Functional characterization of BRCA2 variants of uncertain significance identified in Korean breast cancer patients.

Variants of uncertain significance (VUS) in BRCA2 remain a major challenge in the clinical management of breast cancer, particularly in Asian populations where population-specific reference data are limited. Functional assays may provide complementary biological evidence to support interpretation of these variants. Three BRCA2 missense VUS-c.5969 A > C (p.Asp1990Ala), c.3671G > A (p.Gly1224Asp), and c.5459G > A (p.Cys1820Tyr)-were selected based on their frequency in Korean cohorts and/or strong family history of breast cancer. Full-length BRCA2 constructs harboring each variant were introduced into BRCA2-knockout DLD-1 cells. Functional consequences were evaluated using cellular sensitivity assays following cisplatin treatment and ionizing radiation, response to the PARP inhibitor olaparib, and homologous recombination (HR) efficiency assessed by a GFP-based reporter system. Cells expressing BRCA2 p.Asp1990Ala consistently demonstrated increased sensitivity to cisplatin, ionizing radiation, and olaparib, comparable to pathogenic controls. In addition, GFP-based HR assays revealed a marked reduction in HR repair efficiency in p.Asp1990Ala-expressing cells. In contrast, cells expressing BRCA2 p.Gly1224Asp or p.Cys1820Tyr exhibited DNA damage responses, drug sensitivity profiles, and HR activity similar to wild-type BRCA2 across all assays. Among the three BRCA2 VUS analyzed, p.Asp1990Ala exhibited a pathogenic-like functional phenotype characterized by impaired homologous recombination repair, whereas p.Gly1224Asp and p.Cys1820Tyr appeared functionally neutral. These findings highlight the functional heterogeneity of BRCA2 VUS and support the role of functional assays as complementary tools for variant interpretation in breast cancer.