Functional characterization of BRCA1 variants of unknown significance using homologous recombination repair assays

Inherited mutations in BRCA1 are among the primary causes of hereditary breast and ovarian cancer (HBOC). Genetic testing has identified numerous pathogenic and benign mutations in BRCA1, but also thousands of variants of uncertain significance (VUS) with unclear functional consequences. Accurate cancer risk prediction for carriers of these VUS requires functional assays to assess their pathogenicity. In this study, we analyzed 16 BRCA1 VUS detected at the Hereditary Cancer Genetic Counseling Laboratory in Salamanca, Spain, with the goal of providing additional data to guide their reclassification. Since homologous recombination (HR) is the primary tumor-suppressive function of BRCA1, we employed two complementary HR repair assays to evaluate HR efficiency. The first, an already established assay, uses a HeLa-DR cell line harboring an HR reporter cassette and involves silencing endogenous BRCA1 followed by complementation with the VUS under study. The second, developed in this work, is also based in complementation assays that restore the green fluorescence protein (GFP) gene, and employs a breast cancer cell line with a similar HR reporter system (HCC1937-HR) that lacks BRCA1 expression, allowing direct complementation. Both assays consistently identified five VUS (p.V11G, p.H888Y, p.G1201S, p.Q1395R, and p.F1734L) as pathogenic due to significantly reduced HR efficiency, whereas the remaining 11 VUS were benign in terms of HR function. Notably, two pathogenic variants (p.H888Y, p.G1201S) were located outside the known functional domains of BRCA1. The five HR-deficient variants were further evaluated for their sensitivity to ionizing radiation, which confirmed the deleterious impact on DNA repair for variants p.V11G, p.H888Y, p.G1201S and p.F1734L. Sensitivity to the PARP inhibitor olaparib revealed hypersensitivity only in cells expressing p.V11G and p.F1734L, suggesting variant-specific mechanistic effects with potential therapeutic relevance. In conclusion, recombination-based functional assays complemented with sensitivity assays are effective tools for assessing the pathogenicity of BRCA1 VUS and can provide valuable information to support clinical decision-making. We propose that these assays can be completed within a timeframe compatible with clinical needs, offering critical insights for genetic counseling and personalized treatment strategies.