DESCRIPTION (provided by applicant): Genome-wide association studies (GWASs) have identified multiple disease (trait) associations. While many variants map to 5' cis-regulatory regions (e.g. enhancers), many also localize to the 3'-untranslated region (UTR), suggesting that post-transcriptional mechanisms such as mRNA stability and translatability may play a key role in disease susceptibility. Supporting such a possibility we show that a disease-modifying haplotype in the CC chemokine ligand 2 (CCL2) 3'UTR is associated with allelic expression imbalance (AEI), increased mRNA stability and increased binding to Hu Antigen R (HuR), a RNA stabilizing protein. Notably this haplotype designated as rs1024611G-rs13900T is associated with a number of inflammatory diseases that are associated with monocyte/macrophage recruitment. Our overarching hypothesis is that functional genetic variation in untranslated regions is associated with altered mRNA stability/translatability which determines gene expression and impacts disease susceptibility and pathogenesis. Specific Aim 1 tests the hypothesis that the disease-influencing rs1024611G-containing allele augments CCL2 mRNA expression via post-transcriptional mechanisms, resulting from enhanced binding of the RNA stabilization protein Hu Antigen R (HuR) to the CCL2 3'-UTR. We will rigorously test the hypothesis that the rs13900T-bearing haplotype is linked to mRNA stability and/or altered mRNA translatability mediated via HuR by AEI analyses, RNA mobility shift assays, allele specific RNA immunoprecipitation, RNA interference, overexpression of RNA binding proteins (RBP) and polysome fractionation in primary macrophages and in isogenic macrophages differentiated from iPSC. Significance: rs1024611G-rs13900T haplotype impacts disease pathogenesis in many inflammatory diseases. Specific Aim #2 extends the concepts of aim 1 and examines the functional (AEI) and post-transcriptional impact (differential binding of RBP, mRNA stability/translatability) of SNPs in the 3'-UTR of genes that by GWAS are highly associated with disease susceptibility. These candidate polymorphisms are selected based on their strong disease associations and high probability of regulatory function. The proposed studies will greatly expand our understanding of post-transcriptional mechanisms mediated through genetic variation in inflammatory genes. Innovation, Significance and Clinical Impact: Identification of functional SNPs remains a primary challenge in post-GWAS era. Hitherto, particular emphasis has been placed on genetic variants in promoters and enhancers to ascribe function for GWAS identified genetic variants. The conceptual idea that disease-influencing SNPs affect disease outcomes through post-transcriptional mechanisms is innovative. Such studies are of high translational value as they will facilitate risk-stratification, early intervenion and development of targeted therapies.

PUBLIC HEALTH RELEVANCE: Genetic variants in the genes that are expressed in immune cells modulate their expression and are often associated with increased susceptibility to several common diseases such as atherosclerosis, Crohn's disease, as well as infectious diseases. In this proposal, we aim to understand the mechanisms by which these diseases associated gene variants alter their expression. Understanding such mechanisms will help in risk stratification and identification of novel targets for therapy.