Common variants of the BRCA1 wild-type allele modify the risk of breast cancer in BRCA1 mutation carriers

Abstract

Mutations in the BRCA1 gene substantially increase a woman's lifetime risk of breast cancer. However, there is great variation in this increase in risk with several genetic and non-genetic modifiers identified. The BRCA1 protein plays a central role in DNA repair, a mechanism that is particularly instrumental in safeguarding cells against tumorigenesis. We hypothesized that polymorphisms that alter the expression and/or function of BRCA1 carried on the wild-type (non-mutated) copy of the BRCA1 gene would modify the risk of breast cancer in carriers of BRCA1 mutations. A total of 9874 BRCA1 mutation carriers were available in the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) for haplotype analyses of BRCA1. Women carrying the rare allele of single nucleotide polymorphism rs16942 on the wild-type copy of BRCA1 were at decreased risk of breast cancer (hazard ratio 0.86, 95% confidence interval 0.77-0.95, P = 0.003). Promoter in vitro assays of the major BRCA1 haplotypes showed that common polymorphisms in the regulatory region alter its activity and that this effect may be attributed to the differential binding affinity of nuclear proteins. In conclusion, variants on the wild-type copy of BRCA1 modify risk of breast cancer among carriers of BRCA1 mutations, possibly by altering the efficiency of BRCA1 transcription.

Figure 1.

Haplotypes and LD structure of the BRCA1 gene. Region surrounding BRCA1 on chromosome 17 using HapMap Data Release 27 Phase II and III data, February 2009 build (accessed on 2 September 2010), on NCBI B36 assembly and dbSNP b126. SNPs used in haplotype analyses are shown, relative to their position in the BRCA1 gene.

Figure 2.

Study-specific HRs between rs16942 genotypes on wild-type allele of BRCA1 and breast cancer risk. HRs were calculated in each specific study as described in Materials and Methods. The summary effect estimate for C allele carriers is 0.87 (0.78–0.97), p-heterogeneity 0.91 with a fixed effect model.

Figure 3.

Representative luciferase reporter assays of major BRCA1 haplotype constructs in HeLa cell line. Relative luciferase activity of constructs carrying BRCA1 promoter sequences corresponding to two haplotypes was measured following transient transfection into HeLa cells. The ratio of firefly:Renilla luciferase activity of each promoter construct was normalized against that of the promoter-less pGL3-Basic vector. The pGL3-SV40 vector containing the SV40 early promoter is used as a positive control. Promoter haplotype BRCA1-pHapA was used as reference against which pairwise comparisons were made. Significant differences between haplotype expressions are marked with asterisks (**P = 0.0011). The position of each SNP is given relative to the first base of the initiation codon.

Figure 4.

Representative EMSAs illustrating allelic DNA–protein interactions in the promoter region of BRCA1. Labeled double-stranded oligonucleotide (ds-oligo) probes containing either allele of rs4793204 (A) and rs799908 (B) were incubated with nuclear extracts from HeLa, MCF7 and MDA-MB-231 cells. (A) Lanes 1–6 represent binding to the labeled ds-oligo containing the major allele; lanes 7–12, binding to the ds-oligo containing the minor allele. Lanes 1 and 7, negative control (no extracts). Lanes 3 and 10, competition with unlabeled allelic probes (specific competitors). Lanes 4 and 9, competition with mismatched unlabeled probes. Lanes 5 and 11, competition with a non-specific probe (non-specific competitor). Lanes 6 and 12, pre-incubation with anti-BRG1 antibody. (B) Lanes 1–5 represent binding to the labeled ds-oligo containing the major allele; lanes 6–10, binding to the ds-oligo containing the minor allele. Lanes 1 and 6, negative control (no extracts). Lanes 3 and 9, competition with unlabeled allelic probes (specific competitors). Lanes 4 and 8, competition with mismatched unlabeled probes. Lanes 5 and 10, competition with a non-specific probe (non-specific competitor).