Functional annotation of genetic variants using locally haploid human pluripotent stem cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE233683 | GEO | 2023/12/31
REPOSITORIES: GEO
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Project description:Breast cancer gene 2 (BRCA2) deleterious mutations confer sensitivity to poly(ADP-ribose) polymerase (PARP) inhibition due to its critical role in DNA repair. PARP inhibitor Olaparib is now approved and several other PARP inhibitors are now in different stages of clinical trials. Development of resistance to PARP inhibitors limits their clinical utility. The mechanism of resistance remains not fully understood. Here we show that amplification of mutant BRCA2 confers PARP inhibitor resistance. The amplification of mutant BRCA2 gene copies correlates with an increase in mutant BRCA2 expression and an increase in the levels of its interacting proteins PALB2 and RAD51. In addition, homologous recombination mediated DNA repair is rescued in these cells as evidenced by the formation of RAD51 focus formation. The overexpressed mutant BRCA2 is essential for the observed PARP inhibitor resistance because knockdown of its expression is sufficient to re-sensitize these cells to PARP inhibition. Collectively, our results indicate a new mechanism of resistance to PARP inhibitor in BRCA2 mutant cancer cells
2017-09-02 | GSE86394 | GEO
Project description:Mutations in the tumour suppressor BRCA2 are associated with predisposition to breast and ovarian cancers. BRCA2 has a central role in genome integrity by facilitating the repair of toxic DNA double-strand breaks (DSBs) by homologous recombination. BRCA2 acts by promoting RAD51 nucleofilament formation on resected single-stranded DNA, but how BRCA2 activity is regulated during HR is not fully understood. Here, we delineate a pathway where ATM and ATR kinases phosphorylate a highly conserved region in BRCA2 in response to DNA DSBs. These phosphorylations stimulate the binding of the protein phosphatase PP2A-B56 to BRCA2 through a conserved binding motif. We show that the phosphorylation-dependent formation of the BRCA2-PP2A-B56 complex is required for efficient RAD51 loading to sites of DNA damage and HR-mediated DNA repair. Moreover, we find that several cancer-associated mutations in BRCA2 deregulate the BRCA2-PP2A-B56 interaction and sensitize cells to PARP inhibition. Collectively, our work uncovers PP2A-B56 as a positive regulator of BRCA2 function in HR with clinical implications for BRCA2 and PP2A-B56 mutated cancers.
2021-07-26 | MSV000087884 | MassIVE
Project description:Inhibitors of PARP (PARPis) represent the first clinically approved anticancer agents targeting the DNA damage response. They have been approved as monotherapy and/or in combination and maintenance settings in different tumor types, including high-grade ovarian carcinomas. Their efficacy was first underlined in cells with functional inactivation of BRCA1 and BRCA2 genes and this strong preclinical evidence prompted their clinical development in tumors with BRCA1/BRCA2 mutations. It was later demonstrated that PARPis were very effective in tumors displaying deficiency in homologous recombination (HR) repair beyond BRCA1 and BRCA2 loss of function. In addition, evidence from randomized clinical trials supports their efficacy also in tumors with intact HR repair, although to a lesser extent.
2023-09-14 | E-MTAB-13055 | biostudies-arrayexpress
Project description:Pathogenic germline mutations in BRCA1 or BRCA2 are detected in less than one third of families with a strong history of breast cancer. It is therefore expected that mutations still remain undetected by currently used screening methods. In addition, a growing number of BRCA1/2 sequence variants of unclear pathogen significance are found in the families, constituting an increasing clinical challenge. New methods are therefore needed to improve the detection rate and aid the interpretation of the clinically uncertain variants. In this study we analyzed a series of 33 BRCA1, 22 BRCA2, and 128 sporadic tumors by RNA profiling to investigate the classification potential of RNA profiles to predict BRCA1/2 mutation status. We found that breast tumors from BRCA1 and BRCA2 mutation carriers display characteristic RNA expression patterns, allowing them to be distinguished from sporadic tumors. The majority of BRCA1 tumors were basal-like while BRCA2 tumors were mainly luminal B. Using RNA profiles, we were able to distinguish BRCA1 tumors from sporadic tumors among basal-like tumors with 83% accuracy and BRCA2 from sporadic tumors among luminal B tumors with 89% accuracy. Furthermore, subtype-specific BRCA1/2 gene signatures were successfully validated in two independent data sets with high accuracies. Although additional validation studies are required, indication of BRCA1/2 involvement (“BRCAness”) by RNA profiling could potentially be valuable as a tool for distinguishing pathogenic mutations from benign variants, for identification of undetected mutation carriers, and for selecting patients sensitive to new therapeutics such as PARP inhibitors. Gene expression profiling of 183 breast tumor samples. Breast tumors from hereditary breast cancer patients carrying a pathogenic BRCA1 (n=33) or BRCA2 (n=22) germ-line mutation were included in the study. Serving as a representative control group, primary breast tumor samples (n=128) were randomly selected. The study was conducted using Agilent-029949 Custom SurePrint G3 Human GE 8x60K Microarray platform. For cross-platform validation, a subset of the tumor samples (92 of the 183 samples) were analyzed by our in-house spotted microarray platform.
