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.</div

Patterns and functional implications of rare germline variants across 12 cancer types

Large-scale cancer sequencing data enable discovery of rare germline cancer susceptibility variants. Here we systematically analyse 4,034 cases from The Cancer Genome Atlas cancer cases representing 12 cancer types. We find that the frequency of rare germline truncations in 114 cancer-susceptibility-associated genes varies widely, from 4% (acute myeloid leukaemia (AML)) to 19% (ovarian cancer), with a notably high frequency of 11% in stomach cancer. Burden testing identifies 13 cancer genes with significant enrichment of rare truncations, some associated with specific cancers (for example, RAD51C, PALB2 and MSH6 in AML, stomach and endometrial cancers, respectively). Significant, tumour-specific loss of heterozygosity occurs in nine genes (ATM, BAP1, BRCA1/2, BRIP1, FANCM, PALB2 and RAD51C/D). Moreover, our homology-directed repair assay of 68 BRCA1 rare missense variants supports the utility of allelic enrichment analysis for characterizing variants of unknown significance. The scale of this analysis and the somatic-germline integration enable the detection of rare variants that may affect individual susceptibility to tumour development, a critical step toward precision medicine

DNA repair function scores for 2172 variants in the BRCA1 amino-terminus.

Single nucleotide variants are the most frequent type of sequence changes detected in the genome and these are frequently variants of uncertain significance (VUS). VUS are changes in DNA for which disease risk association is unknown. Thus, methods that classify the functional impact of a VUS can be used as evidence for variant interpretation. In the case of the breast and ovarian cancer specific tumor suppressor protein, BRCA1, pathogenic missense variants frequently score as loss of function in an assay for homology-directed repair (HDR) of DNA double-strand breaks. We previously published functional results using a multiplexed assay for 1056 amino acid substitutions residues 2-192 in the amino terminus of BRCA1. In this study, we have re-assessed the data from this multiplexed assay using an improved analysis pipeline. These new analysis methods yield functional scores for more variants in the first 192 amino acids of BRCA1, plus we report new results for BRCA1 amino acid residues 193-302. We now present the functional classification of 2172 BRCA1 variants in BRCA1 residues 2-302 using the multiplexed HDR assay. Comparison of the functional determinations of the missense variants with clinically known benign or pathogenic variants indicated 93% sensitivity and 100% specificity for this assay. The results from BRCA1 variants tested in this assay are a resource for clinical geneticists for evidence to evaluate VUS in BRCA1

Comparative population distribution of BRCA1 variants under control and experimental conditions.

The distribution of functional scores for each subpopulation of variants (missense, nonsense, synonymous) was analyzed on a plot of functional score (x-axis) vs count (y-axis). Control siRNA treatment is on the left, and BRCA1 3’UTR siRNA treatment (depletion of endogenous BRCA1) is on the right. The functional score scale is log2, and a score of 0 indicates wild-type function. The dotted line on the missense variant plot in the control experiment represents the bottom 1% of the normal distribution modeled on the data. The dotted line in the nonsense variant plot in the transfected BRCA1 3’UTR siRNA experiment is the top 1% of the normal distribution modeled from the data. These lines were used as thresholds for the functional interpretation of LOF.</p

Comparison of BRCA1 variant functional determinations.

A. The number of variants in each pool of BRCA1 codons that were above the read count threshold and the number of variants for which a functional interpretation was made are indicated for the previously published analysis (2018) and the current analysis (2023). In 2018, functional determinations were only published for pools 1 and 2; only depletion scores of 0, 3 or 4 were considered for functional determination in that analysis. In the new analysis, functional determinations are made for all variants, grouped into either functionally normal, loss of function or intermediate. For this table, intermediate is not included in the total for functional calls. B. This scatterplot compares the functional scores for each variant from the previously published analysis (replicates depleted; x-axis) to the new analysis (y-axis). The x-axis represents the number of replicates a variant was depleted in the 2018 analysis [17]. The y-axis displays the re-analysis functional score for the same population of variants. The dotted line signifies the threshold for function, with values above it indicating functionally normal BRCA1 and below indicating loss of function. Colors indicate the color scheme used in the 2018 description. The comparison showed a strong negative correlation, with a Pearson R value of -0.86.</p

New BRCA1 Multiplexed Functional Scores and Additional Outputs.

New BRCA1 Multiplexed Functional Scores and Additional Outputs.</p

Scoring for 18 New BRCA1 Singleton Variants.

Single nucleotide variants are the most frequent type of sequence changes detected in the genome and these are frequently variants of uncertain significance (VUS). VUS are changes in DNA for which disease risk association is unknown. Thus, methods that classify the functional impact of a VUS can be used as evidence for variant interpretation. In the case of the breast and ovarian cancer specific tumor suppressor protein, BRCA1, pathogenic missense variants frequently score as loss of function in an assay for homology-directed repair (HDR) of DNA double-strand breaks. We previously published functional results using a multiplexed assay for 1056 amino acid substitutions residues 2–192 in the amino terminus of BRCA1. In this study, we have re-assessed the data from this multiplexed assay using an improved analysis pipeline. These new analysis methods yield functional scores for more variants in the first 192 amino acids of BRCA1, plus we report new results for BRCA1 amino acid residues 193–302. We now present the functional classification of 2172 BRCA1 variants in BRCA1 residues 2–302 using the multiplexed HDR assay. Comparison of the functional determinations of the missense variants with clinically known benign or pathogenic variants indicated 93% sensitivity and 100% specificity for this assay. The results from BRCA1 variants tested in this assay are a resource for clinical geneticists for evidence to evaluate VUS in BRCA1.</div