A multiplexed homology-directed DNA repair assay reveals the impact of over 1,000 BRCA1 missense substitution variants on protein function
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE116427 | GEO | 2018/06/29
REPOSITORIES: GEO
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Project description: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.
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Project description:A multiplexed homology-directed DNA repair assay reveals the impact of over 1,000 BRCA1 missense substitution variants on protein function
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Project description:In this study, we used multiplexed DNA repair assays of variants in the BRCA1 carboxy-terminus to functionally characterize 2271 variants for homology-directed repair function (HDR) and 1427 variants for cisplatin resistance (CR). We found a high level of consistent results in the two multiplexed functional assays with non-functional variants located within regions of the BRCA1 protein necessary for its tumor suppression activity.
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Project description:Delineating functionally normal variants from functionally abnormal variants in tumor suppressor proteins is critical for cancer surveillance, prognosis, and treatment options. BRCA1 is a protein that has many variants of uncertain significance which are not yet classified as functionally normal or abnormal. In vitro functional assays can be used to identify the functional impact of a variant when the variant has not yet been categorized through clinical observation. Here we employ a homology-directed repair (HDR) reporter assay to evaluate over 300 missense and nonsense BRCA1 variants between amino acid residues 1280 and 1576, which encompasses the coiled-coil and serine cluster domains. Functionally abnormal variants tended to cluster in residues known to interact with PALB2, which is critical for homology-directed repair. Multiplexed results were confirmed by singleton assay and by ClinVar database variant interpretations. Comparison of multiplexed results to designated benign or likely benign or pathogenic or likely pathogenic variants in the ClinVar database yielded 100% specificity and 100% sensitivity of the multiplexed assay. Clinicians can reference the results of this functional assay for help in guiding cancer treatment and surveillance options. These results are the first to evaluate this domain of BRCA1 using a multiplexed approach and indicate the importance of this domain in the DNA repair process.
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Project description: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.
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Project description:BRCA1 (breast cancer 1, early onset) mutations confer a high breast and ovarian cancer risk. Most of BRCA1 cancer-predisposing mutations originate truncated proteins, but missense mutations have also been detected in familial breast and ovarian cancer patients. These variants are rare and their role in cancer predisposition is often difficult to ascertain. Our purpose in the present work was to study the molecular mechanisms affected in human cells by two BRCA1 missense variants both located in the second BRCA1 BRCT domain, M1775R and A1789T. These variants were isolated from familial breast cancer patients and their role in the pathogenesis of breast cancer was also investigated by a study previously performed by our group in yeast cells. Here we present a microarray study to compare the expression profiles of HeLa cells transfected with these two variants and HeLa cells transfected with BRCA1 wild type. Data analysis was performed by evaluating three comparisons: M1775R versus wild-type (M1775RvsWT-contrast), A1789T versus wild-type (A1789TvsWT-contrast) and the mutated BRCT domain versus wild-type (MutvsWT-contrast), obtained by considering the two variants as a unique BRCT mutation. We found 173 differentially expressed genes in MutvsWT-contrast, 201 in M1775RvsWT-contrast and 313 in A1789TvsWT-contrast. Our results showed that the considered BRCA1 variants had an impact on cell processes often deregulated in cancerogenesis, such as cell cycle progression and DNA damage response and repair. Thus, our study further supports the putative role in the pathogenesis of cancer of the M1775R and A1789T BRCA1 variants from a molecular point of view.
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Project description:The Polycomb repressive complex 2 (PRC2) is important for the maintenance of stem cell pluripotency and suppression of cell differentiation by promoting histone H3 lysine 27 trimethylation (H3K27me3) and silencing expression of genes regulating differentiation. Here we show decreased expression of BRCA1 promotes EZH2 re-targeting genome wide and enhances H3K27me3 levels at PRC2 target loci in mouse ES cells. R1 mouse ES cells were transfected with control siRNA and Brca1 specific siRNA. At 48h after transfection, cells were harvested and subjected to ChIP assay with Ezh2 antibody. Brca1 wild-type (AB2.2) and Brca1 deficient (PL2F8) mouse ES cells were harvested and subjected to ChIP assay with Ezh2 antibody. Fragmented DNA obtained from ChIP assay was repaired by DNA Terminator End Repair Kit (Lucigen Corporation, Middleton, WI) and purified by QIAquick PCR purification kit. Then two unidirectional linkers JW102 and JW103 were annealed and ligated to the blunt ends DNA at 16M-BM-0C overnight. The purified products were amplified to 2 M-NM-<g by LM-PCR. NimbleGen Mouse ChIP 3x720K RefSeq Promoter Array (Roche) were used for the hybridization. Relative enrichments were calculated by dividing the normalized level of ChIP DNA to that of input DNA at corresponding locus. Labeling, hybridization and washing were carried out according to manufacturerM-bM-^@M-^Ys instructions. Experiments in AB2.2 and PL2F8 cells were performed in duplicate.
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