Cancer missense mutations in the BRC repeats of BRCA2 protein disrupt RAD51 binding and activity leading to chemotherapeutic sensitivity

Abstract

AbstractBRCA2 is a tumor suppressor gene that maintains genome stability by mediating the high fidelity repair of DNA double-strand breaks (DSBs) through homology-directed repair (HDR). Pathogenic mutations in BRCA2 predispose to breast, ovarian, pancreatic, prostate, and other cancers. Mutations in BRCA2 leading to severe protein truncation predict pathogenicity, however, missense mutations with unknown functional consequences, designated Variants of Uncertain Significance (VUS), comprise 60% of BRCA2 sequence changes deposited in clinical databases. Classifying BRCA2 VUS correctly is critical for relaying clinically actionable information to patients concerning future cancer risk or current treatment options. In this study, we identified and biochemically characterized three BRCA2 VUS located in BRC repeats to determine the impact on canonical HDR functions. Two of the germline variants, S1221P and T1980I, map to conserved residues in BRC2 and BRC7, disrupt RAD51 binding, and are diminished in their ability to stabilize RAD51-ssDNA complexes. We provide supporting cellular evidence that S1221P and T1980I are significantly compromised in their response to chemotherapeutics and ionizing radiation. The third variant, T1346I, lies within the spacer region between BRC2 and BRC3 but remains fully functional. We conclude that T1346I has a neutral impact on BRCA2 function, while S1221P and T1980I are hypomorphic alleles that disrupt the ability of BRCA2 to fully engage and stabilize RAD51 nucleoprotein filaments.Conclusions/Highlights-The BRCA2 missense variants, S1221P in BRC2 and T1980I in BRC7, are hypomorphic alleles which exhibit multiple defects in response to chemotherapeutics, DNA damage, and cellular stress.-The biochemical defects in S1221P and T1980I include disruption of RAD51 binding and failure to stimulate RAD51-ssDNA complex formation.-Identification of a tumor derived somatic BRCA2 missense mutation in the spacer region between BRC2 and BRC3, T1346I, is a benign variant.Potential LimitationsGiven that BRCA2 plays several roles in cellular function, it is possible that the VUS studied disrupt BRCA2 function in ways not measured by the assays described here.Therefore, further experimentation is necessary to validate the observed functionality of the variants studied and discern partial loss of function variants from fully neutral or pathogenic ones.Overexpression of the BRCA2 cDNA may underestimate the functional effect of some variants. However, it is feasible that cDNA-based overexpression of these variants can partially rescue HR and other BRCA2 functions.Caution is warranted when interpreting results from an assay focusing on a single specific biochemical activity to predict pathogenicity of BRCA2. This is extremely challenging for proteins with multiple biochemical and biological functions such as BRCA2. Up to date a small number of pathogenic or non-pathogenic variants have been evaluated and based on. Therefore, discriminating a true intermediate function variant from a neutral or fully pathogenic variant remains difficult and integration of multiple functional assays may be necessary as the presented in this study.Graphical Abstract