2013-06-28 | E-GEOD-40115 | biostudies-arrayexpress
Project description:Pathogenic germline mutations in BRCA1 or BRCA2 are detected in less than one third of families with a strong history of breast cancer. It is therefore expected that mutations still remain undetected by currently used screening methods. In addition, a growing number of BRCA1/2 sequence variants of unclear pathogen significance are found in the families, constituting an increasing clinical challenge. New methods are therefore needed to improve the detection rate and aid the interpretation of the clinically uncertain variants. In this study we analyzed a series of 33 BRCA1, 22 BRCA2, and 128 sporadic tumors by RNA profiling to investigate the classification potential of RNA profiles to predict BRCA1/2 mutation status. We found that breast tumors from BRCA1 and BRCA2 mutation carriers display characteristic RNA expression patterns, allowing them to be distinguished from sporadic tumors. The majority of BRCA1 tumors were basal-like while BRCA2 tumors were mainly luminal B. Using RNA profiles, we were able to distinguish BRCA1 tumors from sporadic tumors among basal-like tumors with 83% accuracy and BRCA2 from sporadic tumors among luminal B tumors with 89% accuracy. Furthermore, subtype-specific BRCA1/2 gene signatures were successfully validated in two independent data sets with high accuracies. Although additional validation studies are required, indication of BRCA1/2 involvement (“BRCAness”) by RNA profiling could potentially be valuable as a tool for distinguishing pathogenic mutations from benign variants, for identification of undetected mutation carriers, and for selecting patients sensitive to new therapeutics such as PARP inhibitors.
2013-06-28 | GSE40115 | GEO
Project description:The pathogenicity of majority of variants identified in cancer-causing genes is unknown due to limited epidemiological data, hence they are considered to be variant of uncertain significance (VUS). To date, Breast Cancer gene-2 (BRCA2) has the highest number of VUSs, which has necessitated the development of several robust functional assays to determine their functional significance. Here we report the use of a humanized-mouse embryonic stem cell (mESC) line expressing a single copy of the human BRCA2 for a CRISPR-Cas9-based high-throughput functional assay. As a proof-of-principle, we have saturated 11 codons encoded by BRCA2 exons 3, 18, 19 and all possible single-nucleotide variants in exon 13 and multiplexed these variants for their functional categorization. Specifically, we used a pool of 180-mer single-stranded donor DNA to generate all possible combination of variants. Using a high throughput sequencing-based approach, we show a significant drop in the frequency of non-functional variants, whereas functional variants are enriched in the pool of the cells. We further demonstrate that variants with partial loss of BRCA2 function are sensitive to the DNA-damaging agents, cisplatin and olaparib, allowing us to discriminate between functional and intermediate variants. We have categorized 599 BRCA2 variants including 93-single nucleotide variants (SNVs) across the 11 codons, of which 28 are reported in ClinVar. We also functionally categorized 252 SNVs from exon 13 into 188 functional, 60 non-functional and 4 intermediate variants, demonstrating that saturation genome editing (SGE) coupled with drug sensitivity assays can enhance functional annotation of BRCA2 VUS.
2023-08-30 | GSE238143 | GEO
Project description:Heterozygous germline mutations in BRCA2 predispose to breast and ovarian cancer. Contrary to non-cancerous cells, where BRCA2 deletion causes cell cycle arrest or cell death, BRCA2 inactivation in tumors is associated with uncontrolled cell proliferation. We set out to investigate this conundrum by exploring modalities of cell adaptation to loss of BRCA2 and focused on genome-wide transcriptome alterations. Human cells in which BRCA2 expression was inhibited using a doxycycline (DOX)-inducible shRNA for 4 or 28 days were subjected to RNA-seq analyses. Gene sets differentially expressed in BRCA2-deficient versus -proficient cells revealed a biphasic response to BRCA2 abrogation. The early, acute response consisted of downregulation of genes involved in cell cycle progression, DNA replication and repair and was associated with cell cycle arrest in G1. Surprisingly, the late, chronic response consisted exclusively of upregulation of innate immune response genes controlled by interferon. Activation of the cGAS-STING pathway detected in these cells further substantiated the concept that long-term BRCA2 abrogation triggers cell-intrinsic immune signaling. Importantly, we found that treatment with PARP inhibitors stimulated the interferon response in cells and tumors lacking BRCA2. We propose that PARP inhibitors suppress growth of BRCA2-deficient cells and tumors, in part, by activating interferon signaling.
