Differences In DNA Repair Capacity Research Articles

Follow-up genotoxicity assessment of Ames-positive/equivocal chemicals using the improved thymidine kinase gene mutation assay in DNA repair-deficient human TK6 cells.

Genotoxicity testing plays an important role in the safety assessment of pharmaceuticals, pesticides and chemical substances. Among the guidelines for various genotoxicity tests, the in vitro genotoxicity test battery comprises the bacterial Ames test and mammalian cell assays. Several chemicals exhibit conflicting results for the bacterial Ames test and mammalian cell genotoxicity studies, which may stem from the differences in DNA repair capacity or metabolism, between different cell types or species. For better understanding the mechanistic implications regarding conflict outcomes between different assay systems, it is necessary to develop in vitro genotoxicity testing approaches with higher specificity towards DNA-damaging reagents. We have recently established an improved thymidine kinase (TK) gene mutation assay (TK assay) i.e. deficient in DNA excision repair system using human lymphoblastoid TK6 cells lacking XRCC1 and XPA (XRCC1-/-/XPA-/-), the core factors of base excision repair (BER) and nucleotide excision repair (NER), respectively. This DNA repair-deficient TK6 cell line is expected to specifically evaluate the genotoxic potential of chemical substances based on the DNA damage. We focussed on four reagents, N-(1-naphthyl)ethylenediamine dihydrochloride (NEDA), p-phenylenediamine (PPD), auramine and malachite green (MG) as the Ames test-positive chemicals. In our assay, assessment using XRCC1-/-/XPA-/- cells revealed no statistically significant increase in the mutant frequencies after treatment with NEDA, PPD and MG, suggesting the chemicals to be non-genotoxic in humans. The observations were consistent with that of the follow-up in vivo studies. In contrast, the mutant frequency was markedly increased in XRCC1-/-/XPA-/- cells after treatment with auramine. The results suggest that auramine is the genotoxic reagent that preferentially induces DNA damages resolved by BER and/or NER in mammals. Taken together, BER/NER-deficient cell-based genotoxicity testing will contribute to elucidate the mechanism of genotoxicity and therefore play a pivotal role in the accurate safety assessment of chemical substances.

772 Age-associated alterations in pro-inflammatory cytokine signaling as well as UV-induced DNA damage contribute to increased basal cell carcinoma (BCC) tumor burden in Ptch1+/-/SKH-1 mice

Human BCC incidence is age-dependent. We have developed Ptch1+/-/SKH-1 mice as a model of human BCC pathogenesis. These mice progressively develop spontaneous BCCs, suggesting per se age-related effects on cancer induction irrespective of aberrant hedgehog signaling. Here, utilizing cohorts of young (2 months) and aged (> 20 months) mice, we tested the hypothesis that chronologically aged skin is more susceptible to ultraviolet radiation (UVR)-induced skin damage and BCC growth as compared to young adult skin. UVR-induced cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts (6-4PPs) were measured in genomic DNA isolated from skin tissues at 24, 48, and 72h post-irradiation (240 mJ/cm2, single exposure). CPD levels peaked at 24h, decreasing gradually and were virtually undetectable after 72h in all cohorts, irrespective of age and sex. A similar pattern was observed for UV-induced 6-4PP in young cohorts. In contrast, skin 6-4PP levels persisted in aged Ptch1+/-/SKH-1 mice, indicating age-related differences in DNA repair capacity. Moreover, circulating levels of inflammatory cytokines known to be altered in cancer and aging (IL1β, IL6, TNFα) were significantly elevated in both aged male and female Ptch1+/-/SKH-1 mice, as well as in UV-irradiated young cohorts. Aged Ptch1+/-/SKH-1 mice subjected to chronic UVR (180 mJ/cm2, 2x/week, 16 weeks) revealed statistically significant increases in tumor burden (UV-irradiated vs. non-irradiated aged cohorts), while we observed < 2-fold increase in BCC burden in UV-irradiated young cohorts vs. non-irradiated controls. These results demonstrate differential susceptibility to UVR-induced BCCs in young and aged Ptch1+/-/SKH-1 mice such that aging enhances BCC burden in Ptch1-deficient skin, and altered DNA damage responses and systemic pro-inflammatory cytokine signaling may contribute to age-dependent enhancement of BCC tumorigenesis in human populations.

