Prospective analysis of DNA damage and repair markers of lung cancer risk from the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial.

Mutagen challenge and DNA repair assays have been used in case-control studies for nearly three decades to assess human cancer risk. The findings still engender controversy because blood was drawn after cancer diagnosis so the results may be biased, a type called 'reverse causation'. We therefore used Epstein-Barr virus-transformed lymphoblastoid cell lines established from prospectively collected peripheral blood samples to evaluate lung cancer risk in relation to three DNA repair assays: alkaline Comet assay, host cell reactivation (HCR) assay with the mutagen benzo[a]pyrene diol epoxide and the bleomycin mutagen sensitivity assay. Cases (n = 117) were diagnosed with lung cancer between 0.3 and 6 years after blood collection and controls (n = 117) were frequency matched on calendar year and age at blood collection, gender and smoking history; all races were included. Case and control status was unknown to laboratory investigators. In unconditional logistic regression analyses, statistically significantly increased lung cancer odds ratios (OR(adjusted)) were observed for bleomycin mutagen sensitivity as quartiles of chromatid breaks/cell [relative to the lowest quartile, OR = 1.2, 95% confidence interval (CI): 0.5-2.5; OR = 1.4, 95% CI: 0.7-3.1; OR = 2.1, 95% CI: 1.0-4.4, respectively, P(trend) = 0.04]. The magnitude of the association between the bleomycin assay and lung cancer risk was modest compared with those reported in previous lung cancer studies but was strengthened when we included only incident cases diagnosed more than a year after blood collection (P(trend) = 0.02), supporting the notion the assay may be a measure of cancer susceptibility. The Comet and HCR assays were unrelated to lung cancer risk.

Inter-individual variation in DNA double-strand break repair in human fibroblasts before and after exposure to low doses of ionizing radiation.

DNA double-strand breaks (DSB) are generally considered the most critical lesion induced by ionizing radiation (IR) and may initiate carcinogenesis and other disease. Using an immunofluorescence assay to simultaneously detect nuclear foci of the phosphorylated forms of histone H2AX and ATM kinase at sites of DSBs, we examined the response of 25 apparently normal and 10 DNA repair-deficient (ATM, ATR, NBN, LIG1, LIG4, and FANCG) primary fibroblast strains irradiated with low doses of (137)Cs gamma-rays. Quiescent G(0)/G(1)-phase cultures were exposed to 5, 10, and 25 cGy and allowed to repair for 24h. The maximum level of IR-induced foci (0.15 foci per cGy, at 10 or 30 min) in the normal strains showed much less inter-individual variation (CV approximately 0.2) than the level of spontaneous foci, which ranged from 0.2-2.6 foci/cell (CV approximately 0.6; mean+/-SD of 1.00+/-0.57). Significantly slower focus formation post-irradiation was observed in seven normal strains, similar to most mutant strains examined. There was variation in repair efficiency measured by the fraction of IR-induced foci remaining 24h post-irradiation, curiously with the strains having slower focus formation showing more efficient repair after 25 cGy. Interestingly, the ranges of spontaneous and residual induced foci levels at 24h in the normal strains were as least as large as those observed for the repair-defective mutant strains. The inter-individual variation in DSB foci parameters observed in cells exposed to low doses of ionizing radiation in this small survey of apparently normal people suggests that hypomorphic genetic variants in genomic maintenance and/or DNA damage signaling and repair genes may contribute to differential susceptibility to cancer induced by environmental mutagens.

Differential expression of TP53 associated genes in Fanconi anemia cells after mitomycin C and hydroxyurea treatment.

