Sequencing-based functional assays for classification of BRCA2 variants in mouse ESCs.

Sequencing of genes, such as BRCA1 and BRCA2, is recommended for individuals with a personal or family history of early onset and/or bilateral breast and/or ovarian cancer or a history of male breast cancer. Such sequencing efforts have resulted in the identification of more than 17,000 BRCA2 variants. The functional significance of most variants remains unknown; consequently, they are called variants of uncertain clinical significance (VUSs). We have previously developed mouse embryonic stem cell (mESC)-based assays for functional classification of BRCA2 variants. We now developed a next-generation sequencing (NGS)-based approach for functional evaluation of BRCA2 variants using pools of mESCs expressing 10-25 BRCA2 variants from a given exon. We use this approach for functional evaluation of 223 variants listed in ClinVar. Our functional classification of BRCA2 variants is concordant with the classification reported in ClinVar or those reported by other orthogonal assays.