Mutational analysis of theFAM175A gene in patients with premature ovarian insufficiency.

The family with sequence similarity 175 member A gene (FAM175A; also known as ABRAXAS1, CCDC98 and ABRA1), a member of the DNA repair family, contributes to the BRCA1 (BRCA1 DNA repair associated)-dependent DNA damage response and is associated with age at natural menopause. However, it remains poorly understood whether sequence variants in FAM175A are causative for premature ovarian insufficiency (POI). The aim of this study was to investigate whether mutations in the gene FAM175A were present in patients with POI. A total of 400 women with idiopathic POI and 498 control women with regular menstruation (306 age-matched women and 192 women over 40 years old) were recruited. After Sanger sequencing of FAM175A, functional experiments were carried out to explore the deleterious effects of the identified variation. DNA damage was subsequently induced by mitomycin C (MMC), and DNA repair capacity and G2-M checkpoint activation were evaluated by examining the phosphorylation level of H2AX (H2A histone family, member X) and the percentage of mitotic cells, respectively. One rare single-nucleotide polymorphism, rs755187051 in gene FAM175A, c.C727G (p.L243V), was identified in two patients but absent in the 498 controls. The functional experiments demonstrated that overexpression of variant p.L243V in HeLa cells resulted in a similar sensitivity to MMC-induced damage compared with cells transfected with wild-type FAM175A. Moreover, after treatment with MMC, there were no differences in DNA repair capacity and G2-M checkpoint activation between the mutant and wild-type genes. Our results suggest that the p.L243V variant of FAM175A may not be causative for POI. The contribution of FAM175A to POI needs further exploration.

Persistence of Gamma-H2AX Foci in Bronchial Cells Correlates with Susceptibility to Radiation Associated Lung Cancer in Mice.

The risk of developing radiation-induced lung cancer differs between different strains of mice, but the underlying cause of the strain differences is unknown. Strains of mice also differ in how quickly they repair radiation-induced DNA double-strand breaks (DSBs). We assayed mouse strains from the CcS/Dem recombinant congenic strain set for their efficacy in repairing DNA DSBs during protracted irradiation. We measured unrepaired γ-H2AX radiation-induced foci (RIF), which persisted after chronic 24-h gamma irradiation, as a surrogate marker for repair efficiency in bronchial epithelial cells for 17 of the CcS/Dem strains and the BALB/c founder strain. We observed a very strong correlation (R2 = 79.18%, P < 0.001) between the level of unrepaired RIF and radiogenic lung cancer incidence measured in the same strains. Interestingly, spontaneous levels of foci in nonirradiated mice also showed good correlation with lung cancer incidence when incidence data from male and female mice were combined. These results suggest that genetic differences in DNA repair capacity largely account for differing susceptibilities to radiation-induced lung cancer among CcS/Dem mouse strains, and that high levels of spontaneous DNA damage are also a relatively good marker of cancer predisposition. In a smaller pilot study, we found that the repair capacity measured in peripheral blood leucocytes also correlated well with radiogenic lung cancer susceptibility, raising the possibility that the assay could be used to detect radiogenic lung cancer susceptibility in humans.

