Scientific Abstract

Proposal No.  IBD-0132R
Principal Investigator:  Jean-Pierre Hugot, M.D., Ph.D.
Applicant Organization:  Naturalia et Biologia (Paris, France)
Project Title:  Identification and genetic analysis of the genes coding for CARD15 interacting proteins
Period of Award:  February 1, 2005 – October 31, 2006

CARD15/NOD2 is the best known inflammatory bowel disease (IBD) susceptibility gene.  It plays a role in Crohn’s disease (CD) and explains about 20% of the genetic predisposition to the disease.  The CARD15/NOD2 protein defines a specific pathway by which the muramyl dipeptide (MDP), a bacterial component, is able to induce the pro-inflammatory NF-κB system.  CARD15/ NOD2 mutations associated with CD are characterized by a defect of this pathway:  the MDP is not able to activate NF-κB.  A better understanding of the disease mechanisms requires a better understanding of the other components of the CARD15/NOD2 pathway, including its regulatory proteins.

We have initiated a yeast two hybrid (Y2H) procedure to identify proteins interacting physically with CARD15/NOD2.  Based on the result of this Y2H screen, we are now exploring the function of the 14 new identified CARD15/NOD2 interacting proteins and on the other hand, testing their respective genes as new candidate for IBD predisposition using a combined approach.

A first set of functional experiments including co-immunoprecipitation, pull down and co-localization experiments is designed to confirm the physical interaction between CARD15/ NOD2 and these new proteins.  Then, using transient transfection experiments, we will analyze the role of these proteins on the CARD15/NOD2 NF-κB pathway.  Finally, the interaction of these new proteins will also be tested with the CARD15/NOD2 mutated proteins by complementary Y2H procedures and other functional analyses in CARD15 knockout mice or in cell lines stably expressing wt or mutated CARD15 proteins.  As a result, we expect to obtain a better understanding of the disease mechanisms and to provide new targets for therapeutic interventions.

Simultaneously, the 14 genes encoding CARD15/NOD2 partners will be tested as new candidate genes for IBD.  Genetic polymorphisms will be searched on a panel of CD and ulcerative colitis (UC) patients.  The nonsynonymous identified polymorphisms will then be tested on a cohort of more than 1000 Caucasian IBD families looking for transmission disequilibrium of a specific allele.  In the case of a positive association, the gene(s) will be further explored by defining the mutational spectrum of the gene and analyzing the genotype/phenotype relationship and the interaction between CARD15/NOD2 and these new genes.

This work will thus help improve our understanding of the genetic predisposition to IBD and disease mechanisms.  Thus, it is expected to contribute to both diagnostic and therapeutic development for IBD.