Characterization and functional analysis of BRCA1 and BRCA2 variants in a cohort of 100 unselected patients undergoing germline screening

Abstract

Germline loss-of-function mutations in BRCA1 and BRCA2 (BRCA) genes are well-established as causative factors for hereditary breast and ovarian cancer (HBOC), conferring an approximately tenfold increased lifetime risk. The identification of BRCA mutations is essential for risk assessment and management in high-risk individuals and cancer patients. In this study, we utilized next-generation sequencing (NGS) to comprehensively analyze the entire coding regions of BRCA genes in a cohort of 100 unselected patients undergoing germline screening (average age ≤45 years and/or family history of related cancers). A total of twenty-two variants were identified, including thirteen pathogenic or likely pathogenic variants (PVs/LPVs) and nine variants of uncertain significance (VUSs). Notably, six novel variants (two in BRCA1 and four in BRCA2) were discovered. Molecular subtyping revealed that breast cancer (BC) patients with BRCA variants were predominantly human epidermal growth factor receptor-2 (HER2)-negative. Using in silico prediction tools, nine VUSs were reclassified, with functional impairment confirmed for the BRCA2 p.W2619C and BRCA2 p.N1023_I1024del variants. Functional assays demonstrated that the BRCA2 p.W2619C variant significantly enhanced cell proliferation, migration, and invasion. Moreover, cells harboring the BRCA2 p.W2619C mutation exhibited increased sensitivity to Olaparib, suggesting potential therapeutic benefit from PARP inhibitors for patients with this variant. Our study identified six novel BRCA mutations and functionally validated the pathogenicity of the BRCA2 p.W2619C variant, thereby advancing the understanding of BRCA-related cancer risk and supporting enhanced genetic counseling and testing strategies for at-risk populations.