Polymorphisms in Nucleotide Excision Repair Genes and DNA Repair ...

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Polymorphisms in Nucleotide Excision Repair Genes and DNA Repair Capacity Phenotype in Sisters Discordant for Breast Cancer Jing Shen,1 Manisha Desai,2 Meenakshi Agrawal,1 David O. Kennedy,1 Ruby T. Senie,3 Regina M. Santella,1 and Mary Beth Terry3 Departments of 1Environmental Health Sciences, 2Biostatistics, and 3Epidemiology, Mailman School of Public Health, Columbia University, New York, New York

Abstract Interindividual differences in DNA repair capacity (DRC) may play a critical role in breast cancer risk. Previously, we determined that DRC measured via removal of in vitro – induced benzo[a]pyrene diolepoxide-DNA adducts in lymphoblastoid cell lines was lower in cases compared with controls among sisters discordant for breast cancer from the Metropolitan New York Registry of Breast Cancer Families. We have now determined genotypes for seven single nucleotide polymorphisms in five nucleotide excision repair genes, including Xeroderma pigmentosum complementation group A (XPA +62T>C), group C (XPC Lys939Gln and Ala499Val), group D (XPD Asp312Asn and Lys751Gln), and group G (XPG His1104Asp) and ERCC1 (8092 C>A) in a total of 160 sister pairs for whom DRC

phenotype data were available. Overall, there were no statistically significant differences in average DRC for most of the genotypes. A final multivariate conditional logistic model, including three single nucleotide polymorphisms (XPA +62T>C, XPC Ala499Val, and XPG His1104Asp) and smoking status, only modestly predicted DRC after adjusting for case-control status and age of blood donation. The overall predictive accuracy was 61% in the model with a sensitivity of 78% and specificity of 39%. These findings suggest that those polymorphisms we have investigated to date in nucleotide excision repair pathway genes explain only a small amount of the variability in DRC. (Cancer Epidemiol Biomarkers Prev 2006;15(9):1614 – 9)

Introduction Elevated levels of DNA damage caused by excessive exposure to carcinogens or inherited deficiencies in repair capacity may increase an individual’s susceptibility to many kinds of human cancers (1, 2). Using different in vitro phenotype assays, several recent studies have shown that DNA repair capacity (DRC) has considerable interindividual variation and that individuals with reduced DRC are at increased risk of developing breast cancer (3-6). DRC measured by the host-cell reactivation assay using both luciferase (luc) and chloramphenicol acetyltransferase (cat) as reporter genes, indicated that women with breast carcinoma had a 22% to 36% reductions in DRC compared with controls (4-6). Those with DRC lower than the median level of controls had a 3-fold increased risk for breast cancer compared with those with higher DRC (5). Our own previous finding also indicated not only that do breast cancer patients have a 24.5% decrease in average DRC compared with their sisters without breast cancer but also that there is an increase in risk with decreasing DRC (odds ratios ranged from 1.2 to 2.4 to 3.0, by quartiles; P trend < 0.01) using an assay measuring the removal of in vitro – induced benzo[a]pyrene diolepoxide (BPDE)-DNA adducts in lymphoblastoid cells (3). For several other types of human cancers (lung, prostate, skin, head, and neck cancers), increased risk is also associated with deficient DRC (7). These phenotype studies are usually good at

Received 3/22/06; revised 5/30/06; accepted 7/11/06. Grant support: NIH grants U01 CA69398, P30 CA13696, and P30 ES09089, the Breast Cancer Research Foundation, and Cancer Prevention and Research Foundation Postdoctoral Research Fellowship (D.O. Kennedy). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Jing Shen, Department of Environmental Health Sciences, Mailman School of Public Health, Columbia University, 701 West 168th Street, Room 505, New York, NY 10032. Phone: 212-305-8158; Fax: 212-305-5328. E-mail: [email protected] Copyright D 2006 American Association for Cancer Research. doi:10.1158/1055-9965.EPI-06-0218

