Scientific Abstract

Proposal No. IBD-0251
Principal Investigator:  Art Petronis, M.D., Ph.D.
Applicant Organization:  Centre for Addiction and Mental Health (Toronto, Ontario, Canada)
Project Title:  Epigenomic studies of twins discordant for Crohn’s disease
Period of Award:  February 1, 2009 – February 28, 2010

Background and rationale: Putative epigenetic misregulation of genes sheds a new light on numerous epidemiological, clinical, and molecular complexities of inflammatory bowel disease. Perfect DNA sequences may be useless or even harmful if not expressed in the appropriate amount, at the right time of the cell cycle, or in the right compartment of the nucleus. Epigenetic modifications of DNA and histones can explain a series of general non-Mendelian features of Crohn’s disease and ulcerative colitis, more so than DNA sequence-based models. Such features include: i) discordance of MZ twins; ii) relatively late age of onset; iii) sexual dimorphism; iv) parental origin effects; v) major fluctuations of the disease course. Apart from the general epigenetic aspects of non-Mendelian irregularities, epigenetic mechanisms may also provide a new perspective on the identification of the molecular effects of environmental and stochastic factors. The epigenetic theory does not reject the role of DNA sequence variation but rather suggests that in complex diseases the contribution of epigenetic factors may be substantial, and that DNA sequence variation should be investigated in parallel with epigenetic regulation. This project is dedicated to a detailed microarray-based epigenetic (DNA methylation) analysis of regulatory regions of the overwhelming majority of protein coding sequences in Crohn’s disease patients and controls.

The main objective is to identify Crohn’s disease specific epigenetic (DNA methylation) differences.

Experimental strategy: Forty Swedish monozygotic and dizygotic twins affected with Crohn’s disease will be compared to i) their unaffected co-twins, and ii) unrelated, unaffected individuals matched for age, sex, and ethnic background. Genomic DNA from three sites of gut (ileum, right colon, and rectum) of all 40 trios of co-twins plus unrelated controls will be subjected to enrichment of unmethylated DNA fraction using methylation sensitive restriction enzymes, adaptor ligation and selective amplification, and interrogated on the Affymetrix microarrays that comprise of over 4.6 million probes tiled through over 25,500 human promoter regions. The microarray data will be analyzed using a battery of bioinformatics tools. Fine characterization of the differentially methylated region(s) will be verified using the bisulfite mapping of modified cytosines using new samples of gut biopsies, peripheral blood leukocytes, and germline (total N= 180 DNA samples).

Innovation and importance: We provide a new interpretation of epidemiological, clinical, and molecular features of inflammatory bowel disease from the epiG point of view. We have also developed a high throughput microarray-based epiG profiling that is a new technology in human morbid biology. Application of this powerful technology to the unique sample of identical and nonidentical twins who are discordant for Crohn’s disease may lead to understanding of fundamental mechanisms of inflammatory bowel disease. The detailed analysis of the promoter in Crohn’s disease would be one of the first large scale projects of this kind in gastroenterological research. Given the relatively slow progress in the traditional genetic and environmental studies, there is little doubt that epigenetics will become a new field of activities in basic and clinical gastroenterology.