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Functional Characterization of Glioma GWAS Variants

胶质瘤 GWAS 变异体的功能表征

基本信息

批准号：
9336270
负责人：
GRAHAM CASEY
金额：
$ 64.9万
依托单位：
UNIVERSITY OF SOUTHERN CALIFORNIA
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-08-23 至 2020-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9336270
关键词：
11q23 20q13 3q26 5p15.33 8q24 9p21 9q21 Alleles Autopsy Biological Biological Assay Biology Brain Brain region CDKN2A gene Candidate Disease Gene ChIP-seq Chromatin Data Data Set Development Distal Enhancers Epidermal Growth Factor Receptor Event Exons Fluorescent in Situ Hybridization Freezing Future Gene Expression Gene Expression Profiling Gene Targeting Genes Genome Genotype Genotype-Tissue Expression Project Glioma Gliomagenesis Goals Guidelines Haplotypes Hypersensitivity Incidence Inherited Intervention Knock-out Linkage Disequilibrium Luciferases Malignant Neoplasms Maps Meta-Analysis Methods Molecular Molecular Conformation Nucleic Acid Regulatory Sequences Pathologic Preventive Quantitative Trait Loci Regulatory Element Research Risk Role Sampling Series Site-Directed Mutagenesis Source Susceptibility Gene TNFRSF6B gene Technology Therapeutic Therapeutic Intervention Translating Universities Validation Variant base brain tissue experience genome editing genome wide association study genome-wide glioma cell line improved insight novel promoter risk variant success tool transcriptome sequencing vector

项目摘要

PROJECT SUMMARY/ABSTRACT The goal of the proposed study is to discern the functional and biological relevance of gliomas risk variants identified through genome wide association studies (GWAS). GWAS have led to the discovery of 8 susceptibility loci in glioma: 8q24.11, 11q23.3, 5p15.33, 9p21.3, 20q13.33, 7p11.2 (2 independent loci) and 3q26.2. None of the GWAS SNPs map to exons and we hypothesize that the associated SNPs are non- functional, are in linkage disequilibrium with casual/functional SNPs, and map to risk enhancers that in turn regulate target gene expression in an allele specific manner. To date no functional/causal variants for glioma GWAS have been identified. However, our team has identified some candidate target genes for 6 of the 8 GWAS loci using expression quantitative trait loci (eQTL) mapping in multiple brain regions and allelic specific gene expression (ASE) analyses. Based on these promising findings, we propose a comprehensive post GWAS analysis of glioma risk loci using a series of complementary approaches. In Aim 1 we will identify candidate target genes of glioma risk loci using data from two sources: publicly available RNA-Seq and genotyping data from multiple brain regions of 400 post-mortem brains of the Genotype-Tissue Expression Project's (GTEx); we will also generate RNA-Seq and genotyping data from an additional 300 pathologically verified, fresh-frozen autopsied normal brain tissues (multiple brain regions) from the University of Miami Brain Bank (UMBB). eQTL mapping, eQTL meta-analyses and eQTL-ASE will be performed, using the UMBB samples as the discovery set and GTEx dataset as a validation dataset. In Aim 2 we will use publicly available ChIP-Seq and DNAse1 hypersensitivity chromatin data to identify candidate regulatory elements within GWAS loci. We will: (a) validate candidate regulatory elements using enhancer/promoter luciferase vector activity assays in multiple glioma cell lines. (b) Assess allele specific effects on enhancer/promoter activity using either site-directed mutagenesis or naturally occurring haplotypes (c). Identify and validate novel candidate target genes of risk enhancers identified in Aim 1 by knocking out risk enhancers using CRISPR-Cas9 gene editing technology in glioma cell lines and assessing changes in target gene expression using RNA-Seq. In Aim 3 we will use existing data and novel data generated through Aims 1 and 2, to identify and validate the physical interaction between risk enhancers and candidate target genes. Interacting loci of candidate risk enhancers will be identified using 4C-Seq using five glioma cell lines, and will be compared to candidate target genes identified through Aims 1 and Aim 2. Any significant interactions, especially those with candidate local and distal target genes identified through Aims 1 and 2 will be further validated by 3C and fluorescent in situ hybridization (FISH). Through these efforts we will develop a mechanistic and biological understanding of glioma risk that will be an essential first step in our translational efforts to develop preventive therapeutics approaches for glioma.

项目总结/文摘