predicting cancer risk but have relatively large variations in risk estimates because of the large assay variation in addition to the relatively small numbers of subjects compared with genotype association studies (2). Because the assays are very time consuming and it is not easy to develop large-scale, highthroughput methods for molecular epidemiologic studies, few subjects are assayed, a major limitation of phenotype assays (3, 5, 8-12). It is likely that single nucleotide polymorphisms (SNP) in coding and regulatory sequences of genes in the nucleotide excision repair (NER) pathway may result in subtle structural alterations in DNA repair enzymes and modulate cancer susceptibility by affecting individual DRC (13-19). Previous studies showed that DRC for removal of BPDE-induced DNA damage measured by the host-cell reactivation assay was modulated by two XPD/ERCC2 polymorphisms (Asp312Asn and Lys751Gln; ref. 20). In other studies, DRC was consistently lower in subjects homozygous for XPC intron 9 poly(AT) or XPD 156Arg, 312Asn, and 751Gln than in subjects with other genotypes, suggesting these polymorphic alleles might have a recessive effect on the DRC phenotype (9, 21). But results for XPD Asp312Asn are inconsistent (22). It may be difficult to detect subtle differences in DRC by studying only one or two SNPs in a very complex pathway. Genotype studies might be improved by using a robust study design and including multiple polymorphisms in the same pathway to estimate risks associated with individual DRC. In the present study, we used sisters discordant for breast cancer from the Metropolitan New York Registry, one of six international collaborating sites (http://www.metronyregistry. org/) of the Breast Cooperative Family Registry (23) to predict the influence of seven SNPs in the NER pathway genes (XPA 5¶ untranslated region +62T>C, XPC Ala499Val and Lys939Gln, XPD Asp312Asn and Lys751Gln, XPG His1104Asp, and ERCC1 8092 C>A) on DRC phenotype. Our aim was to evaluate whether these genetic polymorphisms in NER

Cancer Epidemiol Biomarkers Prev 2006;15(9). September 2006

Cancer Epidemiology, Biomarkers & Prevention 1615 pathway genes could partly explain the variation of individual DRC phenotype. If so, then sets of polymorphisms in the DNA repair pathway, which are easier to measure in large-scale epidemiologic studies, may be a proxy for DNA repair phenotype, which is less easily measured in epidemiologic settings.

Materials and Methods Study Design and Characteristics of Study Population. The Breast Cooperative Family Registry is an international consortium established in 1995 as a resource for research on the epidemiologic, clinical, and genetic aspects of breast cancer (http://epi.grants.cancer.gov/CFR/; ref. 23). The description of the sources of study participants and recruitment and data collection methods as part of the parent Metropolitan New York Registry project have been described in detail elsewhere (3, 24). Briefly, the Metropolitan New York Registry has been recruiting high-risk breast and/or ovarian cancer families from clinical and community settings within the metropolitan New York area since 1995 who met one of the following criteria: (a) one or more members with breast cancer or ovarian cancer diagnosed at A

rs3212986

XPA

NM_000380

9q22.3

Ex1+62T>C, 5¶ UTR

rs1800975

XPC

NM_004628

3p25

Ex9 377C>T/ Ala499Val

rs2228000

5¶-TAGTTCCTCAGT TTCCCG-3¶ (sense) 5¶-TGAGCCAATTCAGCCACT-3¶ (antisense) 5¶-CTACACAGGCTGCTGCTGCTGCT-3¶ (forward probe) 5¶-AAGCCCCGTCGGCCGCCGCCATCTC[T/C]GGCCCAC TCCGAGGACCTAGCTCCC-3¶ (reverse probes) Applied Biosystems assay no. C_482935_1 5¶-GCCTCTGATCCCTCTGATGA-3¶ (sense)

rs2228001

5¶-CATCGCTGCACATTTTCTTG-3¶ (antisense) 5¶-GTAAGGACCCAAGCTTGCCAG-3¶ (forward probe) 5¶-GCCTCAAAACCGAGAAGATG-3¶ (sense)

