Using Multiplexed Functional Assays to Understand the Effects of Genetic Variation Lea M. Starita1,2, Gregory M. Findlay1, Kenneth Matreyek1, Douglas Fowler1,2 , Jay Shendure1,2 1Department of Genome Sciences, University of Washington, Seattle, WA, USA; 2The Brotman Baty Institute for Precision Medicine, Seattle, WA, USA
Variants of uncertain significance (VUS) fundamentally limit the utility of genetic information in a clinical setting. The challenge of VUS is best exemplified by the tumor suppressor gene BRCA1. 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 employed to functionally characterize VUS. To date, however, such approaches have only succeeded in providing reliable interpretations for a modest number of variants. Multiplexed functional assays can provide both the throughput and validity necessary to use create and employ functional data at scale. We employed saturation genome editing to assay all possible single nucleotide variants (SNVs) in 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 functional scores for nearly 4,000 SNVs are bimodally distributed and almost perfectly concordant with established assessments of pathogenicity. We have also performed another multiplexed functional assay on the tumor suppressor PTEN and identified many variants that affect protein stability.
We predict that these functional scores will be directly valuable for the clinical interpretation of cancer risk based on BRCA1 and PTEN 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. Multiplexed functional assays may be an important tool to interpret variants for populations where VUS rates are high.