Fanconi anemia (FA) is a rare, heritable chromosomal instability disease characterized by several congenital defects and cancer predisposition. Functional interactions between specific FA proteins and DNA damage response and repair activities have been reported, but the interplay between these mechanisms for maintaining genomic stability are not well understood. Many DNA damage response proteins are transcriptionally regulated by the tumor suppressor protein p53 (TP53), suggesting an important regulatory role for the DNA damage and stress response pathway. To better understand the association between FA and the DNA damage stress response we analyzed the levels of chromosomal damage and damage mediated gene transcription responses in lymphoblastoid cells derived from normal individuals and patients carrying the most common FA complementation group (FA-A). Chromosomal aberrations were first measured after exposure to mitomicyn C (MMC) or hydroxyurea (HU). Aliquots of the same cell were than assayed for the transcriptional response of 21 DNA damage and stress response genes using quantitative real-time PCR. The FA-A lymphoblastoid cells showed significant increases in the frequency of chromosome aberrations relative to non-FA-A lymphoblastoid lines after MMC treatment. The MMC induced damage was correlated with a general increase in expression of TP53-modulated DNA damage stress response genes involved in processes such as DNA repair, cell cycle progression, and apoptosis. Following HU treatment FA cells showed a decreased induction of CAs with much less transcriptional differences between targeted genes. Overall, the differences between the normal and FA-A cells after genotoxic treatments imply an increased activation and reliance of FA cells on the down-stream activities of TP53 for prevention of cell killing and chromosome damage from interstrand crosslinks but not for general replication arrest and double strand breaks. Furthermore, these results imply a regulatory connection between the FA pathway and activation of TP53 for responding to DNA damage. Alterations in the regulation of the DNA damage response may be related to the complex phenotypes seen in FA patients.

No evidence for differences in DNA damage assessed before and after a cancer diagnosis.

The overwhelming majority of studies that have found increased cancer risk associated with functional deficits in DNA repair used a case-control design, in which measurements were made after cancer diagnosis. However, there are concerns about whether the cancer itself or cancer treatment affected the conclusions (reverse causation bias). We assessed the effect of cancer diagnosis among 26 breast cancer controls who had blood collected during 2001 to 2003 and again in 2005 to 2006 after being diagnosed with cancer. Using the alkaline comet assay, we quantified DNA damage in untreated lymphoblastoid cell lines. Comet distributed moment, olive tail moment, percentage of DNA in tail, and comet tail length were summarized as the geometric mean of 100 cells. For comet distributed moment, olive tail moment, tail DNA, and tail length, the proportions of women with before diagnosis values higher than after diagnosis were 65%, 50%, 50%, and 46%, respectively. We found no significant differences in the before or after diagnosis mean comet values. Median cut-points were determined from the before diagnosis distribution, and we used conditional logistic regression to calculate odds ratios (OR) and upper 95% bounds of the confidence intervals. ORs ranged from 0.6 to 0.9 with upper confidence interval bounds of 1.9 and 2.6, meaning biased ORs above 2.6 are unlikely. We found no evidence that reverse causation bias is an important concern in case-control studies using the comet assay applied to cell lines collected after cancer diagnosis. More work is needed to characterize the effect of cancer diagnosis on other phenotypic assays.

Studies of thioguanine-resistant lymphocytes induced by in vivo irradiation of mice.

The frequency of Hprt-deficient lymphocytes in mice after in vivo gamma irradiation, has been found to vary as a function of time elapsed after exposure and irradiation dose. The frequency of mutant lymphocytes in spleen was determined using an in vitro, clonogenic assay for thioguanine-resistant T-lymphocytes. Mice were exposed to single doses of 0-400 cGy from cesium-137 or to eight daily doses of 50 cGy. The time to maximum-induced mutant frequency was 3 weeks. The dose response was strikingly curvilinear at 3-5 weeks after irradiation, but less precisely defined for 10-53 weeks after exposure, being fit by either linear or quadratic dependence. Three weeks after eight daily 50 cGy exposures, mutant frequency was elevated above controls and mice exposed to 50 cGy (which were not distinct from the nonirradiated controls), but only 17% in that of mice given a single 400 cGy fraction. This fractionation effect and the curvilinearity of the early dose-response curve suggested that saturation of repair increased the yield of mutations at higher acute doses. The decline of spleen mutant frequency in mice observed between 5 and 10 weeks after irradiation may reflect selection against some mutants. The marked variation of mutant frequency, as a function of time after irradiation and of dose rate, emphasize the need to evaluate these variables carefully and consistently in future studies.

