Genome-wide association study in BRCA1 mutation carriers identifies novel loci associated with breast and ovarian cancer risk.

BRCA1-associated breast and ovarian cancer risks can be modified by common genetic variants. To identify further cancer risk-modifying loci, we performed a multi-stage GWAS of 11,705 BRCA1 carriers (of whom 5,920 were diagnosed with breast and 1,839 were diagnosed with ovarian cancer), with a further replication in an additional sample of 2,646 BRCA1 carriers. We identified a novel breast cancer risk modifier locus at 1q32 for BRCA1 carriers (rs2290854, P = 2.7 × 10(-8), HR = 1.14, 95% CI: 1.09-1.20). In addition, we identified two novel ovarian cancer risk modifier loci: 17q21.31 (rs17631303, P = 1.4 × 10(-8), HR = 1.27, 95% CI: 1.17-1.38) and 4q32.3 (rs4691139, P = 3.4 × 10(-8), HR = 1.20, 95% CI: 1.17-1.38). The 4q32.3 locus was not associated with ovarian cancer risk in the general population or BRCA2 carriers, suggesting a BRCA1-specific association. The 17q21.31 locus was also associated with ovarian cancer risk in 8,211 BRCA2 carriers (P = 2×10(-4)). These loci may lead to an improved understanding of the etiology of breast and ovarian tumors in BRCA1 carriers. Based on the joint distribution of the known BRCA1 breast cancer risk-modifying loci, we estimated that the breast cancer lifetime risks for the 5% of BRCA1 carriers at lowest risk are 28%-50% compared to 81%-100% for the 5% at highest risk. Similarly, based on the known ovarian cancer risk-modifying loci, the 5% of BRCA1 carriers at lowest risk have an estimated lifetime risk of developing ovarian cancer of 28% or lower, whereas the 5% at highest risk will have a risk of 63% or higher. Such differences in risk may have important implications for risk prediction and clinical management for BRCA1 carriers.

Evaluation of chromosome 6p22 as a breast cancer risk modifier locus in a follow-up study of BRCA2 mutation carriers.

Several common germline variants identified through genome-wide association studies of breast cancer risk in the general population have recently been shown to be associated with breast cancer risk for BRCA1 and/or BRCA2 mutation carriers. When combined, these variants can identify marked differences in the absolute risk of developing breast cancer for mutation carriers, suggesting that additional modifier loci may further enhance individual risk assessment for BRCA1 and BRCA2 mutation carriers. Recently, a common variant on 6p22 (rs9393597) was found to be associated with increased breast cancer risk for BRCA2 mutation carriers [hazard ratio (HR) = 1.55, 95 % confidence interval (CI) 1.25-1.92, p = 6.0 × 10(-5)]. This observation was based on data from GWAS studies in which, despite statistical correction for multiple comparisons, the possibility of false discovery remains a concern. Here, we report on an analysis of this variant in an additional 6,165 BRCA1 and 3,900 BRCA2 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA). In this replication analysis, rs9393597 was not associated with breast cancer risk for BRCA2 mutation carriers (HR = 1.09, 95 % CI 0.96-1.24, p = 0.18). No association with ovarian cancer risk for BRCA1 or BRCA2 mutation carriers or with breast cancer risk for BRCA1 mutation carriers was observed. This follow-up study suggests that, contrary to our initial report, this variant is not associated with breast cancer risk among individuals with germline BRCA2 mutations.

Common breast cancer susceptibility alleles and the risk of breast cancer for BRCA1 and BRCA2 mutation carriers: implications for risk prediction.

