-RFLP method for HFE gene mutations was performed. RESULTS Only H63D mutation (out of three HFE gene mutations was detected in 8 out of 50 cases. Observed frequency of H63D mutation was 16%. While frequency of C282Y and S65C were 0% each. CONCLUSION The frequency of HFE mutation in -thalassemia major is not very common.

Rapid detection of ERG11 gene mutations in clinical Candida albicans isolates with reduced susceptibility to fluconazole by rolling circle amplification and DNA sequencing

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Ellis David

2009-08-01

Full Text Available Abstract Background Amino acid substitutions in the target enzyme Erg11p of azole antifungals contribute to clinically-relevant azole resistance in Candida albicans. A simple molecular method for rapid detection of ERG11 gene mutations would be an advantage as a screening tool to identify potentially-resistant strains and to track their movement. To complement DNA sequencing, we developed a padlock probe and rolling circle amplification (RCA-based method to detect a series of mutations in the C. albicans ERG11 gene using "reference" azole-resistant isolates with known mutations. The method was then used to estimate the frequency of ERG11 mutations and their type in 25 Australian clinical C. albicans isolates with reduced susceptibility to fluconazole and in 23 fluconazole-susceptible isolates. RCA results were compared DNA sequencing. Results The RCA assay correctly identified all ERG11 mutations in eight "reference" C. albicans isolates. When applied to 48 test strains, the RCA method showed 100% agreement with DNA sequencing where an ERG11 mutation-specific probe was used. Of 20 different missense mutations detected by sequencing in 24 of 25 (96% isolates with reduced fluconazole susceptibility, 16 were detected by RCA. Five missense mutations were detected by both methods in 18 of 23 (78% fluconazole-susceptible strains. DNA sequencing revealed that mutations in non-susceptible isolates were all due to homozygous nucleotide changes. With the exception of the mutations leading to amino acid substitution E266D, those in fluconazole-susceptible strains were heterozygous. Amino acid substitutions common to both sets of isolates were D116E, E266D, K128T, V437I and V488I. Substitutions unique to isolates with reduced fluconazole susceptibility were G464 S (n = 4 isolates, G448E (n = 3, G307S (n = 3, K143R (n = 3 and Y123H, S405F and R467K (each n = 1. DNA sequencing revealed a novel substitution, G450V, in one isolate. Conclusion The sensitive RCA

Thermodynamic framework to assess low abundance DNA mutation detection by hybridization

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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

Genome-scale analysis of aberrant DNA methylation in colorectal cancer

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Hinoue, Toshinori; Weisenberger, Daniel J.; Lange, Christopher P.E.; Shen, Hui; Byun, Hyang-Min; Van Den Berg, David; Malik, Simeen; Pan, Fei; Noushmehr, Houtan; van Dijk, Cornelis M.; Tollenaar, Rob A.E.M.; Laird, Peter W.

2012-01-01

Colorectal cancer (CRC) is a heterogeneous disease in which unique subtypes are characterized by distinct genetic and epigenetic alterations. Here we performed comprehensive genome-scale DNA methylation profiling of 125 colorectal tumors and 29 adjacent normal tissues. We identified four DNA methylation–based subgroups of CRC using model-based cluster analyses. Each subtype shows characteristic genetic and clinical features, indicating that they represent biologically distinct subgroups. A CIMP-high (CIMP-H) subgroup, which exhibits an exceptionally high frequency of cancer-specific DNA hypermethylation, is strongly associated with MLH1 DNA hypermethylation and the BRAFV600E mutation. A CIMP-low (CIMP-L) subgroup is enriched for KRAS mutations and characterized by DNA hypermethylation of a subset of CIMP-H-associated markers rather than a unique group of CpG islands. Non-CIMP tumors are separated into two distinct clusters. One non-CIMP subgroup is distinguished by a significantly higher frequency of TP53 mutations and frequent occurrence in the distal colon, while the tumors that belong to the fourth group exhibit a low frequency of both cancer-specific DNA hypermethylation and gene mutations and are significantly enriched for rectal tumors. Furthermore, we identified 112 genes that were down-regulated more than twofold in CIMP-H tumors together with promoter DNA hypermethylation. These represent ∼7% of genes that acquired promoter DNA methylation in CIMP-H tumors. Intriguingly, 48/112 genes were also transcriptionally down-regulated in non-CIMP subgroups, but this was not attributable to promoter DNA hypermethylation. Together, we identified four distinct DNA methylation subgroups of CRC and provided novel insight regarding the role of CIMP-specific DNA hypermethylation in gene silencing. PMID:21659424

Frequency and Prognostic Relevance of FLT3 Mutations in Saudi Acute Myeloid Leukemia Patients

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Ghaleb Elyamany

2014-01-01

Full Text Available The Fms-like tyrosine kinase-3 (FLT3 is a receptor tyrosine kinase that plays a key role in cell survival, proliferation, and differentiation of hematopoietic stem cells. Mutations of FLT3 were first described in 1997 and account for the most frequent molecular mutations in acute myeloid leukemia (AML. AML patients with FLT3 internal tandem duplication (ITD mutations have poor cure rates the prognostic significance of point mutations; tyrosine kinase domain (TKD is still unclear. We analyzed the frequency of FLT3 mutations (ITD and D835 in patients with AML at diagnosis; no sufficient data currently exist regarding FLT3 mutations in Saudi AML patients. This study was aimed at evaluating the frequency of FLT3 mutations in patients with AML and its significance for prognosis. The frequency of FLT3 mutations in our study (18.56% was lower than many of the reported studies, FLT3-ITD mutations were observed in 14.4%, and FLT3-TKD in 4.1%, of 97 newly diagnosed AML patients (82 adult and 15 pediatric. Our data show significant increase of FLT3 mutations in male more than female (13 male, 5 female. Our results support the view that FLT3-ITD mutation has strong prognostic factor in AML patients and is associated with high rate of relapse, and high leucocytes and blast count at diagnosis and relapse.

HPRT gene mutation frequency and the factor of influence in adult peripheral blood lymphocytes

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Zhao Jingyong; Zheng Siying; Cui Fengmei; Wang Liuyi; Lao Qinhua; Wu Hongliang

2002-01-01

Objective: To study the HPRT gene loci mutation frequencies and the factor of influence in peripheral blood lymphocytes of adult with ages ranging from 21-50. Methods: HPRT gene mutation frequency (GMf) were examined by the technique of multinuclear cell assay. Relation between GMf and years were fitted with a computer. Results: Relation could be described by the following equation: y = 0.7555 + 0.0440x, r = 0.9829. Smoking has influence on GMf and sex hasn't. Conclusion: HPRT gene mutation frequency increases with increasing of age. Increasing rate is 0.00440% per year

Mitochondrial mutations in subjects with psychiatric disorders.

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Adolfo Sequeira

Full Text Available A considerable body of evidence supports the role of mitochondrial dysfunction in psychiatric disorders and mitochondrial DNA (mtDNA mutations are known to alter brain energy metabolism, neurotransmission, and cause neurodegenerative disorders. Genetic studies focusing on common nuclear genome variants associated with these disorders have produced genome wide significant results but those studies have not directly studied mtDNA variants. The purpose of this study is to investigate, using next generation sequencing, the involvement of mtDNA variation in bipolar disorder, schizophrenia, major depressive disorder, and methamphetamine use. MtDNA extracted from multiple brain regions and blood were sequenced (121 mtDNA samples with an average of 8,800x coverage and compared to an electronic database containing 26,850 mtDNA genomes. We confirmed novel and rare variants, and confirmed next generation sequencing error hotspots by traditional sequencing and genotyping methods. We observed a significant increase of non-synonymous mutations found in individuals with schizophrenia. Novel and rare non-synonymous mutations were found in psychiatric cases in mtDNA genes: ND6, ATP6, CYTB, and ND2. We also observed mtDNA heteroplasmy in brain at a locus previously associated with schizophrenia (T16519C. Large differences in heteroplasmy levels across brain regions within subjects suggest that somatic mutations accumulate differentially in brain regions. Finally, multiplasmy, a heteroplasmic measure of repeat length, was observed in brain from selective cases at a higher frequency than controls. These results offer support for increased rates of mtDNA substitutions in schizophrenia shown in our prior results. The variable levels of heteroplasmic/multiplasmic somatic mutations that occur in brain may be indicators of genetic instability in mtDNA.

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

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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. PMID:26308346

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

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Puya G Yazdi

Full Text Available 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.

Maternal inheritance and mitochondrial DNA variants in familial Parkinson's disease

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Pfeiffer Ronald F

2010-04-01

Full Text Available Abstract Background Mitochondrial function is impaired in Parkinson's disease (PD and may contribute to the pathogenesis of PD, but the causes of mitochondrial impairment in PD are unknown. Mitochondrial dysfunction is recapitulated in cell lines expressing mitochondrial DNA (mtDNA from PD patients, implicating mtDNA variants or mutations, though the role of mtDNA variants or mutations in PD risk remains unclear. We investigated the potential contribution of mtDNA variants or mutations to the risk of PD. Methods We examined the possibility of a maternal inheritance bias as well as the association between mitochondrial haplogroups and maternal inheritance and disease risk in a case-control study of 168 multiplex PD families in which the proband and one parent were diagnosed with PD. 2-tailed Fisher Exact Tests and McNemar's tests were used to compare allele frequencies, and a t-test to compare ages of onset. Results The frequency of affected mothers of the proband with PD (83/167, 49.4% was not significantly different from the frequency of affected females of the proband generation (115/259, 44.4% (Odds Ratio 1.22; 95%CI 0.83 - 1.81. After correcting for multiple tests, there were no significant differences in the frequencies of mitochondrial haplogroups or of the 10398G complex I gene polymorphism in PD patients compared to controls, and no significant associations with age of onset of PD. Mitochondrial haplogroup and 10398G polymorphism frequencies were similar in probands having an affected father as compared to probands having an affected mother. Conclusions These data fail to demonstrate a bias towards maternal inheritance in familial PD. Consistent with this, we find no association of common haplogroup-defining mtDNA variants or for the 10398G variant with the risk of PD. However, these data do not exclude a role for mtDNA variants in other populations, and it remains possible that other inherited mitochondrial DNA variants, or somatic mDNA

The spectrum of mutation produced by low dose radiation

International Nuclear Information System (INIS)

Morley, Alexander A.; Turner, David R.

2004-01-01

Inherited mutations are the basis of evolution and acquired mutations in humans are important in ageing, cancer and possibly various forms of tissue degeneration. Mutations are responsible for many of the long-term effects of radiation. However, sensitive direct detection of mutations in humans has been difficult. The aims of the project were to develop methods for the sensitive enumeration of mutations in DNA, to measure mutation frequencies in a wide variety of tissue types and to quantify the mutational effect of direct oxidative damage produced by radiation, at both high and low doses. The project was successful in developing a sensitive method which could detect mutations directly in the genetic material, DNA at a sensitivity of 1 mutated molecule in 1000000000 unmutated molecules. However a number of methodological problems had to be overcome and lack of ongoing funding made it impossible to fulfill all of the aims of the project

Genetic mutation analysis of HBV covalently closed circular DNA in peripheral blood mononuclear cells from chronic hepatitis B patients with nucleos(tide analog-resistant mutations in serum virions

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Zhong-bin LI

2012-06-01

Full Text Available Objective 　To analyze the characteristics of genetic mutations in reverse-transcriptase (RT domain of HBV covalently closed circular DNA (cccDNA in peripheral blood mononuclear cells (PBMCs obtained from chronic hepatitis B (CHB patients with drug-resistant mutations in serum virions during nucleoside/nucleotide analog (NA therapy. Methods 　A total of 30 CHB patients admitted to 302 Hospital of PLA from July 2010 to August 2011 were included in this study. All the patients were confirmed to harbor the drug-resistant mutations in serum virions during an NA therapy longer than 6 months. Total DNA was extracted from PBMCs isolated from 30 whole blood samples at the same time point as that of serum analysis. Plasmid-safe ATP-dependent DNase (PSAD digestion in combination with rolling circle amplification and gap-spanning semi-nested PCR were used to amplify the RT region of HBV cccDNA. NA-resistant-associated mutations were analyzed at nine sites. Results 　HBV cccDNA was efficiently amplified in 16 out of 30 (53.3% PBMC samples, and the detection rate was not correlated with HBeAg-positive rate, serum ALT level or HBV DNA load. Five of 16 (31.3% patients were sustained to have genotype B HBV infection, and 11 of 16 (68.8% were of genotype C HBV infection, and the result was consistent with the genotyping results using serum HBV. Different from drug-resistant mutations detected in the serum virions, the viruses detected in HBV cccDNA of 16 PBMC samples were all wild-type viruses without NA-resistant-associated mutations in RT region. Conclusions 　During NA antiviral treatment, if drug-resistant mutations occur in serum HBV DNA of CHB patients, the dominant species of HBV cccDNA in PBMCs from the same patient is still the original wild-type strains. It is speculated that PBMCs might be the potential "repository" of HBV wild-type strain in vivo.

Endurance exercise rescues progeroid aging and induces systemic mitochondrial rejuvenation in mtDNA mutator mice

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Safdar, Adeel; Bourgeois, Jacqueline M.; Ogborn, Daniel I.; Little, Jonathan P.; Hettinga, Bart P.; Akhtar, Mahmood; Thompson, James E.; Melov, Simon; Mocellin, Nicholas J.; Kujoth, Gregory C.; Prolla, Tomas A.; Tarnopolsky, Mark A.

2011-01-01

A causal role for mitochondrial DNA (mtDNA) mutagenesis in mammalian aging is supported by recent studies demonstrating that the mtDNA mutator mouse, harboring a defect in the proofreading-exonuclease activity of mitochondrial polymerase gamma, exhibits accelerated aging phenotypes characteristic of human aging, systemic mitochondrial dysfunction, multisystem pathology, and reduced lifespan. Epidemiologic studies in humans have demonstrated that endurance training reduces the risk of chronic diseases and extends life expectancy. Whether endurance exercise can attenuate the cumulative systemic decline observed in aging remains elusive. Here we show that 5 mo of endurance exercise induced systemic mitochondrial biogenesis, prevented mtDNA depletion and mutations, increased mitochondrial oxidative capacity and respiratory chain assembly, restored mitochondrial morphology, and blunted pathological levels of apoptosis in multiple tissues of mtDNA mutator mice. These adaptations conferred complete phenotypic protection, reduced multisystem pathology, and prevented premature mortality in these mice. The systemic mitochondrial rejuvenation through endurance exercise promises to be an effective therapeutic approach to mitigating mitochondrial dysfunction in aging and related comorbidities. PMID:21368114

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

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

2016-03-01

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

Mutation induction by ion beams in arabidopsis

International Nuclear Information System (INIS)

Tanaka, Atsushi

1999-01-01

An investigation was made on characteristics of ion beams for the biological effects and the induction of mutation using Arabidopsis plant as a model plant for the molecular genetics. Here, the characteristics of mutation at the molecular level as well as new mutants induced by ion beams were described. The ast and sep1 were obtained from the offspring of 1488 carbon ion-irradiated seeds respectively. The uvi1-uvi4 mutants were also induced from 1280 M 1 lines. Thus, ion beams can induce not only known mutants such as tt, gl and hy but also novel mutants with high frequency. Even in the tt phenotype, two new mutant loci other than known loci were found. In chrysanthemum, several kinds of single, complex or stripped flower-color mutants that have been never induced by γirradiation, indicating that ion beams could produce a variety of mutants with the same phenotype. In conclusion, ion beams for the mutation induction are characterized by 1) to induce mutants with high frequency, 2) to show broad mutation spectrum and 3) to produce novel mutants. For these reasons, chemical mutagens such as EMS and low LET ionizing radiation such as X-rays and γ-rays will predominantly induce many but small modifications or DNA damages on the DNA strands. As the result, several point mutations will be produced on the genome. On the contrary, ion beams as a high LET ionizing radiation will not cause so many but large and irreparable DNA damage locally, resulting in production of limited number of null mutation. (M.N.)

Mutation induction by ion beams in arabidopsis

Energy Technology Data Exchange (ETDEWEB)

Tanaka, Atsushi [Japan Atomic Energy Research Inst., Takasaki, Gunma (Japan). Takasaki Radiation Chemistry Research Establishment

1999-07-01

An investigation was made on characteristics of ion beams for the biological effects and the induction of mutation using Arabidopsis plant as a model plant for the molecular genetics. Here, the characteristics of mutation at the molecular level as well as new mutants induced by ion beams were described. The ast and sep1 were obtained from the offspring of 1488 carbon ion-irradiated seeds respectively. The uvi1-uvi4 mutants were also induced from 1280 M{sub 1} lines. Thus, ion beams can induce not only known mutants such as tt, gl and hy but also novel mutants with high frequency. Even in the tt phenotype, two new mutant loci other than known loci were found. In chrysanthemum, several kinds of single, complex or stripped flower-color mutants that have been never induced by {gamma}irradiation, indicating that ion beams could produce a variety of mutants with the same phenotype. In conclusion, ion beams for the mutation induction are characterized by 1) to induce mutants with high frequency, 2) to show broad mutation spectrum and 3) to produce novel mutants. For these reasons, chemical mutagens such as EMS and low LET ionizing radiation such as X-rays and {gamma}-rays will predominantly induce many but small modifications or DNA damages on the DNA strands. As the result, several point mutations will be produced on the genome. On the contrary, ion beams as a high LET ionizing radiation will not cause so many but large and irreparable DNA damage locally, resulting in production of limited number of null mutation. (M.N.)

Analysis of common deafness gene mutations in deaf people from unique ethnic groups in Gansu Province, China.

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Xu, Bai-Cheng; Bian, Pan-Pan; Liu, Xiao-Wen; Zhu, Yi-Ming; Yang, Xiao-Long; Ma, Jian-Li; Chen, Xing-Jian; Wang, Yan-Li; Guo, Yu-Fen

2014-09-01

The GJB2 gene mutation characteristic of Dongxiang was the interaction result of ethnic background and geographical environment, and Yugur exhibited the typical founder effect. The SLC26A4 gene mutation characteristic of Dongxiang was related to caucasian backgrounds and selection of purpose exons, i.e. ethnic background and the penetrance of ethnic specificity caused the low mtDNA1555A>G mutation frequency in Dongxiang. To determine the prevalence of GJB2 and SLC26A4 genes and mtDNA1555A>G mutations and analyze the ethnic specificity in the non-syndromic sensorineural hearing loss (NSHL) of unique ethnic groups in Gansu Province. Peripheral blood samples were obtained from Dongxiang, Yugur, Bonan, and ethnic Han groups with moderately severe to profound NSHL in Gansu Province. Bidirectional sequencing (or enzyme digestion) was applied to identify the sequence variations. The pathogenic allele frequency of the three gene mutations was different. The frequency of the GJB2 gene among the Dongxiang, Yugur, Bonan, and ethnic Han groups was 9.03%, 12.5%, 5.88%, and 12.17%, respectively. No difference was found between the ethnic groups. The frequencies of the SLC26A4 genes were 3.23%, 8.33%, 0%, and 9.81%, respectively. The mutation frequency of mtDNA1555A>G was 0%, 0%, 0%, and 6.03%, respectively. No difference was found between the ethnic groups, except for the Dongxiang and ethnic Han groups, both in SLC26A4 gene and mtDNA1555A>G.

The SRS2 suppressor of rad6 mutations of Saccharomyces cerevisiae acts by channeling DNA lesions into the RAD52 DNA repair pathway

International Nuclear Information System (INIS)

Schiestl, R.H.; Prakash, S.; Prakash, L.

1990-01-01

rad6 mutants of Saccharomyces cerevisiae are defective in the repair of damaged DNA, DNA damage induced mutagenesis, and sporulation. In order to identify genes that can substitute for RAD6 function, the authors have isolated genomic suppressors of the UV sensitivity of rad6 deletion (rad6Δ) mutations and show that they also suppress the γ-ray sensitivity but not the UV mutagenesis or sporulation defects of rad6. The suppressors show semidominance for suppression of UV sensitivity and dominance for suppression of γ-ray sensitivity. The six suppressor mutations they isolated are all alleles of the same locus and are also allelic to a previously described suppressor of the rad6-1 nonsense mutation, SRS2. They show that suppression of rad6Δ is dependent on the RAD52 recombinational repair pathway since suppression is not observed in the rad6Δ SRS2 strain containing an additional mutation in either the RAD51, RAD52, RAD54, RAD55 or RAD57 genes. Possible mechanisms by which SRS2 may channel unrepaired DNA lesions into the RAD52 DNA repair pathway are discussed

Mutational jackpot events generate effective frequency-dependent selection in adapting populations

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Hallatschek, Oskar

The site-frequency spectrum is one the most easily measurable quantities that characterize the genetic diversity of a population. While most neutral models predict that site frequency spectra should decay with increasing frequency, a high-frequency uptick has been reported in many populations. Anomalies in the high-frequency tail are particularly unsettling because the highest frequencies can be measured with greatest accuracy. Here, we show that an uptick in the spectrum of neutral mutations generally arises when mutant frequencies are dominated by rare jackpot events, mutational events with large descendant numbers. This leads to an effective pattern of frequency-dependent selection (or unstable internal equilibrium at one half frequency) that causes an accumulation of high-frequency polymorphic sites. We reproduce the known uptick occurring for recurrent hitchhiking (genetic draft) as well as rapid adaptation, and (in the future) generalize the shape of the high-frequency tail to other scenarios that are dominated by jackpot events, such as frequent range expansions. We also tackle (in the future) the inverse approach to use the high-frequency uptick for learning about the tail of the offspring number distribution. Positively selected alleles need to surpass, typically, an u NSF Career Award (PoLS), NIH NIGMS R01, Simons Foundation.

Is Increased Low-dose somatic Radiosensitivity Associated with Increased Transgenerational Germline Mutation

International Nuclear Information System (INIS)

Brenner, David J.

2008-01-01

Using single-molecule polymerase chain reaction, the frequency of spontaneous and radiation-induced mutation at an expanded simple tandem repeat (ESTR) locus was studied in DNA samples extracted from sperm and bone marrow of Atm knockout (Atm+/-) heterozygous male mice. The frequency of spontaneous mutation in sperm and bone marrow in Atm+/- males did not significantly differ from that in wild-type BALB/c mice. Acute gamma-ray exposure did not affect ESTR mutation frequency in bone marrow and resulted in similar increases in sperm samples taken from Atm+/- and BALB/c males. Taken together, these results suggest that the Atm haploinsufficiency analyzed in our study does not affect spontaneous and radiation-induced ESTR mutation frequency in mice

The Number of Overlapping AID Hotspots in Germline IGHV Genes Is Inversely Correlated with Mutation Frequency in Chronic Lymphocytic Leukemia.

