DNA Repair Function Scores for 2172 Variants in the BRCA1 Amino-Terminus
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PROVIDER: PRJNA981519 | ENA |
REPOSITORIES: ENA
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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.
2023-06-12 | GSE234482 | GEO
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.
| S-EPMC10449183 | biostudies-literature
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 .Author summaryMost 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 .
| S-EPMC10120616 | biostudies-literature
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.
2022-01-31 | GSE188426 | GEO
Project description:Here we describe a multiplex HDR reporter assay to concurrently measure 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.
2018-06-29 | GSE116427 | GEO
Project description:Amino terminus of cardiac myosin binding protein-C regulates cardiac contractility
| PRJNA716300 | ENA
Project description:Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) regulates cardiac contraction through modulation of actomyosin interactions mediated by the protein’s amino terminal (N’)-region (C0-C2 domains, 358 amino acids). On the other hand, dephosphorylation of cMyBP-C during myocardial injury results in cleavage of the 271 amino acid C0-C1f region and subsequent contractile dysfunction. Yet, our current understanding of amino terminus region of cMyBP-C in the context of regulating thin and thick filament interactions is limited. A novel cardiac-specific transgenic mouse model expressing cMyBP-C, but lacking its C0-C1f region (cMyBP-C delta C0-C1f), displayed dilated cardiomyopathy, underscoring the importance of the N’-region in cMyBP-C. Further exploring the molecular basis for this cardiomyopathy, in vitro studies revealed increased interfilament lattice spacing and rate of tension redevelopment, as well as faster actin-filament sliding velocity within the C-zone of the transgenic sarcomere. Moreover, phosphorylation of the unablated phosphoregulatory sites was increased, likely contributing to normal sarcomere morphology and myoarchitecture. These results led us to hypothesize that restoration of the N’-region of cMyBP-C would return actomyosin interaction to its steady state. Accordingly, we administered recombinant C0-C2 (rC0-C2) to permeabilized cardiomyocytes from transgenic, cMyBP-C null, and human heart failure biopsies, and we found that normal regulation of actomyosin interaction and contractility was restored. Overall, these data provide a unique picture of selective perturbations of the cardiac sarcomere that either lead to injury or adaptation to injury in the myocardium.
2021-03-23 | GSE169340 | GEO
Project description:Brca1 is required for DNA repair by homologous recombination (HR) and normal embryonic development. Here we report that deletion of the DNA damage responsefactor 53BP1 overcomes embryonic lethality in Brca1-nullizygous mice, and rescues HR deficiency, as measured by hypersensitivity to PARP (polyADPribose polymerase) inhibition. However, Brca1,53BP1 double-deficient cells are hypersensitive to DNA interstrand crosslinks (ICLs), indicating that BRCA1 has an additional role in DNA cross-link repair that is distinct from HR. Disruption of the non-homologous end-joining (NHEJ) factor, Ku, promotes DNA repair in Brca1-deficient cells; however deletion of either Ku or 53BP1 exacerbates genomic instability in cells lacking FANCD2, a mediator of the Fanconi Anemia pathway for ICL repair. Brca1 therefore has two separate roles in ICL repair, whereas FANCD2 provides a key activity that can not be bypassed by ablation of 53BP1 or Ku. B cells were stimulated to undergo class switch recombination in vitro. Chromatin from B cells was harvested 72 hours post-stimulation and used for RPA ChIP to study the extent of resection of DNA DSBs.
2012-09-30 | E-GEOD-35698 | biostudies-arrayexpress
Project description:In order to investigate the molecular mechanisms activated by deleterious BRCA1 variants in yeast, the RNA obtained from yeast cells transformed with five variants exhibiting a phenotypic effect, either on proliferation and/or on HR was hybridized on microarrays in comparison with the RNA from cells transformed with wild-type BRCA1
2009-03-31 | E-MEXP-1867 | biostudies-arrayexpress
Project description:Germline and somatic mutations in BRCA1predispose to breast cancer. We found that proteasome inhibitors can selectively kill BRCA1-depleted cells. The toxic response involves a deregulation of the G1/S cell cycle checkpoint via hyperphosphorylation of RB1, 53BP1-mediated arrest at G2/M checkpoint, and ERN1-mediated unfolded protein response, culminating in a TNF receptor-mediated apoptosis. The study new unexpected molecular functions for BRCA1 protein and opens a novel possibility for the treatment of BRCA1-deficient cancers. We used microarrays to detail the global programme of gene expression underlying the response of BRCA1-deficient cells to proteasome inhibitor bortezomib. We aimed to identify genes that are strongly up- or down-regulated with a combination of BRCA1 knockdown and proteasome inhibition, but none of these treatments alone before the onset of apoptosis. HeLa and U2OS cells were transfected either with a non-targeting or anti-BRCA1 siRNAs (siControl or siBRCA1, respectively), treated with bortezomib for 8 hours, after which RNA was extracted for hybridization on Affymetrix microarray. The following treatments have been performed: (T1) siControl; (T2) siControl + 20 nM bortezomib for 8h; (T3) siBRCA1; (T4) siBRCA1 + 20 nM bortezomib for 8h. All samples were used without replicas. However, all genes showing inconsistent expression pattern between the two cell lines were excluded from further consideration. Selected candidate genes were subject to validation by qRT-PCR.
2015-01-01 | E-GEOD-56280 | biostudies-arrayexpress