Polymorphisms in DNA double-strand break repair genes and breast cancer risk in the Nurses' Health Study

Jiali Han1,3,5, Susan E. Hankinson2,4, Hardeep Ranu3, Immaculata De Vivo1,3,4 and David J. Hunter1,2,3,4

1 Department of Nutrition, 2 Department of Epidemiology and 3 Harvard Center for Cancer Prevention, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115, USA and 4 Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, and Harvard Medical School, 181 Longwood Avenue, Boston, MA 02115, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Genetic polymorphisms in double-strand break repair genes may influence DNA repair capacity and, in turn, confer predisposition to breast cancer. We prospectively assessed the associations of candidate polymorphisms G31479A (R188H) in XRCC2, A4541G (5'-UTR), A17893G (IVS5-14) and C18067T (T241 M) in XRCC3, and C299T (5'-UTR) and T1977C (D501D) in Ligase IV with breast cancer risk in a nested case–control study within the Nurses' Health Study (incident cases, n = 1004; controls, n = 1385). We observed no overall associations of these six genotypes with breast cancer risk. Four common haplotypes in XRCC3 accounted for 99% of the chromosomes of the present study population. We observed that Ligase IV 1977C carriers were at increased breast cancer risk if they had a first degree family history of breast cancer (test for interaction, P = 0.01). We observed that the XRCC2 R188H polymorphism modified the association of plasma {alpha}-carotene level and breast cancer risk (test for ordinal interaction, P = 0.03); the significantly decreased risk seen overall for women in the highest quartile of plasma {alpha}-carotene was only present among 188H non-carriers (the top quartile versus the bottom quartile; multivariate odds ratio, 0.55; 95% confidence interval, 0.40–0.75). No significant interactions were seen between any of these polymorphisms and duration or dose of cigarette smoking. The gene–environment interaction data suggest that the subtle effects of some of these variants on breast cancer risk may be magnified in the presence of certain exposures.

Abbreviations: BMI, body mass index; CI, confidence interval; DSB, double-strand breaks; HR, homologous recombination; IR, ionizing radiation; LRT, likelihood ratio test; NHEJ, non-homologous end joining; NHS, Nurses' Health Study; OR, odds ratio; SSB, single-strand breaks; SNP, single nucleotide polymorphism


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Hypersensitivity to ionizing radiation (IR) and deficient repair of radiation-induced DNA damage have been implicated in the development of breast cancer (13). Because DNA double-strand breaks (DSB) are induced by carcinogens such as IR, deficiency in DNA DSB repair may contribute to IR hypersensitivity and breast cancer susceptibility. In addition, the involvement of the two major hereditary breast cancer susceptibility genes, BRCA1 and BRCA2, in the DNA DSB repair pathway also suggests that defects in this pathway may contribute to the development of breast cancer (4,5).

In addition to IR, DSB can be induced by exogenous agents, such as chemicals and chemotherapy, and endogenous reactive oxygen species. DSB can also be generated as products of blocked replication forks and programmed rearrangements (6,7). Repair of DNA DSB is essential to the maintenance of genomic integrity. Homologous recombination (HR) and non-homologous end joining (NHEJ) are two distinct mechanisms in the repair of DSB in mammalian cells. In the HR pathway, the strand exchange is catalyzed by RAD51 and facilitated by RAD52 through direct interaction. Five RAD51 paralogs facilitate the formation of RAD51 foci in two distinct complexes, XRCC3–RAD51C and RAD51B–RAD51C–RAD51D–XRCC2 (8). Hamster cells deficient in XRCC2 or XRCC3 exhibited defects in Rad51 focus formation (9,10), a decrease in HR induced by DSB (11,12), hypersensitivity to radiation, increased spontaneous chromosome aberrations and increased chromosome missegregation (1315), implying critical roles of XRCC2 and XRCC3 in HR. Deficiency in BRCA1 or BRCA2 showed similar phenotypes (1619), suggesting potential roles of XRCC2 and XRCC3 of HR in the development of breast cancer. In the NHEJ pathway, the termini of DSB are bound by the complex of the Ku70 and Ku80 heterodimer and DNA-dependent protein kinase and the break is repaired by the complex of Ligase IV and XRCC4 (6). Inactivation of the Ligase IV gene in mice led to embryonic lethality; these mouse cells showed increased sensitivity to IR and defects in V(D)J joining (20,21).

