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

Abstract

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.

Germline variants in DNA repair genes associated with hereditary breast and ovarian cancer syndrome: analysis of a 21 gene panel in the Brazilian population

“…The BARD1 was the gene that presented more variants associated with HBOC risk. BARD1 form heterodimers with BRCA1 playing an important role as both E3 ubiquitin ligase as homologous repair mediators by recruiting RAD51 to DSB sites [77].…”

“…Variants in this genes have been associated with a deficiency in HR and increased sensitivity to DNA damage, classifying BARD1 as a gene of moderate penetrance to BC and OC [23,[77][78][79]. All three associated variants are described as VUS on ClinVar, but p.Asn255Ser (c.764A > G) and p.Lys423Arg (c.1268A > G) lack studies characterizing their effects on protein functions.…”

“…Regarding p.Lys423Arg variant, it is located in ANK domain which plays an important role in apoptosis activation due to p53 binding [82]. Despite ANK not being related to the DNA repair process, the evaluation of variants located between amino acids 460-560 have shown an HR deficiency demonstrating that this domain is also important to a correct DNA repair [77]. In fact, three in silico tools classified this variant as pathogenic, however, only functional or segregation analyses are required to confirm the suggested pathogenic effect of those variants.…”

Literature Review of BARD1 as a Cancer Predisposing Gene with a Focus on Breast and Ovarian Cancers

“…Twenty-four frameshift and 15 nonsense variants in BARD1 have been reported upstream or within BRCT domains, which could result in losing these domains fully or partially and could affect function (Figure 2, Supplementary Table S1). It has been shown that the BARD1 c. 1793C>T; p.Thr598Ile protein isoform exhibits significantly lower homologous recombination DNA repair activity [118]. This isoform might affect binding to PARP1, as the amino acid change is predicted to alter the protein's surface and not any of the known binding sites involved in DNA repair [118].…”

“…It has been shown that the BARD1 c. 1793C>T; p.Thr598Ile protein isoform exhibits significantly lower homologous recombination DNA repair activity [118]. This isoform might affect binding to PARP1, as the amino acid change is predicted to alter the protein's surface and not any of the known binding sites involved in DNA repair [118].…”