Multiplexed assays reveal effects of missense variants in MSH2 and cancer predisposition.
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ABSTRACT:
SUBMITTER: Nielsen SV
PROVIDER: S-EPMC8061925 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:We performed a massively parallel screen in human HAP1 cells to identify loss-of-function missense variants in the key DNA mismatch repair factor MSH2. Resulting variant loss-of-function (LOF) scores are strongly concordant with previous functional evidence and available variant classification.
2020-12-23 | GSE162130 | GEO
Project description:To validate a high-throughput screening data in human cells using Multiplexed Assays for Variant Effects (MAVE), we performed a high-throughput deep mutational scanning of single nucleotide changes in exon 10 encoding p.G1000 to p.I1037 of the WD40 domain of PALB2 using a cell survival assay in haploid human HAP1 cells. We obtained MAVE scores for 276 single-nucleotide variants, leading to 9 nonsense and 68 synonymous changes, as well as 199 amino acid substitutions. Both variant groups showed an asymmetric distribution that is skewed towards low MAVE scores of nonsense and damaging variants, respectively. These MAVE data included scores for 218 unique single-nucleotide variants, leading to 9 nonsense changes and 209 amino acid substitutions. We observed a good and significant correlation between the outcomes from the MAVE and high-throughput screens (n=179, r=-0,6439, p<0.0001), indicating concordance between the outcomes of high-throughput analysis of PALB2 variants in human and mouse cells.
2024-02-08 | GSE255117 | GEO
Project description:The lack of functional evidence for the majority of missense variants limits their clinical interpretability and poses a key barrier to the broad utility of carrier screening. In Lynch syndrome (LS), one of the most highly prevalent cancer syndromes, nearly 90% of clinically observed missense variants are deemed "variants of uncertain significance" (VUS). To systematically resolve their functional status, we performed a massively parallel screen in human cells to identify loss-of-function missense variants in the key DNA mismatch repair factor MSH2. The resulting functional effect map is substantially complete, covering 94% of the 17,746 possible variants, and is highly concordant (96%) with existing functional data and expert clinicians' interpretations. The large majority (89%) of missense variants were functionally neutral, perhaps unexpectedly in light of its evolutionary conservation. These data provide ready-to-use functional evidence to resolve the ∼1,300 extant missense VUSs in MSH2 and may facilitate the prospective classification of newly discovered variants in the clinic.
| S-EPMC7820803 | biostudies-literature
Project description:Reliable methods for predicting functional consequences of variants in disease genes would be beneficial in the clinical setting. This study was undertaken to predict, and confirm in vitro, splicing aberrations associated with mismatch repair (MMR) variants identified in familial colon cancer patients. Six programs were used to predict the effect of 13 MLH1 and 6 MSH2 gene variants on pre-mRNA splicing. mRNA from cycloheximide-treated lymphoblastoid cell lines of variant carriers was screened for splicing aberrations. Tumors of variant carriers were tested for microsatellite instability and MMR protein expression. Variant segregation in families was assessed using Bayes factor causality analysis. Amino acid alterations were examined for evolutionary conservation and physicochemical properties. Splicing aberrations were detected for 10 variants, including a frameshift as a minor cDNA product, and altered ratio of known alternate splice products. Loss of splice sites was well predicted by splice-site prediction programs SpliceSiteFinder (90%) and NNSPLICE (90%), but consequence of splice site loss was less accurately predicted. No aberrations correlated with ESE predictions for the nine exonic variants studied. Seven of eight missense variants had normal splicing (88%), but only one was a substitution considered neutral from evolutionary/physicochemical analysis. Combined with information from tumor and segregation analysis, and literature review, 16 of 19 variants were considered clinically relevant. Bioinformatic tools for prediction of splicing aberrations need improvement before use without supporting studies to assess variant pathogenicity. Classification of mismatch repair gene variants is assisted by a comprehensive approach that includes in vitro, tumor pathology, clinical, and evolutionary conservation data.
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Project description:1 Novel Missense Variants in Patients with Primary Immunodeficiency and Immune dysregulation
| PRJEB46587 | ENA
Project description:PurposeUp to 30% of patients with Brugada syndrome (BrS) carry loss-of-function (LoF) variants in the cardiac sodium channel gene SCN5A encoding for the protein NaV1.5. Recent studies suggested that NaV1.5 can dimerize, and some variants exert dominant negative effects. In this study, we sought to explore the generality of missense variant NaV1.5 dominant negative effects and their clinical severity.MethodsWe identified 35 LoF variants (<10% of wild type [WT] peak current) and 15 partial LoF variants (10%-50% of WT peak current) that we assessed for dominant negative effects. SCN5A variants were studied in HEK293T cells, alone or in heterozygous coexpression with WT SCN5A using automated patch clamp. To assess the clinical risk, we compared the prevalence of dominant negative vs putative haploinsufficient (frameshift, splice, or nonsense) variants in a BrS consortium and the Genome Aggregation Database population database.ResultsIn heterozygous expression with WT, 32 of 35 LoF and 6 of 15 partial LoF variants showed reduction to <75% of WT-alone peak current, showing a dominant negative effect. Individuals with dominant negative LoF variants had an elevated disease burden compared with the individuals with putative haploinsufficient variants (2.7-fold enrichment in BrS cases, P = .019).ConclusionMost SCN5A missense LoF variants exert a dominant negative effect. This class of variant confers an especially high burden of BrS.
