The considerable uncertainty regarding cancer risks associated with inherited mutations of BRCA2 is due to unknown factors. To investigate whether common genetic variants modify penetrance for BRCA2 mutation carriers, we undertook a two-staged genome-wide association study in BRCA2 mutation carriers. In stage 1 using the Affymetrix 6. 0 platform, 592,163 filtered SNPs genotyped were available on 899 young (<40 years) affected and 804 unaffected carriers of European ancestry. Associations were evaluated using a survival-based score test adjusted for familial correlations and stratified by country of the study and BRCA2*6174delT mutation status. The genomic inflation factor (λ) was 1. 011. The stage 1 association analysis revealed multiple variants associated with breast cancer risk: 3 SNPs had p-values<10−5 and 39 SNPs had p-values<10−4. These variants included several previously associated with sporadic breast cancer risk and two novel loci on chromosome 20 (rs311499) and chromosome 10 (rs16917302). The chromosome 10 locus was in ZNF365, which contains another variant that has recently been associated with breast cancer in an independent study of unselected cases. In stage 2, the top 85 loci from stage 1 were genotyped in 1,264 cases and 1,222 controls. Hazard ratios (HR) and 95% confidence intervals (CI) for stage 1 and 2 were combined and estimated using a retrospective likelihood approach, stratified by country of residence and the most common mutation, BRCA2*6174delT. The combined per allele HR of the minor allele for the novel loci rs16917302 was 0. 75 (95% CI 0. 66–0. 86,) and for rs311499 was 0. 72 (95% CI 0. 61–0. 85,). FGFR2 rs2981575 had the strongest association with breast cancer risk (per allele HR = 1. 28,95% CI 1. 18–1. 39,). These results indicate that SNPs that modify BRCA2 penetrance identified by an agnostic approach thus far are limited to variants that also modify risk of sporadic BRCA2 wild-type breast cancer.
After more than a decade of clinical testing for mutations of BRCA1 and BRCA2, there remains considerable uncertainty regarding cancer risks associated with inherited mutations of these genes. This variable penetrance is most striking for BRCA2 [1]–[4], and it affects medical management [5]. Women with the same BRCA2 mutation may develop breast, ovarian or other cancers at different ages or not at all [6]. In a segregation analysis of families identified through breast cancer cases diagnosed before age 55, the residual familial clustering after accounting for BRCA1 and BRCA2 mutations could be explained by a large number of low penetrance genes with multiplicative effects on breast cancer risk [7], [8]. A candidate gene approach in BRCA2 mutation carriers led to the discovery of loci that modify the penetrance of BRCA2 mutations, such as RAD51 135 G>C [9] and perhaps CASP8 [10], [11] and IGFBP2 [12], if replicated. To investigate whether other common single nucleotide polymorphisms (SNP), copy number variants (CNV), or copy number polymorphisms (CNP) modify penetrance for BRCA2 mutation carriers, we undertook a two-staged genome-wide association study (GWAS) in BRCA2 mutation carriers from the international Consortium for Investigators of Modifiers of BRCA1/2 (CIMBA) and other international studies. We hypothesized that an agnostic search for breast cancer loci in an enriched population of BRCA2 mutation carriers, the first among this high risk population, would provide greater power than a sporadic population of equal number, and would yield associations specific to BRCA2 carriers and/or the general population.
