Massively parallel phenotyping of coding variants in cancer with Perturb-seq
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE161824 | GEO | 2021/10/01
REPOSITORIES: GEO
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Project description:Recent genome sequencing efforts have identified millions of somatic mutations in cancer. However, the functional impact of most variants is poorly understood. Here we characterize 194 somatic mutations identified in primary lung adenocarcinomas using L1000 high-throughput gene-expression assays followed by expression-based variant impact phenotyping (eVIP), a method that uses gene expression changes to distinguish impactful from netural somatic mutations. This series represents the main experiment of the study where 8 replicates of wild-type and mutant ORFs are introduced into A549 cell lines.
2016-06-28 | GSE83744 | GEO
Project description:Recent genome sequencing efforts have identified millions of somatic mutations in cancer. However, the functional impact of most variants is poorly understood. Here we characterize 194 somatic mutations identified in primary lung adenocarcinomas using L1000 high-throughput gene-expression assays followed by expression-based variant impact phenotyping (eVIP), a method that uses gene expression changes to distinguish impactful from netural somatic mutations. This series represents the main experiment of the study where 8 replicates of wild-type and mutant ORFs are introduced into A549 cell lines. An ORF library containing wild-type and mutated versions of genes found to be mutated in lung cancer are introduced in A549 cell lines and measurements are made using the L1000 high-throughput gene-expression assay. These are done with 8 replicate experiemnts. The data are processed through a computational system, that converts raw fluorescence intensities into differential gene expression signatures. The data at each stage of the pre-processing are available: (LXB) - raw, unprocessed flow cytometry data from Luminex scanners. One LXB file is generated for each well of a 384-well plate, and each file contains a fluorescence intensity value for every observed analyte in the well. (Q2NORM) - gene expression profiles of directly measured landmark transcripts. Normalized using invariant set scaling followed by quantile normalization. (Z-SCORES) - signatures with differentially expressed genes computed by robust z-scores for each profile relative to control (relative to plate population as control)
2016-06-28 | E-GEOD-83744 | biostudies-arrayexpress
Project description:While advancements in genome sequencing have identified millions of somatic mutations in cancer, their functional impact is poorly understood. We previously developed the expression-based variant impact phenotyping (eVIP) method to use gene expression data to characterize the function of gene variants. The eVIP method uses a decision tree-based algorithm to predict the functional impact of somatic variants by comparing gene expression signatures induced by introduction of wild-type versus mutant cDNAs in cell lines. The method distinguishes between variants that are gain-of-function, loss-of-function, change-of-function, or neutral. We present eVIP2, software that allows for pathway analysis (eVIP Pathways) and usage with RNA-seq data. To demonstrate the eVIP2 software and approach, we characterized two recurrent frameshift variants in RNF43, a negative regulator of Wnt signaling, frequently mutated in colorectal, gastric and endometrial cancer. RNF43 WT, RNF43 R117fs, RNF43 G659fs, or GFP control cDNA were overexpressed in HEK293T cells. Analysis with eVIP2 predicted that the frameshift at position 117 was a loss-of-function mutation, as expected. The second frameshift at position 659, was, surprisingly, predicted to be a gain-of-function mutation. Additional eVIP Pathways analysis of RNF43 G659fs predicted 10 pathways to be significantly altered, including TNF alpha via NFKB signaling, KRAS signaling, and hypoxia. To validate these predictions, we performed reporter assays and found that all eVIP2 impactful pathways tested in the assay were activated by expression of RNF43 G659fs, but not by expression of RNF43 WT, supporting that RNF43 G659fs is a gain-of-function mutation and its effect on the identified pathways. The eVIP2 method is an important step towards overcoming the current challenge of variant interpretation in the implementation of precision medicine. eVIP2 is available at https://github.com/BrooksLabUCSC/eVIP2