Genetic polymorphisms in DSB repair genes may have subtle effects on DNA repair capacity and confer predisposition to breast cancer. Two case–control studies among European Caucasians provided some preliminary evidence that genetic variants in XRCC2, XRCC3 and Ligase IV may be associated with breast cancer risk (22,23). We prospectively evaluated the associations between candidate polymorphisms in these three genes and breast cancer risk in the Nurses' Health Study (NHS). In addition, because reactive oxygen species and free radicals can cause oxidative DNA damage that can lead to single-strand breaks (SSB) and DSB, oxidative stress due to antioxidant depletion and cigarette smoking may induce excessive strand breaks. Plasma antioxidants and cigarette smoking were evaluated previously in relation to breast cancer risk in the NHS (24; Tamimi et al., submitted for publication). We further investigated the a priori hypothesized gene–environment interaction that genetic variation in the DSB repair pathway may modify the associations of plasma antioxidant status and cigarette smoking with breast cancer risk.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study population
The NHS was established in 1976, when 121 700 female registered nurses between the ages of 30 and 55 completed a self-administered questionnaire on their medical histories and baseline health-related exposures. Updated information has been obtained by questionnaires every 2 years. Incident breast cancers were identified by self-report and confirmed by medical record review. Between 1989 and 1990, blood samples were collected from 32 826 (27%) of the cohort members. Subsequent follow-up has been >98% for this subcohort.

Eligible cases in this study consisted of women with pathologically confirmed incident breast cancer from the subcohort who gave a blood specimen. Cases with a diagnosis after blood collection up to June 1, 1998 with no previously diagnosed cancer except for non-melanoma skin cancer were included. One or two controls were randomly selected among women who gave a blood sample and were free of diagnosed cancer (excluding non-melanoma skin cancer) up to and including the interval in which the case was diagnosed. Controls were matched to cases on year of birth, menopausal status, post-menopausal hormone use, month of blood return, time of day of blood collection and fasting status at blood draw; menopause was defined as previously described (25). The nested case–control study consists of 1004 incident breast cancer cases and 1385 matched controls. The study protocol was approved by the Committee on Use of Human Subjects of the Brigham and Women's Hospital, Boston, MA.

Exposure data
Information regarding breast cancer risk factors was obtained from the 1976 baseline questionnaire, biennial follow-up questionnaires and a questionnaire completed at the time of blood sampling. Menopausal status and use of post-menopausal hormones were assessed at blood draw and updated until date of diagnosis for cases and the equivalent date for matched controls. Information regarding current and past history of cigarette smoking was asked in the 1976 questionnaire and updated every 2 years in subsequent questionnaires. The detailed exposure assessment of cigarette smoking has been described previously (24).

Single nucleotide polymorphism (SNP) identification
The exons and adjacent intronic and non-coding regions of the XRCC2 and XRCC3 genes have been re-sequenced to identify common germline polymorphisms (26,27). The 36 samples for XRCC3 variation screening included 24 Caucasians in a lung cancer study (12 cases and 12 controls) and 12 individuals who were probably healthy Caucasians. Three common (defined as minor allele frequency >5%) polymorphisms were identified in XRCC3: A4541G (5'-UTR), A17893G (IVS5-14) and C18067T (T241 M). Seventy-two healthy individuals of African, Asian or Caucasian origin were used for screening XRCC2 genetic variation. Only one common variant was found in the XRCC2 gene: G31479A (R188H). The genomic sequence of the Ligase IV gene was re-sequenced as part of the NIEHS Environmental Genome Project at the University of Washington (http://egp.gs.washington.edu). The 90 individuals from the NIH DNA Polymorphism Discovery Resource used for screening were from the following ethnic groups: 23 European-Americans, 23 African-Americans, 11 Mexican-Americans, 11 native-Americans and 22 Asian-Americans. However, the ethnicity of individual samples is unknown. Two common polymorphisms, C299T (5'-UTR) and T1977C (D501D), were identified in exons and adjacent non-coding regions. We genotyped these six SNPs in the three DSB repair genes in the present nested case–control study of mostly Caucasian women.