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Yuan, Chaohui; Chu, Charles C; Yan, Xiao-Jie; Bagnara, Davide; Chiorazzi, Nicholas; MacCarthy, Thomas

2017-01-01

The targeting of mutations by Activation-Induced Deaminase (AID) is a key step in generating antibody diversity at the Immunoglobulin (Ig) loci but is also implicated in B-cell malignancies such as chronic lymphocytic leukemia (CLL). AID has previously been shown to preferentially deaminate WRC (W = A/T, R = A/G) hotspots. WGCW sites, which contain an overlapping WRC hotspot on both DNA strands, mutate at much higher frequency than single hotspots. Human Ig heavy chain (IGHV) genes differ in terms of WGCW numbers, ranging from 4 for IGHV3-48*03 to as many as 12 in IGHV1-69*01. An absence of V-region mutations in CLL patients ("IGHV unmutated", or U-CLL) is associated with a poorer prognosis compared to "IGHV mutated" (M-CLL) patients. The reasons for this difference are still unclear, but it has been noted that particular IGHV genes associate with U-CLL vs M-CLL. For example, patients with IGHV1-69 clones tend to be U-CLL with a poor prognosis, whereas patients with IGHV3-30 tend to be M-CLL and have a better prognosis. Another distinctive feature of CLL is that ~30% of (mostly poor prognosis) patients can be classified into "stereotyped" subsets, each defined by HCDR3 similarity, suggesting selection, possibly for a self-antigen. We analyzed >1000 IGHV genes from CLL patients and found a highly significant statistical relationship between the number of WGCW hotspots in the germline V-region and the observed mutation frequency in patients. However, paradoxically, this correlation was inverse, with V-regions with more WGCW hotspots being less likely to be mutated, i.e., more likely to be U-CLL. The number of WGCW hotspots in particular, are more strongly correlated with mutation frequency than either non-overlapping (WRC) hotspots or more general models of mutability derived from somatic hypermutation data. Furthermore, this correlation is not observed in sequences from the B cell repertoires of normal individuals and those with autoimmune diseases.

Electrostatic study of Alanine mutational effects on transcription: application to GATA-3:DNA interaction complex.

Science.gov (United States)

El-Assaad, Atlal; Dawy, Zaher; Nemer, Georges

2015-01-01

Protein-DNA interaction is of fundamental importance in molecular biology, playing roles in functions as diverse as DNA transcription, DNA structure formation, and DNA repair. Protein-DNA association is also important in medicine; understanding Protein-DNA binding kinetics can assist in identifying disease root causes which can contribute to drug development. In this perspective, this work focuses on the transcription process by the GATA Transcription Factor (TF). GATA TF binds to DNA promoter region represented by `G,A,T,A' nucleotides sequence, and initiates transcription of target genes. When proper regulation fails due to some mutations on the GATA TF protein sequence or on the DNA promoter sequence (weak promoter), deregulation of the target genes might lead to various disorders. In this study, we aim to understand the electrostatic mechanism behind GATA TF and DNA promoter interactions, in order to predict Protein-DNA binding in the presence of mutations, while elaborating on non-covalent binding kinetics. To generate a family of mutants for the GATA:DNA complex, we replaced every charged amino acid, one at a time, with a neutral amino acid like Alanine (Ala). We then applied Poisson-Boltzmann electrostatic calculations feeding into free energy calculations, for each mutation. These calculations delineate the contribution to binding from each Ala-replaced amino acid in the GATA:DNA interaction. After analyzing the obtained data in view of a two-step model, we are able to identify potential key amino acids in binding. Finally, we applied the model to GATA-3:DNA (crystal structure with PDB-ID: 3DFV) binding complex and validated it against experimental results from the literature.

Mutation of Mitochondrial DNA G13513A Presenting with Leigh Syndrome, Wolff-Parkinson-White Syndrome and Cardiomyopathy

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Shi-Bing Wang

2008-08-01

Full Text Available Mutation of mitochondrial DNA (mtDNA G13513A, encoding the ND5 subunit of respiratory chain complex I, can cause mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS and Leigh syndrome. Wolff-Parkinson-White (WPW syndrome and optic atrophy were reported in a high proportion of patients with this mutation. We report an 18-month-old girl, with an 11-month history of psychomotor regression who was diagnosed with WPW syndrome and hypertrophic cardiomyopathy, in association with Leigh syndrome. Supplementation with coenzyme Q10, thiamine and carnitine prevented further regression in gross motor function but the patient's heart function deteriorated and dilated cardiomyopathy developed 11 months later. She was found to have a mutation of mtDNA G13513A. We suggest that mtDNA G13513A mutation is an important factor in patients with Leigh syndrome associated with WPW syndrome and/or optic atrophy, and serial heart function monitoring by echocardiography is recommended in this group of patients.

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.

Impact of Somatic Mutations in the D-Loop of Mitochondrial DNA on the Survival of Oral Squamous Cell Carcinoma Patients

Science.gov (United States)

Lin, Jin-Ching; Wang, Chen-Chi; Jiang, Rong-San; Wang, Wen-Yi; Liu, Shih-An

2015-01-01

Objectives The aim of this study was to investigate somatic mutations in the D-loop of mitochondrial DNA (mtDNA) and their impact on survival in oral squamous cell carcinoma patients. Materials and Methods Surgical specimen confirmed by pathological examination and corresponding non-cancerous tissues were collected from 120 oral squamous cell carcinoma patients. The sequence in the D-loop of mtDNA from non-cancerous tissues was compared with that from paired cancer samples and any sequence differences were recognized as somatic mutations. Results Somatic mutations in the D-loop of mtDNA were identified in 75 (62.5%) oral squamous cell carcinoma patients and most of them occurred in the poly-C tract. Although there were no significant differences in demographic and tumor-related features between participants with and without somatic mutation, the mutation group had a better survival rate (5 year disease-specific survival rate: 64.0% vs. 43.0%, P = 0.0266). Conclusion Somatic mutation in D-loop of mtDNA was associated with a better survival in oral squamous cell carcinoma patients. PMID:25906372

Mutagenic DNA repair in Escherichia coli: Pt. 17

International Nuclear Information System (INIS)

Sharif, F.; Bridges, B.A.

1990-01-01

In contrast to the dnaE485 mutation, which is nearly 'dead-stop', dnaE1026 allows DNA synthesis for some time at restrictive temperatures. When bacteria carrying the dnaE486 or dnaE1026 temperature-sensitive mutations were incubated at restrictive temperature after exposure to UV light they showed little or no fixation of mutations as determined by loss of photoreversibility and the mutation frequency fell progressively. These results confirm a role for DnaE protein in UV mutagenesis. A derivative of dnaE1026 carrying the umuC122 allele which blocks normal UV mutagenesis showed induction of mutations when photoreversing light was given to UV-irradiated bacteria after a period of incubation at either permissive or restrictive temperature. The defective DnaE1026 protein is therefore able to carry out the misincorporations postulated to be the first step in the mutagenic process. Its inability to give rise to mutations in umu + bacteria may therefore be attributed to its inability to participate in a later step. In contrast, UV-irradiated dnaE486 umuC122 bacteria did not show mutagenesis when incubated at restrictive temperature before photoreversal, suggesting that the altered DnaE486 protein was not able to carry out the postulate misincorporation step at 43 0 C. DNA polymerase III α-subunit therefore appears to be required for both the misincorporation and bypass steps in the two-step model for UV mutagenesis. (Author)

Detection of DNA oligonucleotides with base mutations by terahertz spectroscopy and microstructures.

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Mingjie Tang

Full Text Available DNA oligonucleotides with a 5-base mutation at the 3'-terminus were investigated by terahertz (THz spectroscopy in a marker-free manner. The four single-stranded oligonucleotides with 17nt have been detected with specificity on a microfluidic chip, and corroborated by spectral measurements with split-ring resonators. The number of hydrogen bonds formed between the oligonucleotide and its surrounding water molecules, deemed a key contribution to the THz absorption of biological solutions, was explored by molecular dynamics simulations to explain the experimental findings. Our work underlies the feasibility of THz spectroscopy combined with microstructures for marker-free detection of DNA, which may form the basis of a prospective diagnostic tool for studying genic mutation.

Sensitive detection of point mutation by electrochemiluminescence and DNA ligase-based assay

Science.gov (United States)

Zhou, Huijuan; Wu, Baoyan

2008-12-01

The technology of single-base mutation detection plays an increasingly important role in diagnosis and prognosis of genetic-based diseases. Here we reported a new method for the analysis of point mutations in genomic DNA through the integration of allele-specific oligonucleotide ligation assay (OLA) with magnetic beads-based electrochemiluminescence (ECL) detection scheme. In this assay the tris(bipyridine) ruthenium (TBR) labeled probe and the biotinylated probe are designed to perfectly complementary to the mutant target, thus a ligation can be generated between those two probes by Taq DNA Ligase in the presence of mutant target. If there is an allele mismatch, the ligation does not take place. The ligation products are then captured onto streptavidin-coated paramagnetic beads, and detected by measuring the ECL signal of the TBR label. Results showed that the new method held a low detection limit down to 10 fmol and was successfully applied in the identification of point mutations from ASTC-α-1, PANC-1 and normal cell lines in codon 273 of TP53 oncogene. In summary, this method provides a sensitive, cost-effective and easy operation approach for point mutation detection.

Effect of combined mutagenic treatments on sensitivity and mutation frequency in rice

International Nuclear Information System (INIS)

Gopinathan Nair, V.

1977-01-01

Rice seeds were subjected to two sets of combination treatments of radiations and NMH. The effects of mutagenic treatments in the M 1 and M 2 generations were recorded and discussed. Mutation frequencies estimated as number of mutations per 100 M 1 years were not higher than the values expected on the basis of additive effects. When estimated as number of mutants per 100 M 2 plants, the frequencies revealed more than additive effects. The synergistic effect on mutant frequencies was due to increase in the segregation ratio of mutants. This effect was more pronounced at the higher dose combinations of fast neutrons and NMH. (author)

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

Nicotinamide starvation and inhibition of poly(ADP-Ribose) synthesis enhance the induced mutation in Chinese hamster V79 cells

International Nuclear Information System (INIS)

Okada, Gensaku; Kaneko, Ichiro; Mitsui, Hideki.

1987-01-01

The effects of nicotinamide (NA) deficiency and added NA and 3-aminobenzamide (3AB) on the cytotoxicity and the induction of mutations in Chinese hamster V79-14 cells were investigated. In NA deficiency the addition of NA (up to 4 mM) and 3AB (up to 7.5 mM) was not cytotoxic. The presence of NA prior to exposure to mitomycin C (MMC) or γ-rays produced a dose-dependent increase in the relative cloning ability of DNA-damaged cells. The lethality of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was significantly potentiated by pre-treatment with 5 mM 3AB, but no potentiation by 3AB was observed for MMC, ultraviolet (UV)-B light, or γ-rays. Among cells pre-cultured in NA-free medium there were increased frequencies of mutations at both the hypoxanthineguanine phosphoribosyltransferase (HGPRT) and the adenine phosphoribosyltransferase (APRT) loci following DNA damage. The enhancing effect by NA deficiency was time-dependent. Incubation with NA prior to DNA damage produced a significant reduction in the frequency of mutations. The addition of 3AB to the nicotinamide adenine dinucleotide (NAD + )-depleted cell cultures before or after the DNA damage also strongly increased the frequency of induced mutations, with increasing concentrations of 3AB up to 5 mM, but the frequency was reduced at higher concentrations. The interaction between NA deficiency and the addition of 3AB appears to act synergistically on mutation induction. A correlation was observed between the potential of inhibiting poly (ADP-ribose) polymerase and the enhancement of mutation frequency. (author)

Mutation frequencies in male mice and the estimation of genetic hazards of radiation in men

International Nuclear Information System (INIS)

Russell, W.L.; Kelly, E.M.

1982-01-01

Estimation of the genetic hazards of ionizing radiation in men is based largely on the frequency of transmitted specific-locus mutations induced in mouse spermatogonial stem cells at low radiation dose rates. The publication of new data on this subject has permitted a fresh review of all the information available. The data continue to show no discrepancy from the interpretation that, although mutation frequency decreases markedly as dose rate is decreased from 90 to 0.8 R/min (1 R = 2.6 x 10/sup -4/ coulombs/kg) there seems to be no further change below 0.8 R/min over the range from that dose rate to 0.0007 R/min. Simple mathematical models are used to compute: (a) a maximum likelihood estimate of the induced mutation frequency at the low dose rates, and (b) a maximum likelihood estimate of the ratio of this to the mutation frequency at high dose rates in the range of 72 to 90 R/min. In the application of these results to the estimation of genetic hazards of radiation in man, the former value can be used to calcualte a doubling dose - i.e., the dose of radiation that induces a mutation frequency equal to the spontaneous frequency. The doubling dose based on the low-dose-rate data compiled here is 110 R. The ratio of the mutation frequency at low dose rate to to that at high dose rate is useful when it becomes necessary to extrapolate from experimental determinations, or from human data, at high dose rates to the expected risk at low dose rates. The ratio derived from the present analysis is 0.33

AIP mutations in Brazilian patients with sporadic pituitary adenomas: a single-center evaluation

Science.gov (United States)

Kasuki, Leandro; de Azeredo Lima, Carlos Henrique; Ogino, Liana; Camacho, Aline H S; Chimelli, Leila; Korbonits, Márta

2017-01-01

Aryl hydrocarbon receptor-interacting protein (AIP) gene mutations (AIPmut) are the most frequent germline mutations found in apparently sporadic pituitary adenomas (SPA). Our aim was to evaluate the frequency of AIPmut among young Brazilian patients with SPA. We performed an observational cohort study between 2013 and 2016 in a single referral center. AIPmut screening was carried out in 132 SPA patients with macroadenomas diagnosed up to 40 years or in adenomas of any size diagnosed until 18 years of age. Twelve tumor samples were also analyzed. Leukocyte DNA and tumor tissue DNA were sequenced for the entire AIP-coding region for evaluation of mutations. Eleven (8.3%) of the 132 patients had AIPmut, comprising 9/74 (12%) somatotropinomas, 1/38 (2.6%) prolactinoma, 1/10 (10%) corticotropinoma and no non-functioning adenomas. In pediatric patients (≤18 years), AIPmut frequency was 13.3% (2/15). Out of the 5 patients with gigantism, two had AIPmut, both truncating mutations. The Y268* mutation was described in Brazilian patients and the K273Rfs*30 mutation is a novel mutation in our patient. No somatic AIP mutations were found in the 12 tumor samples. A tumor sample from an acromegaly patient harboring the A299V AIPmut showed loss of heterozygosity. In conclusion, AIPmut frequency in SPA Brazilian patients is similar to other populations. Our study identified two mutations exclusively found in Brazilians and also shows, for the first time, loss of heterozygosity in tumor DNA from an acromegaly patient harboring the A299V AIPmut. Our findings corroborate previous observations that AIPmut screening should be performed in young patients with SPA. PMID:29074612

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

Science.gov (United States)

2015-09-01

Award Number: W81XWH-12-1-0333 TITLE: Determining the Location of DNA Modification and Mutation Caused by UVB Light in Skin Cancer PRINCIPAL...COVERED 15 Aug 2012 – 14 Aug 2015 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER W81XWH-12-1-0333 Determining the Location of DNA Modification and Mutation ...sequencing libraries generated for both yeast and human cells show pyrimidine bias on the 5’ end, indicating that we are sequencing the dimers

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

Science.gov (United States)

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

2010-01-01

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

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.

A MUTYH germline mutation is associated with small intestinal neuroendocrine tumors

DEFF Research Database (Denmark)

Dumanski, Jan P; Rasi, Chiara; Björklund, Peyman

2017-01-01

. The inactivation of this gene leads to specific increase of G:C- > T:A transversions in DNA sequence and has been shown to cause various cancers in humans and experimental animals. Our results suggest that p.(Gly396Asp) in MUTYH, and potentially other mutations in additional members of the same DNA excision......-generation sequencing of exome- and/or whole-genome of blood DNA, and in selected cases, tumor DNA, from 24 patients from 15 families with the history of SI-NETs. We identified seven candidate mutations in six genes that were further studied using 215 sporadic SI-NET patients. The result was compared with the frequency...

Ultraviolet-irradiated simian virus 40 activates a mutator function in rat cells under conditions preventing viral DNA replication

Energy Technology Data Exchange (ETDEWEB)

Cornelis, J.; Su, Z.Z.; Dinsart, C.; Rommelaere, J. (Universite libre de Bruxelles, Rhode St Genese (Belgium))

The UV-irradiated temperature-sensitive early SV40 mutant tsA209 is able to activate at the nonpermissive temperature the expression of mutator and recovery functions in rat cells. Unirradiated SV40 activates these functions only to a low extent. The expression of these mutator and recovery functions in SV40-infected cells was detected using the single-stranded DNA parvovirus H-1 as a probe. Because early SV40 mutants are defective in the initiation of viral DNA synthesis at the nonpermissive temperature, these results suggest that replication of UV-damaged DNA is not a prerequisite for the activation of mutator and recovery functions in mammalian cells. The expression of the mutator function is dose-dependent, i.e., the absolute number of UV-irradiated SV40 virions introduced per cell determines its level. Implications for the interpretation of mutation induction curves in the progeny of UV-irradiated SV40 in permissive host cells are discussed.

AIP mutations in Brazilian patients with sporadic pituitary adenomas: a single-center evaluation

Directory of Open Access Journals (Sweden)

Paula Bruna Araujo

2017-11-01

Full Text Available Aryl hydrocarbon receptor-interacting protein (AIP gene mutations (AIPmut are the most frequent germline mutations found in apparently sporadic pituitary adenomas (SPA. Our aim was to evaluate the frequency of AIPmut among young Brazilian patients with SPA. We performed an observational cohort study between 2013 and 2016 in a single referral center. AIPmut screening was carried out in 132 SPA patients with macroadenomas diagnosed up to 40 years or in adenomas of any size diagnosed until 18 years of age. Twelve tumor samples were also analyzed. Leukocyte DNA and tumor tissue DNA were sequenced for the entire AIP-coding region for evaluation of mutations. Eleven (8.3% of the 132 patients had AIPmut, comprising 9/74 (12% somatotropinomas, 1/38 (2.6% prolactinoma, 1/10 (10% corticotropinoma and no non-functioning adenomas. In pediatric patients (≤18 years, AIPmut frequency was 13.3% (2/15. Out of the 5 patients with gigantism, two had AIPmut, both truncating mutations. The Y268* mutation was described in Brazilian patients and the K273Rfs*30 mutation is a novel mutation in our patient. No somatic AIP mutations were found in the 12 tumor samples. A tumor sample from an acromegaly patient harboring the A299V AIPmut showed loss of heterozygosity. In conclusion, AIPmut frequency in SPA Brazilian patients is similar to other populations. Our study identified two mutations exclusively found in Brazilians and also shows, for the first time, loss of heterozygosity in tumor DNA from an acromegaly patient harboring the A299V AIPmut. Our findings corroborate previous observations that AIPmut screening should be performed in young patients with SPA.

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

International Nuclear Information System (INIS)

Stanislawska-Sachadyn, Anna; Paszko, Zygmunt; Kluska, Anna; Skasko, Elzibieta; Sromek, Maria; Balabas, Aneta; Janiec-Jankowska, Aneta; Wisniewska, Alicja; Kur, Jozef; Sachadyn, Pawel

2005-01-01

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 μg of Thermus thermophilus his 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

dNTP pool levels modulate mutator phenotypes of error-prone DNA polymerase ε variants.

Science.gov (United States)

Williams, Lindsey N; Marjavaara, Lisette; Knowels, Gary M; Schultz, Eric M; Fox, Edward J; Chabes, Andrei; Herr, Alan J

2015-05-12

Mutator phenotypes create genetic diversity that fuels tumor evolution. DNA polymerase (Pol) ε mediates leading strand DNA replication. Proofreading defects in this enzyme drive a number of human malignancies. Here, using budding yeast, we show that mutator variants of Pol ε depend on damage uninducible (Dun)1, an S-phase checkpoint kinase that maintains dNTP levels during a normal cell cycle and up-regulates dNTP synthesis upon checkpoint activation. Deletion of DUN1 (dun1Δ) suppresses the mutator phenotype of pol2-4 (encoding Pol ε proofreading deficiency) and is synthetically lethal with pol2-M644G (encoding altered Pol ε base selectivity). Although pol2-4 cells cycle normally, pol2-M644G cells progress slowly through S-phase. The pol2-M644G cells tolerate deletions of mediator of the replication checkpoint (MRC) 1 (mrc1Δ) and radiation sensitive (Rad) 9 (rad9Δ), which encode mediators of checkpoint responses to replication stress and DNA damage, respectively. The pol2-M644G mutator phenotype is partially suppressed by mrc1Δ but not rad9Δ; neither deletion suppresses the pol2-4 mutator phenotype. Thus, checkpoint activation augments the Dun1 effect on replication fidelity but is not required for it. Deletions of genes encoding key Dun1 targets that negatively regulate dNTP synthesis, suppress the dun1Δ pol2-M644G synthetic lethality and restore the mutator phenotype of pol2-4 in dun1Δ cells. DUN1 pol2-M644G cells have constitutively high dNTP levels, consistent with checkpoint activation. In contrast, pol2-4 and POL2 cells have similar dNTP levels, which decline in the absence of Dun1 and rise in the absence of the negative regulators of dNTP synthesis. Thus, dNTP pool levels correlate with Pol ε mutator severity, suggesting that treatments targeting dNTP pools could modulate mutator phenotypes for therapy.

Frequencies of aneuploidy and dominant lethal mutations in young female mice induced by low dose γ-rays

International Nuclear Information System (INIS)

Yao Suyan; Zhang Chaoyang; Dai Lianlian; Gao Changwen

1991-01-01

Relationship between aneuploidy, dominant lethal mutations and doses in young feral mice induced by low dose γ-rays was examined. The results suggest that the frequencies of aneuploidy of embryos increased at 0.15 Gy, but increases at over 0.50 Gy after irradiation in groups. The frequencies of aneuploidy and dominant lethal mutations increased with increasing doses and fitted linear relationship. This dose-response relationship of trisomic was not significant. The frequency of dominant lethal mutations induced by 60 Co γ irradiation is 5.59%. The effect of dominant lethal mutation is higher than that of the aneuploidy

Droplet digital PCR-based EGFR mutation detection with an internal quality control index to determine the quality of DNA.