Disparate contributions of the Fanconi anemia pathway and homologous recombination in preventing spontaneous mutagenesis.

Fanconi anemia (FA) is a chromosomal instability disorder in which DNA-damage processing defects are reported for translesion synthesis (TLS), non-homologous end joining (NHEJ) and homologous recombination (HR; both increased and decreased). To reconcile these diverse findings, we compared spontaneous mutagenesis in FA and HR mutants of hamster CHO cells. In the fancg mutant we find a reduced mutation rate accompanied by an increased proportion of deletions within the hprt gene. Moreover, in fancg cells gene amplification at the CAD and dhfr loci is elevated, another manifestation of inappropriate processing of damage during DNA replication. In contrast, the rad51d HR mutant has a greatly elevated rate of hprt mutations, >85% of which are deletions. Our analysis supports the concept that HR faithfully restores broken replication forks, whereas the FA pathway acts more globally to ensure chromosome stability by promoting efficient end joining of replication-derived breaks, as well as TLS and HR.

Exploration of methods to identify polymorphisms associated with variation in DNA repair capacity phenotypes.

Elucidating the relationship between polymorphic sequences and risk of common disease is a challenge. For example, although it is clear that variation in DNA repair genes is associated with familial cancer, aging and neurological disease, progress toward identifying polymorphisms associated with elevated risk of sporadic disease has been slow. This is partly due to the complexity of the genetic variation, the existence of large numbers of mostly low frequency variants and the contribution of many genes to variation in susceptibility. There has been limited development of methods to find associations between genotypes having many polymorphisms and pathway function or health outcome. We have explored several statistical methods for identifying polymorphisms associated with variation in DNA repair phenotypes. The model system used was 80 cell lines that had been resequenced to identify variation; 191 single nucleotide substitution polymorphisms (SNPs) are included, of which 172 are in 31 base excision repair pathway genes, 19 in 5 anti-oxidation genes, and DNA repair phenotypes based on single strand breaks measured by the alkaline Comet assay. Univariate analyses were of limited value in identifying SNPs associated with phenotype variation. Of the multivariable model selection methods tested: the easiest that provided reduced error of prediction of phenotype was simple counting of the variant alleles predicted to encode proteins with reduced activity, which led to a genotype including 52 SNPs; the best and most parsimonious model was achieved using a two-step analysis without regard to potential functional relevance: first SNPs were ranked by importance determined by random forests regression (RFR), followed by cross-validation in a second round of RFR modeling that included ever more SNPs in declining order of importance. With this approach six SNPs were found to minimize prediction error. The results should encourage research into utilization of multivariate analytical methods for epidemiological studies of the association of genetic variation in complex genotypes with risk of common diseases.

Candidate protein biodosimeters of human exposure to ionizing radiation.

PURPOSE: To conduct a literature review of candidate protein biomarkers for individual radiation biodosimetry of exposure to ionizing radiation. MATERIALS AND METHODS: Reviewed approximately 300 publications (1973 - April 2006) that reported protein effects in mammalian systems after either in vivo or in vitro radiation exposure. RESULTS: We found 261 radiation-responsive proteins including 173 human proteins. Most of the studies used high doses of ionizing radiation (>4 Gy) and had no information on dose- or time-responses. The majority of the proteins showed increased amounts or changes in phosphorylation states within 24 h after exposure (range: 1.5- to 10-fold). Of the 47 proteins that are responsive at doses of 1 Gy and below, 6 showed phosphorylation changes at doses below 10 cGy. Proteins were assigned to 9 groups based on consistency of response across species, dose- and time-response information and known role in the radiation damage response. CONCLUSIONS: ATM (Ataxia telengiectasia mutated), H2AX (histone 2AX), CDKN1A (Cyclin-dependent kinase inhibitor 1A), and TP53 (tumor protein 53) are top candidate radiation protein biomarkers. Furthermore, we recommend a panel of protein biomarkers, each with different dose and time optima, to improve individual radiation biodosimetry for discriminating between low-, moderate-, and high-dose exposures. Our findings have applications for early triage and follow-up medical assessments.