2019-12-13 | GSE141963 | GEO
Project description:This SuperSeries is composed of the following subset Series: GSE28239: Identification and functional impact of genomic copy number variants in zebrafish, an important human disease model (Zebrafish Strain CNVs) (expression array) GSE28276: Identification and functional impact of genomic copy number variants in zebrafish, an important human disease model (Zebrafish Strain CNVs) (CGH ZV81M) GSE28278: Identification and functional impact of genomic copy number variants in zebrafish, an important human disease model (Zebrafish Strain CNVs) (CGH ZV81M 2) GSE33962: Extensive genetic diversity and substructuring among zebrafish strains revealed through copy number variant analysis (CGH) Refer to individual Series
2011-12-27 | E-GEOD-28328 | biostudies-arrayexpress
Project description:Background: The identification of causal variants responsible for disease associations from genome-wide association studies (GWAS) facilitates functional understanding of the disease mechanisms implicated by GWAS. One of the earliest GWAS associations to COPD spans an intragenic region within FAM13A, but the causal variants at this loci have not yet been identified. Massively parallel reporter assays (MPRA) can be used to prioritize functional regulatory variants in a high-throughput manner. Methods: We used an integrated approach using fine-mapping in over 10,000 subjects from COPD GWAS studies, two MPRA experiments, traditional reporter assays, chromatin conformation capture, and CRISPR-based gene editing to characterize COPD-associated regulatory variants in FAM13A in human bronchial epithelial cell lines. Results: Conditional genetic association and fine mapping analyses identified two independent COPD association signals in FAM13A. MPRA identified 45 common functional regulatory variants, and six COPD-associated putative functional variants were prioritized for further functional investigation. Three variants demonstrated significant activity in traditional reporter assays, and one variant, rs2013701, was selected for further testing in the endogenous genomic context based on a direction of effect consistent with postulated mechanisms of FAM13A-mediated COPD susceptibility. CRISPR-based genome editing for this variant confirmed allele-specific effects on FAM13A expression and altered rates of cellular proliferation, providing multiple levels of functional characterization for this COPD-associated variant. Conclusions: Comprehensive screening for regulatory variants near FAM13A identified the presence of extensive functional regulatory variation within a 250kb window of FAM13A in HBECs. Focused functional evaluation of the COPD-associated functional variants in LD with the two independent association signals in this region prioritized the common variant rs2013701, for which multiple parallel lines of functional evidence confirm allelic effects on FAM13A regulation.
2019-01-19 | GSE109452 | GEO
Project description:Delineating functionally normal variants from functionally abnormal variants in tumor suppressor proteins is critical for cancer surveillance, prognosis, and treatment options. BRCA1 is a protein that has many variants of uncertain significance which are not yet classified as functionally normal or abnormal. In vitro functional assays can be used to identify the functional impact of a variant when the variant has not yet been categorized through clinical observation. Here we employ a homology-directed repair (HDR) reporter assay to evaluate over 300 missense and nonsense BRCA1 variants between amino acid residues 1280 and 1576, which encompasses the coiled-coil and serine cluster domains. Functionally abnormal variants tended to cluster in residues known to interact with PALB2, which is critical for homology-directed repair. Multiplexed results were confirmed by singleton assay and by ClinVar database variant interpretations. Comparison of multiplexed results to designated benign or likely benign or pathogenic or likely pathogenic variants in the ClinVar database yielded 100% specificity and 100% sensitivity of the multiplexed assay. Clinicians can reference the results of this functional assay for help in guiding cancer treatment and surveillance options. These results are the first to evaluate this domain of BRCA1 using a multiplexed approach and indicate the importance of this domain in the DNA repair process.