In stage 1, genotype data were available for 899 young (<40 years) affected and 804 older (>40 years) unaffected carriers of European ancestry after quality control filtering and removal of ethnic outliers (Figure S1). A total of 592,163 filtered SNPs genotyped using the Affymetrix Genome-Wide Human SNP Array 6. 0 platform passed quality control assessment. In stage 1, comparison of the observed and expected distributions (quantile-quantile plot: Figure S2) showed little evidence for an inflation of the test statistics (genomic inflation factor λ = 1. 01), thereby excluding the possibility of significant hidden population substructure, cryptic relatedness among subjects or differential genotype calling between BRCA2 affected and BRCA2 unaffected carriers. Multiple variants were found to be associated with breast cancer risk (Figure S3): 3 SNPs had p<10−5 and 39 SNPs had p<10−4. The most significant association () was observed for FGFR2 rs2981582 (Table 1), a variant previously shown to be associated with increased risk of BRCA2-related breast cancer [13]. A positive association was also observed with rs3803662 (Table 1), near TOX3, which has also been associated with sporadic breast cancer risk [13]. Using the stage 1 data, we also performed a GSEA as implemented in MAGENTA [14] to evaluate whether a functionally-related set of genes relevant to BRCA2 function (Table S1) was enriched for relative risk associations (see Statistical Methods). The 59 genes selected are related to the Fanconi anemia pathway [15] as well as other pathways reported in the literature to regulate or interact with BRCA1/2 [16]. These showed no enrichment of associations with the breast cancer risk (p = 0. 56). In addition, eight of 125 known cancer susceptibility alleles identified by previous GWAS of other cancers [17] were associated with BRCA2 modification in the current study, a number not greater than expected (Kolmogorv-Smirnov p = 0. 60) by chance alone. Of the 113 most significantly associated SNPs (p<10−3) in our study, three showed significant association (p<0. 05) with BRCA1-associated breast cancer risk in a complimentary GWAS [18]. In the combined stage 1 and stage 2 results, four independent SNPs (pairwise) were associated with increased risk of breast cancer risk with p-values<10−4 (Table 1). Previously identified breast cancer susceptibility loci [13], [19], [20] had the most significant associations among BRCA2 mutation carriers (FGFR2: per allele and TOX3: per allele). Novel loci, rs16917302 on chromosome 10 and rs311499 on chromosome 20, had HRs in stage 2 that were in the same direction as those observed for stage 1 (Figure 1, Table 1), but were smaller in magnitude (HR = 0. 67 (95% CI: 0. 56–0. 80) vs. 0. 85 (95% CI: 0. 70–1. 04) for rs16917302; HR = 0. 60 (95%CI: 0. 50–0. 78) vs. 0. 84 (95%CI: 0. 67–1. 06) for rs311499) perhaps reflecting a “winner' s curse” effect” [21]. The associations for these SNPs were not statistically significant in stage 2 (Table 1). In the combined stage 1 and stage 2 dataset, the C allele of rs16917302 was associated with lower risk of breast cancer (per allele HR = 0. 75,95% CI 0. 66–0. 86; ; Table 1), and the C allele of rs311499 was associated with a reduced risk (per allele HR = 0. 72,95% CI 0. 61–0. 85; ; Table 1). A full list of stage 2 results can be found in Table S2. Using the combined stage 1 and stage 2 data, there was no evidence that the HR for SNP rs16917302 changes with age (p = 0. 63), but there was some evidence that the per-allele HR for rs311499 may increase with age (p = 0. 034). We also examined the association of both high-frequency CNPs and low-frequency CNVs to case-control status using the stage 1 data. After performing standard quality control measures including a minor allele frequency (MAF) threshold of 5%, we identified 191 polymorphisms with reliable genotypes. No associations were found between CNVs and the phenotype; there was no inflation or deflation of the test statistic, and the best p-value was. We similarly assessed less common CNPs, and found neither the overall burden of events (or any subclass thereof, such as large deletions overlapping genes) nor any specific locus associated with breast cancer risk (Figure S4). Because of the prior evidence of significant LD extent around the 6174delT (c. 5946delT) founder mutation in the Ashkenazi Jewish population [22], we explored the potential excess sharing of the genome compared to the BRCA2 region in both Ashkenazi Jewish and non-Jewish European ancestries. Using GERMLINE [23], shared segments of greater than 5 cM were computed based on the imputed genotype dataset. In the BRCA2 region, we observed a significant excess of sharing amongst both Ashkenazi (n = 304) and non-Jewish (n = 1331) individuals compared to samples from an autism study (n = 808) suggesting common founders for BRCA2 mutations. Examining sites across the genome every 2. 5 cM (excluding telomere and centromere regions), we observed possible pairs share segments greater than 5 cM that on average 0. 005% (u = 50. 17, s. d = 55. 5, max = 491) for non-Jewish individuals and 0. 12% (u = 141. 11, s. d = 57. 32, max = 525) for Ashkenazi Jewish individuals. Comparing cases and controls, we did not observe a significant difference in number of pairs of samples sharing segments greater than 5cM across the genome excluding chromosome 13. That is, there was no evidence of overall excess sharing across the genome other than for the BRCA2 locus within the Ashkenazi Jewish and non-Ashkenazi Jewish populations in the study.