Four human FANCG polymorphic variants show normal biological function in hamster CHO cells.

Fanconi anemia (FA) is a rare cancer predisposition disease caused by mutations in at least 12 genes encoding proteins that cooperate to maintain genomic integrity. Variants of FA genes, including FANCG, have been identified in human population screening, but their potential reduction in protein function and role in cancer susceptibility is unclear. To test for possible dysfunction, we constructed plasmids containing four FANCG polymorphisms found in the human population and introduced them in the Fancg-deficient (fancg) KO40 line derived from AA8 hamster CHO cells. Expression of wild-type human FANCG provided fancg cells with complete phenotypic correction as assessed by resistance to the DNA crosslinking agent mitomycin C (MMC), thus providing a sensitive test for detecting the degree of complementation activity for the FANCG variants. We found that all four variants conferred levels of mitomycin C resistance as well as restoration of monoubiquitination of Fancd2, a key indicator of a functional FA protein pathway, similar to those observed in wild-type transfectants. Under the same conditions, the L71P amino acid substitution mutant, identified in an FA patient, gave no complementation. Using this novel system for determining FANCG functionality, we detect no decrement in function of the human FANCG polymorphic variants examined.

DNA damage among thyroid cancer and multiple cancer cases, controls, and long-lived individuals.

Variation in the detection, signaling, and repair of DNA damage contributes to human cancer risk. To assess capacity to modulate endogenous DNA damage among radiologic technologists who had been diagnosed with breast cancer and another malignancy (breast-other, n=42), early-onset breast cancer (early-onset, age or=75% versus below the median, age-adjusted) was most consistently associated with the highest odds ratios in the breast-other, early-onset, and thyroid cancer groups (with risk increased 10-, 5- or 19-fold, respectively, with wide confidence intervals) and decreased risk among the hyper-normal group. For the other three comet measures, risk of breast-other was elevated approximately three-fold. Risk of early-onset breast cancer was mixed and risk of thyroid cancer ranged from null to a two-fold increase. The hyper-normal group showed decreased odds ratios for tail DNA and OTM, but not CDM. DNA damage, as estimated by all comet measures, was relatively unaffected by survival time, reproductive factors, and prior radiation treatment. We detected a continuum of endogenous DNA damage that was highest among cancer cases, less in controls, and suggestively lowest in hyper-normal individuals. Measuring this DNA damage phenotype may contribute to the identification of susceptible sub-groups. Our observations require replication in a prospective study with a large number of pre-diagnostic samples.

A comparison of somatic mutational spectra in healthy study populations from Russia, Sweden and USA.

A comparison of mutation spectra at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene of peripheral blood T-lymphocytes may provide an insight into the aetiology of somatic mutation contributing to carcinogenesis and other diseases. To increase the knowledge of mutation spectra in healthy people, we have analysed HPRT mutant T-cells of 50 healthy Russians originally recruited as controls in a study involving Chernobyl clean-up workers [I.M. Jones, H.Galick, P.Kato et al. (2002) Radiat. Res., 158, 424-442]. Reverse transcriptase-polymerase chain reactions and DNA sequencing identified 161 independent mutations among 176 thioguanine-resistant mutants. Forty mutations affected splicing mechanisms and 27 deletions or insertions of 1-60 nt were identified. Ninety-four single base substitutions were identified, including 62 different mutations at 55 different nucleotide positions, of which 19 had not been reported previously in human T-cells. Comparison of this base substitution spectrum with mutation spectra in a USA [K.J.Burkhart-Schultz, C.L. Thompson and I.M. Jones (1996) Carcinogenesis, 17, 1871-1883] and two Swedish populations [A.Podlutsky, A.-M.Osterholm, S.-M.Hou, A. Hofmaier and B. Lambert (1998) Carcinogenesis, 19, 557-566; A.Podlutsky, S.M.Hou, F.Nyberg, G. Pershagen and B. Lambert (1999) Mutat. Res., 431, 325-39] revealed similarity in the type, frequency and distribution of mutations in the four spectra, consistent with aetiologies inherent in human metabolism. There were 15-19 identical mutations in the three pairwise comparisons of Russian with USA and Swedish spectra. Intriguingly, there were 21 mutations unique to the Russian spectrum, and comparison by the Monte Carlo method of W.T. Adams and T.R. Skopek [(1987) J. Mol. Biol., 194, 391-396] indicated that the Russian spectrum was different from both Swedish spectra (P = 0.007, 0.002), but not different from the USA spectrum (P = 0.07) when Bonferroni correction for multiple comparisons was made (P < 0.008 required for significance). Age and smoking did not account for these differences. Other factors causing mutational differences need to be explored.

