Assessing the Clinical Relevance of BRCA1 RING Domain Variants of Uncertain Significance

The BRCA1 protein serves an essential function in maintaining genomic integrity, to the extent that up to 80% of women carrying a pathogenic BRCA1 variant develop breast cancer (BC). Most of these carriers would benefit from prophylactic care, but genetic screens that uncover variants of uncertain significance (VUSs) do not provide insight on disease risk or clinical decision-making. In accordance with guidelines established by The American College of Molecular Genetics (ACMG) and Association for Molecular Pathology (AMP), this study produced computational and functional evidence to inform the reclassification of BRCA1 VUSs as pathogenic or benign, with a specific focus on the abundant subset of missense variants within the RING domain. A six-feature linear support vector machine (LSVM) specifically trained on BRCA1 RING variants performed well (84% accurate in predicting in vitro binding loss) and provided supporting classification evidence for 322 VUS. A mammalian cell co-immunoprecipitation (co-IP) assay that quantified the binding between variant BRCA RING constructs and endogenous BARD1 provided corroborating strong evidence for nine VUSs and correlated with a homology-directed repair (HDR) assay by Starita et al. (p = 0.04). The combined evidence warrants the reclassification of three VUSs as likely benign (N16S, A17D, and E100D) and one as likely pathogenic (H41P), and underscores the promise of domain-specific approaches for missense VUS reclassification.