In vivo measurements of interindividual differences in DNA glycosylases and APE1 activities

The integrity of our DNA is challenged with at least 100,000 lesions per cell on a daily basis. Failure to repair DNA damage efficiently can lead to cancer, immunodeficiency, and neurodegenerative disease. Base excision repair (BER) recognizes and repairs minimally helix-distorting DNA base lesions induced by both endogenous and exogenous DNA damaging agents. Levels of BER-initiating DNA glycosylases can vary between individuals, suggesting that quantitating and understanding interindividual differences in DNA repair capacity (DRC) may enable us to predict and prevent disease in a personalized manner. However, population studies of BER capacity have been limited because most methods used to measure BER activity are cumbersome, time consuming and, for the most part, only allow for the analysis of one DNA glycosylase at a time. We have developed a fluorescence-based multiplex flow-cytometric host cell reactivation assay wherein the activity of several enzymes [four BER-initiating DNA glycosylases and the downstream processing apurinic/apyrimidinic endonuclease 1 (APE1)] can be tested simultaneously, at single-cell resolution, in vivo. Taking advantage of the transcriptional properties of several DNA lesions, we have engineered specific fluorescent reporter plasmids for quantitative measurements of 8-oxoguanine DNA glycosylase, alkyl-adenine DNA glycosylase, MutY DNA glycosylase, uracil DNA glycosylase, and APE1 activity. We have used these reporters to measure differences in BER capacity across a panel of cell lines collected from healthy individuals, and to generate mathematical models that predict cellular sensitivity to methylmethane sulfonate, H2O2, and 5-FU from DRC. Moreover, we demonstrate the suitability of these reporters to measure differences in DRC in multiple pathways using primary lymphocytes from two individuals.

Differences in DNA Repair Capacity, Cell Death and Transcriptional Response after Irradiation between a Radiosensitive and a Radioresistant Cell Line.

Normal tissue toxicity after radiotherapy shows variability between patients, indicating inter-individual differences in radiosensitivity. Genetic variation probably contributes to these differences. The aim of the present study was to determine if two cell lines, one radiosensitive (RS) and another radioresistant (RR), showed differences in DNA repair capacity, cell viability, cell cycle progression and, in turn, if this response could be characterised by a differential gene expression profile at different post-irradiation times. After irradiation, the RS cell line showed a slower rate of γ-H2AX foci disappearance, a higher frequency of incomplete chromosomal aberrations, a reduced cell viability and a longer disturbance of the cell cycle when compared to the RR cell line. Moreover, a greater and prolonged transcriptional response after irradiation was induced in the RS cell line. Functional analysis showed that 24 h after irradiation genes involved in “DNA damage response”, “direct p53 effectors” and apoptosis were still differentially up-regulated in the RS cell line but not in the RR cell line. The two cell lines showed different response to IR and can be distinguished with cell-based assays and differential gene expression analysis. The results emphasise the importance to identify biomarkers of radiosensitivity for tailoring individualized radiotherapy protocols.

DNA repair in species with extreme lifespan differences.

Differences in DNA repair capacity have been hypothesized to underlie the great range of maximum lifespans among mammals. However, measurements of individual DNA repair activities in cells and animals have not substantiated such a relationship because utilization of repair pathways among animals--depending on habitats, anatomical characteristics, and life styles--varies greatly between mammalian species. Recent advances in high-throughput genomics, in combination with increased knowledge of the genetic pathways involved in genome maintenance, now enable a comprehensive comparison of DNA repair transcriptomes in animal species with extreme lifespan differences. Here we compare transcriptomes of liver, an organ with high oxidative metabolism and abundant spontaneous DNA damage, from humans, naked mole rats, and mice, with maximum lifespans of ~120, 30, and 3 years, respectively, with a focus on genes involved in DNA repair. The results show that the longer-lived species, human and naked mole rat, share higher expression of DNA repair genes, including core genes in several DNA repair pathways. A more systematic approach of signaling pathway analysis indicates statistically significant upregulation of several DNA repair signaling pathways in human and naked mole rat compared with mouse. The results of this present work indicate, for the first time, that DNA repair is upregulated in a major metabolic organ in long-lived humans and naked mole rats compared with short-lived mice. These results strongly suggest that DNA repair can be considered a genuine longevity assurance system.