New insights into the Fanconi anemia pathway from an isogenic FancG hamster CHO mutant.

The Fanconi anemia (FA) proteins overlap with those of homologous recombination through FANCD1/BRCA2, but the biochemical functions of other FA proteins are largely unknown. By constructing and characterizing a null fancg mutant (KO40) of hamster CHO cells, we show that FancG protects cells against a broad spectrum of genotoxic agents. KO40 is consistently hypersensitive to both alkylating agents that produce monoadducts and those that produce interstrand crosslinks. KO40 cells were no more sensitive to mitomycin C (3x) and diepoxybutane (2x) than to 6-thioguanine (5x), ethylnitrosourea (3x), or methyl methanesulfonate (MMS) (3x). These results contrast with the pattern of selective sensitivity to DNA crosslinking agents seen historically with cell lines from FA patients. The hypersensitivity of KO40 to MMS was not associated with a higher level of initial DNA single-strand breaks; nor was there a defect in removing MNU-induced methyl groups from DNA. Both control and MMS-treated synchronized G1-phase KO40 cells progressed through S phase at a normal rate but showed a lengthening of G2 phase compared with wild type. MMS-treated and untreated early S-phase KO40 cells had increased levels of Rad51 foci compared with wild type. Asynchronous KO40 treated with ionizing radiation (IR) exhibited a normal Rad51 focus response, consistent with KO40 having only slight sensitivity to killing by IR. The plating efficiency and doubling time of KO40 cells were nearly normal, and they showed no increase in spontaneous chromosomal aberrations or sister chromatid exchanges. Collectively, our results do not support a role for FancG during DNA replication that deals specifically with processing DNA crosslinks. Nor do they suggest that the main function of the FA protein "pathway" is to promote efficient homologous recombination. We propose that the primary function of FA proteins is to maintain chromosomal continuity by stabilizing replication forks that encounter nicks, gaps, or replication-blocking lesions.

XRCC1 and glutathione-S-transferase gene polymorphisms and susceptibility to radiotherapy-related malignancies in survivors of Hodgkin disease.

BACKGROUND: One of the most serious late effects of treatment for childhood cancer is the occurrence of subsequent malignancy. Survivors of Hodgkin disease (HD), in particular, have been shown to be at high risk of subsequent malignancy, the occurrence of which has been associated strongly with exposure to radiotherapy. METHODS: In the current study, the authors investigated the association between polymorphisms in 3 genes--glutathione-S-transferase M1 (GSTM1), glutathione-S-transferase T1 (GSTT1), and XRCC1, with roles in protection from a variety of DNA-damaging agents-and the risk of subsequent malignancy in 650 survivors of HD enrolled in the Childhood Cancer Survivor Study who had received radiotherapy. RESULTS: Individuals lacking GSTM1 but not GSTT1 were at increased risk of any subsequent malignancy (odds ratio [OR], 1.5; 95% confidence interval [CI], 1.0-2.3), and for subsequent cancer within the radiation field (OR, 1.4; 95% CI, 0.9-2.1). A nonsignificant increased risk of thyroid carcinoma was observed in individuals lacking either GSTM1 (OR, 2.9; 95% CI, 0.8-10.9) or GSTT1 (OR, 3.7; 95% CI, 0.6-23.5). Individuals having the genotype of the arginine/glutamine polymorphism at codon 399 in the XRCC1 gene (R399) showed a nonsignificant increased risk of breast carcinoma compared with those without (OR, 1.4; 95% CI, 0.7-2.7), and a nonsignificant decreased risk against a subsequent thyroid carcinoma (OR, 0.6; 95% CI, 0.2-1.6). No differences in genotype frequencies were observed between survivors with basal cell carcinoma when compared with survivors without a subsequent cancer. CONCLUSIONS: These data illustrated the potential value of incorporating the collection of genomic DNA in longitudinal cohort studies of populations with well defined, potentially carcinogenic exposures. Evaluation of additional genetic polymorphisms in this cohort may help define genes that influence individual sensitivity to radiotherapy.