Science.gov (United States)

Kim, Sung-Su; Choi, Hyun-Jeung; Kim, Jin Ju; Kim, M Sun; Lee, In-Seon; Byun, Bohyun; Jia, Lina; Oh, Myung Ryurl; Moon, Youngho; Park, Sarah; Choi, Joon-Seok; Chae, Seoung Wan; Nam, Byung-Ho; Kim, Jin-Soo; Kim, Jihun; Min, Byung Soh; Lee, Jae Seok; Won, Jae-Kyung; Cho, Soo Youn; Choi, Yoon-La; Shin, Young Kee

2018-01-11

In clinical translational research and molecular in vitro diagnostics, a major challenge in the detection of genetic mutations is overcoming artefactual results caused by the low-quality of formalin-fixed paraffin-embedded tissue (FFPET)-derived DNA (FFPET-DNA). Here, we propose the use of an 'internal quality control (iQC) index' as a criterion for judging the minimum quality of DNA for PCR-based analyses. In a pre-clinical study comparing the results from droplet digital PCR-based EGFR mutation test (ddEGFR test) and qPCR-based EGFR mutation test (cobas EGFR test), iQC index ≥ 0.5 (iQC copies ≥ 500, using 3.3 ng of FFPET-DNA [1,000 genome equivalents]) was established, indicating that more than half of the input DNA was amplifiable. Using this criterion, we conducted a retrospective comparative clinical study of the ddEGFR and cobas EGFR tests for the detection of EGFR mutations in non-small cell lung cancer (NSCLC) FFPET-DNA samples. Compared with the cobas EGFR test, the ddEGFR test exhibited superior analytical performance and equivalent or higher clinical performance. Furthermore, iQC index is a reliable indicator of the quality of FFPET-DNA and could be used to prevent incorrect diagnoses arising from low-quality samples.

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

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Eun Hyun Ahn

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

Mutation frequencies in male mice and the estimation of genetic hazards of radiation in men: (specific-locus mutations/dose-rate effect/doubling dose/risk estimation)

International Nuclear Information System (INIS)

Russell, W.L.; Kelly, E.M.

1982-01-01

Estimation of the genetic hazards of ionizing radiation in men is based largely on the frequency of transmitted specific-locus mutations induced in mouse spermatogonial stem cells at low radiation dose rates. The publication of new data on this subject has permitted a fresh review of all the information available. The data continue to show no discrepancy from the interpretation that, although mutation frequency decreases markedly as dose rate is decreased from 90 to 0.8 R/min (1 R = 2.6 X 10 -4 coulombs/kg) there seems to be no further change below 0.8 R/min over the range from that dose rate to 0.0007 R/min. Simple mathematical models are used to compute: (a) a maximum likelihood estimate of the induced mutation frequency at the low dose rates, and (b) a maximum likelihood estimate of the ratio of this to the mutation frequency at high dose rates in the range of 72 to 90 R/min. In the application of these results to the estimation of genetic hazards of radiation in man, the former value can be used to calculate a doubling dose - i.e., the dose of radiation that induces a mutation frequency equal to the spontaneous frequency. The doubling dose based on the low-dose-rate data compiled here is 110 R. The ratio of the mutation frequency at low dose rate to that at high dose rate is useful when it becomes necessary to extrapolate from experimental determinations, or from human data, at high dose rates to the expected risk at low dose rates. The ratio derived from the present analysis is 0.33

Spectrum and frequency of chlorophyll mutations in urdbean (Vigna mungo L. Hepper) induced by EMS and gamma rays

International Nuclear Information System (INIS)

Sharma, A.K.; Singh, V.P.; Sarma, M.K.

2006-01-01

In mutation breeding experiment, plants with altered characteristics such as chlorophyll changes, sterility, plant lethality etc. could be the marker of the mutability of a variety. In fact, spectrum and frequency of chlorophyll mutations have been studied in the great detail. The chlorophyll mutation is the clear-cut indication of non-directional nature of mutation and possibility of induction of useful mutations. The spectrum and frequency of chlorophyll mutation was estimated by using gamma rays (100, 200, 300 and 400 Gy doses), EMS (0.2, 0.4, 0.6 and 0.8%) and combination of gamma rays (100, 200, 300 400 Gy) with 0.2 % concentration EMS on two cultivars, namely, Pant Urd-19 and Pant Urd-30 of urdbean ( Vigna mungo L. Hepper). Five different types of chlorophyll mutations viz., albina, xantha, viridis, chlorina and maculata were identified in both the cultivars. Almost all the combination treatments produced maximum frequency and wider spectrum of chlorophyll mutations followed by single treatment of gamma rays or EMS. The frequency of chlorophyll mutation increased with higher doses of mutagens but decreased at highest dose. Proc. Nat. Acad. Sci. India. 76(8), I, 2006. 64-68. (author)

Ancient DNA analysis reveals high frequency of European lactase persistence allele (T-13910) in medieval central europe.

Science.gov (United States)

Krüttli, Annina; Bouwman, Abigail; Akgül, Gülfirde; Della Casa, Philippe; Rühli, Frank; Warinner, Christina

2014-01-01

Ruminant milk and dairy products are important food resources in many European, African, and Middle Eastern societies. These regions are also associated with derived genetic variants for lactase persistence. In mammals, lactase, the enzyme that hydrolyzes the milk sugar lactose, is normally down-regulated after weaning, but at least five human populations around the world have independently evolved mutations regulating the expression of the lactase-phlorizin-hydrolase gene. These mutations result in a dominant lactase persistence phenotype and continued lactase tolerance in adulthood. A single nucleotide polymorphism (SNP) at C/T-13910 is responsible for most lactase persistence in European populations, but when and where the T-13910 polymorphism originated and the evolutionary processes by which it rose to high frequency in Europe have been the subject of strong debate. A history of dairying is presumed to be a prerequisite, but archaeological evidence is lacking. In this study, DNA was extracted from the dentine of 36 individuals excavated at a medieval cemetery in Dalheim, Germany. Eighteen individuals were successfully genotyped for the C/T-13910 SNP by molecular cloning and sequencing, of which 13 (72%) exhibited a European lactase persistence genotype: 44% CT, 28% TT. Previous ancient DNA-based studies found that lactase persistence genotypes fall below detection levels in most regions of Neolithic Europe. Our research shows that by AD 1200, lactase persistence frequency had risen to over 70% in this community in western Central Europe. Given that lactase persistence genotype frequency in present-day Germany and Austria is estimated at 71-80%, our results suggest that genetic lactase persistence likely reached modern levels before the historic population declines associated with the Black Death, thus excluding plague-associated evolutionary forces in the rise of lactase persistence in this region. This new evidence sheds light on the dynamic evolutionary

Strikingly different penetrance of LHON in two Chinese families with primary mutation G11778A is independent of mtDNA haplogroup background and secondary mutation G13708A

International Nuclear Information System (INIS)

Wang Huawei; Jia Xiaoyun; Ji Yanli; Kong Qingpeng; Zhang Qingjiong; Yao Yonggang; Zhang Yaping

2008-01-01

The penetrance of Leber's hereditary optic neuropathy (LHON) in families with primary mitochondrial DNA (mtDNA) mutations is very complex. Matrilineal and nuclear genetic background, as well as environmental factors, have been reported to be involved in different affected pedigrees. Here we describe two large Chinese families that show a striking difference in the penetrance of LHON, in which 53.3% and 15.0% of members were affected (P < 0.02), respectively. Analysis of the complete mtDNA genome of the two families revealed the presence of the primary mutation G11778A and several other variants suggesting the same haplogroup status G2a. The family with higher penetrance contained a previously described secondary mutation G13708A, which presents a polymorphism in normal Chinese samples and does not affect in vivo mitochondrial oxidative metabolism as described in a previous study. Evolutionary analysis failed to indicate any putatively pathogenic mutation that cosegregated with G11778A in these two pedigrees. Our results suggest that the variable penetrance of LHON in the two Chinese families is independent of both their mtDNA haplotype background and a secondary mutation G13708A. As a result, it is likely that unknown nuclear gene involvement and/or other factors contribute to the strikingly different penetrance of LHON

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

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

Directory of Open Access Journals (Sweden)

Cotton Richard GH

2003-05-01

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

MethylMeter(®): bisulfite-free quantitative and sensitive DNA methylation profiling and mutation detection in FFPE samples.

Science.gov (United States)

McCarthy, David; Pulverer, Walter; Weinhaeusel, Andreas; Diago, Oscar R; Hogan, Daniel J; Ostertag, Derek; Hanna, Michelle M

2016-06-01

Development of a sensitive method for DNA methylation profiling and associated mutation detection in clinical samples. Formalin-fixed and paraffin-embedded tumors received by clinical laboratories often contain insufficient DNA for analysis with bisulfite or methylation sensitive restriction enzymes-based methods. To increase sensitivity, methyl-CpG DNA capture and Coupled Abscription PCR Signaling detection were combined in a new assay, MethylMeter(®). Gliomas were analyzed for MGMT methylation, glioma CpG island methylator phenotype and IDH1 R132H. MethylMeter had 100% assay success rate measuring all five biomarkers in formalin-fixed and paraffin-embedded tissue. MGMT methylation results were supported by survival and mRNA expression data. MethylMeter is a sensitive and quantitative method for multitarget DNA methylation profiling and associated mutation detection. The MethylMeter-based GliomaSTRAT assay measures methylation of four targets and one mutation to simultaneously grade gliomas and predict their response to temozolomide. This information is clinically valuable in management of gliomas.

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.

Next generation sequencing of DNA-launched Chikungunya vaccine virus

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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.

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.

Role of damage-specific DNA polymerases in M13 phage mutagenesis induced by a major lipid peroxidation product trans-4-hydroxy-2-nonenal

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Janowska, Beata [Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw (Poland); Kurpios-Piec, Dagmara [Department of Biochemistry, Medical University of Warsaw, Banacha 1, 02-097 Warsaw (Poland); Prorok, Paulina [Institute of Genetics and Biotechnology, Warsaw University, Pawinskiego 5a, 02-106 Warsaw (Poland); Szparecki, Grzegorz [Medical University of Warsaw, Zwirki i Wigury 61, 02-097 Warsaw (Poland); Komisarski, Marek [Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw (Poland); Kowalczyk, Pawel [Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University, Pawinskiego 5a, 02-106 Warsaw (Poland); Janion, Celina [Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw (Poland); Tudek, Barbara, E-mail: tudek@ibb.waw.pl [Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw (Poland); Institute of Genetics and Biotechnology, Warsaw University, Pawinskiego 5a, 02-106 Warsaw (Poland)

2012-01-03

One of the major lipid peroxidation products trans-4-hydroxy-2-nonenal (HNE), forms cyclic propano- or ethenoadducts bearing six- or seven-carbon atom side chains to G > C Much-Greater-Than A > T. To specify the role of SOS DNA polymerases in HNE-induced mutations, we tested survival and mutation spectra in the lacZ{alpha} gene of M13mp18 phage, whose DNA was treated in vitro with HNE, and which was grown in uvrA{sup -}Escherichia coli strains, carrying one, two or all three SOS DNA polymerases. When Pol IV was the only DNA SOS polymerase in the bacterial host, survival of HNE-treated M13 DNA was similar to, but mutation frequency was lower than in the strain containing all SOS DNA polymerases. When only Pol II or Pol V were present in host bacteria, phage survival decreased dramatically. Simultaneously, mutation frequency was substantially increased, but exclusively in the strain carrying only Pol V, suggesting that induction of mutations by HNE is mainly dependent on Pol V. To determine the role of Pol II and Pol IV in HNE induced mutagenesis, Pol II or Pol IV were expressed together with Pol V. This resulted in decrease of mutation frequency, suggesting that both enzymes can compete with Pol V, and bypass HNE-DNA adducts in an error-free manner. However, HNE-DNA adducts were easily bypassed by Pol IV and only infrequently by Pol II. Mutation spectrum established for strains expressing only Pol V, showed that in uvrA{sup -} bacteria the frequency of base substitutions and recombination increased in relation to NER proficient strains, particularly mutations at adenine sites. Among base substitutions A:T {yields} C:G, A:T {yields} G:C, G:C {yields} A:T and G:C {yields} T:A prevailed. The results suggest that Pol V can infrequently bypass HNE-DNA adducts inducing mutations at G, C and A sites, while bypass by Pol IV and Pol II is error-free, but for Pol II infrequent.

Diversity and frequency of kdr mutations within Anopheles sinensis populations from Guangxi, China.

Science.gov (United States)

Yang, Chan; Feng, Xiangyang; Huang, Zushi; Li, Mei; Qiu, Xinghui

2016-08-15

Anopheles sinensis is a major vector of malaria in China and its control is under great threat as the development of insecticide resistance. Voltage-gated sodium channel (VGSC) is the target of several classes of insecticides. Genetic mutations of VGSC have been documented to confer knockdown resistance (kdr) to dichlorodiphenyltrichloroethane (DDT) and pyrethroids in mosquitoes. To control this vector efficiently, it is important to know the resistance-associated genetic mutations, their distribution frequencies and genealogical relations. Three hundreds and thirteen (313) adults of An. sinensis collected from nine locations across Guangxi Zhuang Autonomous Region were used. The partial sequence of the An. sinensis voltage gated sodium channel gene (AS-VGSC) containing codon 1014 was sequenced. PHASE2.1 was used to construct the haplotypes of each individual, and the accuracy of haplotypes was further confirmed by clone sequencing. The genealogical relations of kdr mutations in AS-VGSC was analysed using TCS 2.1 and Network 5.0. Sixteen AS-VGSC haplotypes including seven haplotypes carrying non-synonymous mutations at codon 1014, and fifty-five AS-VGSC genotypes were identified from 313 mosquitoes collected from nine geographical locations across Guangxi. The number of haplotypes in each of the nine populations ranged from 5 to 13. The frequency of haplotypes carrying kdr mutations ranged from 2.7 to 80.0 % within the nine populations, of which 1014C was unexpectedly high in the northeast of Guangxi. Genealogical analysis suggested multiple origins of kdr mutations in An. sinensis. Diverse haplotypes of AS-VGSC are distributed in Guangxi. The presence of haplotypes carrying mutations at codon 1014 indicates a risk of pyrethroid and DDT resistance. The kdr mutations show differential distribution geographically, with high frequencies occurred in the northeast of Guangxi. Genealogical analysis suggests multiple origins of kdr mutations in An. sinensis populations

Mutations induced by ultraviolet light

International Nuclear Information System (INIS)

Pfeifer, Gerd P.; You, Young-Hyun; Besaratinia, Ahmad

2005-01-01

The different ultraviolet (UV) wavelength components, UVA (320-400 nm), UVB (280-320 nm), and UVC (200-280 nm), have distinct mutagenic properties. A hallmark of UVC and UVB mutagenesis is the high frequency of transition mutations at dipyrimidine sequences containing cytosine. In human skin cancers, about 35% of all mutations in the p53 gene are transitions at dipyrimidines within the sequence 5'-TCG and 5'-CCG, and these are localized at several mutational hotspots. Since 5'-CG sequences are methylated along the p53 coding sequence in human cells, these mutations may be derived from sunlight-induced pyrimidine dimers forming at sequences that contain 5-methylcytosine. Cyclobutane pyrimidine dimers (CPDs) form preferentially at dipyrimidines containing 5-methylcytosine when cells are irradiated with UVB or sunlight. In order to define the contribution of 5-methylcytosine to sunlight-induced mutations, the lacI and cII transgenes in mouse fibroblasts were used as mutational targets. After 254 nm UVC irradiation, only 6-9% of the base substitutions were at dipyrimidines containing 5-methylcytosine. However, 24-32% of the solar light-induced mutations were at dipyrimidines that contain 5-methylcytosine and most of these mutations were transitions. Thus, CPDs forming preferentially at dipyrimidines with 5-methylcytosine are responsible for a considerable fraction of the mutations induced by sunlight in mammalian cells. Using mouse cell lines harboring photoproduct-specific photolyases and mutational reporter genes, we showed that CPDs (rather than 6-4 photoproducts or other lesions) are responsible for the great majority of UVB-induced mutations. An important component of UVB mutagenesis is the deamination of cytosine and 5-methylcytosine within CPDs. The mutational specificity of long-wave UVA (340-400 nm) is distinct from that of the shorter wavelength UV and is characterized mainly by G to T transversions presumably arising through mechanisms involving oxidized DNA

The frequency of Tay-Sachs disease causing mutations in the Brazilian Jewish population justifies a carrier screening program.

Science.gov (United States)

Rozenberg, R; Pereira, L da V

2001-07-05

Tay-Sachs disease is an autosomal recessive disease characterized by progressive neurologic degeneration, fatal in early childhood. In the Ashkenazi Jewish population the disease incidence is about 1 in every 3,500 newborns and the carrier frequency is 1 in every 29 individuals. Carrier screening programs for Tay-Sachs disease have reduced disease incidence by 90% in high-risk populations in several countries. The Brazilian Jewish population is estimated at 90,000 individuals. Currently, there is no screening program for Tay-Sachs disease in this population. To evaluate the importance of a Tay-Sachs disease carrier screening program in the Brazilian Jewish population by determining the frequency of heterozygotes and the acceptance of the program by the community. Laboratory of Molecular Genetics--Institute of Biosciences--Universidade de São Paulo. 581 senior students from selected Jewish high schools. Molecular analysis of Tay-Sachs disease causing mutations by PCR amplification of genomic DNA, followed by restriction enzyme digestion. Among 581 students that attended educational classes, 404 (70%) elected to be tested for Tay-Sachs disease mutations. Of these, approximately 65% were of Ashkenazi Jewish origin. Eight carriers were detected corresponding to a carrier frequency of 1 in every 33 individuals in the Ashkenazi Jewish fraction of the sample. The frequency of Tay-Sachs disease carriers among the Ashkenazi Jewish population of Brazil is similar to that of other countries where carrier screening programs have led to a significant decrease in disease incidence. Therefore, it is justifiable to implement a Tay-Sachs disease carrier screening program for the Brazilian Jewish population.

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

2014-04-01

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-parent trios and detected two de novo DNMT3A mutations. We identified 11 additional de novo mutations by sequencing DNMT3A in a further 142 individuals with overgrowth. The mutations alter residues in functional DNMT3A domains, and protein modeling suggests that they interfere with domain-domain interactions and histone binding. Similar mutations were not present in 1,000 UK population controls (13/152 cases versus 0/1,000 controls; P < 0.0001). Mutation carriers had a distinctive facial appearance, intellectual disability and greater height. DNMT3A encodes a DNA methyltransferase essential for establishing methylation during embryogenesis and is commonly somatically mutated in acute myeloid leukemia. Thus, DNMT3A joins an emerging group of epigenetic DNA- and histone-modifying genes associated with both developmental growth disorders and hematological malignancies.

Estimation of the frequency of occult mutations for an autosomal recessive disease in the presence of genetic heterogeneity: application to genetic hearing loss disorders.

Science.gov (United States)

Kimberling, William J

2005-11-01

The routine testing for pathologic mutation(s) in a patient's DNA has become the foundation of modern molecular genetic diagnosis. It is especially valuable when the phenotype shows genetic heterogeneity, and its importance will grow as treatments become genotype specific. However, the technology of mutation detection is imperfect and mutations are often missed. This can be especially troublesome when dealing with a recessive disorder where the combination of genetic heterogeneity and missed mutation creates an imprecision in the genotypic assessment of individuals who do not appear to have the expected complement of two pathologic mutations. This article describes a statistical approach to the estimation of the likelihood of a genetic diagnosis under these conditions. In addition to providing a means of testing for missed mutations, it also provides a method of estimating and testing for the presence of genetic heterogeneity in the absence of linkage data. Gene frequencies as well as estimates of sensitivity and specificity can be obtained as well. The test is applied to GJB2 recessive nonsyndromic deafness, Usher syndrome types Ib and IIa, and Pendred-enlarged vestibular aqueduct syndrome. Copyright 2005 Wiley-Liss, Inc.

Analysis of point mutations in an ultraviolet-irradiated shuttle vector plasmid propagated in cells from Japanese xeroderma pigmentosum patients in complementation groups A and F

International Nuclear Information System (INIS)

Yagi, T.; Tatsumi-Miyajima, J.; Sato, M.; Kraemer, K.H.; Takebe, H.

1991-01-01

To assess the contribution to mutagenesis by human DNA repair defects, a UV-treated shuttle vector plasmid, pZ189, was passed through fibroblasts derived from Japanese xeroderma pigmentosum (XP) patients in two different DNA repair complementation groups (A and F). Patients with XP have clinical and cellular UV hypersensitivity, increased frequency of skin cancer, and defects in DNA repair. The XP DNA repair defects represented by complementation groups A (XP-A) and F (XP-F) are more common in Japan than in Europe or the United States. In comparison to results with DNA repair-proficient human cells (W138-VA13), UV-treated pZ189 passed through the XP-A [XP2OS(SV)] or XP-F [XP2YO(SV)] cells showed fewer surviving plasmids (XP-A less than XP-F) and a higher frequency of mutated plasmids (XP-A greater than XP-F). Base sequence analysis of more than 200 mutated plasmids showed the major type of base substitution mutation to be the G:C----A:T transition with all three cell lines. The XP-A and XP-F cells revealed a higher frequency of G:C----A:T transitions and a lower frequency of transversions among plasmids with single or tandem mutations and a lower frequency of plasmids with multiple point mutations compared to the normal line. The spectrum of mutations in pZ189 with the XP-A cells was similar to that with the XP-F cells. Seventy-six to 91% of the single base substitution mutations occurred at G:C base pairs in which the 5'-neighboring base of the cytosine was thymine or cytosine. These studies indicate that the DNA repair defects in Japanese XP patients in complementation groups A and F result in different frequencies of plasmid survival and mutagenesis but in similar types of mutagenic abnormalities despite marked differences in clinical features

Expanded simple tandem repeat (ESTR) mutation induction in the male germline: Lessons learned from lab mice

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Somers, Christopher M. [University of Regina, Department of Biology, 3737 Wascana Parkway, Regina, SK, S4S 0A2 (Canada)]. E-mail: chris.somers@uregina.ca

2006-06-25

Expanded simple tandem repeat (ESTR) DNA loci that are unstable in the germline have provided the most sensitive tool ever developed for investigating low-dose heritable mutation induction in laboratory mice. Ionizing radiation exposures have shown that ESTR mutations occur mainly in pre-meiotic spermatogonia and stem cells. The average spermatogonial doubling dose is 0.62-0.69 Gy for low LET, and 0.18-0.34 Gy for high LET radiation. Chemical alkylating agents also cause significant ESTR mutation induction in pre-meiotic spermatogonia and stem cells, but are much less effective per unit dose than radiation. ESTR mutation induction efficiency is maximal at low doses of radiation or chemical mutagens, and may decrease at higher dose ranges. DNA repair deficient mice (SCID and PARP-1) with elevated levels of single and double-strand DNA breaks have spontaneously elevated ESTR mutation frequencies, and surprisingly do not show additional ESTR mutation induction following irradiation. In contrast, ESTR mutation induction in p53 knock-outs is indistinguishable from that of wild-type mice. Studies of sentinel mice exposed in situ to ambient air pollution showed elevated ESTR mutation frequencies in males exposed to high levels of particulate matter. These studies highlight the application of the ESTR assay for assessing environmental hazards under real-world conditions. All ESTR studies to date have shown untargeted mutations that occur at much higher frequencies than predicted. The mechanism of this untargeted mutation induction is unknown, and must be elucidated before we can fully understand the biological significance of ESTR mutations, or use these markers for formal risk assessment. Future studies should focus on the mechanism of ESTR mutation induction, refining dose responses, and developing ESTR markers for other animal species.

