Functional analysis of BARD1 missense variants in homology-directed repair and damage sensitivity

The BARD1 protein, which heterodimerizes with BRCA1, is encoded by a known breast cancer susceptibility gene. While several BARD1 variants have been identified as pathogenic, many more missense variants exist that do not occur frequently enough to assign a clinical risk. In this paper, whole exome sequencing of over 10,000 cancer samples from 33 cancer types identified from somatic mutations and loss of heterozygosity in tumors 76 potentially cancer-associated BARD1 missense and truncation variants. These variants were tested in a functional assay for homology-directed repair (HDR), as HDR deficiencies have been shown to correlate with clinical pathogenicity for BRCA1 variants. From these 76 variants, 4 in the ankyrin repeat domain and 5 in the BRCT domain were found to be non-functional in HDR. Two known benign variants were found to be functional in HDR, and three known pathogenic variants were non-functional, supporting the notion that the HDR assay can be used to predict the clinical risk of BARD1 variants. The identification of HDR-deficient variants in the ankyrin repeat domain indicates there are DNA repair functions associated with this domain that have not been closely examined. In order to examine whether BARD1-associated loss of HDR function results in DNA damage sensitivity, cells expressing non-functional BARD1 variants were treated with ionizing radiation or cisplatin. These cells were found to be more sensitive to DNA damage, and variations in the residual HDR function of non-functional variants did not correlate with variations in sensitivity. These findings improve the understanding of BARD1 functional domains in DNA repair and support that this functional assay is useful for predicting the cancer association of BARD1 variants. Author summary BARD1 is a breast cancer susceptibility gene encoding a protein that primarily interacts with BRCA1 in DNA repair. Although several BARD1 variants are known to be pathogenic, many more variants do not occur frequently enough to assign a clinical risk. In this paper, we identified 76 potentially cancer-associated BARD1 variants from analysis of over 10,000 tissue samples from people with cancer. It has previously been shown that if a BRCA1 variant cannot repair damaged DNA, then it is likely to cause cancer. We tested BARD1 variants for DNA repair function and identified several non-functional variants that were localized in parts of the BARD1 protein not previously associated with DNA repair. Known benign BARD1 variants were found to be functional and known pathogenic variants were non-functional, showing that examining DNA repair function predicted variant pathogenicity. Cells expressing repair-defective BARD1 variants were also more sensitive to DNA damaging agents. These findings help us better understand how BARD1 is involved in DNA repair and show that examining the DNA repair function of BARD1 variants is useful for predicting their cancer risk.