The known breast cancer susceptibility polymorphisms in FGFR2, TNRC9/TOX3, MAP3K1, LSP1, and 2q35 confer increased risks of breast cancer for BRCA1 or BRCA2 mutation carriers. We evaluated the associations of 3 additional single nucleotide polymorphisms (SNPs), rs4973768 in SLC4A7/NEK10, rs6504950 in STXBP4/COX11, and rs10941679 at 5p12, and reanalyzed the previous associations using additional carriers in a sample of 12,525 BRCA1 and 7,409 BRCA2 carriers. Additionally, we investigated potential interactions between SNPs and assessed the implications for risk prediction. The minor alleles of rs4973768 and rs10941679 were associated with increased breast cancer risk for BRCA2 carriers (per-allele HR = 1.10, 95% CI: 1.03-1.18, P = 0.006 and HR = 1.09, 95% CI: 1.01-1.19, P = 0.03, respectively). Neither SNP was associated with breast cancer risk for BRCA1 carriers, and rs6504950 was not associated with breast cancer for either BRCA1 or BRCA2 carriers. Of the 9 polymorphisms investigated, 7 were associated with breast cancer for BRCA2 carriers (FGFR2, TOX3, MAP3K1, LSP1, 2q35, SLC4A7, 5p12, P = 7 × 10(-11) - 0.03), but only TOX3 and 2q35 were associated with the risk for BRCA1 carriers (P = 0.0049, 0.03, respectively). All risk-associated polymorphisms appear to interact multiplicatively on breast cancer risk for mutation carriers. Based on the joint genotype distribution of the 7 risk-associated SNPs in BRCA2 mutation carriers, the 5% of BRCA2 carriers at highest risk (i.e., between 95th and 100th percentiles) were predicted to have a probability between 80% and 96% of developing breast cancer by age 80, compared with 42% to 50% for the 5% of carriers at lowest risk. Our findings indicated that these risk differences might be sufficient to influence the clinical management of mutation carriers.

Evidence for SMAD3 as a modifier of breast cancer risk in BRCA2 mutation carriers.

INTRODUCTION: Current attempts to identify genetic modifiers of BRCA1 and BRCA2 associated risk have focused on a candidate gene approach, based on knowledge of gene functions, or the development of large genome-wide association studies. In this study, we evaluated 24 SNPs tagged to 14 candidate genes derived through a novel approach that analysed gene expression differences to prioritise candidate modifier genes for association studies. METHODS: We successfully genotyped 24 SNPs in a cohort of up to 4,724 BRCA1 and 2,693 BRCA2 female mutation carriers from 15 study groups and assessed whether these variants were associated with risk of breast cancer in BRCA1 and BRCA2 mutation carriers. RESULTS: SNPs in five of the 14 candidate genes showed evidence of association with breast cancer risk for BRCA1 or BRCA2 carriers (P < 0.05). Notably, the minor alleles of two SNPs (rs7166081 and rs3825977) in high linkage disequilibrium (r² = 0.77), located at the SMAD3 locus (15q22), were each associated with increased breast cancer risk for BRCA2 mutation carriers (relative risk = 1.25, 95% confidence interval = 1.07 to 1.45, P(trend) = 0.004; and relative risk = 1.20, 95% confidence interval = 1.03 to 1.40, P(trend) = 0.018). CONCLUSIONS: This study provides evidence that the SMAD3 gene, which encodes a key regulatory protein in the transforming growth factor beta signalling pathway and is known to interact directly with BRCA2, may contribute to increased risk of breast cancer in BRCA2 mutation carriers. This finding suggests that genes with expression associated with BRCA1 and BRCA2 mutation status are enriched for the presence of common genetic modifiers of breast cancer risk in these populations.

Common variants associated with breast cancer in genome-wide association studies are modifiers of breast cancer risk in BRCA1 and BRCA2 mutation carriers.