2018-12-11 | GSE123631 | GEO
Project description:Introduction: Cis-acting regulatory single nucleotide polymorphisms (SNPs) at specific loci may modulate penetrance of germline mutations at the same loci by introducing different levels of expression of the wild-type allele. We have previously reported that BRCA2 shows differential allelic expression and we hypothesize that the known variable penetrance of BRCA2 mutations might be associated with this mechanism. Methods: We combined haplotype analysis and differential allelic expression of BRCA2 in breast tissue to identify expression haplotypes and candidate cis-regulatory variants. These candidate variants underwent selection based on in-silico predictions for regulatory potential and disruption of transcription factor binding, and were functionally analysed in-vitro and in-vivo in normal and breast cancer cell lines. SNPs tagging the expression haplotypes were correlated with the total expression of several genes in breast tissue measured by Taqman and microarray technologies. The effect of the expression haplotypes on breast cancer risk in BRCA2 mutation carriers was investigated in 2754 carriers. Results: We identified common haplotypes associated with differences in the levels of BRCA2 expression in human breast cells. We characterised three cis-regulatory SNPs located at the promoter and two intronic regulatory elements, which affect the binding of the transcription factors C/EBPα, HMGA1, DBP and ZF5. We showed that the expression haplotypes also correlated with changes in the expression of other genes in normal breast. Furthermore, there was suggestive evidence that the minor allele of SNP rs4942440, which is associated with higher BRCA2 expression, is also associated with a reduced risk of breast cancer (per-allele HR=0.85, 95%CI=0.72-1.00, P-trend=0.048). Conclusion: Our work provides further insights into the role of cis-regulatory variation in the penetrance of disease-causing mutations. We identified small-effect genetic variants associated with allelic expression differences in BRCA2, which could possibly affect the risk in mutation carriers through altering expression levels of the wild-type allele. Total gene expression of normal breast sample from healthy controls. This submission represents transcriptome component of study.
2012-02-01 | E-GEOD-32124 | biostudies-arrayexpress
Project description:Introduction: Cis-acting regulatory single nucleotide polymorphisms (SNPs) at specific loci may modulate penetrance of germline mutations at the same loci by introducing different levels of expression of the wild-type allele. We have previously reported that BRCA2 shows differential allelic expression and we hypothesize that the known variable penetrance of BRCA2 mutations might be associated with this mechanism. Methods: We combined haplotype analysis and differential allelic expression of BRCA2 in breast tissue to identify expression haplotypes and candidate cis-regulatory variants. These candidate variants underwent selection based on in-silico predictions for regulatory potential and disruption of transcription factor binding, and were functionally analysed in-vitro and in-vivo in normal and breast cancer cell lines. SNPs tagging the expression haplotypes were correlated with the total expression of several genes in breast tissue measured by Taqman and microarray technologies. The effect of the expression haplotypes on breast cancer risk in BRCA2 mutation carriers was investigated in 2754 carriers. Results: We identified common haplotypes associated with differences in the levels of BRCA2 expression in human breast cells. We characterised three cis-regulatory SNPs located at the promoter and two intronic regulatory elements, which affect the binding of the transcription factors C/EBPα, HMGA1, DBP and ZF5. We showed that the expression haplotypes also correlated with changes in the expression of other genes in normal breast. Furthermore, there was suggestive evidence that the minor allele of SNP rs4942440, which is associated with higher BRCA2 expression, is also associated with a reduced risk of breast cancer (per-allele HR=0.85, 95%CI=0.72-1.00, P-trend=0.048). Conclusion: Our work provides further insights into the role of cis-regulatory variation in the penetrance of disease-causing mutations. We identified small-effect genetic variants associated with allelic expression differences in BRCA2, which could possibly affect the risk in mutation carriers through altering expression levels of the wild-type allele. Genotype of normal breast sample from healthy controls.
2012-02-01 | E-GEOD-32258 | biostudies-arrayexpress
Project description:Sequencing of genes, such as BRCA1 and BRCA2, is recommended for individuals with a personal or family history of early onset and/or bilateral breast and/or ovarian cancer, or a history of male breast cancer. Such sequencing efforts have resulted in the identification of more than 6000 BRCA2 variants. The functional significance of most variants remains unknown; consequently, they are called variants of uncertain clinical significance (VUS). We have previously developed mouse embryonic stem cell (mESC)-based assays for functional classification of BRCA2 variants. While the assays are reliable, the experimental process is time-consuming. This has impeded the number of variants that can be examined at a time. To overcome this, we have now developed a next-generation sequencing (NGS)-based approach for functional evaluation of BRCA2 variants. In this approach, instead of using clonogenic or XTT-based cell survival assays, pools of mESC expressing 25-30 BRCA2 variants from a given exon are analyzed by NGS. We have also developed a statistical model that utilizes the NGS data to determine the probability of impact on function (PIF) for these variants. While CRISPR/Cas9-based high throughput methods have resulted in the functional characterization of thousands of variants, only a fraction of the variants are included in ClinVar. In contrast, our targeted approach, which involves generating specific variants, resulted in the functional evaluation of 223 variants that are all listed in ClinVar. Our functional classification of BRCA2 variants is concordant with the classification reported in ClinVar or reported by other well-established assays.
2023-11-20 | GSE244578 | GEO