Expanded simple tandem repeat (ESTR) mutation induction in the male germline: Lessons learned from lab mice

International Nuclear Information System (INIS)

Somers, Christopher M.

2006-01-01

Expanded simple tandem repeat (ESTR) DNA loci that are unstable in the germline have provided the most sensitive tool ever developed for investigating low-dose heritable mutation induction in laboratory mice. Ionizing radiation exposures have shown that ESTR mutations occur mainly in pre-meiotic spermatogonia and stem cells. The average spermatogonial doubling dose is 0.62-0.69 Gy for low LET, and 0.18-0.34 Gy for high LET radiation. Chemical alkylating agents also cause significant ESTR mutation induction in pre-meiotic spermatogonia and stem cells, but are much less effective per unit dose than radiation. ESTR mutation induction efficiency is maximal at low doses of radiation or chemical mutagens, and may decrease at higher dose ranges. DNA repair deficient mice (SCID and PARP-1) with elevated levels of single and double-strand DNA breaks have spontaneously elevated ESTR mutation frequencies, and surprisingly do not show additional ESTR mutation induction following irradiation. In contrast, ESTR mutation induction in p53 knock-outs is indistinguishable from that of wild-type mice. Studies of sentinel mice exposed in situ to ambient air pollution showed elevated ESTR mutation frequencies in males exposed to high levels of particulate matter. These studies highlight the application of the ESTR assay for assessing environmental hazards under real-world conditions. All ESTR studies to date have shown untargeted mutations that occur at much higher frequencies than predicted. The mechanism of this untargeted mutation induction is unknown, and must be elucidated before we can fully understand the biological significance of ESTR mutations, or use these markers for formal risk assessment. Future studies should focus on the mechanism of ESTR mutation induction, refining dose responses, and developing ESTR markers for other animal species

Frequency of CNKSR2 mutation in the X-linked epilepsy-aphasia spectrum.

Science.gov (United States)

Damiano, John A; Burgess, Rosemary; Kivity, Sara; Lerman-Sagie, Tally; Afawi, Zaid; Scheffer, Ingrid E; Berkovic, Samuel F; Hildebrand, Michael S

2017-03-01

Synaptic proteins are critical to neuronal function in the brain, and their deficiency can lead to seizures and cognitive impairments. CNKSR2 (connector enhancer of KSR2) is a synaptic protein involved in Ras signaling-mediated neuronal proliferation, migration and differentiation. Mutations in the X-linked gene CNKSR2 have been described in patients with seizures and neurodevelopmental deficits, especially those affecting language. In this study, we sequenced 112 patients with phenotypes within the epilepsy-aphasia spectrum (EAS) to determine the frequency of CNKSR2 mutation within this complex set of disorders. We detected a novel nonsense mutation (c.2314 C>T; p.Arg712*) in one Ashkenazi Jewish family, the male proband of which had a severe epileptic encephalopathy with continuous spike-waves in sleep (ECSWS). His affected brother also had ECSWS with better outcome, whereas the sister had childhood epilepsy with centrotemporal spikes. This mutation segregated in the three affected siblings in an X-linked manner, inherited from their mother who had febrile seizures. Although the frequency of point mutation is low, CNKSR2 sequencing should be considered in families with suspected X-linked EAS because of the specific genetic counseling implications. Wiley Periodicals, Inc. © 2017 International League Against Epilepsy.

Whole-exome sequencing of muscle-invasive bladder cancer identifies recurrent mutations of UNC5C and prognostic importance of DNA repair gene mutations on survival.

Science.gov (United States)

Yap, Kai Lee; Kiyotani, Kazuma; Tamura, Kenji; Antic, Tatjana; Jang, Miran; Montoya, Magdeline; Campanile, Alexa; Yew, Poh Yin; Ganshert, Cory; Fujioka, Tomoaki; Steinberg, Gary D; O'Donnell, Peter H; Nakamura, Yusuke

2014-12-15

Because of suboptimal outcomes in muscle-invasive bladder cancer even with multimodality therapy, determination of potential genetic drivers offers the possibility of improving therapeutic approaches and discovering novel prognostic indicators. Using pTN staging, we case-matched 81 patients with resected ≥pT2 bladder cancers for whom perioperative chemotherapy use and disease recurrence status were known. Whole-exome sequencing was conducted in 43 cases to identify recurrent somatic mutations and targeted sequencing of 10 genes selected from the initial screening in an additional 38 cases was completed. Mutational profiles along with clinicopathologic information were correlated with recurrence-free survival (RFS) in the patients. We identified recurrent novel somatic mutations in the gene UNC5C (9.9%), in addition to TP53 (40.7%), KDM6A (21.0%), and TSC1 (12.3%). Patients who were carriers of somatic mutations in DNA repair genes (one or more of ATM, ERCC2, FANCD2, PALB2, BRCA1, or BRCA2) had a higher overall number of somatic mutations (P = 0.011). Importantly, after a median follow-up of 40.4 months, carriers of somatic mutations (n = 25) in any of these six DNA repair genes had significantly enhanced RFS compared with noncarriers [median, 32.4 vs. 14.8 months; hazard ratio of 0.46, 95% confidence interval (CI), 0.22-0.98; P = 0.0435], after adjustment for pathologic pTN staging and independent of adjuvant chemotherapy usage. Better prognostic outcomes of individuals carrying somatic mutations in DNA repair genes suggest these mutations as favorable prognostic events in muscle-invasive bladder cancer. Additional mechanistic investigation into the previously undiscovered role of UNC5C in bladder cancer is warranted. ©2014 American Association for Cancer Research.

Recurrent APC gene mutations in Polish FAP families

Directory of Open Access Journals (Sweden)

Pławski Andrzej

2007-12-01

Full Text Available Abstract The molecular diagnostics of genetically conditioned disorders is based on the identification of the mutations in the predisposing genes. Hereditary cancer disorders of the gastrointestinal tracts are caused by mutations of the tumour suppressor genes or the DNA repair genes. Occurrence of recurrent mutation allows improvement of molecular diagnostics. The mutation spectrum in the genes causing hereditary forms of colorectal cancers in the Polish population was previously described. In the present work an estimation of the frequency of the recurrent mutations of the APC gene was performed. Eight types of mutations occurred in 19.4% of our FAP families and these constitute 43% of all Polish diagnosed families.

Plasma circulating tumor DNA as an alternative to metastatic biopsies for mutational analysis in breast cancer.

Science.gov (United States)

Rothé, F; Laes, J-F; Lambrechts, D; Smeets, D; Vincent, D; Maetens, M; Fumagalli, D; Michiels, S; Drisis, S; Moerman, C; Detiffe, J-P; Larsimont, D; Awada, A; Piccart, M; Sotiriou, C; Ignatiadis, M

2014-10-01

Molecular screening programs use next-generation sequencing (NGS) of cancer gene panels to analyze metastatic biopsies. We interrogated whether plasma could be used as an alternative to metastatic biopsies. The Ion AmpliSeq™ Cancer Hotspot Panel v2 (Ion Torrent), covering 2800 COSMIC mutations from 50 cancer genes was used to analyze 69 tumor (primary/metastases) and 31 plasma samples from 17 metastatic breast cancer patients. The targeted coverage for tumor DNA was ×1000 and for plasma cell-free DNA ×25 000. Whole blood normal DNA was used to exclude germline variants. The Illumina technology was used to confirm observed mutations. Evaluable NGS results were obtained for 60 tumor and 31 plasma samples from 17 patients. When tumor samples were analyzed, 12 of 17 (71%, 95% confidence interval (CI) 44% to 90%) patients had ≥1 mutation (median 1 mutation per patient, range 0-2 mutations) in either p53, PIK3CA, PTEN, AKT1 or IDH2 gene. When plasma samples were analyzed, 12 of 17 (71%, 95% CI: 44-90%) patients had ≥1 mutation (median 1 mutation per patient, range 0-2 mutations) in either p53, PIK3CA, PTEN, AKT1, IDH2 and SMAD4. All mutations were confirmed. When we focused on tumor and plasma samples collected at the same time-point, we observed that, in four patients, no mutation was identified in either tumor or plasma; in nine patients, the same mutations was identified in tumor and plasma; in two patients, a mutation was identified in tumor but not in plasma; in two patients, a mutation was identified in plasma but not in tumor. Thus, in 13 of 17 (76%, 95% CI 50% to 93%) patients, tumor and plasma provided concordant results whereas in 4 of 17 (24%, 95% CI 7% to 50%) patients, the results were discordant, providing complementary information. Plasma can be prospectively tested as an alternative to metastatic biopsies in molecular screening programs. © The Author 2014. Published by Oxford University Press on behalf of the European Society for Medical Oncology

Strikingly different penetrance of LHON in two Chinese families with primary mutation G11778A is independent of mtDNA haplogroup background and secondary mutation G13708A

Energy Technology Data Exchange (ETDEWEB)

Wang Huawei [Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223 (China)]|[Laboratory for Conservation and Utilization of Bio-resource, Yunnan University, Kunming 650091 (China); Jia Xiaoyun; Ji Yanli [State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060 (China); Kong Qingpeng [State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223 (China); Zhang Qingjiong [State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060 (China)], E-mail: qingjiongzhang@yahoo.com; Yao Yonggang [Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223 (China)]|[State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223 (China)], E-mail: ygyaozh@yahoo.com; Zhang Yaping [Laboratory for Conservation and Utilization of Bio-resource, Yunnan University, Kunming 650091 (China)]|[State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223 (China)

2008-08-25

The penetrance of Leber's hereditary optic neuropathy (LHON) in families with primary mitochondrial DNA (mtDNA) mutations is very complex. Matrilineal and nuclear genetic background, as well as environmental factors, have been reported to be involved in different affected pedigrees. Here we describe two large Chinese families that show a striking difference in the penetrance of LHON, in which 53.3% and 15.0% of members were affected (P < 0.02), respectively. Analysis of the complete mtDNA genome of the two families revealed the presence of the primary mutation G11778A and several other variants suggesting the same haplogroup status G2a. The family with higher penetrance contained a previously described secondary mutation G13708A, which presents a polymorphism in normal Chinese samples and does not affect in vivo mitochondrial oxidative metabolism as described in a previous study. Evolutionary analysis failed to indicate any putatively pathogenic mutation that cosegregated with G11778A in these two pedigrees. Our results suggest that the variable penetrance of LHON in the two Chinese families is independent of both their mtDNA haplotype background and a secondary mutation G13708A. As a result, it is likely that unknown nuclear gene involvement and/or other factors contribute to the strikingly different penetrance of LHON.

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

International Nuclear Information System (INIS)

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

1988-01-01

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

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.

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.

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.

Association between mutation spectra and stable and unstable DNA adduct profiles in Salmonella for benzo[a]pyrene and dibenzo[a,l]pyrene

Energy Technology Data Exchange (ETDEWEB)

DeMarini, David M., E-mail: demarini.david@epa.gov [Integrated Systems Toxicology Division, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711 (United States); Hanley, Nancy M.; Warren, Sarah H.; Adams, Linda D.; King, Leon C. [Integrated Systems Toxicology Division, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711 (United States)

2011-09-01

Highlights: {yields} Benzo[a]pyrene (BP) induces stable DNA adducts and mutations primarily at guanine. {yields} Dibenzo[a,l]pyrene (DBP) induces them primarily at adenine. {yields} BP induces abasic sites, but DBP does not in the Salmonella mutagenicity assay. {yields} Stable DNA adducts alone appear to account for the mutation spectrum of DBP. {yields} Stable DNA adducts and possibly abasic sites account for the mutation spectrum of BP. - Abstract: Benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) are two polycyclic aromatic hydrocarbons (PAHs) that exhibit distinctly different mutagenicity and carcinogenicity profiles. Although some studies show that these PAHs produce unstable DNA adducts, conflicting data and arguments have been presented regarding the relative roles of these unstable adducts versus stable adducts, as well as oxidative damage, in the mutagenesis and tumor-mutation spectra of these PAHs. However, no study has determined the mutation spectra along with the stable and unstable DNA adducts in the same system with both PAHs. Thus, we determined the mutagenic potencies and mutation spectra of BP and DBP in strains TA98, TA100 and TA104 of Salmonella, and we also measured the levels of abasic sites (aldehydic-site assay) and characterized the stable DNA adducts ({sup 32}P-postlabeling/HPLC) induced by these PAHs in TA104. Our results for the mutation spectra and site specificity of stable adducts were consistent with those from other systems, showing that DBP was more mutagenic than BP in TA98 and TA100. The mutation spectra of DBP and BP were significantly different in TA98 and TA104, with 24% of the mutations induced by BP in TA98 being complex frameshifts, whereas DBP produced hardly any of these mutations. In TA104, BP produced primarily GC to TA transversions, whereas DBP produced primarily AT to TA transversions. The majority (96%) of stable adducts induced by BP were at guanine, whereas the majority (80%) induced by DBP were at adenine

The Frequency and Type of K-RAS Mutations in Mexican Patients With Colorectal Cancer: A National Study.

Science.gov (United States)

Cárdenas-Ramos, Susana G; Alcázar-González, Gregorio; Reyes-Cortés, Luisa M; Torres-Grimaldo, Abdiel A; Calderón-Garcidueñas, Ana L; Morales-Casas, José; Flores-Sánchez, Patricia; De León-Escobedo, Raúl; Gómez-Díaz, Antonio; Moreno-Bringas, Carmen; Sánchez-Guillén, Jorge; Ramos-Salazar, Pedro; González-de León, César; Barrera-Saldaña, Hugo A

2017-06-01

Current metastatic colorectal cancer (mCRC) therapy uses monoclonal antibodies against the epidermal growth factor receptor. This treatment is only useful in the absence of K-RAS gene mutations; therefore the study of such mutations is part of a personalized treatment. The aim of this work is to determine the frequency and type of the most common K-RAS mutations in Mexican patients with metastatic disease by nucleotide sequencing. We studied 888 patients with mCRC from different regions of Mexico. The presence of mutations in exon 2, codons 12 and 13, of the K-RAS gene was determined by nucleotide sequencing. Patients exhibited K-RAS gene mutations in 35% (310/888) of cases. Mutation frequency of codons 12 and 13 was 71% (221/310) and 29% (89/310), respectively. The most common mutation (45.7%) in codon 12 was c.35G>A (p.G12D), whereas the one in codon 13 was c.38G>A (p.G13D) (78.7%). Given the frequency of K-RAS mutations in Mexicans, making a genetic study before deciding to treat mCRC patients with monoclonal antibodies is indispensable.

Comparison of somatic mutation frequencies at HGPRT locus induced by radiation and chemical pollutant from energy system

International Nuclear Information System (INIS)

Xu Honglan; Cao Yi; Duan Zhikai; Wu Qiqing; Chen Ying; Zhang Shuxian

1998-12-01

The somatic induction frequencies of mutation at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus induced by 60 Co γ-rays and Benzo-a-pyrene (B(a)P), which are representative of hazardous emission and pollutant from nuclear energy cycle and fossil-fuelled energy cycle respectively, were detected by using forward mutation assay and cloning technique in both V 79 Chinese hamster cells and human peripheral blood T-lymphocytes. Resistant mutants were selected with 6-thioguanine (6-TG). Dose-response curves and mathematical expressions were obtained for mutation frequencies and survival following γ-ray and B(a)P(+S 9 ) treatments. The dose ranges for the two mutagens were compared when they induced the same mutation frequencies. In V 79 /HGPRT assay system, when the mutation frequencies were 5∼35 mutants/10 6 cells the response of γ-rays in the dose range from 0.93∼4.96 Gy at dose rate of 1.16 Gy/min is nearly equivalent to that in the B(a)P dose range from 0.52∼4.27 μg/ml. By using cloning technique in T-lymphocytes, when the mutation frequencies were 1∼14 mutants/10 5 cells the response of γ-rays in the dose range from 0.05∼4.77 Gy at dose rate of 1.03 Gy/min is nearly equivalent to that in the B(a)P dose range from 0.15∼7.36 μg/ml. When the survival fraction is 37%, the mutation frequency induced by B(a)P is higher than that induced by 60 Co γ-rays

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

APOBEC3 cytidine deaminases in double-strand DNA break repair and cancer promotion.

Science.gov (United States)

Nowarski, Roni; Kotler, Moshe

2013-06-15

High frequency of cytidine to thymidine conversions was identified in the genome of several types of cancer cells. In breast cancer cells, these mutations are clustered in long DNA regions associated with single-strand DNA (ssDNA), double-strand DNA breaks (DSB), and genomic rearrangements. The observed mutational pattern resembles the deamination signature of cytidine to uridine carried out by members of the APOBEC3 family of cellular deaminases. Consistently, APOBEC3B (A3B) was recently identified as the mutational source in breast cancer cells. A3G is another member of the cytidine deaminases family predominantly expressed in lymphoma cells, where it is involved in mutational DSB repair following ionizing radiation treatments. This activity provides us with a new paradigm for cancer cell survival and tumor promotion and a mechanistic link between ssDNA, DSBs, and clustered mutations. Cancer Res; 73(12); 3494-8. ©2013 AACR. ©2013 AACR.

Reduction of arsenite-enhanced ultraviolet radiation-induced DNA damage by supplemental zinc

Energy Technology Data Exchange (ETDEWEB)

Cooper, Karen L.; King, Brenee S.; Sandoval, Monica M.; Liu, Ke Jian; Hudson, Laurie G., E-mail: lhudson@salud.unm.edu

2013-06-01

Arsenic is a recognized human carcinogen and there is evidence that arsenic augments the carcinogenicity of DNA damaging agents such as ultraviolet radiation (UVR) thereby acting as a co-carcinogen. Inhibition of DNA repair is one proposed mechanism to account for the co-carcinogenic actions of arsenic. We and others find that arsenite interferes with the function of certain zinc finger DNA repair proteins. Furthermore, we reported that zinc reverses the effects of arsenite in cultured cells and a DNA repair target protein, poly (ADP-ribose) polymerase-1. In order to determine whether zinc ameliorates the effects of arsenite on UVR-induced DNA damage in human keratinocytes and in an in vivo model, normal human epidermal keratinocytes and SKH-1 hairless mice were exposed to arsenite, zinc or both before solar-simulated (ss) UVR exposure. Poly (ADP-ribose) polymerase activity, DNA damage and mutation frequencies at the Hprt locus were measured in each treatment group in normal human keratinocytes. DNA damage was assessed in vivo by immunohistochemical staining of skin sections isolated from SKH-1 hairless mice. Cell-based findings demonstrate that ssUVR-induced DNA damage and mutagenesis are enhanced by arsenite, and supplemental zinc partially reverses the arsenite effect. In vivo studies confirm that zinc supplementation decreases arsenite-enhanced DNA damage in response to ssUVR exposure. From these data we can conclude that zinc offsets the impact of arsenic on ssUVR-stimulated DNA damage in cells and in vivo suggesting that zinc supplementation may provide a strategy to improve DNA repair capacity in arsenic exposed human populations. - Highlights: • Low levels of arsenite enhance UV-induced DNA damage in human keratinocytes. • UV-initiated HPRT mutation frequency is enhanced by arsenite. • Zinc supplementation offsets DNA damage and mutation frequency enhanced by arsenite. • Zinc-dependent reduction of arsenite enhanced DNA damage is confirmed in vivo.

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.

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...

Mutations of the phenylalanine hydroxylase gene in patients with phenylketonuria in Shanxi, China

Directory of Open Access Journals (Sweden)

Yong-An Zhou

2012-01-01

Full Text Available The variation in mutations in exons 3, 6, 7, 11 and 12 of the phenylalanine hydroxylase (PAH gene was investigated in 59 children with phenylketonuria (PKU and 100 normal children. Three single nucleotide polymorphisms were detected by sequence analysis. The mutational frequencies of cDNA 696, cDNA 735 and cDNA 1155 in patients were 96.2%, 76.1% and 7.6%, respectively, whereas in healthy children the corresponding frequencies were 97.0%, 77.3% and 8.3%. In addition, 81 mutations accounted for 61.0% of the mutant alleles. R111X, H64 > TfsX9 and S70 del accounted for 5.1%, 0.8% and 0.8% mutation of alleles in exon 3, whereas EX6-96A > G accounted for 10.2% mutation of alleles in exon 6. R243Q had the highest incidence in exon 7 (12.7%, followed by Ivs7 +2T>A (5.1% and T278I (2.5%. G247V, R252Q, L255S, R261Q and E280K accounted for 0.8% while Y356X and V399V accounted for 5.9% and 5.1%, respectively, in exon 11. R413P and A434D accounted for 5.9% and 2.5%, respectively, in exon 12. Seventy-two variant alleles accounted for the 16 mutations observed here. The mutation characteristics and distributions demonstrated that EX6-96A > G and R243Q were the hot regions for mutations in the PAH gene in Shanxi patients with PKU.

High frequency of mutation G377S in Brazilian type 3 Gaucher disease patients

Directory of Open Access Journals (Sweden)

R. Rozenberg

2006-09-01

Full Text Available Gaucher disease (GD, the most prevalent lysosome storage disorder, presents an autosomal recessive mode of inheritance. It is a paradigm for therapeutic intervention in medical genetics due to the existence of effective enzyme replacement therapy. We report here the analysis of GD in 262 unrelated Brazilian patients, carried out in order to establish the frequency of the most common mutations and to provide prognostic information based on genotype-phenotype correlations. Among 247 type 1 GD patients, mutation N370S was detected in 47% of all the alleles, but N370S/N370S homozygosity was found in only 10% of the patients, a much lower frequency than expected, suggesting that most individuals presenting this genotype may not receive medical attention. Recombinant alleles were detected at a high frequency: 44% of the chromosomes bearing mutation L444P had other mutations derived from the pseudogene sequence, present in 25% of patients. Three neuronopathic type 2 patients were homozygous for L444P, all presenting additional mutations (E326K or recombinant alleles that probably lead to the more severe phenotypes. Six children, classified as type 1 GD patients, had a L444P/L444P genotype, showing that neuronopathic symptoms may only manifest later in life. This would indicate the need for a higher treatment dose during enzyme replacement therapy. Finally, mutation G377S was present in 4 homozygous type 1 patients and also in compound heterozygosity in 5 (42% type 3 patients. These findings indicate that G377S cannot be unambiguously classified as mild and suggest an allele-dose effect for this mutation.

