High-Throughput Functional Evaluation of BRCA2 Variants of Unknown Significance

Abstract

Numerous nontruncating missense variants of the BRCA2 gene have been identified, but there is a lack of convincing evidence, such as familial data, demonstrating their clinical relevance and they thus remain unactionable. To assess the pathogenicity of variants of unknown significance (VUSs) within BRCA2, we developed a novel method, the MANO-B method, for high-throughput functional evaluation utilizing BRCA2-deficient cells and poly(ADP-ribose) polymerase (PARP) inhibitors. The estimated sensitivity and specificity of this assay compared to those of the International Agency for Research on Cancer (IARC) classification system were 95% and 95%, respectively. We classified the pathogenicity of 186 BRCA2 VUSs with our original computational pipeline, resulting in the classification of 126 mutations as "neutral/likely neutral", 23 as "intermediate", and 37 as "deleterious/likely deleterious". We further invented a simplified, on-demand annotation system, the Accurate BRCA Companion Diagnostic (ABCD) test, as a companion diagnostic for PARP inhibitors in patients with unknown BRCA2 VUSs. The ABCD test classification was reproducible and consistent with that of a large-scale MANO-B method.