Abstract 4565: ATM genotyping modulates the risk of radiation-associated breast cancer among atomic-bomb survivors

Abstract Objective: RERF epidemiological studies among atomic-bomb (A-bomb) survivors have demonstrated significantly-increased incidence of selected cancers with increased radiation dose. It is noteworthy that a great deal of inter-individual variation in susceptibility to radiation-associated cancers could not be explained solely by radiation dose and age at exposure, suggesting the possibility that this variation might be partly ascribed to inter-individual differences in DNA repair capacity. Of 11 known single nucleotide polymorphisms (SNPs) of the DNA repair gene ATM, we selected a particular SNP (rs189037; located in the 5′ untranslated region and therefore thought to be associated with ATM gene expression) and examined the association with radiation-associated breast cancer in the context of an RERF A-bomb survivor cohort study. Materials and Methods: A follow-up survey during 1981-2005 of 2,944 female members of the RERF Immunogenome Study Cohort identified a total of 100 breast cancer cases. ATM genotypes (ATM-G/G, -G/A, and -A/A) were determined by the TaqMan assay method for all cohort members. Relative risks (RRs) of breast cancer were estimated for radiation dose, the ATM genotypes, and the combined categories of dose levels and genotypes. Results and Discussion: Disregarding the genotype effects, RR of breast cancer significantly increased with increased radiation dose (RR = 1.51/Gy, 95% CI: 1.25-1.83). For overall radiation dose levels, A-bomb survivors with ATM-G/G homozygote revealed a significantly-increased RR of breast cancer (RR = 1.85, 95%CI: 1.24-2.75), using as reference the risk of those with ATM-A/A homozygotes combined with ATM-G/A heterozygotes. When radiation dose was categorized into three levels (&amp;lt; 5 mGy referred to as “non-exposed,” 5-530 mGy, and &amp;gt;530 mGy), the survivors with ATM-A/A who were exposed to the highest dose level (&amp;gt;530 mGy) showed the highest risk of breast cancer (RR = 4.76, 95%CI: 2.30-9.88), using as reference the risk of “non-exposed” survivors with ATM-T/T or ATM-A/T. These results suggest the possibility that the ATM genotyping may be involved in individually differing susceptibility to radiation-associated breast cancer. Citation Format: Tomonori Hayashi, Yiqun Hu, Kengo Yoshida, Waka Ohishi, Ayumi Hida, Ikue Hayashi, Seishi Kyoizumi, Yoichiro Kusunoki, Kei Nakachi. ATM genotyping modulates the risk of radiation-associated breast cancer among atomic-bomb survivors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4565. doi:10.1158/1538-7445.AM2015-4565

Polymorphisms in DNA repair and multidrug resistance genes among Sindhis of Central India

Polymorphisms in DNA repair and multidrug resistance genes might contribute to interindividual and interethnic differences in DNA repair capacity and drug disposition respectively. In the present study, we determined the allele and genotype frequencies of four single nucleotide polymorphisms (SNPs) located in the DNA repair genes, XRCC1, XRCC3, XPD, OGG1, namely XRCC1 Arg399Gln, XRCC3 Thr241Met, XPD Lys751Gln, and OGG1 Ser326Cys, respectively and two SNPs located in the multidrug resistance gene, ABCB1, namely ABCB1 C3435T and ABCB1 C1236T, in 33–35 healthy and unrelated Sindhi individuals, residing in the Vidarbha region of Central India and compared them with the Maharashtrian population from the same geographical region and some other HapMap populations from the HapMap database. The study findings reveal that the Indian Sindhis are closely related to the Maharashtrians as well as Utah residents with Northern and Western European ancestry and Gujarati Indians in Houston, Texas in the HapMap database.

Enhanced γ-H2AX DNA damage foci detection using multimagnification and extended depth of field in imaging flow cytometry.