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

Science.gov (United States)

Gonzalez-Bosquet, Jesus; Calcei, Jacob; Wei, Jun S; Garcia-Closas, Montserrat; Sherman, Mark E; Hewitt, Stephen; Vockley, Joseph; Lissowska, Jolanta; Yang, Hannah P; Khan, Javed; Chanock, Stephen

2011-01-17

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.

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.

Validation and comparison of two NGS assays for the detection of EGFR T790M resistance mutation in liquid biopsies of NSCLC patients.

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Vollbrecht, Claudia; Lehmann, Annika; Lenze, Dido; Hummel, Michael

2018-04-06

Analysis of circulating cell-free DNA (cfDNA) derived from peripheral blood ("liquid biopsy") is an attractive alternative to identify non-small cell lung cancer (NSCLC) patients with the EGFR T790M mutation eligible for 3rd generation tyrosine kinase inhibitor therapy. We evaluated two PCR-based next generation sequencing (NGS) approaches, one including unique molecular identifiers (UMI), with focus on highly sensitive EGFR T790M mutation detection. Therefore, we extracted and sequenced cfDNA from synthetic plasma samples spiked with mutated DNA at decreasing allele frequencies and from 21 diagnostic NSCLC patients. Data evaluation was performed to determine the limit of detection (LoD), accuracy, specificity and sensitivity of both assays. Considering all tested reference dilutions and mutations the UMI assay performed best in terms of LoD (1% vs. 5%), sensitivity (95.8% vs. 81.3%), specificity (100% vs. 93.8%) and accuracy (96.9% vs. 84.4%). Comparing mutation status of diagnostic samples with both assays showed 81.3% concordance with primary mutation verifiable in 52% of cases. EGFR T790M was detected concordantly in 6/7 patients with allele frequencies from 0.1% to 27%. In one patient, the T790M mutation was exclusively detectable with the UMI assay. Our data demonstrate that both assays are applicable as multi-biomarker NGS tools enabling the simultaneous detection of primary EGFR driver and resistance mutations. However, for mutations with low allelic frequencies the use of NGS panels with UMI facilitates a more sensitive and reliable detection.

A switch from high-fidelity to error-prone DNA double-strand break repair underlies stress-induced mutation.

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Ponder, Rebecca G; Fonville, Natalie C; Rosenberg, Susan M

2005-09-16

Special mechanisms of mutation are induced in microbes under growth-limiting stress causing genetic instability, including occasional adaptive mutations that may speed evolution. Both the mutation mechanisms and their control by stress have remained elusive. We provide evidence that the molecular basis for stress-induced mutagenesis in an E. coli model is error-prone DNA double-strand break repair (DSBR). I-SceI-endonuclease-induced DSBs strongly activate stress-induced mutations near the DSB, but not globally. The same proteins are required as for cells without induced DSBs: DSBR proteins, DinB-error-prone polymerase, and the RpoS starvation-stress-response regulator. Mutation is promoted by homology between cut and uncut DNA molecules, supporting a homology-mediated DSBR mechanism. DSBs also promote gene amplification. Finally, DSBs activate mutation only during stationary phase/starvation but will during exponential growth if RpoS is expressed. Our findings reveal an RpoS-controlled switch from high-fidelity to mutagenic DSBR under stress. This limits genetic instability both in time and to localized genome regions, potentially important evolutionary strategies.

Mutational specificity of γ-rays

International Nuclear Information System (INIS)

Hoebee, Barbara.

1990-01-01

The aim of the study described in this thesis was to get more information on the mutagenic properties of radiation-induced DNA modifications and the possible mechanisms involved in radiation-induced mutagenesis, principally by investigating the kinds of mutations by DNA sequence analysis. The mutations were analyzed after γ-irradiation of recombinant bacteriophage M13 and plasmide pUC DNA in diluted aqueous solutions, followed by transfection or transformation to E. coli cells, in which the damaged DNA molecules are repaired and replicated. Error-prone repair, misrepair or bypass of lesions during replication may lead to the introduction of mutations. Both the M13 and the plasmid DNA used in our mutation studies contain a mutation target sequence, which makes an easy selection and sequence analysis of mutant DNA molecules possible. Under the radiation conditions used, e.g. irradiation of diluted aqueous DNA solutions, only DNA damage occurs introduced by the water derived OH* and H* radicals and the hydrated electrons. By using different gas conditions during irradiation the relative yields of these reaction species can be manipulated, which opens up the opportunity to determine their effects separately. The mutation spectrum obtained in double-stranded (ds) M13DNA after irradiation under oxic conditions and the mutation spectrum obtained under the same conditions and in the same mutation target but cloned in plasmid DNA, are described. The mutation specificity under anoxic conditions in ds M13DNA is given. Results obtained after irradiation of ds M13DNA under N 2 conditions are discussed together with experiments with single-stranded DNA. Similarities and differences between radiation-induced mutation spectra obtained by other groups and those presented in this thesis are discussed. (author). 155 refs.; 134 figs.; 16 tabs

The frequency of Tay-Sachs disease causing mutations in the Brazilian Jewish population justifies a carrier screening program

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Roberto Rozenberg

Full Text Available CONTEXT: Tay-Sachs disease is an autosomal recessive disease characterized by progressive neurologic degeneration, fatal in early childhood. In the Ashkenazi Jewish population the disease incidence is about 1 in every 3,500 newborns and the carrier frequency is 1 in every 29 individuals. Carrier screening programs for Tay-Sachs disease have reduced disease incidence by 90% in high-risk populations in several countries. The Brazilian Jewish population is estimated at 90,000 individuals. Currently, there is no screening program for Tay-Sachs disease in this population. OBJECTIVE: To evaluate the importance of a Tay-Sachs disease carrier screening program in the Brazilian Jewish population by determining the frequency of heterozygotes and the acceptance of the program by the community. SETTING: Laboratory of Molecular Genetics - Institute of Biosciences - Universidade de São Paulo. PARTICIPANTS: 581 senior students from selected Jewish high schools. PROCEDURE: Molecular analysis of Tay-Sachs disease causing mutations by PCR amplification of genomic DNA, followed by restriction enzyme digestion. RESULTS: Among 581 students that attended educational classes, 404 (70% elected to be tested for Tay-Sachs disease mutations. Of these, approximately 65% were of Ashkenazi Jewish origin. Eight carriers were detected corresponding to a carrier frequency of 1 in every 33 individuals in the Ashkenazi Jewish fraction of the sample. CONCLUSION: The frequency of Tay-Sachs disease carriers among the Ashkenazi Jewish population of Brazil is similar to that of other countries where carrier screening programs have led to a significant decrease in disease incidence. Therefore, it is justifiable to implement a Tay-Sachs disease carrier screening program for the Brazilian Jewish population.

Clinical heterogeneity within xeroderma pigmentosum associated with mutations in the DNA repair and transcription gene ERCC3

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Vermeulen, W.; Kleijer, W.J.; Bootsma, D.; Hoeijmakers, J.H.J.; Weeda, G. (Erasmus Univ., Rotterdam (Netherlands)); Scott, R.J.; Rodgers, S.; Mueller, H.J. (Univ. Hospital, Basel (Switzerland)); Cole, J.; Arlett, C.F. (Univ. of Sussex, Brighton (United Kingdom))

1994-02-01

The human DNA excision repair gene ERCC3 specifically corrects the nucleotide excision repair (NER) defect of xeroderma pigmentosum (XP) complementation group B. In addition to its function in NER, the ERCC3 DNA helicase was recently identified as one of the components of the human BTF2/TFIIH transcription factor complex, which is required for initiation of transcription of class II genes. To date, a single patient (XP11BE) has been assigned to this XP group B (XP-B), with the remarkable conjunction of two autosomal recessive DNA repair deficiency disorders: XP and Cockayne syndrome (CS). The intriguing involvement of the ERCC3 protein in the vital process of transcription may provide an explanation for the rarity, severity, and wide spectrum of clinical features in this complementation group. Here the authors report the identification of two new XP-B patients: XPCS1BA and XPCS2BA (siblings), by microneedle injection of the cloned ERCC3 repair gene as well as by cell hybridization. Molecular analysis of the ERCC3 gene in both patients revealed a single base substitution causing a missense mutation in a region that is completely conserved in yeast, Drosophila, mouse, and human ERCC3. As in patient XP11BE, the expression of only one allele (paternal) is detected. The mutation causes a virtually complete inactivation of the NER function of the protein. Despite this severe NER defect, both patients display a late onset of neurologic impairment, mild cutaneous symptoms, and a striking absence of skin tumors even at an age of >40 years. Analysis of the frequency of hprt[sup [minus

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

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

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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

Validation of high-resolution DNA melting analysis for mutation scanning of the CDKL5 gene: identification of novel mutations.

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Raymond, Laure; Diebold, Bertrand; Leroux, Céline; Maurey, Hélène; Drouin-Garraud, Valérie; Delahaye, Andre; Dulac, Olivier; Metreau, Julia; Melikishvili, Gia; Toutain, Annick; Rivier, François; Bahi-Buisson, Nadia; Bienvenu, Thierry

2013-01-01

Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) have been predominantly described in epileptic encephalopathies of female, including infantile spasms with Rett-like features. Up to now, detection of mutations in this gene was made by laborious, expensive and/or time consuming methods. Here, we decided to validate high-resolution melting analysis (HRMA) for mutation scanning of the CDKL5 gene. Firstly, using a large DNA bank consisting to 34 samples carrying different mutations and polymorphisms, we validated our analytical conditions to analyse the different exons and flanking intronic sequences of the CDKL5 gene by HRMA. Secondly, we screened CDKL5 by both HRMA and denaturing high performance liquid chromatography (dHPLC) in a cohort of 135 patients with early-onset seizures. Our results showed that point mutations and small insertions and deletions can be reliably detected by HRMA. Compared to dHPLC, HRMA profiles are more discriminated, thereby decreasing unnecessary sequencing. In this study, we identified eleven novel sequence variations including four pathogenic mutations (2.96% prevalence). HRMA appears cost-effective, easy to set up, highly sensitive, non-toxic and rapid for mutation screening, ideally suited for large genes with heterogeneous mutations located along the whole coding sequence, such as the CDKL5 gene. Copyright © 2012 Elsevier B.V. All rights reserved.

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.

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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.

Mutation breeding in ornamental plants

International Nuclear Information System (INIS)

Datta, S.K.

1990-01-01

Full text: Mutation induction produced a large number of new promising varieties in ornamental species. 37 new mutants of Chrysanthemum and 14 of rose have been developed by mutations and released for commercialisation. The mutations in flower colour/shape were detected as chimeras in M 1 V 1 , M 1 V 2 , M 1 V 3 generations. The mutation frequency varied with the cultivar and exposure to gamma rays. Comparative analysis of original cultivars and their respective induced mutants on cytomorphological, anatomical and biochemical characters are being carried out for better understanding of the mechanism involved in the origin and evolution of somatic flower colour/shape mutations. Cytological analysis with reference to chromosomal aberrations, chromosome number, ICV, INV and DNA content gave no differences between the original and mutant cultivars. Analysis of florets/petal pigments by TLC and spectrophotometric methods indicated both qualitative and quantitative changes. (author)

MELAS syndrome associated with both A3243G-tRNALeu mutation and multiple mitochondrial DNA deletions.

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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.

Modification of radiation-induced sex-linked recessive lethal mutation frequency by tocopherol

International Nuclear Information System (INIS)

Beckman, C.; Roy, R.M.; Sproule, A.

1982-01-01

The present study evaluates the effect of supplementing culture medium with α-tocopherol acetate on the yield of sex-linked recessive lethal mutants induced by X-irradiation in mature sperm of Drosophila. Although tocopherol treatment of males had no impact on the yield of mutations, a drastic reduction in mutation frequency was observed when irradiated males were mated to females raised and subsequently maintained on tocopherol-enriched diet. (orig./MG)

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

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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.

Sun exposure causes somatic second-hit mutations and angiofibroma development in tuberous sclerosis complex

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Tyburczy, Magdalena E.; Wang, Ji-an; Li, Shaowei; Thangapazham, Rajesh; Chekaluk, Yvonne; Moss, Joel; Kwiatkowski, David J.; Darling, Thomas N.

2014-01-01

Tuberous sclerosis complex (TSC) is characterized by the formation of tumors in multiple organs and is caused by germline mutation in one of two tumor suppressor genes, TSC1 and TSC2. As for other tumor suppressor gene syndromes, the mechanism of somatic second-hit events in TSC tumors is unknown. We grew fibroblast-like cells from 29 TSC skin tumors from 22 TSC subjects and identified germline and second-hit mutations in TSC1/TSC2 using next-generation sequencing. Eighteen of 22 (82%) subjects had a mutation identified, and 8 of the 18 (44%) subjects were mosaic with mutant allele frequencies of 0 to 19% in normal tissue DNA. Multiple tumors were available from four patients, and in each case, second-hit mutations in TSC2 were distinct indicating they arose independently. Most remarkably, 7 (50%) of the 14 somatic point mutations were CC>TT ultraviolet ‘signature’ mutations, never seen as a TSC germline mutation. These occurred exclusively in facial angiofibroma tumors from sun-exposed sites. These results implicate UV-induced DNA damage as a cause of second-hit mutations and development of TSC facial angiofibromas and suggest that measures to limit UV exposure in TSC children and adults should reduce the frequency and severity of these lesions. PMID:24271014

Mutagenesis and repair of DNA

International Nuclear Information System (INIS)

Janion, C.; Grzesiuk, E.; Fabisiewicz, A.; Tudek, B.; Ciesla, J.; Graziewicz, M.; Wojcik, A.; Speina, E.

1998-01-01

Full text. The discovery that the mfd gene codes for a transcription-coupling repair factor (TRCF) prompted us to re-investigate the MFD (mutation frequency decline) phenomenon in E.coli K-12 strain when mutations were induced by ultraviolet light, halogen light or MMS-treatment. These studies revealed that: (i) the process of MFD involves the proofreading activity of DNA pol III and the mismatch repair system, as well as, TRCF and the UvrABC-excinuclease (ii) a semi-rich plate test may be replaced by a rich liquid medium, (iii) the T-T pyrimidine dimers are the lesions excised with the highest activity, and (iv) overproduction of UmuD(D'C) proteins leads to a great increase in mutant frequency in irradiated and MMS-treated cells. The role of mismatch repair (MR) in MMS-induced mutagenesis is obscured by the fact that the spectra of mutational specificity are different in bacteria proficient and deficient in MR. It has been found that transposons Tn10 (and Tn5) when inserted into chromosomal DNA of E. coli influence the phenotype lowering the survival and frequency of mutations induced by UV or halogen light irradiation. This is connected with a deficiency of UmuD(D') and UmuC proteins. Transformation of bacteria with plasmids bearing the umuD(D')C genes, suppresses the effects of the transposon insertion, a phenomenon which has not been described before. Single-stranded DNA of M13mp18 phage was oxidized in vitro by a hydroxyl radical generating system including hypoxanthine/xanthine oxidase/Fe3+/EDTA, and it was found that Fapy-Ade, Fapy-Gua, 8-oxyAde and thymine glycol were the main products formed. Replication of the oxidized template by T7 phage DNA polymerase, Klenow fragment of polymerase I, or polymerase beta from bovine thymus has revealed that oxidized pyrimidines are stronger blockers than oxidized purines for T7 phage and Klenow fragment polymerases and the blocking potency depends on the neighboring bases and on the type of polymerase. Studies of

A mild mutator phenotype arises in a mouse model for malignancies associated with neurofibromatosis type 1

International Nuclear Information System (INIS)

Garza, Rene; Hudson, Robert A.; McMahan, C. Alex; Walter, Christi A.; Vogel, Kristine S.

2007-01-01

Defects in genes that control DNA repair, proliferation, and apoptosis can increase genomic instability, and thus promote malignant progression. Although most tumors that arise in humans with neurofibromatosis type 1 (NF1) are benign, these individuals are at increased risk for malignant peripheral nerve sheath tumors (MPNST). To characterize additional mutations required for the development of MPNST from benign plexiform neurofibromas, we generated a mouse model for these tumors by combining targeted null mutations in Nf1 and p53, in cis. CisNf1+/-; p53+/- mice spontaneously develop PNST, and these tumors exhibit loss-of-heterozygosity at both the Nf1 and p53 loci. Because p53 has well-characterized roles in the DNA damage response, DNA repair, and apoptosis, and because DNA repair genes have been proposed to act as modifiers in NF1, we used the cisNf1+/-; p53+/- mice to determine whether a mutator phenotype arises in NF1-associated malignancies. To quantitate spontaneous mutant frequencies (MF), we crossed the Big Blue mouse, which harbors a lacI transgene, to the cisNf1+/-; p53+/- mice, and isolated genomic DNA from both tumor and normal tissues in compound heterozygotes and wild-type siblings. Many of the PNST exhibited increased mutant frequencies (MF = 4.70) when compared to normal peripheral nerve and brain (MF = 2.09); mutations occurred throughout the entire lacI gene, and included base substitutions, insertions, and deletions. Moreover, the brains, spleens, and livers of these cisNf1+/-; p53+/- animals exhibited increased mutant frequencies when compared to tissues from wild-type littermates. We conclude that a mild mutator phenotype arises in the tumors and tissues of cisNf1+/-; p53+/- mice, and propose that genomic instability influences NF1 tumor progression and disease severity

Mutation Rate Variation is a Primary Determinant of the Distribution of Allele Frequencies in Humans.

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Arbel Harpak

2016-12-01

Full Text Available The site frequency spectrum (SFS has long been used to study demographic history and natural selection. Here, we extend this summary by examining the SFS conditional on the alleles found at the same site in other species. We refer to this extension as the "phylogenetically-conditioned SFS" or cSFS. Using recent large-sample data from the Exome Aggregation Consortium (ExAC, combined with primate genome sequences, we find that human variants that occurred independently in closely related primate lineages are at higher frequencies in humans than variants with parallel substitutions in more distant primates. We show that this effect is largely due to sites with elevated mutation rates causing significant departures from the widely-used infinite sites mutation model. Our analysis also suggests substantial variation in mutation rates even among mutations involving the same nucleotide changes. In summary, we show that variable mutation rates are key determinants of the SFS in humans.

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

DEFF Research Database (Denmark)

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

2013-01-01

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

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

Molecular analysis on germline mutation caused by low-dose irradiation

International Nuclear Information System (INIS)

Uchiyama, R.; Fujikawa, K.; Nishimura, M.; Adzuma, H.; Shimada, Y.; Yamauchi, M.

2003-01-01

Full text: Genetic heterogeneity and a low frequency of germline mutation at single-copy gene loci have limited the direct measurement of germline mutation in human populations. Two conflicting results have been reported for the effect of ionizing radiation on germline mutation in human populations. A study conducted on the first-generation progeny of the survivors of the atomic bombs at Hiroshima and Nagasaki found no significant increase in germline mutations. On the other hand, a significant increase in germline mutation was reported among the human population in the Belarus area after the Chernobyl accident in 1986. We investigated the germline mutation at the molecular level using experimental mouse strains with different genetic backgrounds to assess the risk of ionizing radiation on human populations. The C3H male parents were exposed to X ray (0, 0.3, 1, and 3Gy) and mated with unexposed C57BL females after two weeks interval, so as to detect the germline mutation occurred at the spermatid stage. Genomic DNA samples were prepared from the both parents and F1s, and the genomic DNA sequences were compared between parents and offspring at the specific genomic gene loci, such as adenine phosphoribosyl transferase (aprt) gene and cytidine triphosphate synthetase (ctps) gene, using the automated DNA sequencer. Also hypervariable Pc-1 (Ms6-hm) minisatellite repeat locus was analyzed by using Southern blot hybridization technique. Our preliminary results indicated that the changes of the restriction DNA fragment length in offspring did not reflect the occurrence of the mutation, such as point mutation, insertion, and deletion, in the genomic gene loci including the intervening sequence (intron)

Contribution of TARDBP mutations to sporadic amyotrophic lateral sclerosis.

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Daoud, H; Valdmanis, P N; Kabashi, E; Dion, P; Dupré, N; Camu, W; Meininger, V; Rouleau, G A

2009-02-01

Mutations in the TARDBP gene, which encodes the TAR DNA binding protein (TDP-43), have been described in individuals with familial and sporadic amyotrophic lateral sclerosis (ALS). We screened the TARDBP gene in 285 French sporadic ALS patients to assess the frequency of TARDBP mutations in ALS. Six individuals had potentially deleterious mutations of which three were novel including a Y374X truncating mutation and P363A and A382P missense mutations. This suggests that TARDBP mutations may predispose to ALS in approximately 2% of the individuals followed in this study. Our findings, combined with those from other collections, brings the total number of mutations in unrelated ALS patients to 17, further suggesting that mutations in the TARDBP gene have an important role in the pathogenesis of ALS.

Ancient DNA analysis reveals high frequency of European lactase persistence allele (T-13910 in medieval central europe.

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Annina Krüttli

Full Text Available Ruminant milk and dairy products are important food resources in many European, African, and Middle Eastern societies. These regions are also associated with derived genetic variants for lactase persistence. In mammals, lactase, the enzyme that hydrolyzes the milk sugar lactose, is normally down-regulated after weaning, but at least five human populations around the world have independently evolved mutations regulating the expression of the lactase-phlorizin-hydrolase gene. These mutations result in a dominant lactase persistence phenotype and continued lactase tolerance in adulthood. A single nucleotide polymorphism (SNP at C/T-13910 is responsible for most lactase persistence in European populations, but when and where the T-13910 polymorphism originated and the evolutionary processes by which it rose to high frequency in Europe have been the subject of strong debate. A history of dairying is presumed to be a prerequisite, but archaeological evidence is lacking. In this study, DNA was extracted from the dentine of 36 individuals excavated at a medieval cemetery in Dalheim, Germany. Eighteen individuals were successfully genotyped for the C/T-13910 SNP by molecular cloning and sequencing, of which 13 (72% exhibited a European lactase persistence genotype: 44% CT, 28% TT. Previous ancient DNA-based studies found that lactase persistence genotypes fall below detection levels in most regions of Neolithic Europe. Our research shows that by AD 1200, lactase persistence frequency had risen to over 70% in this community in western Central Europe. Given that lactase persistence genotype frequency in present-day Germany and Austria is estimated at 71-80%, our results suggest that genetic lactase persistence likely reached modern levels before the historic population declines associated with the Black Death, thus excluding plague-associated evolutionary forces in the rise of lactase persistence in this region. This new evidence sheds light on the dynamic

Mutational screening of the USH2A gene in Spanish USH patients reveals 23 novel pathogenic mutations

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Diaz-Llopis Manuel

2011-10-01

Full Text Available Abstract Background Usher Syndrome type II (USH2 is an autosomal recessive disorder, characterized by moderate to severe hearing impairment and retinitis pigmentosa (RP. Among the three genes implicated, mutations in the USH2A gene account for 74-90% of the USH2 cases. Methods To identify the genetic cause of the disease and determine the frequency of USH2A mutations in a cohort of 88 unrelated USH Spanish patients, we carried out a mutation screening of the 72 coding exons of this gene by direct sequencing. Moreover, we performed functional minigene studies for those changes that were predicted to affect splicing. Results As a result, a total of 144 DNA sequence variants were identified. Based upon previous studies, allele frequencies, segregation analysis, bioinformatics' predictions and in vitro experiments, 37 variants (23 of them novel were classified as pathogenic mutations. Conclusions This report provide a wide spectrum of USH2A mutations and clinical features, including atypical Usher syndrome phenotypes resembling Usher syndrome type I. Considering only the patients clearly diagnosed with Usher syndrome type II, and results obtained in this and previous studies, we can state that mutations in USH2A are responsible for 76.1% of USH2 disease in patients of Spanish origin.