Laboratory assays
In order to maximize power we included all available samples with plasma antioxidant levels, including samples left unpaired due to either subsequent exclusions (of either the cases or controls) or laboratory issues. Plasma samples of 994 cases and 995 controls were assessed using reversed phase high performance liquid chromatography methods described by El-Sohemy et al. (28) to determine the concentrations of {alpha}-carotene, ß-carotene, ß-cryptoxanthin, lycopene, lutein/zeaxanthin, {alpha}-tocopherol and {gamma}-tocopherol. All case–control pairs were assayed together; the samples were ordered randomly within each pair. Plasma quality control samples were interspersed to assess laboratory precision. The coefficient of variation for each antioxidant was <10%. Laboratory personnel were blind to case–control status and the identity of replicate samples (Tamimi et al., submitted for publication).

Genotyping was performed by the 5'-nuclease assay (TaqMan®), using the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA), in 384-well format. TaqMan® primers and probes were designed using the Primer Express® Oligo Design software v2.0 (Applied Biosystems). Laboratory personnel were blind to case–control status and blind quality control samples were inserted to validate genotyping procedures; concordance for the blind samples was 100%. Primers, probes and conditions for genotyping assays are available upon request.

Statistical analysis
Conditional logistic regression was employed to calculate odds ratio (OR) and 95% confidence interval (CI) to assess the risk of breast cancer according to genotype. Unconditional logistic regression was utilized in interaction analyses to increase statistical power. Plasma levels of antioxidants were categorized into quartiles, with cut-off points based on the batch-specific distribution of control subjects. Weighted median values for each quartile were based on the batch-specific medians of controls weighted by the proportion of subjects in each batch. Alcohol consumption was based on the 1990 dietary questionnaire; the 1986 questionnaire was used for individuals who did not provide this information on the 1990 questionnaire. We assessed Hardy–Weinberg equilibrium by using a {chi}2 test. We inferred haplotypes using the ARLEQUIN 2.0 software package by Excoffier et al. (http://lgb.unige.ch/arlequin/). We used a {chi}2 test to determine the P value for difference in haplotype frequencies between cases and controls.

Tests for trend were conducted by assigning the weighted median values for quartiles of plasma antioxidant levels among controls to both cases and controls as continuous variables. To test statistical significance of interactions between the environmental exposures and the genotype, we first used a likelihood ratio test (LRT) to compare nested models that included terms for all combinations of the genotype and levels of environmental exposure to the models with indicator variables for the main effects only (nominal LRT). We also modeled genotypes as ordinal variables and environmental exposures as continuous variables as described for trend tests to assess the statistical significance of interactions by LRT test of a single interaction term (ordinal LRT). For dichotomous exposure and genotype variables, these two LRT tests yielded the same results. All P values were two-sided.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The mean age at diagnosis of cases was 61.8 years and that of controls 62.6 years. There were 8.9% pre-menopausal and 84.9% post-menopausal women and 6.1% women with uncertain menopausal status at diagnosis. Cases and controls had similar mean body mass index (BMI) at blood draw (25.4 versus 25.5 kg/m2) and weight gain since age 18 years (11.8 versus 11.4 kg). Compared with controls, cases had similar ages at menarche (12.5 versus 12.6 years), first birth (24.8 versus 24.8 years) and menopause (48.2 versus 48.0 years). The proportion of women with a first degree family history of breast cancer was higher among cases (20.9 versus 14.8%). Cases were more likely to have a history of benign breast disease (65.0 versus 49.6%) and a longer duration of post-menopausal hormone use (38.0 versus 26.9% current users for >=5 years). Self-reported major ethnicity was similar between cases versus controls: Southern European, 17.5 versus 17.8%; Scandinavian, 8.6 versus 7.6%; other Caucasian, 63.1 versus 61.3%; Asians, Hispanics and African-Americans comprised ~1% of cases and controls.

