Using Integrative Genomics To Identify and Characterize Emphysema-Associated eQTL

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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.