Accurate and rapid methods for the detection of DNA damage foci in eukaryotic cells are central to DNA repair studies, which identify differences in DNA repair capacity in cell lines. Such assays have been important in delineating mechanisms of DNA repair in human cells. Previously we were the first to demonstrate the use of imaging flow cytometry for the detection of γ‐H2AX foci in cells exposed to ionizing radiation causing the induction of DNA strand breaks. In this report we extend these studies and show an enhancement of foci quantitation and image resolution using next generation imaging flow cytometry with the Amnis ImagestreamX Mark II. We demonstrate using cell lines derived from normal individuals, and DNA double strand break repair defective cells that the number of foci observed is significantly increased when using 60× as compared to 40× magnification. Also, foci numbers and resolution is further increased with the application of the focus stacking (Extended Depth of Field–EDF) capacity activated. This report represents the first such demonstration of multimagnification and EDF for the enhanced quantitation of DNA damage in cells and provides a level of resolution, which near matches in situ microscopy methods for the detection of γ‐H2AX foci. © 2015 The Authors. Published by Wiley Periodicals Inc. on behalf of ISAC.

Genetic code evolution reveals the neutral emergence of mutational robustness, and information as an evolutionary constraint.

The standard genetic code (SGC) is central to molecular biology and its origin and evolution is a fundamental problem in evolutionary biology, the elucidation of which promises to reveal much about the origins of life. In addition, we propose that study of its origin can also reveal some fundamental and generalizable insights into mechanisms of molecular evolution, utilizing concepts from complexity theory. The first is that beneficial traits may arise by non-adaptive processes, via a process of “neutral emergence”. The structure of the SGC is optimized for the property of error minimization, which reduces the deleterious impact of point mutations. Via simulation, it can be shown that genetic codes with error minimization superior to the SGC can emerge in a neutral fashion simply by a process of genetic code expansion via tRNA and aminoacyl-tRNA synthetase duplication, whereby similar amino acids are added to codons related to that of the parent amino acid. This process of neutral emergence has implications beyond that of the genetic code, as it suggests that not all beneficial traits have arisen by the direct action of natural selection; we term these “pseudaptations”, and discuss a range of potential examples. Secondly, consideration of genetic code deviations (codon reassignments) reveals that these are mostly associated with a reduction in proteome size. This code malleability implies the existence of a proteomic constraint on the genetic code, proportional to the size of the proteome (P), and that its reduction in size leads to an “unfreezing” of the codon – amino acid mapping that defines the genetic code, consistent with Crick’s Frozen Accident theory. The concept of a proteomic constraint may be extended to propose a general informational constraint on genetic fidelity, which may be used to explain variously, differences in mutation rates in genomes with differing proteome sizes, differences in DNA repair capacity and genome GC content between organisms, a selective pressure in the evolution of sexual reproduction, and differences in translational fidelity. Lastly, the utility of the concept of an informational constraint to other diverse fields of research is explored.

Glutathione depletion sensitizes cisplatin- and temozolomide-resistant glioma cells in vitro and in vivo

Malignant glioma is a severe type of brain tumor with a poor prognosis and few options for therapy. The main chemotherapy protocol for this type of tumor is based on temozolomide (TMZ), albeit with limited success. Cisplatin is widely used to treat several types of tumor and, in association with TMZ, is also used to treat recurrent glioma. However, several mechanisms of cellular resistance to cisplatin restrict therapy efficiency. In that sense, enhanced DNA repair, high glutathione levels and functional p53 have a critical role on cisplatin resistance. In this work, we explored several mechanisms of cisplatin resistance in human glioma. We showed that cellular survival was independent of the p53 status of those cells. In addition, in a host-cell reactivation assay using cisplatin-treated plasmid, we did not detect any difference in DNA repair capacity. We demonstrated that cisplatin-treated U138MG cells suffered fewer DNA double-strand breaks and DNA platination. Interestingly, the resistant cells carried higher levels of intracellular glutathione. Thus, preincubation with the glutathione inhibitor buthionine sulfoximine (BSO) induced massive cell death, whereas N-acetyl cysteine, a precursor of glutathione synthesis, improved the resistance to cisplatin treatment. In addition, BSO sensitized glioma cells to TMZ alone or in combination with cisplatin. Furthermore, using an in vivo model the combination of BSO, cisplatin and TMZ activated the caspase 3–7 apoptotic pathway. Remarkably, the combined treatment did not lead to severe side effects, while causing a huge impact on tumor progression. In fact, we noted a remarkable threefold increase in survival rate compared with other treatment regimens. Thus, the intracellular glutathione concentration is a potential molecular marker for cisplatin resistance in glioma, and the use of glutathione inhibitors, such as BSO, in association with cisplatin and TMZ seems a promising approach for the therapy of such devastating tumors.