Six polymorphisms in three DNA DSB repair genes (XRCC2, XRCC3 and Ligase IV) were genotyped. The genotype distributions of these six polymorphisms among cases and controls were in Hardy–Weinberg equilibrium. The associations of these six polymorphisms and breast cancer risk are presented in Table I. We did not observe an association of the XRCC2 R188H polymorphism with breast cancer risk. As compared with non-carriers, women with one XRCC3 4541G allele were at a marginally significantly increased breast cancer risk (multivariate OR, 1.20; 95% CI, 0.99–1.46). However, a gene dosage effect was not apparent; women with two 4541G alleles had a multivariate risk of 0.85 (95% CI, 0.54–1.35). XRCC3 A17893G and C18067T were not associated with breast cancer risk. Similarly, we observed no difference in breast cancer risk according to Ligase IV C299T or T1977C genotype. There were four common haplotypes inferred from the three SNPs in the XRCC3 gene, which accounted for 99% of the chromosomes of the present study population of mostly Caucasian women (Table II). Three rare haplotypes were also observed. There was no significant difference in frequency distribution in cases and controls for any inferred haplotype.


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Table I. Polymorphisms in XRCC2, XRCC3 and Ligase IV and breast cancer risk

 

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Table II. XRCC3 inferred haplotypes in cases and controlsa

 
We examined the potential interactions between genotypes and first degree family history of breast cancer (Table III). None of the variant genotypes (carriers versus non-carriers) was significantly associated with breast cancer risk in the first degree family history negative stratum. We observed a significant interaction between Ligase IV T1977C and first degree family history of breast cancer (P = 0.01, test for interaction). The multivariate OR for joint effect of first degree family history and carriage of 1977C was 2.35 (95% CI, 1.56–3.53). Among 1977C non-carriers, the first degree family history of breast cancer was associated with breast cancer risk (multivariate OR, 1.37; 95% CI, 1.05–1.80), whereas the multivariate OR was 0.88 (95% CI, 0.72–1.08) for carriage of the 1977C allele in the first degree family history negative stratum. Of note, in control only analysis, unlike the other polymorphisms, carriage of one or two Ligase IV 1977C alleles was significantly inversely associated with first degree family history of breast cancer (age-adjusted P = 0.02). There were no significant interactions for other polymorphisms with a first degree family history of breast cancer.


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Table III. Breast cancer risk by first degree family history of breast cancer and genotype

 
Among carotenoids, plasma {alpha}-carotene level was significantly inversely associated with breast cancer risk in this nested case–control study (Tamimi et al., submitted for publication). We assessed whether the association between plasma {alpha}-carotene level and breast cancer risk differed by genotype (Table IV). As compared with the reference group of XRCC2 188H non-carriers in the lowest plasma {alpha}-carotene quartile, the reduction in risk (45%) was statistically significant among non-carriers in the highest quartile (multivariate OR, 0.55; 95% CI, 0.40–0.75). This inverse relation was not present among XRCC2 188H carriers. The multivariate OR remained significant after additionally controlling for plasma ß-carotene, ß-cryptoxanthin, lycopene, {alpha}-tocopherol, {gamma}-tocopherol and lutein/zeaxanthin, one at a time or all simultaneously. The inverse association between plasma {alpha}-carotene level and breast cancer risk was significantly stronger among non- carriers than carriers (P = 0.03, test for ordinal interaction). However, including the cross-classified interaction terms did not significantly increase the goodness fit of the model (P = 0.21, test for nominal interaction). No suggestive evidence of effect modification of any of the other polymorphisms was observed on the relation of plasma {alpha}-carotene with breast cancer risk (data not shown).