In this GWAS of BRCA2 mutation carriers, the first in this high risk population, we found previously identified breast cancer susceptibility loci modified risk of BRCA2-associated breast cancer with similar magnitude of association. Although FGFR2 (rs2981575) was the only locus to reach genome-wide statistical significance, novel loci, rs16917302 and rs10509168 were each associated with breast cancer risk. rs16917302 is located on chromosome 10, in the zinc finger protein 365 gene (ZNF365). A recent multistage GWAS of 15,992 sporadic breast cancer cases and 16,891 controls also observed an inverse association (per allele OR = 0. 82,95% CI 0. 82–0. 91,) between breast cancer risk and rs10509168, a SNP 18kb from rs16917302 (pairwise) and located in intron 4 of ZNF365 [24]. Of the 3,659 cases and 4,897 controls in phase 1 of that study, imputation revealed that the locus identified in our BRCA2 study, rs16917302, was significantly associated with risk for breast cancer (p = 0. 02) (Easton DF, personal communication). The second novel SNP in the current study, rs311499, is located on chromosome 20, within a region containing several possible candidate genes including GMEB2, SRMS, PTK6, STMN3, and TNFRSF6. The functional significance of both of these regions with breast carcinogenesis is unknown; further research is warranted. There was some evidence that the HR associated with rs311499 may change with age. We also observed that the stage 1 HR for this SNPs was larger in magnitude compared to the stage 2 HR, consistent with a winner' s curse effect [21]. Since stage 1 of our experiment included mostly BRCA2 mutation carriers diagnosed at a young age, and stage 2 mutation carriers diagnosed an older age, the “winner' s curse” and age-specific effects are confounded and may be difficult to distinguish. Fitting the age-dependent HR model for SNP rs311499 using the stage 2 data yielded no significant variation in the HR by age (p = 0. 47), but the sample size for this analysis was relatively small. Future larger studies should aim to clarify this. Mutations in known genes (BRCA1, BRCA2, TP53, CHEK2, PTEN, and ATM) explain only 20–25% of the familial clustering of breast cancer; the residual familial clustering may be explained by the existence of multiple common, low-penetrance alleles (‘polygenes’) [25]. Perhaps because the majority of BRCA2-associated breast tumors are estrogen receptor (ER) -positive, as are the majority of non-hereditary breast cancers [26], risk alleles for sporadic breast cancer are more likely to be modifiers of risk of BRCA2-associated hereditary breast cancer. Of the seven GWAS-identified breast cancer-associated SNPs examined in a BRCA2 background [13], [19], [20], SNPS in FGFR2 (rs2981575), TOX3 (rs3803662), MAP3K1 (rs889312), and LSP1 (rs3817198) have been shown to modify BRCA2 penetrance, in contrast with BRCA1 tumors, in which only two of these same SNPs (based on a 2 degrees of freedom model) modified risk of these largely ER-negative tumors [26]. As previously noted [13], [20], the stage 1 HRs among BRCA2 mutation carriers, reported here, were nearly identical to odds ratio estimates observed in sporadic breast cancer studies, consistent with a simple multiplicative interaction between the BRCA2 mutant alleles and the common susceptibility SNPs. If replicated, the two additional SNPs identified here would only explain about 1. 7% of the variance in breast cancer risk among BRCA2 mutation carriers. Taken together, the combined effects of all the common and putative risk modifiers in this study only account for ∼4% of the variance of BRCA2 mutations, compared with 1. 1% for the single RAD51 135 G>C variant, which is rare and biologically-linked to BRCA2 function, as shown by candidate gene studies [9]. Thus, the common alleles that modify risk in BRCA1 and BRCA2 backgrounds appear to have comparable associated risks in sporadic ER-positive and ER-negative tumors, respectively [18]. While individual SNPs are unlikely to be used to guide radiographic screening and risk-reducing surgical strategies, the combined effect of these SNPs may ultimately be used for the tailor management of subsets of BRCA mutation carriers [5]. While we took great efforts to collect all of the possible known BRCA2 mutation carriers, there were insufficient numbers to stratify by race and BRCA2 mutations with the exception of BRCA2*6174delT mutations. Due to the small numbers of women of non-European ancestry who have participated