The anti-cancer actions of O6-methylguanine-DNA-methyltransferase in relation to colon polyps

BackgroundGenetic variability in DNA repair genes may contribute to differences in DNA repair capacity and susceptibility to colon polyps and cancer. In this study, we examined the role of MGMT polymorphisms in colon polyps formation. MethodsPCR–SSCP analysis was performed included 254 patients with colon polyps and 330 controls. ResultsThe homozygous F84F genotype was significantly more prevalent in study group than in controls. The polymorphic allele 84F was more frequent appeared in group of older patients and in group of smoking patients. On the other hand, there were no association between 84F and gender, size of polyps, cancer family history. ConclusionsWe concluded that high frequency of 84F allele in the group of patients may suggest the role of the MGMT variant in colon polyps etiology.

Inter-individual variation in nucleotide excision repair pathway is modulated by non-synonymous polymorphisms in ERCC4 and MBD4 genes

Inter-individual differences in DNA repair capacity (DRC) may lead to genome instability and, consequently, modulate individual cancer risk. Among the different DNA repair pathways, nucleotide excision repair (NER) is one of the most versatile, as it can eliminate a wide range of helix-distorting DNA lesions caused by ultraviolet light irradiation and chemical mutagens. We performed a genotype–phenotype correlation study in 122 healthy subjects in order to assess if any associations exist between phenotypic profiles of NER and DNA repair gene single nucleotide polymorphisms (SNPs). Individuals were genotyped for 768 SNPs with a custom Illumina Golden Gate Assay, and peripheral blood mononuclear cells (PBMCs) of the same subjects were tested for a NER comet assay to measure DRC after challenging cells by benzo(a)pyrene diolepoxide (BPDE).We observed a large inter-individual variability of NER capacity, with women showing a statistically significant lower DRC (mean±SD: 6.68±4.76; p=0.004) than men (mean±SD: 8.89±5.20). Moreover, DRC was significantly lower in individuals carrying a variant allele for the ERCC4 rs1800124 non-synonymous SNP (nsSNP) (p=0.006) and significantly higher in subjects with the variant allele of MBD4 rs2005618 SNP (p=0.008), in linkage disequilibrium (r2=0.908) with rs10342 nsSNP.Traditional in silico docking approaches on protein–DNA and protein–protein interaction showed that Gly875 variant in ERCC4 (rs1800124) decreases the DNA–protein interaction and that Ser273 and Thr273 variants in MBD4 (rs10342) indicate complete loss of protein–DNA interactions. Our results showed that NER inter-individual capacity can be modulated by cross-talk activity involving nsSNPs in ERCC4 and MBD4 genes, and they suggested to better investigate SNP effect on cancer risk and response to chemo- and radiotherapies.

DNA Repair Genes XRCC1, XRCC3, XPD, and OGG1 Polymorphisms among the Central Region Population of Saudi Arabia

DNA repair is one of the central defense mechanisms against mutagenic exposures. Inherited SNPs of DNA repair genes may contribute to variations in DNA repair capacity and susceptibility to cancer. Due to the presence of these variants, inter-individual and ethnic differences in DNA repair capacity have been established in various populations. Saudi Arabia harbors enormous genetic and cultural diversity. In the present study we aimed to determine the genotype and allele frequencies of XRCC1 Arg399Gln (rs25487), XRCC3 Thr241Met (rs861539), XPD Lys751Gln (rs13181), and OGG1 Ser326Cys (rs1052133) gene polymorphisms in 386 healthy individuals residing in the central region of Saudi Arabia and compare them with HapMap and other populations. The genotype and allele frequencies of the four DNA repair gene loci in central Saudi population showed a distinctive pattern. Furthermore, comparison of polymorphisms in these genes with other populations also showed a unique pattern for the central Saudi population. To the best of our knowledge, this is the first report that deals with these DNA repair gene polymorphisms among the central Saudi population.