Recent studies have identified single nucleotide polymorphisms (SNPs) that significantly modify breast cancer risk in BRCA1 and BRCA2 mutation carriers. Since these risk modifiers were originally identified as genetic risk factors for breast cancer in genome-wide association studies (GWASs), additional risk modifiers for BRCA1 and BRCA2 may be identified from promising signals discovered in breast cancer GWAS. A total of 350 SNPs identified as candidate breast cancer risk factors (P < 1 x 10(-3)) in two breast cancer GWAS studies were genotyped in 3451 BRCA1 and 2006 BRCA2 mutation carriers from nine centers. Associations with breast cancer risk were assessed using Cox models weighted for penetrance. Eight SNPs in BRCA1 carriers and 12 SNPs in BRCA2 carriers, representing an enrichment over the number expected, were significantly associated with breast cancer risk (P(trend) < 0.01). The minor alleles of rs6138178 in SNRPB and rs6602595 in CAMK1D displayed the strongest associations in BRCA1 carriers (HR = 0.78, 95% CI: 0.69-0.90, P(trend) = 3.6 x 10(-4) and HR = 1.25, 95% CI: 1.10-1.41, P(trend) = 4.2 x 10(-4)), whereas rs9393597 in LOC134997 and rs12652447 in FBXL7 showed the strongest associations in BRCA2 carriers (HR = 1.55, 95% CI: 1.25-1.92, P(trend) = 6 x 10(-5) and HR = 1.37, 95% CI: 1.16-1.62, P(trend) = 1.7 x 10(-4)). The magnitude and direction of the associations were consistent with the original GWAS. In subsequent risk assessment studies, the loci appeared to interact multiplicatively for breast cancer risk in BRCA1 and BRCA2 carriers. Promising candidate SNPs from GWAS were identified as modifiers of breast cancer risk in BRCA1 and BRCA2 carriers. Upon further validation, these SNPs together with other genetic and environmental factors may improve breast cancer risk assessment in these populations.

Common variants in LSP1, 2q35 and 8q24 and breast cancer risk for BRCA1 and BRCA2 mutation carriers.

Genome-wide association studies of breast cancer have identified multiple single nucleotide polymorphisms (SNPs) that are associated with increased breast cancer risks in the general population. In a previous study, we demonstrated that the minor alleles at three of these SNPs, in FGFR2, TNRC9 and MAP3K1, also confer increased risks of breast cancer for BRCA1 or BRCA2 mutation carriers. Three additional SNPs rs3817198 at LSP1, rs13387042 at 2q35 and rs13281615 at 8q24 have since been reported to be associated with breast cancer in the general population, and in this study we evaluated their association with breast cancer risk in 9442 BRCA1 and 5665 BRCA2 mutation carriers from 33 study centres. The minor allele of rs3817198 was associated with increased breast cancer risk only for BRCA2 mutation carriers [hazard ratio (HR) = 1.16, 95% CI: 1.07-1.25, P-trend = 2.8 x 10(-4)]. The best fit for the association of SNP rs13387042 at 2q35 with breast cancer risk was a dominant model for both BRCA1 and BRCA2 mutation carriers (BRCA1: HR = 1.14, 95% CI: 1.04-1.25, P = 0.0047; BRCA2: HR = 1.18 95% CI: 1.04-1.33, P = 0.0079). SNP rs13281615 at 8q24 was not associated with breast cancer for either BRCA1 or BRCA2 mutation carriers, but the estimated association for BRCA2 mutation carriers (per-allele HR = 1.06, 95% CI: 0.98-1.14) was consistent with odds ratio estimates derived from population-based case-control studies. The LSP1 and 2q35 SNPs appear to interact multiplicatively on breast cancer risk for BRCA2 mutation carriers. There was no evidence that the associations vary by mutation type depending on whether the mutated protein is predicted to be stable or not.

Common breast cancer-predisposition alleles are associated with breast cancer risk in BRCA1 and BRCA2 mutation carriers.