Many amino acid substitution variants identified in DNA repair genes during human population screenings are predicted to impact protein function.

Over 520 different amino acid substitution variants have been previously identified in the systematic screening of 91 human DNA repair genes for sequence variation. Two algorithms were employed to predict the impact of these amino acid substitutions on protein activity. Sorting Intolerant from Tolerant (SIFT) classified 226 of 508 variants (44%) as "Intolerant." Polymorphism Phenotyping (PolyPhen) classed 165 of 489 amino acid substitutions (34%) as "Probably or possibly damaging." Another 9-15% of the variants were classed as "Potentially intolerant or damaging." The results from the two algorithms are highly associated, with concordance in predicted impact observed for approximately 62% of the variants. Twenty-one to thirty-one percent of the variant proteins are predicted to exhibit reduced activity by both algorithms. These variants occur at slightly lower individual allele frequency than do the variants classified as "Tolerant" or "Benign." Both algorithms correctly predicted the impact of 26 functionally characterized amino acid substitutions in the APE1 protein on biochemical activity, with one exception. It is concluded that a substantial fraction of the missense variants observed in the general human population are functionally relevant. These variants are expected to be the molecular genetic and biochemical basis for the associations of reduced DNA repair capacity phenotypes with elevated cancer risk.

Bilirubin UDP-glucuronosyltransferase 1A1 (UGT1A1) gene promoter polymorphisms and HPRT, glycophorin A, and micronuclei mutant frequencies in human blood.

A dinucleotide repeat polymorphism (5-, 6-, 7-, or 8-TA units) has been identified within the promoter region of UDP-glucuronosyltransferase 1A1 (UGT1A1) gene. The 7-TA repeat allele has been associated with elevated serum bilirubin levels that cause a mild hyperbilirubinemia (Gilbert's syndrome). Studies suggest that promoter transcriptional activity of UGT1A1 is inversely related to the number of TA repeats, and that unconjugated bilirubin concentration increases directly with the number of TA repeat elements. Because bilirubin is a known antioxidant, we hypothesized that UGT1A1 repeats associated with higher bilirubin may be protective against oxidative damage. We examined the effect of UGT1A1 genotype on somatic mutant frequency in the hypoxanthine-guanine phosphoribosyl-transferase (HPRT) gene in human lymphocytes and the glycophorin A (GPA) gene of red blood cells (both N0, NN mutants), and the frequency of lymphocyte micronuclei (both kinetochore (K)-positive or micronuclei K-negative) in 101 healthy smoking and nonsmoking individuals. As hypothesized, genotypes containing 7- and 8-TA displayed marginally lower GPA_NN mutant frequency relative to 5/5, 5/6, 6/6 genotypes ( [Formula: see text] ). In contrast, our analysis showed that lower expressing UGT1A1 alleles (7- and 8-TA) were associated with modestly increased HPRT mutation frequency ( [Formula: see text] ), while the same low-expression genotypes were not significantly associated with micronuclei frequencies (K-positive or K-negative) when compared to high-expression genotypes (5- and 6-TA). We found weak evidence that UGT1A1 genotypes containing 7- and 8-TA were associated with increased GPA_NØ mutant frequency relative to 5/5, 5/6, 6/6 genotypes ( [Formula: see text] ). These data suggest that UGT1A1 genotype may modulate somatic mutation of some types, in some cell lineages, by a mechanism not involving bilirubin antioxidant activity. More detailed studies examining UGT1A1 promoter variation, oxidant/antioxidant balance and genetic damage will be needed.

