RAD51 135G-->C modifies breast cancer risk among BRCA2 mutation carriers: results from a combined analysis of 19 studies

Abstract

RAD51 is an important component of double-stranded DNA-repair mechanisms that interacts with both BRCA1 and BRCA2. A single-nucleotide polymorphism (SNP) in the 5' untranslated region (UTR) of RAD51, 135G-->C, has been suggested as a possible modifier of breast cancer risk in BRCA1 and BRCA2 mutation carriers. We pooled genotype data for 8,512 female mutation carriers from 19 studies for the RAD51 135G-->C SNP. We found evidence of an increased breast cancer risk in CC homozygotes (hazard ratio [HR] 1.92 [95% confidence interval {CI} 1.25-2.94) but not in heterozygotes (HR 0.95 [95% CI 0.83-1.07]; P=.002, by heterogeneity test with 2 degrees of freedom [df]). When BRCA1 and BRCA2 mutation carriers were analyzed separately, the increased risk was statistically significant only among BRCA2 mutation carriers, in whom we observed HRs of 1.17 (95% CI 0.91-1.51) among heterozygotes and 3.18 (95% CI 1.39-7.27) among rare homozygotes (P=.0007, by heterogeneity test with 2 df). In addition, we determined that the 135G-->C variant affects RAD51 splicing within the 5' UTR. Thus, 135G-->C may modify the risk of breast cancer in BRCA2 mutation carriers by altering the expression of RAD51. RAD51 is the first gene to be reliably identified as a modifier of risk among BRCA1/2 mutation carriers.

Figure 1.

RAD51 135G→C variant and alternative splicing within the 5′ UTR. A, Schematic representation and sequence of 5′ RAD51 exons. Exons are represented by hatched boxes (in 5′ UTR) and unblackened boxes (in coding region). Major splicing patterns are shown by blue connecting lines above (for isoform 1) and below (for isoform 2) the gene scheme. ATG is the translation initiation codon. The nucleotide sequence of the full-length 5′ UTR is in blue, the 5′ UTR sequence alternatively spliced as part of intron 1 is in italics, and the canonical motif of the alternative 5′ splice site within the 5′ UTR is underlined. B, Results of the RT-PCR performed with the primers shown in panel A in lymphoblastoid cell lines from carriers of three genotypes of the RAD51 135G→C variant. A predominant RAD51 transcript with the longest 5′ UTR (isoform 1, with full-length 5′ UTR of length 257 nt [GenBank accession number NM_002875]) and a less abundant transcript, with the shortest 5′ UTR (isoform 2, with truncated 5′ UTR of length 153 nt [GenBank accession number AK131299]), as well as several minor RAD51 transcript isoforms with intermediate 5′ UTR lengths characterized by sequencing, were detected.

Figure 2.

Relative levels of the RAD51 isoforms 1 and 2 transcripts by genotype, measured by quantitative RT-PCR in lymphoblastoid cell lines established from individuals with different RAD51 135G→C genotypes. The relative expression level of each RAD51 isoform across the three 135G/C variant genotypes was normalized by the geometric mean of the expression level of the reference housekeeping genes (in experiment 1, G6PD; in experiment 2, GAPDH and ACTB) and are given in arbitrary units relative to the mean level for the GG genotype. Two replicates were performed for each experiment. The nonparametric Kruskal-Wallis tests were performed to investigate differences in the distributions of the isoform levels across the genotypes.