Germline mutations in BRCA1 and BRCA2 confer high risks of breast cancer. However, evidence suggests that these risks are modified by other genetic or environmental factors that cluster in families. A recent genome-wide association study has shown that common alleles at single nucleotide polymorphisms (SNPs) in FGFR2 (rs2981582), TNRC9 (rs3803662), and MAP3K1 (rs889312) are associated with increased breast cancer risks in the general population. To investigate whether these loci are also associated with breast cancer risk in BRCA1 and BRCA2 mutation carriers, we genotyped these SNPs in a sample of 10,358 mutation carriers from 23 studies. The minor alleles of SNP rs2981582 and rs889312 were each associated with increased breast cancer risk in BRCA2 mutation carriers (per-allele hazard ratio [HR] = 1.32, 95% CI: 1.20-1.45, p(trend) = 1.7 x 10(-8) and HR = 1.12, 95% CI: 1.02-1.24, p(trend) = 0.02) but not in BRCA1 carriers. rs3803662 was associated with increased breast cancer risk in both BRCA1 and BRCA2 mutation carriers (per-allele HR = 1.13, 95% CI: 1.06-1.20, p(trend) = 5 x 10(-5) in BRCA1 and BRCA2 combined). These loci appear to interact multiplicatively on breast cancer risk in BRCA2 mutation carriers. The differences in the effects of the FGFR2 and MAP3K1 SNPs between BRCA1 and BRCA2 carriers point to differences in the biology of BRCA1 and BRCA2 breast cancer tumors and confirm the distinct nature of breast cancer in BRCA1 mutation carriers.

Contribution of germline BRCA1 and BRCA2 sequence alterations to breast cancer in Northern India.

BACKGROUND: A large number of distinct mutations in the BRCA1 and BRCA2 genes have been reported worldwide, but little is known regarding the role of these inherited susceptibility genes in breast cancer risk among Indian women. We investigated the distribution and the nature of BRCA1 and BRCA2 germline mutations and polymorphisms in a cohort of 204 Indian breast cancer patients and 140 age-matched controls. METHOD: Cases were selected with regard to early onset disease (< or =40 years) and family history of breast and ovarian cancer. Two hundred four breast cancer cases along with 140 age-matched controls were analyzed for mutations. All coding regions and exon-intron boundaries of the BRCA1 and BRCA2 genes were screened by heteroduplex analysis followed by direct sequencing of detected variants. RESULTS: In total, 18 genetic alterations were identified. Three deleterious frame-shift mutations (185delAG in exon 2; 4184del4 and 3596del4 in exon 11) were identified in BRCA1, along with one missense mutation (K1667R), one 5'UTR alteration (22C>G), three intronic variants (IVS10-12delG, IVS13+2T>C, IVS7+38T>C) and one silent substitution (5154C>T). Similarly three pathogenic protein-truncating mutations (6376insAA in exon 11, 8576insC in exon19, and 9999delA in exon 27) along with one missense mutation (A2951T), four intronic alterations (IVS2+90T>A, IVS7+75A>T, IVS8+56C>T, IVS25+58insG) and one silent substitution (1593A>G) were identified in BRCA2. Four previously reported polymorphisms (K1183R, S1613G, and M1652I in BRCA1, and 7470A>G in BRCA2) were detected in both controls and breast cancer patients. Rare BRCA1/2 sequence alterations were observed in 15 out of 105 (14.2%) early-onset cases without family history and 11.7% (4/34) breast cancer cases with family history. Of these, six were pathogenic protein truncating mutations. In addition, several variants of uncertain clinical significance were identified. Among these are two missense variants, one alteration of a consensus splice donor sequence, and a variant that potentially disrupts translational initiation. CONCLUSION: BRCA1 and BRCA2 mutations appear to account for a lower proportion of breast cancer patients at increased risk of harboring such mutations in Northern India (6/204, 2.9%) than has been reported in other populations. However, given the limited extent of reported family history among these patients, the observed mutation frequency is not dissimilar from that reported in other cohorts of early onset breast cancer patients. Several of the identified mutations are unique and novel to Indian patients.

Understanding germ-line mutations in BRCA1.

