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：
9159686
负责人：
GRAHAM CASEY
金额：
$ 65.67万
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/9159686
关键词：
5p15.33 9p21.3 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 Preventive Quantitative Trait Loci RNA Regulatory Element Research Risk Role Sampling Series Single Nucleotide Polymorphism Map 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.

项目摘要/摘要拟议的研究的目的是辨别神经胶质瘤风险变体的功能和生物学相关性通过基因组广泛的关联研究（GWAS）确定。 GWAS导致发现了8 神经胶质瘤中的敏感位点：8Q24.11，11q23.3，5p15.33，9p21.3，20q13.33，7p11.2（2个独立基因座）和 3Q26.2。 GWAS SNP映射没有到外显子，我们假设相关的SNP是非 - 功能性，与休闲/功能性SNP处于连锁不平衡，并将其映射到风险增强器中以特定于等位基因的方式调节靶基因表达。迄今为止，没有用于神经胶质瘤的功能/因果变异已经确定了GWAS。但是，我们的团队已经确定了8个候选目标基因 GWAS基因座使用表达定量性状基因座（EQTL）映射在多个大脑区域和等位基因特定区域基因表达（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 GWAS分析神经胶质瘤的风险基因座使用一系列补充方法。在目标1中，我们将确定使用来自两个来源的数据，可神经胶质瘤的候选靶基因风险基因座：公开可用的RNA-Seq和来自400个基因型 - 组织表达的400个验尸大脑的多个大脑区域的基因分型数据项目（GTEX）；我们还将从300个病理上生成RNA-seq和基因分型数据经过验证的，新鲜的液体尸检正常脑组织（多个大脑区域）来自迈阿密大学的大脑银行（UMBB）。将使用UMBB执行EQTL映射，EQTL荟萃分析和eqtl-ase 示例作为发现集，将GTEX数据集作为验证数据集。在AIM 2中，我们将使用公开可用 CHIP-SEQ和DNASE1高敏性染色质数据，以识别GWAS中的候选调节元素基因座。我们将：（a）使用增强子/启动子荧光素酶矢量活性验证候选调节元件多神经胶质瘤细胞系的测定。（b）使用任何一个评估对增强子/启动子活动的特定影响位置定向的诱变或天然发生的单倍型（C）。识别和验证新型候选目标 AIM 1中确定的风险增强基因通过使用CRISPR-CAS9基因编辑淘汰风险增强子来确定的风险增强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AIM 1和2生成的现有数据和新数据，以识别和验证物理风险增强子与候选靶基因之间的相互作用。候选风险增强者的互动基因座将使用五个神经胶质瘤细胞系使用4C-SEQ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。任何重大互动，尤其是那些与候选人本地和通过目标1和2鉴定的远端靶基因将通过3C进一步验证3C和原位荧光杂交（鱼）。通过这些努力，我们将对胶质瘤风险将是我们转化努力开发预防性治疗的重要第一步神经胶质瘤的方法。