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...

A comparative investigation of DNA strand breaks, sister chromatid exchanges and K-ras gene mutations induced by cadmium salts in cultured human cells

International Nuclear Information System (INIS)

Mouron, Silvana Andrea; Grillo, Claudia Alejandra; Dulout, Fernando Noel; Golijow, Carlos Daniel

2004-01-01

Cadmium (Cd) is a toxic heavy metal of continuing occupational and environmental concern with a wide variety of adverse effects. Several studies have shown that cadmium produces DNA strand breaks, DNA-protein cross-links, oxidative DNA damage, chromosomal aberrations, dysregulation of gene expression resulting in enhanced proliferation, depressed apoptosis and/or altered DNA repair. This study was undertaken to investigate the ability of cadmium chloride (CdCl 2 ) and cadmium sulphate (CdSO 4 ) to induce point mutations in codon 12 of the K-ras protooncogene assessed by polymerase chain reaction-single strand conformation polymorphisms (PCR-SSCP) and RFLP-enriched PCR methods. Also their genotoxic effects were analyzed by the comet assay and sister chromatid exchanges test. The human lung fibroblast cell line MRC-5 was used for the experiments. Sister chromatid exchanges assay (SCEs) frequencies were significantly increased in cells exposed to cadmium salts in relation to controls (p < 0.001). Despite the slow increment observed in the three comet parameters considered when cells were treated with cadmium chloride, significant differences between groups were only found in the variable comet moment (CM) (p < 0.005). On the other hand, when cells were exposed to cadmium sulphate, the Kruskal-Wallis test showed highly significant differences between groups for migration, tail moment and comet moment parameters (p < 0.001). Nevertheless, a null or weak point mutation induction in K-ras protooncogene was detected using polymerase chain reaction-low ionic strength-single strand conformation polymorphisms (PCR-LIS-SSCP) and RFLP-enriched PCR methods when cells were treated with cadmium salts. Thus, inorganic cadmium produces genotoxicity in human lung fibroblast MRC-5 cells, in the absence of significant point mutation of the K-ras gene

Identification of three novel NHS mutations in families with Nance-Horan syndrome.

Science.gov (United States)

Huang, Kristen M; Wu, Junhua; Brooks, Simon P; Hardcastle, Alison J; Lewis, Richard Alan; Stambolian, Dwight

2007-03-27

Nance-Horan Syndrome (NHS) is an infrequent and often overlooked X-linked disorder characterized by dense congenital cataracts, microphthalmia, and dental abnormalities. The syndrome is caused by mutations in the NHS gene, whose function is not known. The purpose of this study was to identify the frequency and distribution of NHS gene mutations and compare genotype with Nance-Horan phenotype in five North American NHS families. Genomic DNA was isolated from white blood cells from NHS patients and family members. The NHS gene coding region and its splice site donor and acceptor regions were amplified from genomic DNA by PCR, and the amplicons were sequenced directly. We identified three unique NHS coding region mutations in these NHS families. This report extends the number of unique identified NHS mutations to 14.

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.

Frequency and spectrum of mutations induced by gamma irradiation in single, double and triple dwarf wheats

International Nuclear Information System (INIS)

Dhonukshe, B.L.

1981-01-01

Induced mutation studies were carried with three dwarf wheat varieties viz., ''Sonalika'', ''Chhoti Lerma'' and ''Hira'', considered to be single, double and trible dwarfs, respectively. Gamma-rays were used as a source of irradiation. Frequency of chlorophyll mutations were comparatively low and the spectrum was narrow. Chlorophyll mutations were altogether absent in the variety ''Sonalika''. A very wide spectrum of viable mutations affecting stem, leaf, ear growth habit, maturity and fertility characteristics was observed in the M 2 . The cumulative frequency of all the mutants together was quite high, which varied with the varieties. There were varietal differences in the composition and width of the spectrum induced by gamma-rays. The dwarf mutants having desirable leaf and spike characters were isolated in all the three varieties. (author)

Somatic point mutations in mtDNA control region are influenced by genetic background and associated with healthy aging: a GEHA study

DEFF Research Database (Denmark)

Rose, Giuseppina; Romeo, Giuseppe; Dato, Serena

2010-01-01

and of mortality risk in the elderly. Our study provides new evidence on the relevance of mtDNA somatic mutations in aging and longevity and confirms that the occurrence of specific point mutations in the mtDNA control region may represent a strategy for the age-related remodelling of organismal functions....

Mitochondrial DNA polymerase editing mutation, PolgD257A, reduces the diabetic phenotype of Akita male mice by suppressing appetite.

Science.gov (United States)

Fox, Raymond; Kim, Hyung-Suk; Reddick, Robert L; Kujoth, Gregory C; Prolla, Tomas A; Tsutsumi, Shuichi; Wada, Youichiro; Smithies, Oliver; Maeda, Nobuyo

2011-05-24

Diabetes and the development of its complications have been associated with mitochondrial DNA (mtDNA) dysfunction, but causal relationships remain undetermined. With the objective of testing whether increased mtDNA mutations exacerbate the diabetic phenotype, we have compared mice heterozygous for the Akita diabetogenic mutation (Akita) with mice homozygous for the D257A mutation in mitochondrial DNA polymerase gamma (Polg) or with mice having both mutations (Polg-Akita). The Polg-D257A protein is defective in proofreading and increases mtDNA mutations. At 3 mo of age, the Polg-Akita and Akita male mice were equally hyperglycemic. Unexpectedly, as the Polg-Akita males aged to 9 mo, their diabetic symptoms decreased. Thus, their hyperglycemia, hyperphagia and urine output declined significantly. The decrease in their food intake was accompanied by increased plasma leptin and decreased plasma ghrelin, while hypothalamic expression of the orexic gene, neuropeptide Y, was lower and expression of the anorexic gene, proopiomelanocortin, was higher. Testis function progressively worsened with age in the double mutants, and plasma testosterone levels in 9-mo-old Polg-Akita males were significantly reduced compared with Akita males. The hyperglycemia and hyperphagia returned in aged Polg-Akita males after testosterone administration. Hyperglycemia-associated distal tubular damage in the kidney also returned, and Polg-D257A-associated proximal tubular damage was enhanced. The mild diabetes of female Akita mice was not affected by the Polg-D257A mutation. We conclude that reduced diabetic symptoms of aging Polg-Akita males results from appetite suppression triggered by decreased testosterone associated with damage to the Leydig cells of the testis.

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...

Analysis of mutant frequencies and mutation spectra in hMTH1 knockdown TK6 cells exposed to UV radiation

Energy Technology Data Exchange (ETDEWEB)

Fotouhi, Asal [Center for Radiation Protection Research, Department of Molecular Biosciences, Wenner-Gren Institute, Stockholm University (Sweden); Hagos, Winta Woldai [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Ilic, Marina; Wojcik, Andrzej; Harms-Ringdahl, Mats [Center for Radiation Protection Research, Department of Molecular Biosciences, Wenner-Gren Institute, Stockholm University (Sweden); Gruijl, Frank de [Department of Dermatology, Leiden University Medical Center, Leiden (Netherlands); Mullenders, Leon; Jansen, Jacob G. [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Haghdoost, Siamak, E-mail: Siamak.Haghdoost@su.se [Center for Radiation Protection Research, Department of Molecular Biosciences, Wenner-Gren Institute, Stockholm University (Sweden)

2013-11-15

Highlights: • hMTH1 protects cells from mutagenesis induced by UVA and UVB, but not UVC. • No protective role of hMTH1 in cell survival post UVB and UVC irradiation. • hMTH1 prevents induction of transition-type mutations at AT and GC post UVA irradiation. • 2-OH-dATP rather than 8-oxo-dGTP in the nucleotide pool likely contributes in UVA-induced mutations. - Abstract: Ultraviolet radiation is a highly mutagenic agent that damages the DNA by the formation of mutagenic photoproducts at dipyrimidine sites and by oxidative DNA damages via reactive oxygen species (ROS). ROS can also give rise to mutations via oxidation of dNTPs in the nucleotide pool, e.g. 8-oxo-dGTP and 2-OH-dATP and subsequent incorporation during DNA replication. Here we show that expression of human MutT homolog 1 (hMTH1) which sanitizes the nucleotide pool by dephosphorylating oxidized dNTPs, protects against mutagenesis induced by long wave UVA light and by UVB light but not by short wave UVC light. Mutational spectra analyses of UVA-induced mutations at the endogenous Thymidine kinase gene in human lymphoblastoid cells revealed that hMTH1 mainly protects cells from transitions at GC and AT base pairs.

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)

Mitochondrial DNA sequence characteristics modulate the size of the genetic bottleneck.

Science.gov (United States)

Wilson, Ian J; Carling, Phillipa J; Alston, Charlotte L; Floros, Vasileios I; Pyle, Angela; Hudson, Gavin; Sallevelt, Suzanne C E H; Lamperti, Costanza; Carelli, Valerio; Bindoff, Laurence A; Samuels, David C; Wonnapinij, Passorn; Zeviani, Massimo; Taylor, Robert W; Smeets, Hubert J M; Horvath, Rita; Chinnery, Patrick F

2016-03-01

With a combined carrier frequency of 1:200, heteroplasmic mitochondrial DNA (mtDNA) mutations cause human disease in ∼1:5000 of the population. Rapid shifts in the level of heteroplasmy seen within a single generation contribute to the wide range in the severity of clinical phenotypes seen in families transmitting mtDNA disease, consistent with a genetic bottleneck during transmission. Although preliminary evidence from human pedigrees points towards a random drift process underlying the shifting heteroplasmy, some reports describe differences in segregation pattern between different mtDNA mutations. However, based on limited observations and with no direct comparisons, it is not clear whether these observations simply reflect pedigree ascertainment and publication bias. To address this issue, we studied 577 mother-child pairs transmitting the m.11778G>A, m.3460G>A, m.8344A>G, m.8993T>G/C and m.3243A>G mtDNA mutations. Our analysis controlled for inter-assay differences, inter-laboratory variation and ascertainment bias. We found no evidence of selection during transmission but show that different mtDNA mutations segregate at different rates in human pedigrees. m.8993T>G/C segregated significantly faster than m.11778G>A, m.8344A>G and m.3243A>G, consistent with a tighter mtDNA genetic bottleneck in m.8993T>G/C pedigrees. Our observations support the existence of different genetic bottlenecks primarily determined by the underlying mtDNA mutation, explaining the different inheritance patterns observed in human pedigrees transmitting pathogenic mtDNA mutations. © The Author 2016. Published by Oxford University Press.

Mutagenic effect of radionuclides incorporated into DNA of Drosophila melanogaster. Progress report, 1978-1979

International Nuclear Information System (INIS)

Lee, W.R.

1979-01-01

Current progress in studies on the mutagenic effect of 3 H incorporated into the DNA of Drosophila melanogaster is reported. It was shown that selected 3 H precursors incorporated into DNA are metabolized. The forms (metabolites) of tritium found in the DNA molecules and the mutation frequencies resulting therefrom were identified. An alcohol dehydrogenase system was developed for recovering mutations that is capable of distinguishing between base changes and chain breakage events that may lead to the formation of deletions

Detection of MPLW515L/K mutations and determination of allele frequencies with a single-tube PCR assay.

Science.gov (United States)

Takei, Hiraku; Morishita, Soji; Araki, Marito; Edahiro, Yoko; Sunami, Yoshitaka; Hironaka, Yumi; Noda, Naohiro; Sekiguchi, Yuji; Tsuneda, Satoshi; Ohsaka, Akimichi; Komatsu, Norio

2014-01-01

A gain-of-function mutation in the myeloproliferative leukemia virus (MPL) gene, which encodes the thrombopoietin receptor, has been identified in patients with essential thrombocythemia and primary myelofibrosis, subgroups of classic myeloproliferative neoplasms (MPNs). The presence of MPL gene mutations is a critical diagnostic criterion for these diseases. Here, we developed a rapid, simple, and cost-effective method of detecting two major MPL mutations, MPLW515L/K, in a single PCR assay; we termed this method DARMS (dual amplification refractory mutation system)-PCR. DARMS-PCR is designed to produce three different PCR products corresponding to MPLW515L, MPLW515K, and all MPL alleles. The amplicons are later detected and quantified using a capillary sequencer to determine the relative frequencies of the mutant and wild-type alleles. Applying DARMS-PCR to human specimens, we successfully identified MPL mutations in MPN patients, with the exception of patients bearing mutant allele frequencies below the detection limit (5%) of this method. The MPL mutant allele frequencies determined using DARMS-PCR correlated strongly with the values determined using deep sequencing. Thus, we demonstrated the potential of DARMS-PCR to detect MPL mutations and determine the allele frequencies in a timely and cost-effective manner.

Detection of MPLW515L/K mutations and determination of allele frequencies with a single-tube PCR assay.

Directory of Open Access Journals (Sweden)

Hiraku Takei

Full Text Available A gain-of-function mutation in the myeloproliferative leukemia virus (MPL gene, which encodes the thrombopoietin receptor, has been identified in patients with essential thrombocythemia and primary myelofibrosis, subgroups of classic myeloproliferative neoplasms (MPNs. The presence of MPL gene mutations is a critical diagnostic criterion for these diseases. Here, we developed a rapid, simple, and cost-effective method of detecting two major MPL mutations, MPLW515L/K, in a single PCR assay; we termed this method DARMS (dual amplification refractory mutation system-PCR. DARMS-PCR is designed to produce three different PCR products corresponding to MPLW515L, MPLW515K, and all MPL alleles. The amplicons are later detected and quantified using a capillary sequencer to determine the relative frequencies of the mutant and wild-type alleles. Applying DARMS-PCR to human specimens, we successfully identified MPL mutations in MPN patients, with the exception of patients bearing mutant allele frequencies below the detection limit (5% of this method. The MPL mutant allele frequencies determined using DARMS-PCR correlated strongly with the values determined using deep sequencing. Thus, we demonstrated the potential of DARMS-PCR to detect MPL mutations and determine the allele frequencies in a timely and cost-effective manner.

Experiences from treatment-predictive KRAS testing; high mutation frequency in rectal cancers from females and concurrent mutations in the same tumor

DEFF Research Database (Denmark)

Jönsson, Mats; Ekstrand, Anna; Edekling, Thomas

2009-01-01

. METHODS: We used a real-time PCR based method to determine KRAS mutations in 136 colorectal cancers with mutations identified in 53 (39%) tumors. RESULTS: KRAS mutations were significantly more often found in rectal cancer (21/38, 55%) than in colon cancer (32/98, 33%) (P = 0.02). This finding...... was explained by marked differences mutation rates in female patients who showed mutations in 33% of the colon cancers and in 67% of the rectal cancers (P = 0.01). Concurrent KRAS mutations were identified in three tumors; two colorectal cancers harbored Gly12Asp/Gly13Asp and Gly12Cys/Gly13Asp and a third tumor...... carried Gly12Cys/Gly12Asp in an adenomatous component and additionally acquired Gly12Val in the invasive component. CONCLUSION: The demonstration of a particularly high KRAS mutation frequency among female rectal cancer patients suggests that this subset is the least likely to respond to anti...

Mutation frequencies of the cytochrome CYP2D6 gene in Parkinson disease patients and in families

Energy Technology Data Exchange (ETDEWEB)

Lucotte, G.; Turpin, J.C. [CHR, Reims (France); Gerard, N. [INSERM, Paris (France)] [and others

1996-07-26

The frequencies of five mutations of the debrisoquine 4-hydroxylase (CYP2D6) gene (mutations D6-A, B, C, D, and T), corresponding to poor metabolizer (PM) phenotypes, were determined by restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR) in 47 patients with Parkinson disease, and compared with the findings in 47 healthy controls. These mutant alleles were about twice as frequent among patients as in controls, with an approximate relative risk ratio of 2.12 (95% confidence interval, 1.41-2.62). There seem to be no significant differences in frequencies of mutant genotypes in patients among gender and modalities of response with levodopa therapy; but frequency of the mutations was slightly enhanced after age-at-onset of 60 years. Mutations D6-B, D, and T were detected in 7 patients belonging to 10 Parkinson pedigrees. 25 refs., 1 fig., 2 tabs.

Guanine holes are prominent targets for mutation in cancer and inherited disease.

Directory of Open Access Journals (Sweden)

Albino Bacolla

Full Text Available Single base substitutions constitute the most frequent type of human gene mutation and are a leading cause of cancer and inherited disease. These alterations occur non-randomly in DNA, being strongly influenced by the local nucleotide sequence context. However, the molecular mechanisms underlying such sequence context-dependent mutagenesis are not fully understood. Using bioinformatics, computational and molecular modeling analyses, we have determined the frequencies of mutation at G • C bp in the context of all 64 5'-NGNN-3' motifs that contain the mutation at the second position. Twenty-four datasets were employed, comprising >530,000 somatic single base substitutions from 21 cancer genomes, >77,000 germline single-base substitutions causing or associated with human inherited disease and 16.7 million benign germline single-nucleotide variants. In several cancer types, the number of mutated motifs correlated both with the free energies of base stacking and the energies required for abstracting an electron from the target guanines (ionization potentials. Similar correlations were also evident for the pathological missense and nonsense germline mutations, but only when the target guanines were located on the non-transcribed DNA strand. Likewise, pathogenic splicing mutations predominantly affected positions in which a purine was located on the non-transcribed DNA strand. Novel candidate driver mutations and tissue-specific mutational patterns were also identified in the cancer datasets. We conclude that electron transfer reactions within the DNA molecule contribute to sequence context-dependent mutagenesis, involving both somatic driver and passenger mutations in cancer, as well as germline alterations causing or associated with inherited disease.

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.

Mitochondrial DNA mutations in preneoplastic lesions of the gastrointestinal tract: A biomarker for the early detection of cancer

Directory of Open Access Journals (Sweden)

Montgomery Elizabeth A

2006-12-01

Full Text Available Abstract Background Somatic mutations of mitochondrial DNA (mtDNA are common in many human cancers. We have described an oligonucleotide microarray ("MitoChip" for rapid sequencing of the entire mitochondrial genome (Zhou et al, J Mol Diagn 2006, facilitating the analysis of mtDNA mutations in preneoplastic lesions. We examined 14 precancerous lesions, including seven Barrett esophagus biopsies, with or without associated dysplasia; four colorectal adenomas; and three inflammatory colitis-associated dysplasia specimens. In all cases, matched normal tissues from the corresponding site were obtained as germline control. MitoChip analysis was performed on DNA obtained from cryostat-embedded specimens. Results A total of 513,639 bases of mtDNA were sequenced in the 14 samples, with 490,224 bases (95.4% bases assigned by the automated genotyping software. All preneoplastic lesions examined demonstrated at least one somatic mtDNA sequence alteration. Of the 100 somatic mtDNA alterations observed in the 14 cases, 27 were non-synonymous coding region mutations (i.e., resulting in an amino acid change, 36 were synonymous, and 37 involved non-coding mtDNA. Overall, somatic alterations most commonly involved the COI, ND4 and ND5 genes. Notably, somatic mtDNA alterations were observed in preneoplastic lesions of the gastrointestinal tract even in the absence of histopathologic evidence of dysplasia, suggesting that the mitochondrial genome is susceptible at the earliest stages of multistep cancer progression. Conclusion Our findings further substantiate the rationale for exploring the mitochondrial genome as a biomarker for the early diagnosis of cancer, and confirm the utility of a high-throughput array-based platform for this purpose from a clinical applicability standpoint.

Deletion mutations of bacteriophage

International Nuclear Information System (INIS)

Ryo, Yeikou

1975-01-01

Resolution of mutation mechanism with structural changes of DNA was discussed through the studies using bacteriophage lambda. One of deletion mutations inductions of phage lambda is the irradiation of ultraviolet ray. It is not clear if the inductions are caused by errors in reparation of ultraviolet-induced damage or by the activation of int gene. Because the effective site of int gene lies within the regions unnecessary for existing, it is considered that int gene is connected to deletion mutations induction. A certain system using prophage complementarity enables to detect deletion mutations at essential hereditary sites and to solve the relations of deletion mutations with other recombination system, DNA reproduction and repairment system. Duplication and multiplication of hereditary elements were discussed. If lambda deletion mutations of the system, which can control recombination, reproduction and repairment of added DNA, are constructed, mutations mechanism with great changes of DNA structure can be solved by phage lambda. (Ichikawa, K.)

Mutation induction by and mutational interaction between monochromatic wavelength radiations in the near-ultraviolet and visible ranges

International Nuclear Information System (INIS)

Tyrrell, R.M.

1980-01-01

The induction of mutations (reversion to tryptophan independence) by various UV (254, 313, 334 and 365 nm) and visible (405 and 434 nm) wavelengths was measured in exponential phase populations of Escherichia coli B/r thy trp and B/r thy trp uvr A by assay of irradiated populations on semi-enriched media. No mutations were induced in the repair proficient strain at wavelengths longer than 313 nm. Mutations were induced to the excisionless strain at wavelengths as long as 405 nm but less than expected from the known amount of DNA damage induced. Irradiation at the long wavelenths (434, 405, 365 and 334 nm) suppressed the appearance of 254- or 313 nm-induced mutations in the repair competent strain but not in the excision deficient strain. The relative dose-requirement for mutation suppression was related to the relative efficiency of these wavelengths in inducing growth delay. These results suggest that the growth delay induced by near-UV and visible wavelenghts allows more time for the 'error-free' excision repair process to act on the potentially mutagenic lesions induced by 254- and 313-nm radiations, thereby reducing the mutation frequency observed in the repair-proficient strain. The level of near-UV mutation induced in the excision deficient strain is lower than expected from the DNA damage known to be induced. It is possible that near-UV radiation induces a class of lethal lesions that are not susceptible to error-prone repair. (author)

A threshold in the dose-response relationship for X-ray induced somatic mutation frequency in drosophila melanogaster

International Nuclear Information System (INIS)

Koana, Takao; Sakai, Kazuo; Okada, M.O.

