Using Integrative Genomics To Identify and Characterize Emphysema-Associated eQTL

项目摘要：慢性阻塞性肺疾病(COPD)是一种进行性、衰弱的疾病 迫切需要哪种新的、治疗疾病的方法。由于人类支持的药物靶点 遗传证据更有可能导致FDA批准的治疗方法，基因组的功能特征- 广联研究(GWAS)基因座是翻译研究的重点。我们小组发挥了带头作用 在COPD Gwas中，迄今为止最大的COPD Gwas已经确定了82个显著基因座，其中大多数具有 没有在功能上被定性。在这个项目的第一阶段，我们结合了肺气肿GWAS的结果 通过对表达数量性状基因座(EQTL)的研究，采用GWAS-eQTL共定位的方法对 COPD GAs为靶基因。这种方法使我们能够优先考虑TGFB2和ACVR1B进行功能研究 在呼吸道上皮细胞和肺成纤维细胞中发现了这些基因座的功能变异。但是，&gt；50% 部分由于固有的限制，COPD的Gwas基因座还没有表现出与eQTL的强烈共定位 EQTL依赖于基因水平的表达量化，而不反映替代的影响 拼接。选择性剪接是Gwas基因座的一种重要功能机制，可能与 与eQTL一样重要。在这个项目的下一阶段，我们建议识别与COPD相关的新基因 改变剪接的变体(SQTL)及其异构体特异性效应的特征。在目标1中，我们将表演 利用两个RNA-seq资源在全基因组范围内发现剪接QTL(SQTL) COPD基因研究中4515名受试者的血液RNA-seq和肺RNA-seq的受试者 肺组织研究联盟(LTRC)的1078名受试者。使用协同定位方法，我们将 鉴定COPD相关基因变异体改变其剪接的新的COPD Gwas靶基因。在……里面 目的2，我们将识别COPD患者差异表达和差异使用的异构体。 来自LTRC中1,078例COPD患者和对照的短读取肺组织RNA-seq的异构体定量。 然后，我们将生成牛津纳米孔技术(ONT)针对10个COPD Gwas基因的长读RNA-seq 在80名患有COPD的LTRC受试者和80名对照组中，使用靶向浓缩方法。在《目标3》中，我们将 结合精细作图和功能研究确定改变初级剪接的COPD GWA变异体 肺细胞。首先，我们将使用靶向长读rna-seq来表征细胞类型的特定异构体。 四种原代肺细胞的COPD GWA靶基因图谱。然后我们将从功能上验证罚款- 使用剪接报告分析绘制了COPD Gwas变异体，我们将表征这些变异体的影响 按Marsico基因选择的呼吸道上皮细胞中COPD相关细胞表型的变异 肺研究所细胞库。我们的多学科研究团队在慢性阻塞性肺疾病遗传学方面拥有必要的专业知识 而基因组学、分子生物学、长读测序、剪接和RNA生物学都是完成这一重要任务的关键 该项目旨在确定与选择性剪接有关的新的COPD Gwas靶基因。

Project Summary: Chronic obstructive pulmonary disease (COPD) is a progressive, debilitating disease for which new, disease-modifying treatments are desperately needed. Since drug targets supported by human genetic evidence are more likely to lead to FDA-approved treatments, functional characterization of genome- wide association study (GWAS) loci is a translational research priority. Our group has played a leading role in COPD GWAS, and the largest COPD GWAS to date has identified 82 significant loci, most of which have not been functionally characterized. In the first phase of this project, we combined emphysema GWAS results with expression quantitative trait locus (eQTL) studies, using GWAS-eQTL colocalization methods to identify COPD GWAS target genes. This approach allowed us to prioritize TGFB2 and ACVR1B for functional studies in airway epithelial cells and lung fibroblasts that identified functional variants in these loci. However, >50% of COPD GWAS loci have not yet shown strong colocalization with eQTLs, due in part to inherent limitations of eQTLs which depend on gene-level expression quantifications that do not reflect effects of alternative splicing. Alternative splicing is an important functional mechanism for GWAS loci, perhaps equally as important as eQTLs. In the next phase of this project, we propose to identify novel COPD-associated genetic variants that alter splicing (sQTLs) and characterize their isoform-specific effects. In Aim 1, we will perform genome-wide discovery of splicing QTLs (sQTLs) using two RNA-seq resources with large numbers of subjects with COPD – blood RNA-seq from 4,515 subjects in the COPDGene Study and lung RNA-seq from 1,078 subjects in the Lung Tissue Research Consortium (LTRC). Using colocalization methods, we will identify novel COPD GWAS target genes whose splicing is altered by COPD-associated genetic variants. In Aim 2, we will identify differentially expressed and differentially used isoforms in COPD using estimated isoform quantifications from short read lung tissue RNA-seq in 1,078 COPD cases and controls in the LTRC. We will then generate Oxford Nanopore Technologies (ONT) long read RNA-seq for 10 COPD GWAS genes in 80 LTRC subjects with COPD and 80 controls using a targeted enrichment approach. In Aim 3, we will combine fine mapping and functional studies to identify COPD GWAS variants that alter splicing in primary lung cells. First, we will use targeted long read RNA-seq to characterize the cell-type specific isoform profiles of COPD GWAS target genes in four primary lung cell types. We will then functionally validate fine- mapped COPD GWAS variants using splicing reporter assays, and we will characterize the effects of these variants on COPD-related cellular phenotypes in airway epithelial cells selected by genotype from the Marsico Lung Institute cell bank. Our multi-disciplinary research team has the requisite expertise in COPD genetics and genomics, molecular biology, long read sequencing, splicing and RNA biology to complete this important project to identify novel COPD GWAS target genes involved in alternative splicing.