| S-EPMC9262418 | biostudies-literature
Project description:Using a high throughput splicing reporter assay, we tested 1,080 single nucleotide variants in POU1F1, a key transcription factor essential for pituitary development. Our saturation splicing effect map identifies 96 splice disruptive variants, including 14 synonymous variants, of which 8 were found in unrelated patients diagnosed with hypopituitarism.
2021-04-22 | GSE172504 | GEO
Project description:Missense DNA variants have variable effects upon protein function. Consequently, interpreting their pathogenicity is challenging, especially when they are associated with disease variability. To determine the degree to which functional assays inform interpretation, we analyzed 48 CFTR missense variants associated with variable expressivity of cystic fibrosis (CF). We assessed function in a native isogenic context by evaluating CFTR mutants that were stably expressed in the genome of a human airway cell line devoid of endogenous CFTR expression. 21 of 29 variants associated with full expressivity of the CF phenotype generated <10% wild-type CFTR (WT-CFTR) function, a conservative threshold for the development of life-limiting CF lung disease, and five variants had moderately decreased function (10% to ?25% WT-CFTR). The remaining three variants in this group unexpectedly had >25% WT-CFTR function; two were higher than 75% WT-CFTR. As expected, 14 of 19 variants associated with partial expressivity of CF had >25% WT-CFTR function; however, four had minimal to no effect on CFTR function (>75% WT-CFTR). Thus, 6 of 48 (13%) missense variants believed to be disease causing did not alter CFTR function. Functional studies substantially refined pathogenicity assignment with expert annotation and criteria from the American College of Medical Genetics and Genomics and Association for Molecular Pathology. However, four algorithms (CADD, REVEL, SIFT, and PolyPhen-2) could not differentiate between variants that caused severe, moderate, or minimal reduction in function. In the setting of variable expressivity, these results indicate that functional assays are essential for accurate interpretation of missense variants and that current prediction tools should be used with caution.
| S-EPMC5992123 | biostudies-literature
Project description:The diffusion of next-generation sequencing technologies has revolutionized research and diagnosis in the field of rare Mendelian disorders, notably via whole-exome sequencing (WES). However, one of the main issues hampering achievement of a diagnosis via WES analyses is the extended list of variants of unknown significance (VUS), mostly composed of missense variants. Hence, improved solutions are needed to address the challenges of identifying potentially deleterious variants and ranking them in a prioritized short list. We present MISTIC (MISsense deleTeriousness predICtor), a new prediction tool based on an original combination of two complementary machine learning algorithms using a soft voting system that integrates 113 missense features, ranging from multi-ethnic minor allele frequencies and evolutionary conservation, to physiochemical and biochemical properties of amino acids. Our approach also uses training sets with a wide spectrum of variant profiles, including both high-confidence positive (deleterious) and negative (benign) variants. Compared to recent state-of-the-art prediction tools in various benchmark tests and independent evaluation scenarios, MISTIC exhibits the best and most consistent performance, notably with the highest AUC value (> 0.95). Importantly, MISTIC maintains its high performance in the specific case of discriminating deleterious variants from benign variants that are rare or population-specific. In a clinical context, MISTIC drastically reduces the list of VUS (<30%) and significantly improves the ranking of "causative" deleterious variants. Pre-computed MISTIC scores for all possible human missense variants are available at http://lbgi.fr/mistic.
| S-EPMC7394404 | biostudies-literature
Project description:Lynch syndrome (LS) predisposes patients to cancer and is caused by germline mutations in the DNA mismatch repair (MMR) genes. Identifying the deleterious mutation, such as a frameshift or nonsense mutation, is important for confirming an LS diagnosis. However, discovery of a missense variant is often inconclusive. The effects of these variants of uncertain significance (VUS) on disease pathogenesis are unclear, though understanding their impact on protein function can help determine their significance. Laboratory functional studies performed to date have been limited by their artificial nature. We report here an in-cellulo functional assay in which we engineered site-specific MSH2 VUS using clustered regularly interspaced short palindromic repeats-Cas9 gene editing in human embryonic stem cells. This approach introduces the variant into the endogenous MSH2 loci, while simultaneously eliminating the wild-type gene. We characterized the impact of the variants on cellular MMR functions including DNA damage response signaling and the repair of DNA microsatellites. We classified the MMR functional capability of eight of 10 VUS providing valuable information for determining their likelihood of being bona fide pathogenic LS variants. This human cell-based assay system for functional testing of MMR gene VUS will facilitate the identification of high-risk LS patients.
| S-EPMC6810757 | biostudies-literature