2004-01-01

The dose-response relationship of ionizing radiation and its stochastic effects has been thought to be linear without any thresholds for a long time. The basic data for this model was obtained from mutational assays using germ cells of male fruit fly Drosophila melanogaster. However, cancer-causing activity should be examined more appropriately in somatic cells than in germ cells. In this paper, we examined the dose-response relationship of X-ray irradiation and somatic mutation in drosophila, and found a threshold at approximately 1 Gy in the DNA repair proficient flies. In the repair deficient siblings, the threshold was smaller and the inclination of the dose-response curve was five times steeper. These results suggest that the dose-response relationship between X-ray irradiation and somatic mutation has a threshold, and that the DNA repair function contributes to its formation. (author)

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

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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.

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

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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.

The spontaneous chlorophyll mutation frequency in barley

DEFF Research Database (Denmark)

Jørgensen, Jørgen Helms; Jensen, Hans Peter

1986-01-01

A total of 1866 barley plants were progeny tested in the greenhouse. Twenty-five plants segregated for newly arisen, spontaneous chlorophyll mutant genes. Among the total of 470,129 seedlings screened there were 79 mutants (1.7 .+-. 0.6 .times. 10-4). The data are added to data from three similar...... materials and the resulting estimate of the chlorophyll mutant frequency is 1.6 .times. 10-4 in about 1.43 million seedlings. The estimate of the chlorophyll mutation rate per generation is close to 67.3 .times. 10-4 per diploid genome or in the order of 6 .times. 10-7 per locus and haploid genome....

Genotype-Phenotype Correlation of Maternally Inherited Disorders due to Mutations in Mitochondrial DNA

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Peterus Thajeb

2006-09-01

Full Text Available Mitochondrial disorders are heterogeneous systemic ailments that are most often caused by maternal inheritance of a variety of mutations of the mitochondrial (mt DNA. Paternal inheritance and somatic mutation are rare. The disorders are well recognized not only for the genotypic heterogeneity, but also the phenotypic variation among the affected members of a single family. The genotype-phenotype correlation of the diversity of the syndromic and non-syndromic features of mitochondrial disorders are discussed. Some aspects of the molecular mechanisms of this heterogeneity, and the histopathologic findings are highlighted.

PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases.

OpenAIRE

Cline, J; Braman, J C; Hogrefe, H H

1996-01-01

The replication fidelities of Pfu, Taq, Vent, Deep Vent and UlTma DNA polymerases were compared using a PCR-based forward mutation assay. Average error rates (mutation frequency/bp/duplication) increased as follows: Pfu (1.3 x 10(-6)) < Deep Vent (2.7 x 10(-6)) < Vent (2.8 x 10(-6)) < Taq (8.0 x 10(-6)) < < exo- Pfu and UlTma (approximately 5 x 10(-5)). Buffer optimization experiments indicated that Pfu fidelity was highest in the presence of 2-3 mM MgSO4 and 100-300 microM each dNTP and at p...

PMS2 gene mutational analysis: direct cDNA sequencing to circumvent pseudogene interference.

Science.gov (United States)

Wimmer, Katharina; Wernstedt, Annekatrin

2014-01-01

The presence of highly homologous pseudocopies can compromise the mutation analysis of a gene of interest. In particular, when using PCR-based strategies, pseudogene co-amplification has to be effectively prevented. This is often achieved by using primers designed to be parental gene specific according to the reference sequence and by applying stringent PCR conditions. However, there are cases in which this approach is of limited utility. For example, it has been shown that the PMS2 gene exchanges sequences with one of its pseudogenes, named PMS2CL. This results in functional PMS2 alleles containing pseudogene-derived sequences at their 3'-end and in nonfunctional PMS2CL pseudogene alleles that contain gene-derived sequences. Hence, the paralogues cannot be distinguished according to the reference sequence. This shortcoming can be effectively circumvented by using direct cDNA sequencing. This approach is based on the selective amplification of PMS2 transcripts in two overlapping 1.6-kb RT-PCR products. In addition to avoiding pseudogene co-amplification and allele dropout, this method has also the advantage that it allows to effectively identify deletions, splice mutations, and de novo retrotransposon insertions that escape the detection of most DNA-based mutation analysis protocols.

Somatic mutations affect key pathways in lung adenocarcinoma

Science.gov (United States)

Ding, Li; Getz, Gad; Wheeler, David A.; Mardis, Elaine R.; McLellan, Michael D.; Cibulskis, Kristian; Sougnez, Carrie; Greulich, Heidi; Muzny, Donna M.; Morgan, Margaret B.; Fulton, Lucinda; Fulton, Robert S.; Zhang, Qunyuan; Wendl, Michael C.; Lawrence, Michael S.; Larson, David E.; Chen, Ken; Dooling, David J.; Sabo, Aniko; Hawes, Alicia C.; Shen, Hua; Jhangiani, Shalini N.; Lewis, Lora R.; Hall, Otis; Zhu, Yiming; Mathew, Tittu; Ren, Yanru; Yao, Jiqiang; Scherer, Steven E.; Clerc, Kerstin; Metcalf, Ginger A.; Ng, Brian; Milosavljevic, Aleksandar; Gonzalez-Garay, Manuel L.; Osborne, John R.; Meyer, Rick; Shi, Xiaoqi; Tang, Yuzhu; Koboldt, Daniel C.; Lin, Ling; Abbott, Rachel; Miner, Tracie L.; Pohl, Craig; Fewell, Ginger; Haipek, Carrie; Schmidt, Heather; Dunford-Shore, Brian H.; Kraja, Aldi; Crosby, Seth D.; Sawyer, Christopher S.; Vickery, Tammi; Sander, Sacha; Robinson, Jody; Winckler, Wendy; Baldwin, Jennifer; Chirieac, Lucian R.; Dutt, Amit; Fennell, Tim; Hanna, Megan; Johnson, Bruce E.; Onofrio, Robert C.; Thomas, Roman K.; Tonon, Giovanni; Weir, Barbara A.; Zhao, Xiaojun; Ziaugra, Liuda; Zody, Michael C.; Giordano, Thomas; Orringer, Mark B.; Roth, Jack A.; Spitz, Margaret R.; Wistuba, Ignacio I.; Ozenberger, Bradley; Good, Peter J.; Chang, Andrew C.; Beer, David G.; Watson, Mark A.; Ladanyi, Marc; Broderick, Stephen; Yoshizawa, Akihiko; Travis, William D.; Pao, William; Province, Michael A.; Weinstock, George M.; Varmus, Harold E.; Gabriel, Stacey B.; Lander, Eric S.; Gibbs, Richard A.; Meyerson, Matthew; Wilson, Richard K.

2009-01-01

Determining the genetic basis of cancer requires comprehensive analyses of large collections of histopathologically well-classified primary tumours. Here we report the results of a collaborative study to discover somatic mutations in 188 human lung adenocarcinomas. DNA sequencing of 623 genes with known or potential relationships to cancer revealed more than 1,000 somatic mutations across the samples. Our analysis identified 26 genes that are mutated at significantly high frequencies and thus are probably involved in carcinogenesis. The frequently mutated genes include tyrosine kinases, among them the EGFR homologue ERBB4; multiple ephrin receptor genes, notably EPHA3; vascular endothelial growth factor receptor KDR; and NTRK genes. These data provide evidence of somatic mutations in primary lung adenocarcinoma for several tumour suppressor genes involved in other cancers—including NF1, APC, RB1 and ATM—and for sequence changes in PTPRD as well as the frequently deleted gene LRP1B. The observed mutational profiles correlate with clinical features, smoking status and DNA repair defects. These results are reinforced by data integration including single nucleotide polymorphism array and gene expression array. Our findings shed further light on several important signalling pathways involved in lung adenocarcinoma, and suggest new molecular targets for treatment. PMID:18948947

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

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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.

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

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Xin Cai

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

Studies on induced mutation frequency in Catharanthus roseus (L.) G. Don by gamma rays and EMS individually and in combination

International Nuclear Information System (INIS)

Venkateswarlu, M.; Susheelamma, B.N.; Kumar, P.; Subhash, K.

1988-01-01

Seeds of pink flowered (PF) and white flowered (WF) Catharanthus roseus were soaked in distilled water for 24 h and treated with gamma rays and 0.1% EMS separately and in combination. Six types of chlorophyll mutations, viz., xantha, albina, chlorina, viridis, maculata and tigrina were recovered to M 2 generation of both forms. The frequency of chlorophyll mutations was found to be dependent on the dose, of gamma rays and duration of treatment with EMS. Higher frequency of chlorophyll mutations was noticed in PF, which is mutagenically more sensitive than WF. It was also noticed that the combination treatments of gamma rays and EMS enhanced the frequency of chlorophyll mutations

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

Science.gov (United States)

Hassim, Farzanah; Papadopoulos, Andrea O; Kana, Bavesh D; Gordhan, Bhavna G

2015-09-01

Hydroxyl radical (OH) among reactive oxygen species cause damage to nucleobases with thymine being the most susceptible, whilst in contrast, the singlet oxygen ((1)02) targets only guanine bases. The high GC content of mycobacterial genomes predisposes these organisms to oxidative damage of guanine. The exposure of cellular DNA to OH and one-electron oxidants results in the formation of two main degradation products, the pro-mutagenic 8-oxo-7,8-dihydroguanine (8-oxoGua) and the cytotoxic 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua). These lesions are repaired through the base excision repair (BER) pathway and we previously, demonstrated a combinatorial role for the mycobacterial Endonuclease III (Nth) and the Nei family of DNA glycosylases in mutagenesis. In addition, the formamidopyrimidine (Fpg/MutM) and MutY DNA glycosylases have also been implicated in mutation avoidance and BER in mycobacteria. In this study, we further investigate the combined role of MutY and the Fpg/Nei DNA glycosylases in Mycobacterium smegmatis and demonstrate that deletion of mutY resulted in enhanced sensitivity to oxidative stress, an effect which was not exacerbated in Δfpg1 Δfpg2 or Δnei1 Δnei2 double mutant backgrounds. However, combinatorial loss of the mutY, fpg1 and fpg2 genes resulted in a significant increase in mutation rates suggesting interplay between these enzymes. Consistent with this, there was a significant increase in C → A mutations with a corresponding change in cell morphology of rifampicin resistant mutants in the Δfpg1 Δfpg2 ΔmutY deletion mutant. In contrast, deletion of mutY together with the nei homologues did not result in any growth/survival defects or changes in mutation rates. Taken together these data indicate that the mycobacterial mutY, in combination with the Fpg DNA N-glycosylases, plays an important role in controlling mutagenesis under oxidative stress. Copyright © 2015 Elsevier B.V. All rights reserved.

HFE gene C282Y, H63D and S65C mutations frequency in the Transylvania region, Romania.

Science.gov (United States)

Trifa, Adrian P; Popp, Radu A; Militaru, Mariela S; Farcaş, Marius F; Crişan, Tania O; Gana, Ionuţ; Cucuianu, Andrei; Pop, Ioan V

2012-06-01

HFE-associated haemochromatosis is one of the most frequent autosomal recessive disorders in the Caucasian population. Although most of the cases are homozygous individuals for the C282Y mutation, another two mutations, H63D and S65C, have been reported to be associated with milder forms of the disease. This study was a first attempt to evaluate the distribution of these HFE gene mutations in the Transylvania region. Two-hundred and twenty-five healthy, unrelated volunteers originating from the Transylvania region, Romania, were screened for the HFE gene C282Y, H63D and S65C mutations, using molecular genetics assays (Polymerase Chain Reaction-Restriction Fragments Length Polymorphism). For the C282Y mutation, 7 heterozygotes (3.1%) were found, but no homozygous individual. In the case of the H63D mutation, 40 heterozygotes (17.8%) and 4 homozygotes (1.75%) for the mutant allele were evidenced. We found a compound heterozygous genotype (C282Y/H63D) in one individual (0.45%). Thus, the allele frequencies of the C282Y and H63D were 1.75% and 10.9%, respectively. Three individuals (1.3%) were found to harbour the S65C mutation in a heterozygous state, but none in a homozygous state: the allele frequency of the mutant allele was 0.75%. The distribution of the HFE gene C282Y, H63D and S65C mutations found in our group matches the tendencies observed in other European countries: a decreasing gradient from Northern to Southern Europe for the C282Y mutation; high frequency for the H63D mutation, and low frequency for the S65C mutation in most of the countries.

Population data and mutation rates of 19 STR loci in seven provinces from China based on Goldeneye™ DNA ID System 20A.

Science.gov (United States)

Liu, Qiu-Ling; Chen, Ye-Fei; Huang, Xiao-Ling; Liu, Kai-Yan; Zhao, Hu; Lu, De-Jian

2017-05-01

Short tandem repeat (STR) analysis is a primary tool in forensic casework. Population data and mutation rates of STRs are very important for paternity testing and forensic genetics. However, the population data and mutation rates of STRs in Han nationality based on large samples have still not been fully described in China. In this study, the allelic frequencies, forensic parameters, and mutation rate of 19 STR loci (D19S433, D5S818, D21S11, D18S51, D6S1043, D3S1358, D13S317, D7S820, D16S539, CSFIPO, PentaD, vWA, D8S1179, TPOX, Penta E, TH01, D12S391, D2S1338, and FGA) based on the Goldeneye™ DNA ID System 20A in Southern China Han nationality among seven provinces were investigated. Furthermore, population stratification of Southern China Han nationality among seven provinces was established. The multidimensional scaling (MDS) plot based on genetic distances (Fst) showed that the studied populations can be clustered into two major groups. However, relationships among populations were weak (Fst < 0.0043). A total of 376 cases of mutation were detected from the 19 selected loci in 15,396 meioses. The average mutation rate for the 19 loci was estimated to be 1.3 × 10 -3 per meiosis. The mutation was mainly single step; the paternal mutation rate was higher than the maternal; and paternal mutation rate increases with paternal age.

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...

Mutational effects of γ-rays and carbon ion beams on Arabidopsis seedlings

International Nuclear Information System (INIS)

Yoshihara, Ryohei; Nozawa, Shigeki; Hase, Yoshihiro; Sakamoto, Ayako N.; Narumi, Issay; Hidema, Jun

2013-01-01

To assess the mutational effects of radiation on vigorously proliferating plant tissue, the mutation spectrum was analyzed with Arabidopsis seedlings using the plasmid-rescue method. Transgenic plants containing the Escherichia coli rpsL gene were irradiated with γ-rays and carbon ion beams (320-MeV 12 C 6+ ), and mutations in the rpsL gene were analyzed. Mutant frequency increased significantly following irradiation by γ-rays, but not by 320-MeV 12 C 6+ . Mutation spectra showed that both radiations increased the frequency of frameshifts and other mutations, including deletions and insertions, but only γ-rays increased the frequency of total base substitutions. These results suggest that the type of DNA lesions which cause base substitutions were less often induced by 320-MeV 12 C 6+ than by γ-rays in Arabidopsis seedlings. Furthermore, γ-rays never increased the frequencies of G:C to T:A or A:T to C:G transversions, which are caused by oxidized guanine; 320-MeV 12 C 6+ , however, produced a slight increase in both transversions. Instead, γ-rays produced a significant increase in the frequency of G:C to A:T transitions. These results suggest that 8-oxoguanine has little effect on mutagenesis in Arabidopsis cells. (author)

Molecular analysis of radiation-induced mutations in vitro

International Nuclear Information System (INIS)

Kronenberg, A.

1996-01-01

This review will focus on the nature of specific locus mutations detected in mammalian cells exposed in vitro to different types of ionizing radiations. Ionizing radiation has been shown to produce a wide variety of heritable alterations in DNA. These range from single base pair substitutions to stable loss or translocation of large portions of whole chromosomes. Data will be reviewed for certain test systems that reveal different mutation spectra. Techniques for the analysis of molecular alterations include applications of the polymerase chain reaction, some of which may be coupled with DNA sequence analysis, and a variety of hybridization-based techniques. The complexity of large scale rearrangements is approached with cytogenetic techniques including high resolution banding and various applications of the fluorescence in situ hybridization (FISH) technique. Radiation-induced mutant frequencies and mutation spectra are a function of the linkage constraints on the recovery of viable mutants for a given locus and test system. 44 refs

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

Science.gov (United States)

2013-09-01

reads were then processed to determine the dinucleotide composition on the 5’ end by separating the Watson and Crick strands, and the dinucleotide...AD_________________ Award Number: W81XWH-12-1-0333 TITLE: Determining the Location of DNA ...COVERED 15 August 2012 – 14 August 2013 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Determining the Location of DNA Modification and Mutation Caused

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

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...

Identification of a novel mutation in WFS1 in a family affected by low-frequency hearing impairment

Energy Technology Data Exchange (ETDEWEB)

Kunz, Juergen; Marquez-Klaka, Ben; Uebe, Steffen; Volz-Peters, Anja; Berger, Roswitha; Rausch, Peter

2003-04-09

Previously we confirmed linkage of autosomal dominantly inherited low-frequency sensorineural hearing impairment (LFSNHI) in a German family to the genetic locus DFNA6/DFNA14 on chromosome 4p16.3 close to the markers D4S432 and D4S431. Analysis of data from the Human Genome Project, showed that WFS1 is located in this region. Mutations in WFS1 are known to be responsible for Wolfram syndrome (DIDMOAD, MIM no. 606201), which follows an autosomal recessive trait. Studies in low-frequency hearing loss families showed that mutations in WFS1 were responsible for the phenotype. In all affected family members analysed, we detected a missense mutation in WFS1 (K705N) and therefore confirm the finding that the majority of mutations responsible for LFSNHI are missense mutations which localise to the C-terminal domain of the protein.

Identification of a novel mutation in WFS1 in a family affected by low-frequency hearing impairment

International Nuclear Information System (INIS)

Kunz, Juergen; Marquez-Klaka, Ben; Uebe, Steffen; Volz-Peters, Anja; Berger, Roswitha; Rausch, Peter

2003-01-01

Previously we confirmed linkage of autosomal dominantly inherited low-frequency sensorineural hearing impairment (LFSNHI) in a German family to the genetic locus DFNA6/DFNA14 on chromosome 4p16.3 close to the markers D4S432 and D4S431. Analysis of data from the Human Genome Project, showed that WFS1 is located in this region. Mutations in WFS1 are known to be responsible for Wolfram syndrome (DIDMOAD, MIM no. 606201), which follows an autosomal recessive trait. Studies in low-frequency hearing loss families showed that mutations in WFS1 were responsible for the phenotype. In all affected family members analysed, we detected a missense mutation in WFS1 (K705N) and therefore confirm the finding that the majority of mutations responsible for LFSNHI are missense mutations which localise to the C-terminal domain of the protein

DNA Damage, Mutagenesis and Cancer

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Ashis K. Basu

2018-03-01

Full Text Available A large number of chemicals and several physical agents, such as UV light and γ-radiation, have been associated with the etiology of human cancer. Generation of DNA damage (also known as DNA adducts or lesions induced by these agents is an important first step in the process of carcinogenesis. Evolutionary processes gave rise to DNA repair tools that are efficient in repairing damaged DNA; yet replication of damaged DNA may take place prior to repair, particularly when they are induced at a high frequency. Damaged DNA replication may lead to gene mutations, which in turn may give rise to altered proteins. Mutations in an oncogene, a tumor-suppressor gene, or a gene that controls the cell cycle can generate a clonal cell population with a distinct advantage in proliferation. Many such events, broadly divided into the stages of initiation, promotion, and progression, which may occur over a long period of time and transpire in the context of chronic exposure to carcinogens, can lead to the induction of human cancer. This is exemplified in the long-term use of tobacco being responsible for an increased risk of lung cancer. This mini-review attempts to summarize this wide area that centers on DNA damage as it relates to the development of human cancer.

Abnormal ultraviolet mutagenic spectrum in plasmid DNA replicated in cultured fibroblasts from a patient with the skin cancer-prone disease, xeroderma pigmentosum

International Nuclear Information System (INIS)

Seetharam, S.; Protic-Sabljic, M.; Seidman, M.M.; Kraemer, K.H.

1987-01-01

A shuttle vector plasmid, pZ189, was utilized to assess the types of mutations that cells from a patient with xeroderma pigmentosum, complementation group D, introduce into ultraviolet (UV) damaged, replicating DNA. Patients with xeroderma pigmentosum have clinical and cellular UV hypersensitivity, increased frequency of sun-induced skin cancer, and deficient DNA repair. In comparison to UV-treated pZ189 replicated in DNA repair-proficient cells, there were fewer surviving plasmids, a higher frequency of plasmids with mutations, fewer plasmids with two or more mutations in the marker gene, and a new mutagenic hotspot. The major type of base substitution mutation was the G:C to A:T transition with both cell lines. These results, together with similar findings published earlier with cells from a xeroderma pigmentosum patient in complementation group A, suggest that isolated G:C to A:T somatic mutations may be particularly important in generation of human skin cancer by UV radiation

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.

DNA-based watermarks using the DNA-Crypt algorithm

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Barnekow Angelika

2007-05-01

Full Text Available Abstract Background The aim of this paper is to demonstrate the application of watermarks based on DNA sequences to identify the unauthorized use of genetically modified organisms (GMOs protected by patents. Predicted mutations in the genome can be corrected by the DNA-Crypt program leaving the encrypted information intact. Existing DNA cryptographic and steganographic algorithms use synthetic DNA sequences to store binary information however, although these sequences can be used for authentication, they may change the target DNA sequence when introduced into living organisms. Results The DNA-Crypt algorithm and image steganography are based on the same watermark-hiding principle, namely using the least significant base in case of DNA-Crypt and the least significant bit in case of the image steganography. It can be combined with binary encryption algorithms like AES, RSA or Blowfish. DNA-Crypt is able to correct mutations in the target DNA with several mutation correction codes such as the Hamming-code or the WDH-code. Mutations which can occur infrequently may destroy the encrypted information, however an integrated fuzzy controller decides on a set of heuristics based on three input dimensions, and recommends whether or not to use a correction code. These three input dimensions are the length of the sequence, the individual mutation rate and the stability over time, which is represented by the number of generations. In silico experiments using the Ypt7 in Saccharomyces cerevisiae shows that the DNA watermarks produced by DNA-Crypt do not alter the translation of mRNA into protein. Conclusion The program is able to store watermarks in living organisms and can maintain the original information by correcting mutations itself. Pairwise or multiple sequence alignments show that DNA-Crypt produces few mismatches between the sequences similar to all steganographic algorithms.

DNA-based watermarks using the DNA-Crypt algorithm.

