Accurate classification of BRCA1 variants with saturation genome editing
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE117159 | GEO | 2018/08/01
REPOSITORIES: GEO
Similar Datasets
Project description:Accurate classification of BRCA1 variants with saturation genome editing
| PRJNA481326 | ENA
Project description:Variants of uncertain significance fundamentally limit the clinical utility of genetic information. The challenge they pose is epitomized by BRCA1, a tumour suppressor gene in which germline loss-of-function variants predispose women to breast and ovarian cancer. Although BRCA1 has been sequenced in millions of women, the risk associated with most newly observed variants cannot be definitively assigned. Here we use saturation genome editing to assay 96.5% of all possible single-nucleotide variants (SNVs) in 13 exons that encode functionally critical domains of BRCA1. Functional effects for nearly 4,000 SNVs are bimodally distributed and almost perfectly concordant with established assessments of pathogenicity. Over 400 non-functional missense SNVs are identified, as well as around 300 SNVs that disrupt expression. We predict that these results will be immediately useful for the clinical interpretation of BRCA1 variants, and that this approach can be extended to overcome the challenge of variants of uncertain significance in additional clinically actionable genes.
| S-EPMC6181777 | 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: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: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: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: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:We employed saturation genome editing (SGE) to assess the functional consequences of synonymous, missense, and nonsense variants across KARS1 exon 2. Deep DNA sequencing of fixed-amplicon PCR products targeting the endogenous KARS1 Exon 2 locus was used to determine variant frequencies as part of a larger study to identify a set of reproducible enrichment scores indicating effects of variants on KARS1 function.
2024-05-31 | GSE232150 | GEO
Project description:We employed saturation genome editing (SGE) to assess the functional consequences of synonymous, missense, and nonsense variants across KARS1 exon 2. Deep DNA sequencing of fixed-amplicon PCR products targeting the endogenous KARS1 Exon 2 locus was used to determine variant frequencies as part of a larger study to identify a set of reproducible enrichment scores indicating effects of variants on KARS1 function.
2024-05-31 | GSE232151 | GEO