Using Integrative Genomics To Identify and Characterize Emphysema-Associated eQTL

项目概述：慢性阻塞性肺疾病（COPD）是一种进行性、使人衰弱的疾病， 哪些新的疾病治疗方法是迫切需要的。由于药物靶点由人类支持， 基因证据更有可能导致FDA批准的治疗，基因组的功能表征， 广泛关联研究（GWAS）基因座是翻译研究的优先事项。我们小组起了带头作用 在COPD GWAS中，迄今为止最大的COPD GWAS已经确定了82个重要位点，其中大多数具有 没有功能特征。在该项目的第一阶段，我们结合了肺气肿GWAS结果， 与表达数量性状基因座（eQTL）的研究，采用GWAS-eQTL共定位方法， COPD GWAS靶基因。这种方法使我们能够优先考虑TGFB 2和ACVR 1B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。然而，>50% 的COPD GWAS位点尚未显示出与eQTL的强共定位，部分原因是固有的局限性 依赖于基因水平表达定量的eQTL，不反映替代效应。 拼接选择性剪接是GWAS基因座的一种重要功能机制，可能与 重要的eQTL。在该项目的下一阶段，我们建议鉴定新的COPD相关遗传学。 改变剪接的变体（sQTL）并表征其异构体特异性效应。在目标1中，我们将 使用两个具有大量的RNA序列的RNA-seq资源，在全基因组范围内发现剪接QTL（sQTL）。 COPD受试者-来自COPD基因研究中4，515名受试者的血液RNA-seq和来自 肺组织研究联盟（LTRC）中的1，078例受试者。使用colocalization方法，我们将 鉴定新COPD GWAS靶基因，其剪接被COPD相关遗传变异体改变。在 目的2，我们将通过评估COPD中差异表达和差异使用的亚型， 来自LTRC中1，078例COPD病例和对照的短读肺组织RNA-seq的同种型定量。 然后，我们将生成10个COPD GWAS基因的Oxford Nanopore Technologies（ONT）长读段RNA-seq 在80名患有COPD的LTRC受试者和80名对照者中使用靶向富集方法。在目标3中，我们 联合收割机精细定位和功能研究，以确定COPD GWAS变异，改变原发性 肺细胞首先，我们将使用靶向长读段RNA-seq来表征细胞类型特异性同种型 在四种主要肺细胞类型中的COPD GWAS靶基因谱。我们将在功能上验证精细- 使用剪接报告基因检测绘制COPD GWAS变异体，我们将描述这些变异体的作用。 通过来自Marsico的基因型选择的气道上皮细胞中COPD相关细胞表型的变体 肺脏研究所细胞库。我们的多学科研究团队在COPD遗传学方面具有必要的专业知识 以及基因组学、分子生物学、长读段测序、剪接和RNA生物学来完成这一重要的 该项目旨在鉴定参与选择性剪接的新型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.