We propose the dissection the function of Genes and genetic variants discovered in Crohn's disease (CD) using systems approaches. Recent linkage and association studies of CD have conclusively identified a number of genetic risk factors including CARD15, IBD5, IL23R, ATG16L1, IRGM, MST1 and NKX2.3. Among the novel genetic discoveries, ATG16L1 and IRGM are central regulators of autophagy, identifying a key role in disease pathogenesis for this previously unsuspected pathway. As part of the preliminary studies for this proposal we have demonstrated that ATG16L1 is essential for Classical and Salmonella induced autophagy. We also demonstrate that a 20- kilobase insertion/deletion polymorphism upstream of IRGM is associated with altered IRGM expression and is tightly linked to CD associated SNPs. These findings provide the first example of a structural variant influencing disease risk via expression of a nearby gene. We will integrate all available GWAS data, discover specifically causal coding and regulatory variants via high throughput sequencing, define the impact of these variants on gene expression (locally and globally), and assemble the set of variants into a systems level model involving potential genetic interactions on their influence on risk or gene expression. Given the involvement of two genes central to the autophagy response, we propose to apply systems approaches, synthetic biology and unique genetic tools to dissect mechanisms involved in the pathogenesis of CD. This proposal probes the function or consequences of, and pathways that interact with, human mutations that have been validated as playing a causal role in Crohn's disease. The broad goals of this proposal will pursued through studies of three specific aims:
Aim 1 : To define specific variants associated with Crohn's disease suitable for functional analysis.
Aim 2 : To identify small molecules that cause different cellular effects in epithelial cells and lymphoblastoid cells bearing mutations that are implicated in Crohn's disease.
Aim 3 : To delineate proteins required for ATG16L1 and IRGM autophagy response to bacteria using RNAi loss of function analysis
Crohn's disease genetics has made dramatic progress in the past year with more than ten new genes identified using genome-wide association approaches. In order to translate these findings into a functional understanding of the biology of disease, we propose detailed functional evaluation of the impact of these associated variants on protein coding and gene expression and the biological impact of these changes in the colon and immune system. Through use of chemical screens and specific gene knockdown experiments, we will uncover the fundamental biological changes associated with these mutations and explore which may be therapeutically reversible.
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