Vol. 10, 955–960, September 2001 Cancer Epidemiology, Biomarkers & Prevention 955 A Single Nucleotide Polymorphism in the 5Ј Untranslated Region of RAD51 and Risk of Cancer among BRCA1/2 Mutation Carriers1 Wendy W. Wang, Amanda B. Spurdle, statistically significant association of the 135 “c” allele Prema Kolachana, Betsy Bove, Baruch Modan, with the risk of breast cancer (adjusted odds ratio, 3.2; Sarah M. Ebbers, Graeme Suthers, Margaret A. Tucker, 95% confidence limit, 1.4–40). BRCA1/2 mutation David J. Kaufman, Michele M. Doody, Robert E. Tarone, carriers with ovarian cancer were only about one half as Mary Daly, Hanoch Levavi, Heather Pierce, likely to carry the RAD51:135 g3c SNP. Analysis of the Angela Chetrit, kConFab,2 ABCFS/CFRBCS,3 AJBCS,4 RAD51:135 g3c SNP in 738 subjects from an Israeli NISOC,5 Galit Hirsh Yechezkel, ovarian cancer case-control study was consistent with a Georgia Chenevix-Trench, Kenneth Offit, lower risk of ovarian cancer among BRCA1/2 mutation Andrew K. Godwin, and Jeffery P. Struewing6 carriers with the “c” allele. We have identified a RAD51 ؅ Laboratory of Population Genetics [W. W., S. M. E., D. J. K., J. P. S.], Genetic 5 untranslated region SNP that may be associated with Epidemiology Branch [M. A. T.], Radiation Epidemiology Branch [M. M. D.], an increased risk of breast cancer and a lower risk of and Biostatistics Branch [R. E. T.], National Cancer Institute, NIH, Bethesda, ovarian cancer among BRCA2 mutation carriers. The Maryland 20892; Queensland Institute of Medical Research, Brisbane biochemical basis of this risk modifier is currently QLD 4029, Australia [A. B. S., G. C-T.]; South Australian Clinical Genetics Service, Adelaide 5005, Australia [G. S.]; Memorial Sloan Kettering Cancer unknown. Center, New York, New York 10021 [P. K., H. P., K. O.]; Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111 [B. B., M. D., A. K. G.]; and Chaim Introduction Sheba Medical Center, Tel Hashomer, Israel 52641 [B. M., H. L., A. C., G. H. Y.] Genetic factors are important in breast and ovarian cancer, but less than 10% are attributable to the inheritance of mutations in a single gene, such as BRCA1 and BRCA2 (1). Although rela- Abstract tively uncommon, female carriers of mutations in the BRCA1 RAD51 colocalizes with both BRCA1 and BRCA2, and and BRCA2 genes are estimated to have breast cancer risks of genetic variants in RAD51 would be candidate BRCA1/2 37% to over 85% by age 70 and ovarian cancer risks approx- modifiers. We searched for RAD51 polymorphisms by imately one half as high (2–5). Both BRCA1 and BRCA2 appear sequencing 20 individuals. We compared the to confer similarly high risks of female breast cancer, whereas polymorphism allele frequencies between female BRCA1/2 ovarian cancer risk is higher for BRCA1 mutation carriers and mutation carriers with and without breast or ovarian male breast cancer risk is largely confined to BRCA2 carriers (1, cancer and between population-based ovarian cancer 6). The heterogeneity of risk in breast and ovarian cancer cases with BRCA1/2 mutations to cases and controls between different families segregating mutations in the same without mutations. We discovered two single nucleotide gene and the estimated average risk of breast cancer of about polymorphisms (SNPs) at positions 135 g3c and 172 g3t 50% both suggest the existence of important environmental or of the 5؅ untranslated region. In an initial group of genetic modifiers of risk. Some potential genetic modifiers BRCA1/2 mutation carriers, 14 (21%) of 67 breast cancer have been identified, including rare polymorphisms in H-ras cases carried a “c” allele at RAD51:135 g3c, whereas 8 for ovarian cancer (7) and variation in the androgen receptor (7%) of 119 women without breast cancer carried this gene for breast cancer risk (8). allele. In a second set of 466 mutation carriers from three The precise mechanisms by which mutations in BRCA1/2 centers, the association of RAD51:135 g3c with breast predispose to breast and ovarian cancer are unknown. They are cancer risk was not confirmed. Analyses restricted to the large proteins that share little sequence similarity with each 216 BRCA2 mutation carriers, however, showed a other or with other known sequences. Both appear to be in- volved in double-strand DNA break repair, particularly because of their association with RAD51 (9). RAD51 is a homologue of the Escherichia coli recA protein and is involved in recombi- Received 12/31/00; revised 5/11/01; accepted 6/11/01. nation and the repair of double-strand DNA breaks (10). The costs of publication of this article were defrayed in part by the payment of RAD51 shares 60% protein identity with the yeast scRad51, page charges. This article must therefore be hereby marked advertisement in which has been studied extensively (10, 11). The relative con- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. sistency of the findings of BRCA1 and BRCA2 associating 1 Supported by the Koodish Foundation, the Queensland Cancer Fund, the Evan M. Frankel Foundation, and the Gloria and Sidney Danziger Foundation. with RAD51, their possible complementary roles as DNA dam- 2 Kathleen Cuningham Foundation Consortium for Research into Familial Breast age repair proteins, and the availability of a model organism Cancer. system in which to investigate findings led us to consider 3 Australian Breast Cancer Family Study/Cooperative Family Registry for Breast RAD51 as a candidate modifier locus. Cancer Studies. 