DNA Repair Gene Polymorphisms at XRCC1, XRCC3, XPD, and OGG1 Loci in the Hyderabad Population of India

DNA repair is one of the crucial defense mechanism against mutagenic exposure. Inherited SNPs of DNA repair genes may contribute to variation in DNA repair capacity and susceptibility to cancer. Due to the presence of these variants, inter-individual and ethnic differences in DNA repair capacity have been established in various populations. India harbors enormous genetic and cultural diversity. In the present study we aimed to determine the genotypes and allele frequencies of XRCC1 Arg399Gln (rs25487), XRCC3 Thr241Met (rs861539), XPD Lys751Gln (rs13181), and OGG1 Ser326Cys (rs1052133) gene polymorphisms in 186 healthy individuals residing in the Hyderabad region of India and to compare them with HapMap and other populations. The genotype and allele frequency distribution at the four DNA repair gene loci among Hyderabad population of India revealed a characteristic pattern. Comparison of these gene polymorphisms with other populations revealed a distinctiveness of Hyderabad population from the Deccan region of India. To the best of our knowledge, this is the first report of such DNA repair gene polymorphisms in the Deccan Indian population.

DNA Repair Gene Polymorphisms at XRCC1 (Arg194Trp, Arg280His, and Arg399Gln) in a Healthy Tunisian Population: Interethnic Variation and Functional Prediction

The genetic polymorphisms in DNA repair genes might affect the repair activities of the enzymes, predisposing individuals to cancer risk. Due to these genetic variants, interethnic differences in DNA repair capacity were observed in various populations. Hence, our study aimed to determine the prevalence of three nonsynonymous single-nucleotide polymorphisms (SNPs) in an X-ray repair cross-complementation group 1 gene (XRCC1) (Arg194Trp, Arg280His, and Arg399Gln) in a healthy Tunisian population (TUN) and to compare that with HapMap ( www.hapmap.org ) populations. Also, we predicted their eventual functional effect based on the protein conservation analysis by Sorting Intolerant From Tolerant (SIFT; http://sift.jcvi.org/www/SIFT_dbSNP.html ) software. The genotypes of 154 healthy individuals were determined by the polymerase chain reaction-restriction fragment length polymorphism. Tunisians showed a relative relatedness with Caucasians (European ancestry) for Arg194Trp and Arg399Gln that may be explained by the strategic geographic location of Tunisia in the Mediterranean, allowing exchanges with European countries. However, a characteristic pattern was observed in Arg280His polymorphism, which could be explained by the high inbreeding rate in TUN. The analysis of protein conservation showed that the three amino acid substitutions were predicted as damaged. The results presented here provide the first report on XRCC1 polymorphisms about Tunisians and may establish baseline database for our future clinical and genetic studies.

The MBD4 Glu346Lys Polymorphism Is Associated With the Risk of Cervical Cancer in a Chinese Population