2023-10-13 | GSE237142 | GEO
Project description:Mutational inactivation of TP53 is a common event in cancer. Germline mutations in TP53 that inactivate this protein also occur in Li Fraumeni syndrome, which predisposes to early-onset cancer. In addition, there are dozens of other germline variants in TP53 that do not completely inactivate the function of this protein. In many cases studies have shown strong support for an impact of these lesser-functioning hypomorphs with increased cancer risk in humans and mouse models; however, the majority of these hypomorphs have yet to be categorized as pathogenic in clinical genetics databases. There is thus need for a functional assay to distinguish lesser-functioning hypomorphic p53 variants from wild type p53, or benign, fully-functional, variants. We report the surprising finding that two different African-centric genetic hypomorphs of p53, which occur in distinct functional domains of the protein, share common activities. We show that the Pro47Ser variant in the transactivation domain and the Tyr107His variant in the DNA binding domain both share increased propensity to misfold into a conformation specific for mutant p53. Moreover, cells and tissues with these variants show increased NF-B activity. We have identified a common gene signature from unstressed lymphocyte cell lines that is shared between these two, and other, genetic missense hypomorphs of TP53. We show that this gene signature successfully distinguishes wild type p53 and a benign p53 variant from lesser-functioning hypomorphic variants. These findings should allow us to better understand how hypomorphic variants contribute to cancer risk, and to better inform cancer risk in hypomorph carriers.
2023-03-01 | GSE209837 | GEO
Project description:The most recent genome-wide association study (GWAS) of cutaneous melanoma identified 54 risk-associated loci, but functional variants and their target genes for most have not been established. Here, we performed massively parallel reporter assays (MPRA) using malignant melanoma and normal melanocyte cells and further integrated multi-layer annotation to systematically prioritize functional variants and susceptibility genes from these GWAS loci. Of 1,992 risk-associated variants tested in MPRA, we identified 285 from 42 loci (78% of the known loci) displaying significant allelic transcriptional activities in either cell type (FDR < 1%). We further characterized MPRA-significant variants by motif prediction, epigenomic annotation, and statistical/functional fine-mapping to create integrative variant scores, which prioritized one to six plausible candidate variants per locus for the 42 loci and nominated a single variant for 43% of these loci. Overlaying the MPRA-significant variants with genome-wide significant expression or methylation quantitative trait loci (QTLs) from melanocytes or melanomas identified candidate susceptibility genes for 60% of variants (172 of 285 variants). CRISPRi of top-scoring variants validated their cis-regulatory effect on the eQTL target genes, MAFF (22q13.1) and GPRC5A (12p13.1). Finally, we identified 36 melanoma-specific and 45 melanocyte-specific MPRA-significant variants, a subset of which are linked to cell-type-specific target genes. Analyses of transcription factor availability in MPRA datasets and variant-transcription factor interaction in eQTL datasets highlighted the roles of transcription factors in cell-type-specific variant functionality. In conclusion, MPRA along with variant scoring effectively prioritized plausible candidates for most melanoma GWAS loci and highlighted cellular contexts where the susceptibility variants are functional.
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Project description:The most recent genome-wide association study (GWAS) of cutaneous melanoma identified 54 risk-associated loci, but functional variants and their target genes for most have not been established. Here, we performed massively parallel reporter assays (MPRA) using malignant melanoma and normal melanocyte cells and further integrated multi-layer annotation to systematically prioritize functional variants and susceptibility genes from these GWAS loci. Of 1,992 risk-associated variants tested in MPRA, we identified 285 from 42 loci (78% of the known loci) displaying significant allelic transcriptional activities in either cell type (FDR < 1%). We further characterized MPRA-significant variants by motif prediction, epigenomic annotation, and statistical/functional fine-mapping to create integrative variant scores, which prioritized one to six plausible candidate variants per locus for the 42 loci and nominated a single variant for 43% of these loci. Overlaying the MPRA-significant variants with genome-wide significant expression or methylation quantitative trait loci (QTLs) from melanocytes or melanomas identified candidate susceptibility genes for 60% of variants (172 of 285 variants). CRISPRi of top-scoring variants validated their cis-regulatory effect on the eQTL target genes, MAFF (22q13.1) and GPRC5A (12p13.1). Finally, we identified 36 melanoma-specific and 45 melanocyte-specific MPRA-significant variants, a subset of which are linked to cell-type-specific target genes. Analyses of transcription factor availability in MPRA datasets and variant-transcription factor interaction in eQTL datasets highlighted the roles of transcription factors in cell-type-specific variant functionality. In conclusion, MPRA along with variant scoring effectively prioritized plausible candidates for most melanoma GWAS loci and highlighted cellular contexts where the susceptibility variants are functional.
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