Sequencing-based Functional Assays for Classification of BRCA2 Variants in Mouse ES Cells
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ABSTRACT:
PROVIDER: PRJNA1023599 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR26272800_1.fastq.gz | Fastqsanger.gz | |||
| SRR26272800_2.fastq.gz | Fastqsanger.gz | |||
| SRR26272900_1.fastq.gz | Fastqsanger.gz | |||
| SRR26272900_2.fastq.gz | Fastqsanger.gz | |||
| SRR26272801_1.fastq.gz | Fastqsanger.gz |
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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
Project description:Germline BRCA2 loss-of function (LOF) variants identified by clinical genetic testing predispose to breast, ovarian, prostate and pancreatic cancer. However, variants of uncertain significance (VUS) (n>5000) limit the clinical use of testing results. Thus, there is an urgent need for functional characterization and clinical classification of all BRCA2 variants. Here we report on comprehensive saturation genome editing-based functional characterization of 99% of all possible single nucleotide variants (SNVs) in the BRCA2 DNA Binding Domain hotspot for pathogenic missense variants that is encoded by exons 15 to 26. The assay was based on deep sequence analysis of HAP1 haploid cells endogenously targeted using a CRISPR/cas9 knockin approach. A total of 6959 SNVs were characterized for effects on cell survival. The assay was validated relative to nonsense and synonymous variants, ClinVar pathogenic/likely pathogenic and benign/likely benign variants and homology directed repair cell based DNA repair assay results, all of which showed >94% sensitivity and specificity. Variants were assigned posterior probabilities of pathogenicity using a VarCall two component Bayesian mixture model and were further grouped according to ACMG strength of evidence under the PS3/BS3 rule resulting in Benign Strong (n=5430), Benign Moderate (n=190), Benign Supporting (n=61), VUS (122), Pathogenic Strong (n=1021), Pathogenic Moderate (n=88), and Pathogenic Supporting (n=47). Breast cancer case-control association studies showed that pooled SNVs encoding functionally pathogenic missense variants were associated with increased risks of breast (odds ratio (OR)3.81, 95%CI: 2.88-5.07) and ovarian cancer (OR 5.93, 95%CI: 4.12-8.52). The functional data were also combined with other sources of information in the ClinGen BRCA1/2 VCEP ACMG/AMP-classification model. A total of 785 SNVs, including 261 missense SNVs, were classified as pathogenic or likely pathogenic, while 5566 SNVs, including 3786 missense SNVs, were classified as benign or likely benign. These classified variants can now be used for risk assessment and clinical care of variant carriers.
2024-09-30 | GSE270424 | 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:BRCA1 and BRCA2 classification of ovarian cancer
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Project description:Saturation genome editing-based functional evaluation and clinical 2 classification of BRCA2 single nucleotide variants
| PRJNA1126616 | ENA
Project description:Inactivating germline monoallelic mutations in the tumor suppressor BRCA2 predispose to breast and ovarian cancer. BRCA2 is essential in DNA repair via homologous recombination (HR). Tumorigenesis in this setting is thought to require the inactivation of the second allele; however, this event is not always observed suggesting haploinsufficiency. We investigated this question recreating two BRCA2 pathogenic truncating variants in heterozygosis in breast epithelial cells. One prompted sensitivity to PARP inhibitors (PARPi) and reduced HR capacity unlike the other. The other resulted in transcriptomic and epigenetic changes including histone H4 acetylation levels and NF-kB activation that we link to PCAF acetyl transferase. These changes impacted on cell migration and proliferation in 2D and 3D, processes that were also deregulated in tumors. Our work demonstrates that distinct truncating variants of BRCA2 are not phenotypically equivalent. It also identifies PCAF as shaping BRCA2-related gene expression. These findings may have implications in cancer prevention.
2024-11-21 | GSE272335 | GEO
Project description:Inactivating germline BRCA1 and BRCA2 mutations confer a defect in homologous recombination DNA repair which was found to leave traces in tumor DNA copy number aberration (CNA) profiles. In analogy to previously trained breast cancer CNA classifiers that predicted association with BRCA1 and BRCA2 mutated cancer and benefit of high dose double strand break inducing chemotherapy, we trained BRCA1 and BRCA2 classifiers on CNA profiles of 50 BRCA1 mutated, 10 BRCA2 mutated and 13 non-familial ovarian cancers and investigated whether tumor type and mutation type independent classifiers could be trained. The cross validated area under the curve of the receiver/operator characteristic curve of ovarian cancer BRCA1 and BRCA2 classifiers were 0.67 (95% CI: 0.55-0.78) and 0.91 (95% CI: 0.79-1). These classifiers identified the majority of the samples with germline and somatic BRCA1 and BRCA2 mutations and BRCA1 promoter hypermethylation in the Cancer Genome Atlas (TCGA) dataset. Combining tumor type or mutated gene did not yield higher AUCs than single gene classifiers, although the ovarian BRCA1+BRCA2 classifier identified most BRCA1 and -2 mutated cases, including those in the TCGA dataset, and a combined breast and ovarian cancer BRCA1 classifier may improve response prediction to double strand break inducing chemotherapy.
2021-12-31 | GSE111688 | GEO
Project description:Accurate interpretation of genetic variation is a critical step towards realizing the potential of precision medicine. Sequencing-based genetic tests have uncovered a vast array ofBRCA2sequence variants. Due to limited clinical, familial and/or epidemiological data, thousands of variants are considered to be variants of uncertain significance (VUS). Here we have utilized CRISPR-Cas9-based saturation genome editing (SGE) in a humanized-mouse embryonic stem cell line. We have categorized nearly all possible missense single nucleotide variants (SNVs) encompassing the C-terminal DNA binding domain ofBRCA2.We have generated the function scores for 6270 SNVs, covering 95.5% of possible SNVs in exons 15-26 spanning residues 2479-3216, including 1069 unique missense VUS, with 81% functional and 14% found to be nonfunctional. Our classification aligns strongly with pathogenicity data from ClinVar, orthogonal functional assays and computational meta predictors. Our statistical classifier exhibits 92.2% sensitivity and 96% specificity in distinguishing clinically benign and pathogenic variants recorded in ClinVar. Furthermore, we offer proactive evidence for 617 SNVs being non-functional and 3396 SNVs being functional demonstrated by impact on cell growth and response to DNA-damaging drugs like cisplatin and olaparib. This classification serves as a valuable resource for interpreting unidentified variants in the population and for physicians and genetic counselors assessingBRCA2VUSs in patients.
2024-09-27 | GSE248438 | GEO
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: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