<em>RAD51</em> and Breast Cancer Susceptibility: No Evidence for Rare Variant Association in the Breast Cancer Family Registry Study

<div><h3>Background</h3><p>Although inherited breast cancer has been associated with germline mutations in genes that are functionally involved in the DNA homologous recombination repair (HRR) pathway, including <em>BRCA1</em>, <em>BRCA2</em>, <em>TP53</em>, <em>ATM</em>, <em>BRIP1</em>, <em>CHEK2</em> and <em>PALB2,</em> about 70% of breast cancer heritability remains unexplained. Because of their critical functions in maintaining genome integrity and already well-established associations with breast cancer susceptibility, it is likely that additional genes involved in the HRR pathway harbor sequence variants associated with increased risk of breast cancer. <em>RAD51</em> plays a central biological function in DNA repair and despite the fact that rare, likely dysfunctional variants in three of its five paralogs, <em>RAD51C, RAD51D,</em> and <em>XRCC2,</em> have been associated with breast and/or ovarian cancer risk, no population-based case-control mutation screening data are available for the <em>RAD51</em> gene. We thus postulated that <em>RAD51</em> could harbor rare germline mutations that confer increased risk of breast cancer.</p> <h3>Methodology/Principal Findings</h3><p>We screened the coding exons and proximal splice junction regions of the gene for germline sequence variation in 1,330 early-onset breast cancer cases and 1,123 controls from the Breast Cancer Family Registry, using the same population-based sampling and analytical strategy that we developed for assessment of rare sequence variants in <em>ATM</em> and <em>CHEK2.</em> In total, 12 distinct very rare or private variants were characterized in <em>RAD51</em>, with 10 cases (0.75%) and 9 controls (0.80%) carrying such a variant. Variants were either likely neutral missense substitutions (3), silent substitutions (4) or non-coding substitutions (5) that were predicted to have little effect on efficiency of the splicing machinery.</p> <h3>Conclusion</h3><p>Altogether, our data suggest that <em>RAD51</em> tolerates so little dysfunctional sequence variation that rare variants in the gene contribute little, if anything, to breast cancer susceptibility.</p> </div

Rare genetic variants of <i>RAD51</i> identified in the BCFR.

*<p>A protein multiple sequence alignment (PMSA) including 15 sequences from Human to Drosophila (Dmel) was used to obtain scores for Align-GVGD and for SIFT (Median sequence conservation of 4.32 substitutions per position).</p><p>NA, not applicable.</p

Stratified analyses of the <i>RAD51</i> −135G/C SNP on breast cancer risk in the BCFR.

a<p>Test for the difference in C allele frequency between cases and controls.</p>b<p>Results of the logistic regression assuming a log-additive model with study center and age included in the regression model as covariates in the combined analysis, and with race/ethnicity, study center and age as covariates in the stratified analysis.</p

Association between <i>RAD52</i> SNP rs10849605 and UADT cancer risk.

<p>Squares represent ORs, size of the square represents the inverse of the variance of the log ORs, horizontal lines represent 95% CIs. The solid vertical line indicates OR = 1 and the dashed vertical line the overall OR under the log-additive model. p_het is the p-value for heterogeneity between the different subgroups. I2 is the % of observed variation across subgroups (negative I2 were set to 0).</p

Distribution of individuals by <i>RAD52</i> expression.

<p>Individuals were ordered by unsupervised clustering based on <i>RAD52</i> expression levels. Heatmap represents the scaled RPKM normalized values with higher expression levels represented in red and lower expression levels in blue. The individuals carrying a copy number gain (log₂(ratio) > 0.5) of <i>RAD52</i> are highlighted in green (light yellow otherwise). <i>RAD52</i> gain carriers seem to have the same high expression pattern and cluster together. Particularly in LUAD one of the 3 gain carriers has the highest <i>RAD52</i> expression level.</p

Demographic characteristics of the cases and controls included in the genetic susceptibility study of <i>RAD52</i>/rs10849605.

<p>OR, CI and p-values represent the risk of UADT in each substrata, adjusted for sex and study specific country of origin.</p><p>Demographic characteristics of the cases and controls included in the genetic susceptibility study of <i>RAD52</i>/rs10849605.</p

eQTL analysis.

<p>Boxplots showing the effect of the genotype for the SNP <i>RAD52</i> rs10849605 on <i>RAD52</i> tumor expression levels in HNSC, LUSC and LUAD. The risk allele (C) significantly increases <i>RAD52</i> expression levels (p = 9x10⁻⁴ and 8x10⁻⁴ respectively) in both squamous cancers but not in lung adenocarcinoma (p = 0.75). In contrast, there was no evidence for association between rs10849605 and expression levels of other genes in the 12p13.33 region (Table D in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117639#pone.0117639.s001" target="_blank">S1 File</a>).</p