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.

A Multiplex Homology-Directed DNA Repair Assay Reveals the Impact of More Than 1,000 BRCA1 Missense Substitution Variants on Protein Function.

Loss-of-function pathogenic variants in BRCA1 confer a predisposition to breast and ovarian cancer. Genetic testing for sequence changes in BRCA1 frequently reveals a missense variant for which the impact on cancer risk and on the molecular function of BRCA1 is unknown. Functional BRCA1 is required for the homology-directed repair (HDR) of double-strand DNA breaks, a critical activity for maintaining genome integrity and tumor suppression. Here, we describe a multiplex HDR reporter assay for concurrently measuring the effects of hundreds of variants of BRCA1 for their role in DNA repair. Using this assay, we characterized the effects of 1,056 amino acid substitutions in the first 192 residues of BRCA1. Benchmarking these results against variants with known effects on DNA repair function or on cancer predisposition, we demonstrate accurate discrimination of loss-of-function versus benign missense variants. We anticipate that this assay can be used to functionally characterize BRCA1 missense variants at scale, even before the variants are observed in results from genetic testing.

HDAC10 as a potential therapeutic target in ovarian cancer.

OBJECTIVE: We analyzed histone deacetylase 10 (HDAC10) for function in the context of the DNA damage response in BRCA1-null ovarian cancer cells as well as evaluated the potential of general HDAC inhibitors in primary ovarian carcinoma cells. HDAC10 had previously been shown to be highly stimulatory to the process of homology directed repair in HeLa cells, and in this study we investigated whether HDAC10 could impact in vitro the response to anticancer therapies. We hypothesized that the loss of HDAC10 would sensitize cells to platinum therapy. METHODS: We combined informatics analysis of large DNA sequencing datasets from ovarian cancer tumors with tissue culture based assays of primary and established cell lines to test for sensitivity to platinum therapy if HDAC10 activity was inhibited or depleted. RESULTS: Using The Cancer Genome Atlas (TCGA) dataset, we found that deep deletions in HDAC10 occurred in 5-10% of ovarian cancer tumors. From the TCGA data we found that low HDAC10 mRNA levels correlated with platinum sensitivity of the tumors. Cell proliferation and DNA damage assays in a BRCA1-null ovarian carcinoma cell line demonstrated reduced DNA repair capacity and sensitization of platinum therapy. Similarly, primary ovarian carcinoma cells demonstrated a sensitization to platinum therapies when treated with HDAC inhibitors. CONCLUSIONS: From the results of this study, we suggest that the inhibition of HDAC10 may potentiate the effects of platinum therapies in ovarian tumors.

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 . AUTHOR SUMMARY: Most missense substitutions in BRCA1 are variants of unknown significance (VUS), and individuals with a VUS in BRCA1 cannot know from genetic information alone whether this variant predisposes to breast or ovarian cancer. We apply a multiplexed functional assay for homology directed repair of DNA double strand breaks to assess variant impact on this important BRCA1 protein function. We analyzed 2172 variants in the amino-terminus of BRCA1 and demonstrate that variants that are known as pathogenic have a loss of function in the DNA repair assay. Conversely, variants that are known to be benign are functionally normal in the multiplexed assay. We suggest that these functional determinations of BRCA1 variants can be used to augment the information that clinical cancer geneticists provide to patients who have a VUS in BRCA1 .