Ex16+211A>C/ Lys939Gln XPD (ERCC2)

XPG (ERCC)

NM_000400

NM_000123

19q13.3

13q33

Ex10 16G>A/ Asp312Asn

rs1799793

Ex23+61A>C/ lys751Gln

rs13181

5¶-CTGCCTCAGTTT GCCTTCTC-3¶ (antisense) 5¶-GGGCGCTCAGCTCACAGCT-3¶ (reverse probe) 5¶-CGGGGCTCACCCTGCAGCACTTCGT[C/T]GGGCAGC ACGGGGTTGGCCAGGTGG-3¶ (reverse probes) Applied Biosystems assay no. C_3145050_10 5¶-CCCTCTCCCTTTCCTCTGTT-3¶ (sense)

rs17655

5¶-GGCAAGACTCAGGAGTCACC-3¶ (antisense) 5¶-CTGAGCAATCTGCTCTATCCTCT -3¶ (reverse probe) 5¶-ACGAAAGAATACATGCGGTGGA-3¶ (sense)

Ex15 344G>C/ Asp1104His

5¶-ATCTGGCGGTCACGAGGAC-3¶ (antisense) 5¶-CCTCTCAGAATCTGATGGATCTTCAAGTGAA-3¶ (forward probe) Abbreviations: NCBI, National Center for Biotechnology Information; UTR, untranslated region.


1616 Polymorphisms in NER Genes and DRC Phenotype Table 2. Comparison of breast cancer cases and unaffected sister controls by selected variables Selected variables Age at blood donation, mean (SD) Smoking status, n (%) Nonsmoker Ever smoker No. cigarette daily, mean (SD) BMI, mean (SD) DRC, mean (SD) DRC c >35.1% (higher) V35.1% (low)

Cases (n = 160)

Controls (n = 160)

P*

49.4 (10.0)

49.8 (10.8)

0.38

94 66 15.2 25.7 26.5

86 74 14.8 25.4 35.4

0.37

(58.8) (41.2) (12.1) (5.8) (22.5)

51 (32.7) 105 (67.3)

(53.8) (46.2) (10.7) (4.5) (24.3)

0.94 0.50 60 years were more likely to have poorer DNA repair, although the point estimate was not statistically significant. We observed a positive association between smoking and DRC phenotype (Table 4) in agreement with another study finding smoking associated with DRC (40). Compared with previous studies, our study has several advantages. One strength was the family-based design using sisters from the same families as controls, which can potentially reduce confounding from population admixture as well as lifestyle factors that cluster within families (41). The study included nuclear families that were not selected based on a population-based scheme and may limit the generalizability of the findings (24). The study sample is of moderate size limiting our statistical power. Given our sample size and the range of frequencies for our genotypes, we were only able to detect associations 2-fold or higher between genotype and phenotype. Other factors possibly predicting individual DRC phenotype, such as dietary antioxidants, were not examined in the current study (42). These results should therefore be viewed as preliminary until confirmed in larger studies. In conclusion, specific SNPs in the NER pathway genes (XPA +62T>C, XPC Ala499Val, and XPG His1104Asp) and ever smoking only modestly predict individual variation in DRC; overall predictive accuracy (61%) was relatively low (sensitivity, 78%; specificity, 39%). These NER pathway SNPs that we selected are commonly assessed in epidemiologic studies and their poor prediction for DNA repair phenotype may offer some evidence of why studies only using SNPs have been inconsistent. It may still be possible that an extensive panel of DNA repair SNPs may predict phenotype, but our findings suggest that the small subsets of SNPs are likely a poor proxy for phenotype. The results obtained from discordant sister pairs in a breast cancer family registry should serve as a stimulus for further studies on the complex molecular events that affect individual DRC phenotype.

4. 5. 6.

7. 8.

9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

22. 23.

24. 25. 26. 27. 28.

Acknowledgments We thank Irina Gurvich for the processing, storage, and inventory of biospecimens for the study.

29. 30.

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