Using Integrative Genomics To Identify and Characterize Emphysema-Associated eQTL

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.

Grant Number: 5R01HL124233-09
NIH Institute/Center: NIH
Principal Investigator: Peter Castaldi