Functional Categorization of <i>BRCA1</i> Variants of Uncertain Clinical Significance in Homologous Recombination Repair Complementation Assays.

Abstract

Because <i>BRCA1</i> is a high-risk breast/ovarian cancer susceptibility gene, <i>BRCA1</i> sequence variants of uncertain clinical significance (VUS) complicate genetic counseling. As most VUS are rare, reliable classification based on clinical and genetic data is often impossible. However, all pathogenic <i>BRCA1</i> variants analyzed result in defective homologous recombination DNA repair (HRR). Thus, <i>BRCA1</i> VUS may be categorized based on their functional impact on this pathway. Two hundred thirty-eight <i>BRCA1</i> VUS-comprising most <i>BRCA1</i> VUS known in the Netherlands and Belgium-were tested for their ability to complement <i>Brca1-</i>deficient mouse embryonic stem cells in HRR, using cisplatin and olaparib sensitivity assays and a direct repeat GFP (DR-GFP) HRR assay. Assays were validated using 25 known benign and 25 known pathogenic <i>BRCA1</i> variants. For assessment of pathogenicity by a multifactorial likelihood analysis method, we collected clinical and genetic data for functionally deleterious VUS and VUS occurring in three or more families. All three assays showed 100% sensitivity and specificity (95% confidence interval, 83%-100%). Out of 238 VUS, 45 showed functional defects, 26 of which were deleterious in all three assays. For 13 of these 26 variants, we could calculate the probability of pathogenicity using clinical and genetic data, resulting in the identification of 7 (likely) pathogenic variants. We have functionally categorized 238 <i>BRCA1</i> VUS using three different HRR-related assays. Classification based on clinical and genetic data alone for a subset of these variants confirmed the high sensitivity and specificity of our functional assays.