Science.gov (United States)

Heider, Dominik; Barnekow, Angelika

2007-05-29

The aim of this paper is to demonstrate the application of watermarks based on DNA sequences to identify the unauthorized use of genetically modified organisms (GMOs) protected by patents. Predicted mutations in the genome can be corrected by the DNA-Crypt program leaving the encrypted information intact. Existing DNA cryptographic and steganographic algorithms use synthetic DNA sequences to store binary information however, although these sequences can be used for authentication, they may change the target DNA sequence when introduced into living organisms. The DNA-Crypt algorithm and image steganography are based on the same watermark-hiding principle, namely using the least significant base in case of DNA-Crypt and the least significant bit in case of the image steganography. It can be combined with binary encryption algorithms like AES, RSA or Blowfish. DNA-Crypt is able to correct mutations in the target DNA with several mutation correction codes such as the Hamming-code or the WDH-code. Mutations which can occur infrequently may destroy the encrypted information, however an integrated fuzzy controller decides on a set of heuristics based on three input dimensions, and recommends whether or not to use a correction code. These three input dimensions are the length of the sequence, the individual mutation rate and the stability over time, which is represented by the number of generations. In silico experiments using the Ypt7 in Saccharomyces cerevisiae shows that the DNA watermarks produced by DNA-Crypt do not alter the translation of mRNA into protein. The program is able to store watermarks in living organisms and can maintain the original information by correcting mutations itself. Pairwise or multiple sequence alignments show that DNA-Crypt produces few mismatches between the sequences similar to all steganographic algorithms.

DNA-based watermarks using the DNA-Crypt algorithm

Science.gov (United States)

Heider, Dominik; Barnekow, Angelika

2007-01-01

Background The aim of this paper is to demonstrate the application of watermarks based on DNA sequences to identify the unauthorized use of genetically modified organisms (GMOs) protected by patents. Predicted mutations in the genome can be corrected by the DNA-Crypt program leaving the encrypted information intact. Existing DNA cryptographic and steganographic algorithms use synthetic DNA sequences to store binary information however, although these sequences can be used for authentication, they may change the target DNA sequence when introduced into living organisms. Results The DNA-Crypt algorithm and image steganography are based on the same watermark-hiding principle, namely using the least significant base in case of DNA-Crypt and the least significant bit in case of the image steganography. It can be combined with binary encryption algorithms like AES, RSA or Blowfish. DNA-Crypt is able to correct mutations in the target DNA with several mutation correction codes such as the Hamming-code or the WDH-code. Mutations which can occur infrequently may destroy the encrypted information, however an integrated fuzzy controller decides on a set of heuristics based on three input dimensions, and recommends whether or not to use a correction code. These three input dimensions are the length of the sequence, the individual mutation rate and the stability over time, which is represented by the number of generations. In silico experiments using the Ypt7 in Saccharomyces cerevisiae shows that the DNA watermarks produced by DNA-Crypt do not alter the translation of mRNA into protein. Conclusion The program is able to store watermarks in living organisms and can maintain the original information by correcting mutations itself. Pairwise or multiple sequence alignments show that DNA-Crypt produces few mismatches between the sequences similar to all steganographic algorithms. PMID:17535434

Escherichia coli DnaE Polymerase Couples Pyrophosphatase Activity to DNA Replication.

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Fabio Lapenta

Full Text Available DNA Polymerases generate pyrophosphate every time they catalyze a step of DNA elongation. This elongation reaction is generally believed as thermodynamically favoured by the hydrolysis of pyrophosphate, catalyzed by inorganic pyrophosphatases. However, the specific action of inorganic pyrophosphatases coupled to DNA replication in vivo was never demonstrated. Here we show that the Polymerase-Histidinol-Phosphatase (PHP domain of Escherichia coli DNA Polymerase III α subunit features pyrophosphatase activity. We also show that this activity is inhibited by fluoride, as commonly observed for inorganic pyrophosphatases, and we identified 3 amino acids of the PHP active site. Remarkably, E. coli cells expressing variants of these catalytic residues of α subunit feature aberrant phenotypes, poor viability, and are subject to high mutation frequencies. Our findings indicate that DNA Polymerases can couple DNA elongation and pyrophosphate hydrolysis, providing a mechanism for the control of DNA extension rate, and suggest a promising target for novel antibiotics.

HFE gene mutations in coronary atherothrombotic disease

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Calado R.T.

2000-01-01

Full Text Available Although iron can catalyze the production of free radicals involved in LDL lipid peroxidation, the contribution of iron overload to atherosclerosis remains controversial. The description of two mutations in the HFE gene (Cys282Tyr and His63Asp related to hereditary hemochromatosis provides an opportunity to address the question of the association between iron overload and atherosclerosis. We investigated the prevalence of HFE mutations in 160 survivors of myocardial infarction with angiographically demonstrated severe coronary atherosclerotic disease, and in 160 age-, gender- and race-matched healthy control subjects. PCR amplification of genomic DNA followed by RsaI and BclI restriction enzyme digestion was used to determine the genotypes. The frequency of the mutant Cys282Tyr allele was identical among patients and controls (0.022; carrier frequency, 4.4%, whereas the mutant His63Asp allele had a frequency of 0.143 (carrier frequency, 27.5% in controls and of 0.134 (carrier frequency, 24.5% in patients. Compound heterozygotes were found in 2 of 160 (1.2% controls and in 1 of 160 (0.6% patients. The finding of a similar prevalence of Cys282Tyr and His63Asp mutations in the HFE gene among controls and patients with coronary atherothrombotic disease, indirectly questions the possibility of an association between hereditary hemochromatosis and atherosclerosis.

Evaluation the frequency of factor V Leiden mutation in pregnant women with preeclampsia syndrome in an Iranian population

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Azadeh Azinfar

2012-01-01

Full Text Available Background: Role of genetic factors in etiology of preeclampsia is not confirmed yet.Objective: Gene defect frequency varies in different geographic areas as well as ethnic groups. In this study, the role of factor V Leiden mutation in the pathogenesis of preeclampsia syndrome among the pregnant population of northern shore of Persian Gulf in Iran, were considered.Materials and Methods: Between Jan. 2008 and Dec. 2009, in a nested case control study, pregnant women with preeclampsia (N=198 as cases and healthy (N=201 as controls were enrolled in the study. DNA were extracted from 10 CC peripheral blood and analyzed for presence of factor V Leiden mutation in these subjects. The maternal and neonatal outcomes of pregnancy according to the distribution of factor V Leiden were also compared among cases.Results: In total, 17(8.6% of cases and 2(1% of controls showed the factor V Leiden mutation. The incidence of factor V Leiden was typically higher in preeclamptic women than control group (OR: 9.34 %95 CI: 2.12-41.01. There was no difference in incidence rate of preterm delivery< 37 weeks (OR: 1.23 %95 CI: 0.38-4.02, very early preterm delivery<32 weeks (OR: 1.00 %95 CI: 0.12-8.46, intra uterine fetal growth restriction (IUGR (OR: 1.32 %95 CI: 0.15-11.30 ,and the rate of cesarean section (OR: 0.88 %95 CI: 0.29-2.62 among cases based on the prevalence of factor V Leiden mutation.Conclusion: The pregnant women with factor V Leiden mutation are prone for preeclampsia syndrome during pregnancy, but this risk factor was not correlated to pregnancy complications in the studied women

PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases.

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Cline, J; Braman, J C; Hogrefe, H H

1996-09-15

The replication fidelities of Pfu, Taq, Vent, Deep Vent and UlTma DNA polymerases were compared using a PCR-based forward mutation assay. Average error rates (mutation frequency/bp/duplication) increased as follows: Pfu (1.3 x 10(-6)) Pfu and UlTma (approximately 5 x 10(-5)). Buffer optimization experiments indicated that Pfu fidelity was highest in the presence of 2-3 mM MgSO4 and 100-300 microM each dNTP and at pH 8.5-9.1. Under these conditions, the error rate of exo- Pfu was approximately 40-fold higher (5 x 10(-5)) than the error rate of Pfu. As the reaction pH was raised from pH 8 to 9, the error rate of Pfu decreased approximately 2-fold, while the error rate of exo- Pfu increased approximately 9-fold. An increase in error rate with pH has also been noted for the exonuclease-deficient DNA polymerases Taq and exo- Klenow, suggesting that the parameters which influence replication error rates may be similar in pol l- and alpha-like polymerases. Finally, the fidelity of 'long PCR' DNA polymerase mixtures was examined. The error rates of a Taq/Pfu DNA polymerase mixture and a Klentaq/Pfu DNA polymerase mixture were found to be less than the error rate of Taq DNA polymerase, but approximately 3-4-fold higher than the error rate of Pfu DNA polymerase.

Genetic variation among the Mapuche Indians from the Patagonian region of Argentina: mitochondrial DNA sequence variation and allele frequencies of several nuclear genes.

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Ginther, C; Corach, D; Penacino, G A; Rey, J A; Carnese, F R; Hutz, M H; Anderson, A; Just, J; Salzano, F M; King, M C

1993-01-01

DNA samples from 60 Mapuche Indians, representing 39 maternal lineages, were genetically characterized for (1) nucleotide sequences of the mtDNA control region; (2) presence or absence of a nine base duplication in mtDNA region V; (3) HLA loci DRB1 and DQA1; (4) variation at three nuclear genes with short tandem repeats; and (5) variation at the polymorphic marker D2S44. The genetic profile of the Mapuche population was compared to other Amerinds and to worldwide populations. Two highly polymorphic portions of the mtDNA control region, comprising 650 nucleotides, were amplified by the polymerase chain reaction (PCR) and directly sequenced. The 39 maternal lineages were defined by two or three generation families identified by the Mapuches. These 39 lineages included 19 different mtDNA sequences that could be grouped into four classes. The same classes of sequences appear in other Amerinds from North, Central, and South American populations separated by thousands of miles, suggesting that the origin of the mtDNA patterns predates the migration to the Americas. The mtDNA sequence similarity between Amerind populations suggests that the migration throughout the Americas occurred rapidly relative to the mtDNA mutation rate. HLA DRB1 alleles 1602 and 1402 were frequent among the Mapuches. These alleles also occur at high frequency among other Amerinds in North and South America, but not among Spanish, Chinese or African-American populations. The high frequency of these alleles throughout the Americas, and their specificity to the Americas, supports the hypothesis that Mapuches and other Amerind groups are closely related.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Somatic mutation frequencies in the stamen hairs of stable and mutable clones of Tradescantia after acute gamma-ray treatments with small doses

International Nuclear Information System (INIS)

Ichikawa, Sadao; Takahashi, C.S.

1977-01-01

Young inflorescences of two different Tradescantia clones heterozygous for flower and stamen-hair color, one stable (KU 9) and the other spontaneously mutable (KU 20), were irradiated acutely with small doses (approx. 3 to 50 R) of 60 Co gamma-rays. Somatic mutation frequencies from blue to pink in the stamen hairs scored on post-irradiation days 10 to 16 increased essentially linearly with increasing gamma-ray dose in both clones. Despite about a 5-fold difference in spontaneous mutation frequency per hair found between the two clones, the dose-response curves of pink mutations determined were similar to each other, giving average mutation frequencies of 1.51 and 1.41 pink-mutant events per 1000 hairs per R for KU 9 and KU 20, respectively. These frequencies are comparable to earlier results obtained from acute irradiation treatments of other clones with higher doses. The doubling dose of pink mutation (the radiation dose making the mutation frequency double the spontaneous level) was calculated to be 2.09 R for KU 9, and this low doubling dose must be given full attention. On the other hand, the doubling dose for KU 20 (calculated to be 10.4 R) is of questionable value, being greatly subject to change because of the diversely variable spontaneous mutation frequency of this clone

Photoreactivation of conversion and de novo suppressor mutation in Escherichia coli

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Bockrath, R C; Plamer, J E [Indiana Univ., Indianapolis (USA). Dept. of Microbiology

1977-04-01

Studies of mutagenesis and photoreactivation in various E.coli strains have shown that conversion mutation of a mutant containing an amber suppressor to one containing an ochre suppressor is sensitive to photoreactivation. Direct photoreactivation by photoreactivating light (PRL) after uv mutagenesis reduced mutation frequencies by a factor of about 2 for each minute of exposure during the first 5 to 8 min of exposure for cells with normal repair capacity. Conversion and potential de novo suppressor mutations were about equally sensitive. For conversion, the sensitivities to PRL were identical in the repair-normal and excisions-repair-deficient strains. For de novo suppressor mutation, the rate of mutation frequency reduction by PRL in the repair-deficient strain was about one-half that in the other strains. The results suggest that ultraviolet radiation produces both de novo suppressor mutation and conversion at the sup(E,B) locus by photoreversible pyrimidine dimers in the DNA. The causative dimers could be Thy()Cyt dimers in the transcribed strand or the non-transcribed strand, respectively.

Mechanisms of mtDNA segregation and mitochondrial signalling in cells with the pathogenic A3243G mutation

NARCIS (Netherlands)

Jahangir Tafrechi, Roshan Sakineh

2008-01-01

Using newly developed single cell A3243G mutation load assays a novel mechanism of mtDNA segregation was identified in which the multi-copy mtDNA nucleoid takes a central position. Furthermore, likely due to low level changes in gene expression, no genes or gene sets could be identified with gene

Multi-center analysis of glucocerebrosidase mutations in Parkinson disease

Science.gov (United States)

Sidransky, Ellen; Nalls, Michael A.; Aasly, Jan O.; Aharon-Peretz, Judith; Annesi, Grazia; Barbosa, Egberto Reis; Bar-Shira, Anat; Berg, Daniela; Bras, Jose; Brice, Alexis; Chen, Chiung-Mei; Clark, Lorraine N.; Condroyer, Christel; De Marco, Elvira Valeria; Dürr, Alexandra; Eblan, Michael J.; Fahn, Stanley; Farrer, Matthew; Fung, Hon-Chung; Gan-Or, Ziv; Gasser, Thomas; Gershoni-Baruch, Ruth; Giladi, Nir; Griffith, Alida; Gurevich, Tanya; Januario, Cristina; Kropp, Peter; Lang, Anthony E.; Lee-Chen, Guey-Jen; Lesage, Suzanne; Marder, Karen; Mata, Ignacio F.; Mirelman, Anat; Mitsui, Jun; Mizuta, Ikuko; Nicoletti, Giuseppe; Oliveira, Catarina; Ottman, Ruth; Orr-Urtreger, Avi; Pereira, Lygia V.; Quattrone, Aldo; Rogaeva, Ekaterina; Rolfs, Arndt; Rosenbaum, Hanna; Rozenberg, Roberto; Samii, Ali; Samaddar, Ted; Schulte, Claudia; Sharma, Manu; Singleton, Andrew; Spitz, Mariana; Tan, Eng-King; Tayebi, Nahid; Toda, Tatsushi; Troiano, André; Tsuji, Shoji; Wittstock, Matthias; Wolfsberg, Tyra G.; Wu, Yih-Ru; Zabetian, Cyrus P.; Zhao, Yi; Ziegler, Shira G.

2010-01-01

Background Recent studies indicate an increased frequency of mutations in the gene for Gaucher disease, glucocerebrosidase (GBA), among patients with Parkinson disease. An international collaborative study was conducted to ascertain the frequency of GBA mutations in ethnically diverse patients with Parkinson disease. Methods Sixteen centers participated, including five from the Americas, six from Europe, two from Israel and three from Asia. Each received a standard DNA panel to compare genotyping results. Genotypes and phenotypic data from patients and controls were analyzed using multivariate logistic regression models and the Mantel Haenszel procedure to estimate odds ratios (ORs) across studies. The sample included 5691 patients (780 Ashkenazi Jews) and 4898 controls (387 Ashkenazi Jews). Results All 16 centers could detect GBA mutations, L444P and N370S, and the two were found in 15.3% of Ashkenazi patients with Parkinson disease (ORs = 4.95 for L444P and 5.62 for N370S), and in 3.2% of non-Ashkenazi patients (ORs = 9.68 for L444P and 3.30 for N370S). GBA was sequenced in 1642 non-Ashkenazi subjects, yielding a frequency of 6.9% for all mutations, demonstrate that limited mutation screens miss half the mutant alleles. The presence of any GBA mutation was associated with an OR of 5.43 across studies. Clinically, although phenotypes varied, subjects with a GBA mutation presented earlier, and were more likely to have affected relatives and atypical manifestations. Conclusion Data collected from sixteen centers demonstrate that there is a strong association between GBA mutations and Parkinson disease. PMID:19846850

Prevalence of Germline Mutations in Genes Engaged in DNA Damage Repair by Homologous Recombination in Patients with Triple-Negative and Hereditary Non-Triple-Negative Breast Cancers.

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Pawel Domagala

Full Text Available This study sought to assess the prevalence of common germline mutations in several genes engaged in the repair of DNA double-strand break by homologous recombination in patients with triple-negative breast cancers and hereditary non-triple-negative breast cancers. Tumors deficient in this type of DNA damage repair are known to be especially sensitive to DNA cross-linking agents (e.g., platinum drugs and to poly(ADP-ribose polymerase (PARP inhibitors.Genetic testing was performed for 36 common germline mutations in genes engaged in the repair of DNA by homologous recombination, i.e., BRCA1, BRCA2, CHEK2, NBN, ATM, PALB2, BARD1, and RAD51D, in 202 consecutive patients with triple-negative breast cancers and hereditary non-triple-negative breast cancers.Thirty five (22.2% of 158 patients in the triple-negative group carried mutations in genes involved in DNA repair by homologous recombination, while 10 (22.7% of the 44 patients in the hereditary non-triple-negative group carried such mutations. Mutations in BRCA1 were most frequent in patients with triple-negative breast cancer (18.4%, and mutations in CHEK2 were most frequent in patients with hereditary non-triple-negative breast cancers (15.9%. In addition, in the triple-negative group, mutations in CHEK2, NBN, and ATM (3.8% combined were found, while mutations in BRCA1, NBN, and PALB2 (6.8% combined were identified in the hereditary non-triple-negative group.Identifying mutations in genes engaged in DNA damage repair by homologous recombination other than BRCA1/2 can substantially increase the proportion of patients with triple-negative breast cancer and hereditary non-triple-negative breast cancer who may be eligible for therapy using PARP inhibitors and platinum drugs.

The frequency pattern of dumpy mutations induced by x-rays in the successive stages of oocytes of Drosophila

International Nuclear Information System (INIS)

Miyamoto, Tomio; Nakao, Yoshio

1978-01-01

The frequency patterns of the different kinds of dumpy mutations induced by x-rays (1,500 and 3,000R) in the successive stages of oocyte development were investigated by transferring the inseminated females daily to fresh vials for 12 days. Under this transferring procedure, the first egg-laying period represents oocytes irradiated when they are at stage 14, and the subsequent ones represent progressively earlier stages of oocyte development at the time of irradiation. The results obtained indicate that (1) the overall yield of complete dumpy mutations recovered in the first six day egg-laying periods (1st-6th day) are relatively higher than that in the subsequent six day periods (7th-12th day), showing a response pattern with a higher mutation frequency in the early egg-laying periods and a lower frequency in the late egg-laying periods; (2) the frequency patterns for the exceptions of the ol and lv types and those of ov and olv types through the sampling periods are practically similar to those observed in the total dumpy mutations; (3) a somewhat peculiar frequency pattern, which seems to be variable by dose, is found in the yield of the o and v exceptions. At the exposure level of 1,500R, no definite difference in response pattern for these exceptions is found, though a response pattern which is practically similar to that observed in the total dumpy mutations is found at the exposure level of 3,000R. The foregoing findings seem to indicate that the different kinds of dumpy exceptions are affected at various degrees by the difference in the cell stages of oocyte. This may possibly suggest that these exceptions are different from each other in the nature of their mutations, that is to say, whether they are associated with chromosome breakage events or not. (auth.)

Involvement of specialized DNA polymerases Pol II, Pol IV and DnaE2 in DNA replication in the absence of Pol I in Pseudomonas putida

International Nuclear Information System (INIS)

Sidorenko, Julia; Jatsenko, Tatjana; Saumaa, Signe; Teras, Riho; Tark-Dame, Mariliis; Horak, Rita; Kivisaar, Maia

2011-01-01

The majority of bacteria possess a different set of specialized DNA polymerases than those identified in the most common model organism Escherichia coli. Here, we have studied the ability of specialized DNA polymerases to substitute Pol I in DNA replication in Pseudomonas putida. Our results revealed that P. putida Pol I-deficient cells have severe growth defects in LB medium, which is accompanied by filamentous cell morphology. However, growth of Pol I-deficient bacteria on solid rich medium can be restored by reduction of reactive oxygen species in cells. Also, mutants with improved growth emerge rapidly. Similarly to the initial Pol I-deficient P. putida, its adapted derivatives express a moderate mutator phenotype, which indicates that DNA replication carried out in the absence of Pol I is erroneous both in the original Pol I-deficient bacteria and the adapted derivatives. Analysis of the spectra of spontaneous Rif r mutations in P. putida strains lacking different DNA polymerases revealed that the presence of specialized DNA polymerases Pol II and Pol IV influences the frequency of certain base substitutions in Pol I-proficient and Pol I-deficient backgrounds in opposite ways. Involvement of another specialized DNA polymerase DnaE2 in DNA replication in Pol I-deficient bacteria is stimulated by UV irradiation of bacteria, implying that DnaE2-provided translesion synthesis partially substitutes the absence of Pol I in cells containing heavily damaged DNA.

Frequency of CDH1 germline mutations in gastric carcinoma coming from high- and low-risk areas: metanalysis and systematic review of the literature

International Nuclear Information System (INIS)

Corso, Giovanni; Marrelli, Daniele; Pascale, Valeria; Vindigni, Carla; Roviello, Franco

2012-01-01

The frequency of E-cadherin germline mutations in countries with different incidence rates for gastric carcinoma has not been well established. The goal of this study was to assess the worldwide frequency of CDH1 germline mutations in gastric cancers coming from low- and high-risk areas. English articles using MEDLINE access (from 1998 to 2011). Search terms included CDH1, E-cadherin, germline mutation, gastric cancer, hereditary, familial and diffuse histotype. The study included all E-cadherin germline mutations identified in gastric cancer patients; somatic mutations and germline mutations reported in other tumors were excluded. The method of this study was scheduled in accordance with the 'PRISMA statement for reporting systematic reviews and meta-analyses'. Countries were classified as low- or middle/high risk-areas for gastric carcinoma incidence. Statistical analysis was performed to correlate the CDH1 mutation frequency with gastric cancer incidence areas. A total of 122 E-cadherin germline mutations have been identified; the majority (87.5%) occurred in gastric cancers coming from low-risk areas. In high-risk areas, we identified 16 mutations in which missense mutations were predominant. (68.8%). We verified a significant association between the mutation frequency and the gastric cancer risk area (p < 0.001: overall identified mutations in low- vs. middle/high-risk areas). E-cadherin genetic screenings performed in low-risk areas for gastric cancer identified a higher frequency of CDH1 germline mutations. This data could open new approaches in the gastric cancer prevention test; before proposing a proband candidate for the CDH1 genetic screening, geographic variability, alongside the family history should be considered