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.

项目概要/摘要拟议研究的目的是辨别神经胶质瘤风险变异的功能和生物学相关性通过全基因组关联研究（GWAS）确定。 GWAS 发现了 8 个胶质瘤易感位点：8q24.11、11q23.3、5p15.33、9p21.3、20q13.33、7p11.2（2 个独立位点）和 3q26.2。没有一个 GWAS SNP 映射到外显子，我们假设相关的 SNP 是非功能性，与休闲/功能性 SNP 连锁不平衡，并映射到风险增强剂，而风险增强剂反过来以等位基因特异性方式调节靶基因表达。迄今为止，神经胶质瘤尚无功能性/因果性变异 GWAS 已被确定。然而，我们的团队已经为这 8 个基因中的 6 个确定了一些候选靶基因。使用表达数量性状位点 (eQTL) 映射多个脑区域和等位基因特异性的 GWAS 位点基因表达（ASE）分析。基于这些有希望的发现，我们提出了一个全面的帖子使用一系列补充方法对神经胶质瘤风险位点进行 GWAS 分析。在目标 1 中，我们将确定使用来自两个来源的数据确定神经胶质瘤风险位点的候选靶基因：公开的 RNA-Seq 和来自 400 个死后大脑的多个大脑区域的基因型组织表达的基因分型数据项目（GTEx）；我们还将从另外 300 个病理学样本中生成 RNA 测序和基因分型数据来自迈阿密大学脑部的经过验证的新鲜冷冻尸检正常脑组织（多个脑区域）银行（UMBB）。将使用 UMBB 进行 eQTL 作图、eQTL 荟萃分析和 eQTL-ASE 样本作为发现集，GTEx数据集作为验证数据集。在目标 2 中，我们将使用公开可用的 ChIP-Seq 和 DNAse1 超敏染色质数据可识别 GWAS 中的候选调控元件基因座。我们将：（a）使用增强子/启动子荧光素酶载体活性验证候选调控元件在多种神经胶质瘤细胞系中进行测定。 (b) 使用任一方法评估等位基因对增强子/启动子活性的特异性影响定点诱变或自然发生的单倍型（c）。识别并验证新的候选目标通过使用 CRISPR-Cas9 基因编辑敲除风险增强子，在目标 1 中鉴定出风险增强子的基因神经胶质瘤细胞系中的技术并使用 RNA-Seq 评估靶基因表达的变化。在目标 3 中，我们将使用现有数据和通过目标 1 和 2 生成的新数据来识别和验证物理风险增强剂和候选靶基因之间的相互作用。候选风险增强剂的相互作用位点将使用 5 个神经胶质瘤细胞系使用 4C-Seq 进行鉴定，并将与候选目标基因进行比较通过目标 1 和目标 2 确定。任何重要的互动，尤其是与候选人当地和通过目标 1 和 2 确定的远端靶基因将通过 3C 和荧光原位进一步验证杂交（FISH）。通过这些努力，我们将对以下问题产生机械和生物学的理解：神经胶质瘤风险，这将是我们开发预防性疗法转化努力中重要的第一步神经胶质瘤的治疗方法。