RAD51C germline mutations in breast and ovarian cancer cases from high-risk families

Abstract

BRCA1 and BRCA2 are the most well-known breast cancer susceptibility genes. Additional genes involved in DNA repair have been identified as predisposing to breast cancer. One such gene, RAD51C, is essential for homologous recombination repair. Several likely pathogenic RAD51C mutations have been identified in BRCA1- and BRCA2-negative breast and ovarian cancer families. We performed complete sequencing of RAD51C in germline DNA of 286 female breast and/or ovarian cancer cases with a family history of breast and ovarian cancers, who had previously tested negative for mutations in BRCA1 and BRCA2. We screened 133 breast cancer cases, 119 ovarian cancer cases, and 34 with both breast and ovarian cancers. Fifteen DNA sequence variants were identified; including four intronic, one 5' UTR, one promoter, three synonymous, and six non-synonymous variants. None were truncating. The in-silico SIFT and Polyphen programs were used to predict possible pathogenicity of the six non-synonomous variants based on sequence conservation. G153D and T287A were predicted to be likely pathogenic. Two additional variants, A126T and R214C alter amino acids in important domains of the protein such that they could be pathogenic. Two-hybrid screening and immunoblot analyses were performed to assess the functionality of these four non-synonomous variants in yeast. The RAD51C-G153D protein displayed no detectable interaction with either XRCC3 or RAD51B, and RAD51C-R214C displayed significantly decreased interaction with both XRCC3 and RAD51B (p<0.001). Immunoblots of RAD51C-Gal4 activation domain fusion peptides showed protein levels of RAD51C-G153D and RAD51C-R214C that were 50% and 60% of the wild-type, respectively. Based on these data, the RAD51C-G153D variant is likely to be pathogenic, while the RAD51C- R214C variant is hypomorphic of uncertain pathogenicity. These results provide further support that RAD51C is a rare breast and ovarian cancer susceptibility gene.

Figure 1. Effect of RAD51C point mutations on the interaction with XRCC3 and RAD51B.

(A) Yeast two-hybrid assays were performed with XRCC3 in the DNA-binding domain vector and either wild-type or site-specifically mutated RAD51C in the activating domain vector. (B) Yeast two-hybrid assays were performed with RAD51B in the DNA-binding domain vector and the RAD51C variants in the activating domain vector. Results from liquid ONPG assays are the average of 5–7 different transformants performed in triplicate, with the standard error of the mean. * : P<0.05 and ** :P≤0.001 using the student T-test.

Figure 2. Immunoblots of RAD51C in the yeast strains used for the two-hybrid analysis

. The upper panel shows the immunoblot blot analysis of the yeast strains containing human XRCC3 and RAD51C while the lower panel shows the same immunoblots from the yeast strains used in the RAD51B and RAD51C analysis. The RAD51C-Gal4 fusion peptides were identified using an anti-Gal4 monoclonal antibody. The same lysates were probed with an antibody against β-actin as a control for equal loading of protein.

Figure 3. Pedigrees of cases carrying the R214C and G153D mutations.

(A) R214C: African-American proband diagnosed with stage IIA, ER/PR positive, Her2/Neu positive, infiltrating ductal carcinoma of the breast at age 42 years. (B) G153D: Non-Hispanic Caucasian proband diagnosed with breast cancer at age 60 years and with stage IV, serous carcinoma of the ovary at age 79 years.