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Table IV. Breast cancer risk by plasma {alpha}-carotene level and XRCC2 R188H

 
We evaluated whether the genetic variants modified the effect of dose or duration of cigarette smoking on the risk of breast cancer. Whether exposure was defined at diagnosis, or 10 years prior to diagnosis, we observed no significant interactions between any genotype and current or past smoking behavior (data not shown).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We evaluated the polymorphisms in the XRCC2 and XRCC3 genes of the homologous recombination repair pathway in relation to breast cancer risk in this nested case–control study. None of these polymorphisms was significantly associated with breast cancer risk. Two published studies have examined the association between XRCC2 G31479A (R188H) and breast cancer risk (22,23). In a case–control study of 2205 cases and 1826 controls, Kuschel et al. observed a significantly increased risk of breast cancer for women homozygous for XRCC2 188H (OR, 2.6; 95% CI, 1.0–6.7) (22). This significant association was partially due to an under-representation of the H/H genotype among controls (6 women with the H/H genotype were observed, 10.7 were expected; Hardy–Weinberg {chi}2 = 2.46, P = 0.12). Among 521 cases and 895 controls, Rafii et al. reported that women with the 188H/H genotype had an OR of 2.14 (95% CI, 0.65–7.07), with H allele carriers at a borderline significantly increased risk of breast cancer (OR, 1.30; 95% CI, 0.96–1.75) (23). In our study, the multivariate OR for women with two H alleles was much lower (OR, 1.33), although the 95% CI included the odds ratios reported in the two previous studies. We observed that women with at least one H allele had a multivariate OR of 1.10 (95% CI, 0.85–1.42), lower than, but compatible with, the result reported by Rafii et al. The functional effect of this variant on cell survival following mitomycin C-induced DNA interstrand cross-linking has been studied in in vitro experiments (23). Substitution by the amino acid alanine or deletion of 188R in XRCC2 showed a substantial effect on the survival of XRCC2-deficient cells, whereas the naturally occurring 188H allele displayed a much smaller difference in survival from the wild-type, suggesting that 188H had only a subtle effect on damage sensitivity (23).

For the XRCC3 gene, we observed no significant elevation in risk for any polymorphism, but our data were compatible with the observations of Kuschel et al. (22) that women with one XRCC3 4541G allele were at borderline significantly increased risk of breast cancer (OR, 1.1, 95% CI, 1.0–1.2) and the positive association was not further increased in women who were homozygous for the 4541G allele (OR, 0.9; 95% CI, 0.7–1.3). Kuschel et al. (22) observed an inverse association for the XRCC3 17893G allele (OR for 17893G homozygotes, 0.8; 95% CI, 0.6–1.0) and a positive association for the XRCC3 18067 T allele (OR for 18067T homozygotes, 1.3; 95% CI, 1.1–1.6) with breast cancer risk. In contrast, we did not observe material associations between these two alleles and breast cancer risk. Jacobsen et al. also did not observe any association of C 18067T with breast cancer risk (426 cases and 424 controls) (29). No difference was found in the ability of the XRCC3 18067T (241 M) allele and the wild-type allele to complement the DSB repair defect or correct the hypersensitivity to mitomycin C of the XRCC3-deficient cells (30), which is consistent with the previous finding of no association between the XRCC3 241 M allele and radiation-induced G2 phase delay (1,31). The association of the XRCC3 241 M allele with higher bulky DNA adduct levels (32,33) suggests a possible role of XRCC3 in repairing bulky DNA adducts. In addition to breast cancer, XRCC3 T241 M has been evaluated in relation to other cancer sites. No association was found between XRCC3 T241 M and lung cancer (34,35), squamous cell carcinoma of the head and neck (36) and basal cell carcinoma (29). Significantly positive associations of XRCC3 T241 M with melanoma and bladder cancer were reported previously (32,37). However, the results were not subsequently confirmed in larger studies (38,39).