in the individual studies represented here, the current analysis was based only on women who had genetic backgrounds consistent with HapMap CEU samples. While we expect that SNPs identified among women of European ancestry might also be applicable to women of other genetic backgrounds, additional research in these populations will be needed. Similarly, the observed associations represented across all types of mutations, and specifically a weighted average of BRCA2*6174delT and non-delT mutations. It is possible that the observed associations may only modify the penetrance of specific BRCA2 mutations due to differential effects on function or differences in genetic background. Our analysis was stratified on the basis of the most common BRCA2 mutation, BRCA2*6174delT, which is prevalent in individuals with an Ashkenazi Jewish ancestry. Large numbers of mutation carriers will be necessary to calculate mutation-specific estimates. In addition, there was a drop-out of SNPs in the two phases of this study. While we were able to achieve a representative coverage of the genome, it is also possible that additional studies using denser arrays may provide further information. As expected, we observed associations with some of the major common genetic variants seen in genome-wide scans of breast cancer in a non-BRCA1/2 mutation background. However, we found no evidence for loci with stronger effects than FGFR2. Although we observed an association with a novel locus at ZNF365 that appears also to be a risk factor for sporadic breast cancer, overall, our results suggest that there are no common variants with major effects (i. e. , OR>2. 0) that are specific in BRCA2 carriers. Similarly, in a recent report of SNPs from sporadic breast cancer GWAS genotyped in a restricted set of BRCA1/2 carriers [27], loci in LOC134997 (rs9393597: per allele HR = 1. 55,95% CI 1. 25–1. 92,) and FBXL7 (rs12652447: HR = 1. 37,95% CI 1. 16–1. 62,) were associated with BRCA2 breast cancer risk with p-values weaker than FGFR2 reported here (per allele), although the magnitudes of the associations were slightly stronger than FGFR2 (HR = 1. 28). Although these SNPs were not in our genotyped panel of SNPs at stage 1, imputation results indicate that SNP rs9393597 has a p-value of 0. 008 and SNP rs12652447 a p-value of 0. 04 for association with breast cancer risk for the BRCA2 mutation carriers in our stage1. However, there is substantial overlap between our study and the study of Wang et al. [27]. Replication in larger datasets will be necessary to precisely estimate the magnitude of the associations of suspected loci identified from our study, candidate gene analysis [10]–[12], and other selection approaches [27]. It is of interest, however, that when utilizing an agnostic approach in BRCA2 mutation carriers in this study, the major determinants of risk variation in mutation carriers are those that also modify risk in subsets of sporadic, BRCA1/2 wild type, breast cancer. However, it remains possible that unique variants with smaller effects, or rarer variants (not evaluated in this experiment), may be specific modifiers of breast cancer risk in BRCA2 carriers. Their detection would require study populations much larger than the current analysis, which is presently the largest such cohort assembled.
The risk of breast cancer associated with BRCA2 mutations varies widely. To determine whether common genetic variants modify the penetrance of BRCA2 mutations, we conducted the first genome-wide association study of breast cancer among women with BRCA2 mutations using a two-stage approach. The major finding of the study is that only those loci known to be associated with breast cancer risk in the general population, including FGFR2 (rs2981575), modified BRCA2-associated risk in our high-risk population. Two novel loci, on chromosomes 10 in ZNF365 (rs16917302) and chromosome 20 (rs311499), were shown to modify risk in BRCA2 mutation carriers, although not at a genome-wide level of significance. However, the ZNF365 locus has recently independently been associated with breast cancer risk in sporadic tumors, highlighting the potential significance of this zinc finger-containing gene in breast cancer pathogenesis. Our results indicate that it is unlikely that other common variants have a strong modifying effect on BRCA2 penetrance.
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Common Genetic Variants and Modification of Penetrance of BRCA2-Associated Breast Cancer