Scientific Abstract

Proposal No. IBD-0259
Principal Investigator: David Boone, M.D.
Applicant Organization: The University of Chicago (Illinois, U.S.A.)
Project Title: Functional consequences of autophagy mutations in Crohn’s disease 
Period of Award: December 1, 2008 – February 28, 2010

Genome wide scans (GWS) have implicated a polymorphism (SNP) in the gene ATG16L1 in Crohn’s disease (CD). ATG16L1 is involved in the cellular process of autophagy, thus implicating autophagy in the pathology of CD. The SNP in ATG16L1 falls within the coding sequence of ATG16L1 and is predicted to cause an amino acid change in the C-terminal half of human ATG16L1 protein (T300A). Our objective is to determine the functional consequences of this SNP in ATG16L1. Using somatic cell gene targeting, we will generate human intestinal epithelial cells (IEC) that either lack ATG16L1 or have the T300A mutation engineered into the endogenous gene sequence. This approach will allow us to interrogate the function of ATG16L1 and the effects of the ATG16L1 T300A mutation in autophagy in human cells. Autophagy is a cellular process that involves sequestration of cytosolic material in an autophagosome and destruction of its contents following fusion with the lysosome. Autophagy is thus employed by cells during times of nutrient deprivation to reclaim amino acids from cellular organelles. Autophagy is also involved in the turnover of large proteins or protein complexes, cell death, antigen presentation, and defenses against intracellular pathogens. Alterations in any or all of these processes may account for a functional connection between the ATG16L1 T300A mutation and susceptibility to CD. Our hypothesis is that ATG16L1 is required for autophagy in human cells and that the ATG16L1 T300A mutation alters autophagy and susceptibility of IEC to infection by intracellular bacteria. We will test this by generating ATG16L1+/-, ATG16L1-/-, and ATG16L1∆T300A HCT116 cells by somatic gene targeting and using these cells to address the following specific aims, namely: Determine the requirement for ATG16L1 and the effects of the ATG16L1 mutation on (1) autophagy induced by nutrient deprivation; (2) autophagy regulation by TNFR, TLR and NOD2 ligands; and (3) autophagy in response to IEC infection by intracellular pathogens.

These studies will rapidly provide key information on whether the CD-associated mutation in the human ATG16L1 gene has a functional consequence for autophagy, whether the mutation increases or decreases autophagy in human cells or how autophagy defects might contribute to CD.