High throughput functional studies of IBD-associated GWAS variants

PROJECT SUMMARY/ABSTRACT The Inflammatory bowel diseases (IBD), Crohn’s disease (CD) and ulcerative colitis (UC), are chronic inflammatory diseases of the gastrointestinal tract with no cure. Genome-wide association studies (GWAS) have found >250 genomic loci associated with IBD, but variant contributions to mechanisms driving IBD pathogenesis and disease prognosis remain unclear. Each locus typically contains tens to hundreds of variants, the vast majority of which are in non-coding regions suggesting a role in gene regulation. For most loci, the causal variant, the affected regulatory element, and the target gene being regulated are unknown. We hypothesize that regulatory variants contribute to IBD phenotypes by altering gene transcriptional programs driving phenotypic heterogeneity. We propose to identify putative casual regulatory variants using two, orthogonal, high-throughput analyses. Aim 1: Regulatory quantitative trait loci (QTL) for chromatin accessibility (caQTL) and transcription factor binding (tfQTL) associate genetic variation with alterations in regulatory activity. For variants in GWAS loci, these analyses will identify regulatory variants with potential contributions to regulation in disease-relevant cell types and tissues. Aim 2: Alternatively, massively parallel reporter assays (MPRA) systematically interrogate allelic effects on transcriptional regulation of thousands of genetic variants. MPRA using vectors of human DNA regulatory elements containing IBD associated variants of interest can be performed in mouse cells or organs due to the well-established conservation of transcription factor motifs between human and mouse. We will use MPRA to determine variants that alter regulatory activity in colon, ileum, and mesenteric lymph nodes under both normal and LPS-stimulated inflammatory states. Aim 3: Integrating results from QTL and MPRA assays, we will select high confidence putative IBD regulatory variants for intestinal epithelial cell focused functional validation in patient derived 2D intestinal monolayer systems. The long-term goals of this project are: 1) To fill the gap between our ability to detect genetic, gene regulatory, and gene expression variation linked to IBD and our ability to explain how that variation ultimately contributes to IBD; and 2) To provide a unique data resource for IBD investigators to access for their own studies.