Marshall, Megan L.

Abstract Pathogenic protein-truncating variants of RAD51C, which plays an integral role in promoting DNA damage repair, increase the risk of breast and ovarian cancer. A large number of RAD51C missense variants of uncertain significance (VUS) have been identified, but the effects of the majority of these variants on RAD51C function and cancer predisposition have not been established. Here, analysis of 173 missense variants by a homology-directed repair (HDR) assay in reconstituted RAD51C−/− cells identified 30 nonfunctional (deleterious) variants, including 18 in a hotspot within the ATP-binding region. The deleterious variants conferred sensitivity to cisplatin and olaparib and disrupted formation of RAD51C/XRCC3 and RAD51B/RAD51C/RAD51D/XRCC2 complexes. Computational analysis indicated the deleterious variant effects were consistent with structural effects on ATP-binding to RAD51C. A subset of the variants displayed similar effects on RAD51C activity in reconstituted human RAD51C-depleted cancer cells. Case–control association studies of deleterious variants in women with breast and ovarian cancer and noncancer controls showed associations with moderate breast cancer risk [OR, 3.92; 95% confidence interval (95% CI), 2.18–7.59] and high ovarian cancer risk (OR, 14.8; 95% CI, 7.71–30.36), similar to protein-truncating variants. This functional data supports the clinical classification of inactivating RAD51C missense variants as pathogenic or likely pathogenic, which may improve the clinical management of variant carriers. Significance: Functional analysis of the impact of a large number of missense variants on RAD51C function provides insight into RAD51C activity and information for classification of the cancer relevance of RAD51C variants.

Abstract Purpose: The identification of variants of uncertain significance (VUS) in the BRCA1 and BRCA2 genes by hereditary cancer testing poses great challenges for the clinical management of variant carriers. The ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology) variant classification framework, which incorporates multiple sources of evidence, has the potential to establish the clinical relevance of many VUS. We sought to classify the clinical relevance of 133 single-nucleotide substitution variants encoding missense variants in the DNA-binding domain (DBD) of BRCA2 by incorporating results from a validated functional assay into an ACMG/AMP-variant classification model from a hereditary cancer–testing laboratory. Experimental Design: The 133 selected VUS were evaluated using a validated homology-directed double-strand DNA break repair (HDR) functional assay. Results were combined with clinical and genetic data from variant carriers in a rules-based variant classification model for BRCA2. Results: Of 133 missense variants, 44 were designated as non-functional and 89 were designated as functional in the HDR assay. When combined with genetic and clinical information from a single diagnostic laboratory in an ACMG/AMP-variant classification framework, 66 variants previously classified by the diagnostic laboratory were correctly classified, and 62 of 67 VUS (92.5%) were reclassified as likely pathogenic (n = 22) or likely benign (n = 40). In total, 44 variants were classified as pathogenic/likely pathogenic, 84 as benign/likely benign, and 5 remained as VUS. Conclusions: Incorporation of HDR functional analysis into an ACMG/AMP framework model substantially improves BRCA2 VUS re-classification and provides an important tool for determining the clinical relevance of individual BRCA2 VUS.