Availability of benign missense variant “truthsets” for validation of functional assays: current status and a novel systematic approach

Multiplex assays of variant effect (MAVEs) provide promising new sources of functional evidence, potentially empowering improved classification of germline genomic variants, particularly rare missense variants, which are commonly assigned as VUS (variants of uncertain significance). However, paradoxically, quantification of clinically applicable evidence strengths for MAVEs requires construction of “truthsets” comprising missense variants already robustly classified as pathogenic and benign. In this study, we demonstrate how benign truthset size is the primary driver of applicable functional evidence towards pathogenicity (PS3). We demonstrate, when using existing ClinVar classifications as a source of benign missense truthset variants, for only 19.8% (23/116) of established cancer susceptibility genes was a PS3 evidence strength of “strong” attainable when simulating validation for a hypothetical new MAVE (applying also favourable assumption of perfect concordance). We describe a “proactive-systematic” framework for benign truthset construction, in which all possible missense variants in a gene of interest are concurrently assessed for assignation of (likely) benignity via established ACMG/AMP combination rules including population frequency, in silico evidence codes and case-control signal. We apply this framework to eight hereditary breast and ovarian cancer genes, demonstrating that proactive-systematically generated benign missense truthsets allow maximum application of PS3 at greater (or equivalent) strength – reaching “moderate” for CHEK2 and “strong” for the other seven genes – than those derived from ClinVar ≥2* classifications alone. We propose, given many genes have few existing benign-classified missense variants, application of this proactive-systematic framework to disease genes more broadly will be important for leveraging full value from MAVEs.