Functional Characterization of Glioma GWAS Variants

项目总结/摘要 这项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）鉴定。GWAS已经发现了8个 脑胶质瘤易感基因座：8q24.11、11q23.3、5p15.33、9p21.3、20q13.33、7p11.2（2个独立基因座）， 3q26.2.没有GWAS单核苷酸多态性映射到外显子，我们假设相关的单核苷酸多态性是非- 功能性，与偶然/功能性SNP连锁不平衡，并映射到风险增强子， 以等位基因特异性方式调节靶基因表达。迄今为止，没有神经胶质瘤的功能性/因果性变体 GWAS已被识别。然而，我们的团队已经为8个中的6个确定了一些候选靶基因 GWAS基因座使用多个脑区域中的表达数量性状基因座（eQTL）作图和等位基因特异性 基因表达（ASE）分析。基于这些令人鼓舞的发现，我们提出了一个全面的职位， 使用一系列互补方法的胶质瘤风险位点的GWAS分析。在目标1中，我们将确定 神经胶质瘤风险基因座的候选靶基因，使用来自两个来源的数据：公开可用的RNA-Seq和 来自400个死后脑的多个脑区域的基因型-组织表达的基因型数据 项目的（GTEx）;我们还将从另外300个病理学 来自迈阿密大学的经验证的新鲜冷冻尸检正常脑组织（多个脑区） Bank（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DNAse 1超敏染色质数据用于鉴定GWAS中的候选调控元件 的位点我们将：（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的测定。(b)评估等位基因对增强子/启动子活性的特异性影响， 定点诱变或天然存在的单倍型（c）。识别和验证新的候选目标 通过使用CRISPR-Cas9基因编辑敲除风险增强子在Aim 1中鉴定的风险增强子基因 技术在胶质瘤细胞系中的应用，并使用RNA-Seq.在目标3中， 将使用现有数据和通过目标1和2产生的新数据，以确定和验证物理 风险增强子和候选靶基因之间的相互作用。候选风险增强子的相互作用基因座将 使用4C-Seq使用五种胶质瘤细胞系进行鉴定，并将其与候选靶基因进行比较 通过目标1和目标2确定。任何重要的互动，特别是与当地候选人和 通过目标1和2鉴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，我们将发展一个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治疗方法的重要第一步 神经胶质瘤的治疗方法

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.