4 Australian Jewish Breast Cancer Study. 5 National Israeli Study of Ovarian Cancer. Materials and Methods 6 To whom requests for reprints should addressed, at Laboratory of Population Genetics, Building 41/D702, 41 Library Drive, Bethesda, MD 20892-5060. Polymorphisms in RAD51 were investigated by complete se- Phone: (301) 435-8953; Fax: (301) 435-8963; E-mail: [email protected]. quence analysis of the gene in 10 individuals who had devel- Downloaded from cebp.aacrjournals.org on September 26, 2021. © 2001 American Association for Cancer Research. 956 RAD51 SNP and Cancer Risk in BRCA1/2 Mutation Carriers oped both breast cancer and at least one other primary cancer score of 1.3 for linkage to BRCA1). The remaining mutations obtained from a national cohort of radiological technologists were nonsense, frameshift, splice junction, or large genomic participating in a prospective epidemiological study (12), and rearrangements. Among group (a) subjects, the two most com- the 5ЈUTR7 was sequenced in an additional 10 individuals mon mutations were BRCA1:185delAG (n ϭ 216) and BRCA2: (subjects 01 and 02 from each of the Center d’Etude du Poly- 6174delT (n ϭ 124). morphisme Humain families 1331, 1332, 1347, 1362, and Laboratory Analysis. To identify RAD51 polymorphisms, be- 1413; Coriell Institute, Camden, NJ). Sequencing these subjects cause the genomic structure of RAD51 was unpublished, we 3 3 revealed a g c polymorphism at position 135 and a g tat extracted total RNA from lymphoblastoid cell lines, performed position 172 of the published cDNA (GenBank accession no. reverse transcription-PCR, and sequenced the cDNA in over- D14134.1) lapping fragments (conditions and primer sequences available Two groups were studied to test for allelic association, on request). The two 5ЈUTR SNPs identified could be detected including: (a) a series of female BRCA1/2 mutation carriers; in one single-strand conformational polymorphism/heterodu- and (b) Jewish subjects from an Israeli ovarian cancer case- plex analysis assay using genomic DNA as template and the control study. All of the subjects with individually identifiable primers 21F (5Ј-ccgagccctaaggagagtgcggcgcttc-3Ј) and 232R samples gave informed consent for participation. Study group (5Ј-tactcggtccgcagcgctcctctctccag-3Ј). Thirty-five samples (a) included all of the known female BRCA1 and/or BRCA2 were sequenced and analyzed with single-strand conforma- heterozygous mutation carriers from each of four study centers. tional polymorphism/heteroduplex analysis to verify the corre- Disease associations for the two SNPs were first assessed in lation between the observed banding patterns and the sequences two groups of subjects from the NCI. One group was from the at the two SNPs. Over 97% of the samples could be scored for Washington Ashkenazi Study, a community-based sample of both loci by blinded review by two individuals. The RAD51: 86 females who carried the185delAG or 5382 insC mutations in 135 g3c SNP was assayed in some subjects using a standard BRCA1 and/or the 6174delT mutation in BRCA2 (3). The sec- PCR and BstNI digestion specific for the wild-type “g” allele, ond NCI group consisted of 100 female members of 27 breast/ using the primers 64F (5Ј-gggaactgcaactcatctgggtt) and 170R ovarian cancer families with identified BRCA1 or BRCA2 mu- (5Ј-tgg cac gcg ccc gac) at 200 nM concentration to amplify a tations from the NCI Genetic Epidemiology Branch family 107-bp product. The population allele frequencies for the poly- registry (13). morphisms were estimated by genotyping 40 unrelated Ashke- To replicate the findings with RAD51:135 g3c, study nazi spouses and 62 non-Jewish spouses from the NCI registry group (a) was expanded to include subjects from three addi- families and 79 males from the Washington Ashkenazi Study. tional centers. Subjects from the Fox Chase Cancer Center were The RAD51:135c allele frequency was 0.05 among Jews and recruited from high-risk and breast cancer clinics at the Fox 0.07 among non-Jews, and the RAD51:172t allele frequencies Chase Cancer Center and affiliated hospitals in the Delaware were 0.43 and 0.41, respectively. The genotype frequencies did Valley, as well as through self- and physician-referral. Subjects not deviate significantly from Hardy-Weinberg equilibrium. from Australia were either members of multiple-case families ascertained in family cancer clinics by the Kathleen Cuningham Statistical Analysis.