A high-throughput functional complementation assay for classification of BRCA1 missense variants

Abstract

Mutations in BRCA1 and BRCA2 account for the majority of hereditary breast and ovarian cancers, and therefore sequence analysis of both genes is routinely performed in patients with early-onset breast cancer. Besides mutations that clearly abolish protein function or are known to increase cancer risk, a large number of sequence variants of uncertain significance (VUS) have been identified. Although several functional assays for BRCA1 VUS have been described, thus far it has not been possible to perform a high-throughput analysis in the context of the full-length protein. We have developed a relatively fast and easy cDNA-based functional assay to classify BRCA1 VUS based on their ability to functionally complement BRCA1-deficient mouse embryonic stem cells. Using this assay we have analyzed 74 unclassified BRCA1 missense mutants, of which all predicted pathogenic variants are confined to the BRCA1 RING and BRCT domains.