We also evaluated the polymorphisms in the Ligase IV gene of the NHEJ pathway in relation to breast cancer risk in this study. Our data were compatible with the results of Kuschel et al. (22) that the Ligase IV 1977 C/C genotype was associated with a decreased risk of breast cancer (OR, 0.7; 95% CI, 0.4–1.0) (22). No functional data are available for this polymorphism.

Reduced repair of radiation-induced DNA damage has been observed in breast cancer cases and their female relatives compared with healthy women without a family history of breast cancer (2,3,40). Because BRCA1 and BRCA2 are involved in HR repair, variation in DNA DSB repair capacity due to other genetic polymorphisms in the HR or NHEJ pathway may modify BRCA1/2-related breast cancer risk. We observed some suggestion that the Ligase IV 1977C allele was associated with increased breast cancer risk among women with a first degree family history of breast cancer. The significantly inverse association of carriage of the Ligase IV 1977C allele with a first degree family history of breast cancer among controls partially accounted for the significant interaction between the Ligase IV 1977C allele and a first degree family history of breast cancer on the risk.

Oxidative DNA damage by reactive oxygen species and free radicals can lead to SSB and DSB. The antioxidant potential of carotenoids could prevent oxidative DNA damage (4144). Among carotenoids, plasma {alpha}-carotene level was significantly inversely associated with breast cancer risk in this nested case–control study (Tamimi et al., submitted for publication). We observed a suggestion that the XRCC2 R188H polymorphism modified the association of plasma {alpha}-carotene level with breast cancer risk. A high plasma level of {alpha}-carotene was particularly beneficial among XRCC2 188H non-carriers, presumably with slightly stronger DNA repair capacity than 188H carriers (23); the significantly decreased risk was only seen among 188H non-carriers in the highest quartile. This suggests that the enhanced DNA repair capacity of XRCC2 188H non-carriers may only be apparent in the context of low DNA damage level and abolished by excessive DNA damage. In addition, in the analysis of the interaction between the XRCC2 R188H genotype and plasma {alpha}-carotene, the multivariate ORs did not change materially after controlling for other plasma antioxidants one at a time or all simultaneously, suggesting that {alpha}-carotene may be the most relevant carotenoid in preventing oxidative DNA damage and DSB.

Oxidative stress due to cigarette smoking can induce oxidative DNA damage, SSB and DSB. However, we did not observe any clear and statistically significant interactions between any of the polymorphisms and dose or duration of smoking, whether defined at diagnosis or 10 years prior to diagnosis to allow for latency. Overall, in this cohort there is no evidence for any strong relation of cigarette smoking with breast cancer risk (24) and we did not observe a clear relation in any of the groups defined by DNA repair genotype.

The prospective design, blood sample collection before case diagnosis, relatively large number of incident cases and high follow-up rates strengthen the validity of this study. We observed no overall associations of these six genotypes in the three DSB repair genes with breast cancer risk. The gene–environment interaction data suggested that the subtle effects of some variants on breast cancer risk were magnified in the presence of certain levels of exposure. In view of the large number of potential interactions we tested, these findings should be interpreted with caution. Additional studies are warranted to confirm these potential gene–environment interactions.


    Notes
 
5 To whom correspondence should be addressed at: Harvard Center for Cancer Prevention, Harvard School of Public Health, Room 105, Building II, 665 Huntington Avenue, Boston, MA 02115, USA Email: jhan{at}hsph.harvard.edu Back


    Acknowledgments
 
We thank Robert O'Brien, Pati Soule and Alicia Whittington for their laboratory assistance, Rong Chen for her programming support and Drs Rulla Tamimi and Hannia Campos for plasma carotenoid analyses. We are also indebted to the participants in the Nurses' Health Study for their dedication and commitment. This work was supported by NIH grants CA65725, CA87969 and CA49449.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received June 2, 2003; revised September 29, 2003; accepted October 14, 2003.