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:
PROVIDER: PRJNA478453 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR7451400.fastq.gz | Fastqsanger.gz | |||
| SRR7451410.fastq.gz | Fastqsanger.gz | |||
| SRR7451420.fastq.gz | Fastqsanger.gz | |||
| SRR7451391.fastq.gz | Fastqsanger.gz | |||
| SRR7451401.fastq.gz | Fastqsanger.gz |
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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: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.
2012-06-01 | E-MTAB-761 | biostudies-arrayexpress
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.
2023-10-13 | GSE237142 | GEO
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:In order to investigate whether carriers of BRCA1 heterozygous mutation show altered gene expression profiles, we used a whole genome microarray expression profiling of a panel of lymphoblastoid cell lines (LCLs) harboring either mutated or WT BRCA1 (control). We were especially interested whether we find differences in gene expression between carriers of missense versus truncating mutation in BRCA1 gene. We evaluated gene expression of a panel of 21 non-treated lymphoblastoid cell lines (LCLs). Among them, 6 LCLs with WT BRCA1, 5 LCLs carrying missense mutation in BRCA1, and 10 LCLs harboring truncating mutation in BRCA1 were included in this study.
2015-12-31 | E-GEOD-60396 | biostudies-arrayexpress
Project description:The functional consequences of missense variants in disease genes are difficult to predict. We assessed if gene expression profiles could distinguish between BRCA1 or BRCA2 pathogenic truncating and missense mutation carriers and familial breast cancer cases whose disease was not attributable to BRCA1 or BRCA2 mutations (BRCAX cases). 72 cell lines from affected women in high-risk breast-ovarian families were assayed after exposure to ionising irradiation, including 23 BRCA1 carriers, 22 BRCA2 carriers, and 27 BRCAX individuals. A subset of 10 BRCAX individuals carried rare BRCA1/2 sequence variants considered to be of low clinical significance (LCS). BRCA1 and BRCA2 mutation carriers had similar expression profiles, with some subclustering of missense mutation carriers. The majority of BRCAX individuals formed a distinct cluster, but BRCAX individuals with LCS variants had expression profiles similar to BRCA1/2 mutation carriers. Gaussian Process Classifier predicted BRCA1, BRCA2 and BRCAX status with a maximum of 62% accuracy, and prediction accuracy decreased with inclusion of BRCAX samples carrying an LCS variant, and inclusion of pathogenic missense carriers. Similarly, prediction of mutation status with gene lists derived using Support Vector Machines was good for BRCAX samples without an LCS variant (82-94%), poor for BRCAX with an LCS (40-50%), and improved for pathogenic BRCA1/2 mutation carriers when the gene list used for prediction was appropriate to mutation effect being tested (71-100%). This study indicates that mutation effect, and presence of rare variants possibly associated with a low risk of cancer, must be considered in the development of array-based assays of variant pathogenicity. Keywords: cell type comparison, stress response
2008-03-21 | GSE10905 | 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.
2023-06-12 | GSE234482 | GEO
Project description:In order to investigate whether carriers of BRCA1 heterozygous mutation show altered miRNA expression profiles, we established a panel of lymphoblastoid cell lines (LCLs) harboring either mutated or WT BRCA1 (control) and investigated their miRNA expression profiles. We were especially interested whether we find differences in miRNA expression between carriers of missense versus truncating mutation in BRCA1 gene. We evaluated the miRNA expression in a panel of 21 non-treated lymphoblastoid cell lines (LCLs). Among them, 6 LCLs with WT BRCA1, 5 LCLs carrying missense mutation in BRCA1, and 10 LCLs harboring truncating mutation in BRCA1 were included in this study.
2015-12-31 | E-GEOD-60444 | biostudies-arrayexpress
Project description:Nuclear deubiquitinase BAP1 (BRCA1-Associated Protein 1) is a core component of multiprotein complexes that promote transcription by reversing the ubiquitination of histone 2A (H2A). BAP1 is a tumor suppressor gene whose germline loss-of-function variants predispose to cancer. To our knowledge, there are very rare examples of different germline variants in the same gene causing either a NDD or a tumor predisposition syndrome. Here, we report a series of 11 de novo germline heterozygous missense BAP1 variants associated with a rare syndromic neurodevelopmental disorder (NDD). Functional analysis showed that most of the variants cannot rescue the consequences of BAP1 inactivation, suggesting a loss-of-function mechanism. In T cells isolated from two affected children, H2A deubiquitination was impaired in matching peripheral blood mononuclear cells, histone H3 K27 acetylation ChIP-seq indicated that these BAP1 variants induced genome-wide chromatin state alterations, with enrichment for regulatory regions surrounding genes of the ubiquitin-proteasome system (UPS). Altogether, these results define a clinical syndrome caused by rare germline missense BAP1 variants that alter chromatin remodeling through abnormal histone ubiquitination and lead to transcriptional dysregulation of developmental genes.
2021-12-10 | GSE190394 | GEO
Project description:In order to investigate whether carriers of BRCA1 heterozygous mutation show altered gene expression profiles, we used a whole genome microarray expression profiling of a panel of lymphoblastoid cell lines (LCLs) harboring either mutated or WT BRCA1 (control). We were especially interested whether we find differences in gene expression between carriers of missense versus truncating mutation in BRCA1 gene.
2015-12-31 | GSE60396 | GEO