Functional Characterization of Glioma GWAS Variants

项目摘要/摘要 这项拟议的研究的目标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导致了8个 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)和 3q26.2。没有一个GWASNPs映射到外显子，我们假设相关的SNPs是非 功能性，与偶然/功能性SNP连锁不平衡，并映射到风险增强剂，进而 以等位基因特异性的方式调节目标基因的表达。到目前为止，还没有脑胶质瘤的功能性/因果变异 已经确定了GWAs。然而，我们的团队已经为8个基因中的6个确定了一些候选靶基因 利用表达数量性状基因座(EQTL)定位多个脑区和等位基因特异性的GWASs基因座 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职位 使用一系列互补的方法对胶质瘤危险基因进行分析。在目标1中，我们将确定 使用两个来源的数据作为胶质瘤风险基因的候选靶基因：公开可用的RNA-Seq和 400例死后脑多个脑区的基因分型数据--组织表达 项目(GTEx)；我们还将从另外300个病理数据中生成RNA-Seq和基因分型数据 来自迈阿密大学大脑的经过验证的新鲜冷冻的正常脑组织(多个脑区) 银行(UMBB)。将使用UMBB进行eQTL定位、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Aim 2中，我们将使用公开可用的 ChIP-Seq和DNase1超敏染色质数据，以确定GWAS内的候选调控元件 精神错乱。我们将：(A)使用增强子/启动子荧光素酶载体活性来验证候选调控元件 在多个胶质瘤细胞系中进行检测。(B)评估等位基因特异性对增强子/启动子活性的影响 定点突变或自然发生的单倍型(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中确定的风险增强子的基因 技术在胶质瘤细胞系中的应用，并使用RNA-Seq评估靶基因表达的变化。在《目标3》中我们 将使用通过目标1和2生成的现有数据和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基因座将 使用5个胶质瘤细胞系使用4C-Seq进行鉴定，并将与候选靶基因进行比较 通过目标1和目标2确定。任何重要的交互，特别是与候选本地和 通过AIMS 1和AIMS 2确定的远端靶基因将进一步通过3C和荧光原位验证 杂交(鱼)。通过这些努力，我们将从机械和生物学的角度理解 神经胶质瘤风险将是我们开发预防性治疗方法的翻译努力的重要第一步 神经胶质瘤的手术入路。

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.