<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

Distribution of p.Thr141Ile, p.Ser7Ser and p.Ser11Ser by race/ethnicity.

<p>Distribution of p.Thr141Ile, p.Ser7Ser and p.Ser11Ser by race/ethnicity.</p

Analysis of potentially pathogenic <i>ABRAXAS</i> in-frame deletion or rare missense substitutions.

<p>Analysis of potentially pathogenic <i>ABRAXAS</i> in-frame deletion or rare missense substitutions.</p

Distribution of <i>ABRAXAS</i> rare variants (<i>i</i>.<i>e</i>. with a minor allele frequency<1% in the Exome Variant Server (EVS)) identified in the BCFR.

<p>Distribution of <i>ABRAXAS</i> rare variants (<i>i</i>.<i>e</i>. with a minor allele frequency<1% in the Exome Variant Server (EVS)) identified in the BCFR.</p

p.Gly39val and p.Thr141Ile ABRAXAS mutants have defects in gamma-H2AX formation.

<p>(A) Typical DNA damage foci of ABRAXAS in shABRAXAS (shABX145) MCF7 cells complemented with ABRAXAS-HA-Flag, ABRAXAS-HA-Flag pThr141Ile, or ABRAXAS-HA-Flag pGly39Val. The anti-Flag antibody was used to monitor ABRAXAS foci formation (green), anti-gamma-H2AX (red) and the merge picture is depicted. In blue, DAPI staining. (B) Quantification of gamma-H2AX foci formation in MCF7 cells after neocarzinostatin treatment and release. P-values were obtained with a Wilcoxon’s Test with N = 100 cells from four independent experiments.</p

Stratified analyses of the common SNP rs13125836 (c. 1117G>A, p.Asp373Asn) on breast cancer risk in the BCFR.

<p>Stratified analyses of the common SNP rs13125836 (c. 1117G>A, p.Asp373Asn) on breast cancer risk in the BCFR.</p

ABRAXAS multiple-sequence alignment.

<p>Substitution designations are indicated above the corresponding human reference sequence residue. Amino acid symbols are colored to represent standard Dayhoff groupings.</p

Distribution of cases and controls by study center and by ethnicity in the BCFR.

<p>Distribution of cases and controls by study center and by ethnicity in the BCFR.</p