Methyl-CpG binding domain 4 (MBD4) protein functions as a DNA repair enzyme and plays an important role in maintaining genome integrity and carcinogenesis. The polymorphisms in the MBD4 gene may be associated with differences in DNA repair capacity and thereby influence an individual's susceptibility to cervical cancer. To verify this hypothesis, we examined the potential association between the MBD4 Glu346Lys polymorphism (rs140693, G>A) and the risk of cervical cancer in a Chinese population. We genotyped the MBD4 Glu346Lys polymorphism in 146 cervical cancer cases and 320 healthy female subjects using polymerase chain reaction-based restriction fragment length polymorphism method. Unconditional logistic regression analysis was used to estimate the association between the genotypes and the risk of cervical cancer. We observed a significantly decreased risk of cervical cancer associated with the heterozygous Lys/Glu genotype (odds ratio [OR], 0.60; 95% confidence interval [CI], 0.36-0.99; P = 0.046) and the homozygous Glu/Glu genotype (OR, 0.52; 95% CI, 0.30-0.89; P = 0.018), compared with the Lys/Lys homozygotes. Moreover, the reduced cervical cancer risk was more predominant among younger subjects or human papillomavirus-positive individuals carrying Glu/Glu genotypes (OR, 0.33; 95% CI, 0.14-0.78, P = 0.011; and OR, 0.27; 95% CI, 0.09-0.75, P = 0.013, respectively). The MBD4 codon 346 polymorphism may play a role in cervical cancer susceptibility in the Chinese population. Further larger case-control and functional studies are needed to validate these findings.

An interethnic variability and a functional prediction of DNA repair gene polymorphisms: the example of XRCC3 (p.Thr241&gt;Met) and XPD (p.Lys751&gt;Gln) in a healthy Tunisian population

Genetic polymorphisms in DNA repair genes might influence the repair activities of the enzymes predisposing individuals to cancer risk. Owing to the presence of these genetic variants, interethnic differences in DNA repair capacity have been observed in various populations. The present study was undertaken to determine the allele and genotype frequencies of two common non-synonymous SNPs, XRCC3 p.Thr241>Met (C > T, rs861539) and XPD p.Lys751>Gln (T > G, rs13181) in a healthy Tunisian population and to compare them with HapMap ( http://www.hapmap.org/ ) populations. Also, we predicted their eventual functional effect based on bioinformatics tools. The genotypes of 154 healthy and unrelated individuals were determined by PCR-RFLP procedure. Our findings showed a close relatedness with Caucasians from European ancestry which might be explained by the strategic geographic location of Tunisia in the Mediterranean, thus allowing exchanges with Europeans countries. The in silico predictions showed that p.Thr241>Met substitution in XRCC3 protein was predicted as possibly damaging, indicating that it is likely to have functional consequences as well. To the best of our knowledge, this is the first study in this regard in Tunisia. So, these data could provide baseline database and help us to explore the relationship of XRCC3 and XPD polymorphisms with both cancer risk and DNA repair variability in our population.

DNA damage and nucleotide excision repair capacity in healthy individuals

Interindividual differences in DNA repair capacity (DRC) represent an important source of variability in genome integrity and thus influence health risk. In the last decade, DRC measurement has attracted attention as a potential biomarker in cancer prediction. Aim of the present exploratory study was to characterize the variability in DNA damage and DRC on 100 healthy individuals and to identify biological, lifestyle, or genetic factors modulating these parameters. The ultimate goal was to obtain reference data from cancer-free population, which may constitute background for further investigations on cancer patients. The endogenous DNA damage was measured as a level of DNA single-strand breaks and DRC, specific for nucleotide excision repair (NER), was evaluated using modified comet assay, following the challenge of peripheral blood mononuclear cells with benzo[a]pyrene diolepoxide. Additionally, genetic polymorphisms in NER genes (XPA, XPC, XPD, and XPG) were assessed. We have observed a substantial interindividual variability for both examined parameters. DNA damage was significantly affected by gender and alcohol consumption (P = 0.003 and P = 0.012, respectively), whereas DRC was associated with family history of cancer (P = 0.012). The stratification according to common variants in NER genes showed that DNA damage was significantly modulated by the presence of the variant T allele of XPC Ala499Val polymorphism (P = 0.01), while DRC was modulated by the presence of the A allele of XPA G23A polymorphism (P = 0.048). Our results indicate the range of endogenous DNA single-strand breaks and capacity of NER in healthy volunteers as well as the role of potentially relevant confounders. Environ. Mol. Mutagen. 2011. © 2011 Wiley-Liss, Inc.