Germ-line mutations in BRCA1 account for the majority of familial breast and ovarian cancer cases and development of cancer in individuals who carry such mutations requires somatic inactivation of the normal allele. BRCA1 is highly polymorphic with more than 1,200 distinct documented variants. Approximately 70% of reported variants lead to absence of full-length BRCA1 protein, through loss of expression or protein truncation, and are suspected to predispose to cancer. These include regulatory mutations, splice site alterations, large rearrangements, large and small deletions or insertions, and nonsense mutations. However, characterizing the remaining missense alterations as either deleterious (cancer-associated mutations) or neutral variants is more complex, as the functional significance of the respective amino acid substitution is not straightforward to evaluate. In addition, many missense variants have been identified only once in defined ethnic groups and represent alleles with very low frequency. Most often, little information is available about segregation of the variant with disease in families, and assessment of disease risk for low frequency alleles through association studies is problematic, requiring a large number of samples stratified and matched by ethnicity. The fact that a significant proportion of BRCA1 variants remain unclassified represents a gap in risk assessment, such that individuals undergoing genetic testing will receive noninformative test results. An approach for assessing the potential clinical significance of missense variants is to combine available genetic data with functional and structural studies. Here we review the available information on BRCA1 variants and explore ways in which we can analyze unclassified variants.

Are ATM mutations 7271T-->G and IVS10-6T-->G really high-risk breast cancer-susceptibility alleles?

Two mutations of the ATM gene were recently suggested to confer breast cancer risks similar to mutations of BRCA1 or BRCA2. Here, we set out to confirm these findings in 961 families with non-BRCA1/BRCA2 breast cancer from diverse geographical regions. We did not detect the ATM 7271T-->G mutation in any family. The ATM IVS10-6T-->G mutation was detected in eight families, which was similar to its frequency among population-matched control individuals (pooled Mantel-Haenszel odds ratio = 1.60; 95% confidence interval = 0.48 to 5.35; P = 0.44). Bayesian analysis of linkage in the ATM IVS10-6T-->G-positive families showed an overall posterior probability of causality for this mutation of 0.008. We conclude that the ATM IVS10-6T-->G mutation does not confer a significantly elevated breast cancer risk and that ATM 7271T-->G is a rare event in familial breast cancer.

BRCA1 and BRCA2 in Indian breast cancer patients.

Incidence of breast cancer in Indian women is not as high as in Western countries, nonetheless age-adjusted incidence rates (AAR) have risen from 17.9 to 24.9 per 100,000 from 1965 to 1985. Although these rates are still approximately one quarter to one third of incidence rates in North America and Europe, respectively, due to the large population of women at risk, nearly 80,000 new cases were diagnosed in India in 2000. Although identification of BRCA1 and BRCA2 has greatly increased our understanding of breast cancer genetics in populations of Western European descent, the role of these genes in Indian populations remains unexplored. Analysis of a series of 20 breast cancer patients from North India with either family history of breast and/or ovarian cancer (2 or more affected first degree relatives) or early age of onset (<35 years) led to identification of two novel splice variants (331+1G>T; 4476+2T>C) in BRCA1 (10%). In addition, two BRCA2 missense variants were each identified in more than one patient (two unrelated individuals each) and likely represent population-specific polymorphisms.

Evaluation of linkage of breast cancer to the putative BRCA3 locus on chromosome 13q21 in 128 multiple case families from the Breast Cancer Linkage Consortium.

The known susceptibility genes for breast cancer, including BRCA1 and BRCA2, only account for a minority of the familial aggregation of the disease. A recent study of 77 multiple case breast cancer families from Scandinavia found evidence of linkage between the disease and polymorphic markers on chromosome 13q21. We have evaluated the contribution of this candidate "BRCA3" locus to breast cancer susceptibility in 128 high-risk breast cancer families of Western European ancestry with no identified BRCA1 or BRCA2 mutations. No evidence of linkage was found. The estimated proportion (alpha) of families linked to a susceptibility locus at D13S1308, the location estimated by Kainu et al. [(2000) Proc. Natl. Acad. Sci. USA 97, 9603-9608], was 0 (upper 95% confidence limit 0.13). Adjustment for possible bias due to selection of families on the basis of linkage evidence at BRCA2 did not materially alter this result (alpha = 0, upper 95% confidence limit 0.18). The proportion of linked families reported by Kainu et al. (0.65) is excluded with a high degree of confidence in our dataset [heterogeneity logarithm of odds (HLOD) at alpha = 0.65 was -11.0]. We conclude that, if a susceptibility gene does exist at this locus, it can only account for a small proportion of non-BRCA1/2 families with multiple cases of early-onset breast cancer.