Saturation genome editing of 11 codons and exon 13 of BRCA2 determines pathogenicity of variants
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PROVIDER: PRJNA998036 | ENA |
REPOSITORIES: ENA
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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:The unknown pathogenicity of a significant number of variants found in cancer-related genes is attributed to limited epidemiological data, resulting in their classification as 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 the response of these variants to the DNA-damaging agents, cisplatin and olaparib, allowing us to use cellular survival and drug response as parameters for variant classification. Using this approach, 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 and 60 non-functional variants, demonstrating that saturation genome editing (SGE) coupled with drug sensitivity assays can enhance functional annotation of BRCA2 VUS.
| S-EPMC10529611 | biostudies-literature
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:Saturation genome Editing of BRCA2 variants from C-terminal DNA binding domain
| PRJNA1044005 | ENA
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:Saturation genome editing-based functional evaluation and clinical 2 classification of BRCA2 single nucleotide variants
| PRJNA1126616 | ENA
Project description:Deleterious alterations of the gene encoding BRCA2, a protein involved in homologous recombination (HR), contribute to the pathogenesis of a subset of ovarian, breast, prostate and pancreatic cancers as well as their increased sensitivity to platinating agents and poly(ADP-ribose) polymerase inhibitors (PARPis). While secondary mutations that restore a functional BRCA2 protein are detected in a fraction of cases that become drug resistant, the causes of resistance in other cases are poorly understood. Here we characterized clones derived from the BRCA2-mutant ovarian cancer line PEO1 by PARPi selection. Colony forming assays demonstrated cross-resistance of these clones to multiple PARPis, cisplatin and doxorubicin. Despite persistence of the signature 4965C>G (p.Y1655X) BRCA2 nonsense mutation, HR was restored in the resistant cells. Immunoprecipitation followed by immunoblotting or mass spectrometry demonstrated expression of low levels of BRCA2 protein, including peptides distal to the premature termination codon. Reporter assays demonstrated readthrough selective for the UAG stop codon in the resistant clones but not parental PEO1 cells. Mass spectrometry and immunoblotting provided evidence for readthrough of stop codons, particularly UAGs, in additional proteins in the resistant clones. BRCA2 gene interruption either 5’ or 3’ to the stop codon restored sensitivity of the resistant clones to PARPis and cisplatin, implicating the low level BRCA2 expression in resistance. Accordingly, the present results identify low level readthrough of nonsense codons as a potential mechanism of drug resistance that might need to be considered when assessing the impact of BRCA2 nonsense mutations.
2024-09-01 | GSE243142 | GEO
Project description:Saturation mutagenesis reveals manifold determinants of exon definition
| PRJNA415408 | ENA
Project description:A pathogenic mutation in BRCA2 significantly increases the risk of breast and ovarian cancers making it imperative to examine the functional consequences of variants of uncertain clinical significance. Variants that are predicted to result in a truncated protein are unambiguously classified as pathogenic. We have previously shown how a pathogenic splice site variant known to generate a premature termination codon (PTC) in exon 9 and a nonsense mutation at exon 7, can generate functional BRCA2 by skipping exons 4-7 and restoring the reading frame. Using a well-established mouse embryonic stem cell-based assay, we functionally characterize here one splice site mutation and 11 pathogenic BRCA2 variants that are either nonsense mutation or generate PTC in different exons ranging from exons 4 to 7. Our study shows that five variants can restore the open reading frame by exon skipping and generate a functional protein. This suggests further need to exercise prudence when classifying clearly pathogenic variants.
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Project description:This SuperSeries is composed of the following subset Series: GSE32124: Effects of BRCA2 cis-regulation in normal breast and cancer risk amongst BRCA2 mutation carriers [expression data] GSE32258: Effects of BRCA2 cis-regulation in normal breast and cancer risk amongst BRCA2 mutation carriers [SNP data] Refer to individual Series
2012-02-01 | E-GEOD-32259 | biostudies-arrayexpress