Multiplexed functional analysis of BRCA1 and BARD1 missense variants in DNA repair ABSTRACT Variants of unknown significance (VUS) are, in general, missense variants for which the interpretation of phenotypic impact is trapped in the void between pathogenic and benign. Any of these amino acid substitutions could cause major damage to the structure or molecular function of the encoded protein and therefore significantly impact disease risk, or it could have no effect all. Nowhere is the problem of VUS more apparent than in genetic testing for hereditary breast and ovarian cancer where published VUS rates range from 2-42% depending on the company doing testing/variant calling and number of genes included on the panel. For BRCA1 alone, there are 1020 VUS listed in the clinical genetics database, ClinVar. To address the problem of VUS interpretation, which is required to make genetic test results more useful for more patients, functional assays could be used to understand how each variant affects protein function. However, performing a post hoc functional assay for each variant as it is discovered is impossible at the current rate of accumulation. Here we propose to use deep mutational scanning to determine the functional impact of all possible missense variants in BRCA1 and BARD1 on their function in DNA repair in human cells. Our approach, developed in collaboration between our labs at Ohio State University and the University of Washington, measures the functional capacity of hundreds of protein variants in parallel in a homology directed DNA double strand break repair assay. The outcome of this project will have two deliverables: The first is an understanding of the sequence–function relationships, at single amino acid resolution, of two tumor suppressors in their role protecting the genome from DNA double strand breaks. The second outcome is a “look up table” for the functional impact of any possible missense variant in BRCA1 and BARD1 that can be used by clinical geneticists to aid interpretation for variants that have been identified previously and those that have not yet been seen in the clinic.