Accurate functional classification of thousands of <i>BRCA1</i> variants with saturation genome editing

Abstract

                Variants of uncertain significance (VUS) fundamentally limit the utility of genetic information in a clinical setting. The challenge of VUS is epitomized by
                BRCA1
                , a tumor suppressor gene integral to DNA repair and genomic stability. Germline
                BRCA1
                loss-of-function (LOF) variants predispose women to early-onset breast and ovarian cancers. Although
                BRCA1
                has been sequenced in millions of women, the risk associated with most newly observed variants cannot be definitively assigned. Data sharing attenuates this problem but it is unlikely to solve it, as most newly observed variants are exceedingly rare. In lieu of genetic evidence, experimental approaches can be used to functionally characterize VUS. However, to date, functional studies of
                BRCA1
                VUS have been conducted in a
                post hoc
                , piecemeal fashion. Here we employ saturation genome editing to assay 96.5% of all possible single nucleotide variants (SNVs) in 13 exons that encode functionally critical domains of BRCA1. Our assay measures cellular fitness in a haploid human cell line whose survival is dependent on intact
                BRCA1
                function. The resulting function scores for nearly 4,000 SNVs are bimodally distributed and almost perfectly concordant with established assessments of pathogenicity. Sequence-function maps enhanced by parallel measurements of variant effects on mRNA levels reveal mechanisms by which loss-of-function SNVs arise. Hundreds of missense SNVs critical for protein function are identified, as well as dozens of exonic and intronic SNVs that compromise
                BRCA1
                function by disrupting splicing or transcript stability. We predict that these function scores will be directly useful for the clinical interpretation of cancer risk based on
                BRCA1
                sequencing. Furthermore, we propose that this paradigm can be extended to overcome the challenge of VUS in other genes in which genetic variation is clinically actionable.