Functional Analysis of BARD1 Missense Variants in Homology-Directed Repair of DNA Double Strand Breaks

Abstract

Genes associated with hereditary breast and ovarian cancer (HBOC) are often sequenced in search of mutations that are predictive of susceptibility to these cancer types, but the sequence results are frequently ambiguous due to detection of missense substitutions for which the clinical impact is unknown. The BARD1 protein is the heterodimeric partner of BRCA1 and is included on clinical gene panels for testing for susceptibility to HBOC. Like BRCA1, it is required for homology-directed DNA repair (HDR). We measured the HDR function of 29 BARD1 missense variants, 27 culled from clinical test results and two synthetic variants. 23 of the assayed variants were functional for HDR; of these, four are known neutral variants. Three variants showed intermediate function, and three others were defective in HDR. When mapped to BARD1 domains, residues crucial for HDR were located in the N- and C- termini of BARD1. In the BARD1 RING domain, critical residues mapped to the zinc-coordinating amino acids and to the BRCA1-BARD1 binding interface, highlighting the importance of interaction between BRCA1 and BARD1 for HDR activity. Based on these results, we propose that the HDR assay is a useful complement to genetic analyses to classify BARD1 variants of unknown clinical significance.

Differential requirements for DNA repair proteins in immortalized cell lines using alternative lengthening of telomere mechanisms

“…These results show that some HR proteins are required for ALT in some but not all ALT cell lines and that collectively BARD1 and BRCA2 may be disposable for ALT activity. BRCA1 function in ALT seems to be independent of BARD1, striking in that these two proteins function interdependently in HR …”

“…BRCA1 function in ALT seems to be independent of BARD1, striking in that these two proteins function interdependently in HR. 37,72,73 WRN, a RecQ-like DNA helicase similar in structure to BLM but with an exonuclease domain, is a protein that prevents aberrant HR (Table 2) and can be required for ALT processes. 20,61 Reduced expression of WRN in Saos-2, U-2 OS, and VA-13 cells (Figure 2A,C,E) was associated with a small but not significant increase in C-circles in Saos-2 and U-2 OS (P 5 0.116; P 5 0.199, respectively) ( Figure 2B,D).…”

“…7,12,26,80 Our data show that depletion of BARD1, the heterodimeric protein partner of BRCA1, has no effect on C-circles, even though depletion of BARD1 strongly impacts HDR. 37,81,82 The HR protein BRCA2, a known binding partner of RAD51, 58,83 also had no effect on C-circles. In contrast, PALB2, a protein that bridges BRCA1 and BRCA2, 59,60 significantly decreases C-circles in Saos-2 cells but not in the two other cell lines tested.…”

BRCA1-Interacting Protein OLA1 Requires Interaction with BARD1 to Regulate Centrosome Number

“…The BRCA1/BARD1 ubiquitinates centrosomal proteins, including g-tubulin; it is thus feasible that OLA1 regulates the BRCA1/BARD1 E3 ubiquitin ligase. BRCA1/BARD1 functions in homology-directed DNA repair, whereas mutations of V695L and S761N in BARD1 do not affect homology-directed DNA repair (46,47). In BRCA mutation carriers, centrosome amplification is observed even in normal mammary tissue (48).…”

A multiplexed homology-directed DNA repair assay reveals the impact of ~1,700 BRCA1 variants on protein function

“…More importantly, our results are concordant with known cancer-predisposing mutants of BRCA1, including perfect positive predictive value for identifying known mutations damaging to protein function. This assay can be repurposed to measure the effect of variants in other proteins in the HDR pathway that are also hereditary breast and ovarian cancer tumor suppressors, such as BRCA2 28–30 and BARD1 31,32 . As genetic testing for cancer risk becomes more common and additional genes are added to testing panels, the number of rare missense variants that inevitably become VUS will continue to increase.…”