Second cancers after radiotherapy: any evidence for radiation-induced genomic instability?

Do second primary cancers in humans arise from radiation-induced somatic genomic instability after radiotherapy for the first malignancy? The amount of truly pertinent human information on this issue is sparse, leading to the conclusion that we cannot confirm or refute that instability induction by radiation is involved. However, the in vitro findings of radiation-induced genomic instability through bystander effects or increased mutation rates in cell progeny of apparently normal but irradiated cells are provocative and their transferability to human in vivo biology deserves further investigation. We describe possible animal and human studies to stimulate ideas, but the collaborative commitment of multiple large institutions to tumor tissue procurement and retrieval will be essential. In addition, detecting the temporal progression of genomic instability and identifying the salient genetic events as being radiation-induced will be pivotal. Execution of some of the studies suggested is not possible now, but applying next-generation methods could bring the concepts to fruition. As nearly one in 10 cancer diagnoses are second (or higher) malignancies, it is important to understand the contribution of radiotherapy to second cancer induction and pursue well-coordinated efforts to determine the role of induced genomic instability.

Challenges and complexities in estimating both the functional impact and the disease risk associated with the extensive genetic variation in human DNA repair genes.

Individual risk and the population incidence of disease result from the interaction of genetic susceptibility and exposure. DNA repair is an example of a cellular process where genetic variation in families with extreme predisposition is documented to be associated with high disease likelihood, including syndromes of premature aging and cancer. Although the identification and characterization of new genes or variants in cancer families continues to be important, the focus of this paper is the current status of efforts to define the impact of polymorphic amino acid substitutions in DNA repair genes on individual and population cancer risk. There is increasing evidence that mild reductions in DNA repair capacity, assumed to be the consequence of common genetic variation, affect cancer predisposition. The extensive variation being found in the coding regions of DNA repair genes and the large number of genes in each of the major repair pathways results in complex genotypes with potential to impact cancer risk in the general population. The implications of this complexity for molecular epidemiology studies, as well as concepts that may make these challenges more manageable, are discussed. The concepts include both experimental and computational approaches that could be employed to develop predictors of disease susceptibility based on DNA repair genotype, focusing initially on studies to assess functional impact on individual proteins and pathways and then on molecular epidemiology studies to assess exposure-dependent health risk. In closing, we raise some of the non-technical challenges to the utilization of the full richness of the genetic variation to reduce disease occurrence and ultimately improve health care.

Identification of 127 amino acid substitution variants in screening 37 DNA repair genes in humans.

The repair of damaged DNA requires the function of multiple proteins in generally damage-specific, nonredundant pathways. The relationship of DNA repair to cancer susceptibility is obvious in "cancer families," in which low frequency, high penetrance, loss-of-function variant alleles of genes with roles in the repair of damaged DNA have been associated with a high risk of disease. More important for the cancer incidence in the general population, many individuals exhibit reduced (60-75% of normal) repair capacity phenotypes that have been associated with several-fold increases in individual cancer risk. In a program to identify the molecular basis for the variation in repair capacity and the elevated cancer susceptibility, we have identified 127 amino acid substitution variants in resequencing 37 DNA repair genes in 36-164 unrelated individuals. Over 50% of the substitutions are exchanges of amino acid residues with dissimilar physical or chemical properties, at sites at which the common residue is identical in the human and mouse proteins. Five additional sequence changes resulting in proteins with altered termination of translation and one amino acid insertion variant were detected. The variant allele frequencies average 0.047, with individual variant allele frequencies ranging from <0.01 to 0.43. Homozygous variant individuals and individuals with multiple amino acid substitutions in a gene were observed. Most individuals exhibited variation in multiple genes in a repair pathway. Ten variant alleles accounted for 52% of the genetic variation among individuals, but a striking 23% of the total variation is associated with 108 variants with allele frequencies of less than 5%. Screening generally healthy individuals generates a catalogue of common variants that is a resource for molecular epidemiology studies endeavoring to use a genotype to phenotype paradigm to estimate the role of genetic variation and individual susceptibility in disease risk from environmental and lifestyle exposures in the general population of the United States.