Final Progress Report
Proposal No. IBD-0055
Principal Investigator: Derek P. Jewell, BM, BCh, DPhil
Applicant Organization: University of Oxford (United Kingdom)
Project Title: Identification of the susceptibility gene for colonic Crohn's disease by targeted positional cloning and microarray expression studies
Period of Award: March 1, 2003 - February 29, 2004
A. Summary of project aims.
The aim of this study is to identify the key genes involved in disease initiation and early inflammation in colonic Crohn’s disease (CD) by combining two molecular approaches, namely gene expression and positional cloning. Using the human ileostomy model, we have determined changes in gene expression in both peripheral blood mononuclear cells and colonic mucosal tissue in the early phase of reactivation of colonic disease using a customized oligonucleotide array. A key element of this study has been analysis of control subjects who have had a loop ileostomy for rectal cancer, thereby allowing the discrimination of changes specific to Crohn’s colitis.
B. Accomplishments towards meeting those aims.
This report details our progress to June 2004 and represents an interim rather than a final report. Patient recruitment to this study has been slower than initially anticipated and we therefore aim to present the results at the BMRP annual investigator meeting in February 2005.
1. Patient recruitment
Inclusion criteria for the patient arm of this study were: UK caucasoids with disease confined to the colon; ileostomy in place for more than six months; and quiescent disease for more than three months. Between October 2002 and June 2004, a total of 39 Crohn’s disease patients with a loop ileostomy were identified from the Oxford IBD database. Twenty-three patients were unsuitable for recruitment (Figure 1). Letters of invitation were sent to the remaining 16 patients and nine agreed to participate. To date, seven patients with Crohn’s disease have participated, of whom five have successfully completed the study. Two patients were unable to complete the study because we were unable to access the right colon via the efferent limb of the ileostomy. Two patients who had stomas formed earlier this year are booked for study in July and August 2004
Control subjects were ethnically matched individuals who had a defunctioning loop ileostomy for non-inflammatory bowel disease (rectal cancer or intestinal dysmotility). Letters of invitation were sent to 39 patients and six agreed to participate. To date, five control patients have participated, of which four have successfully completed the study. One patient was unable to complete the study because of unacceptable discomfort during the procedure. One further patient who had a stoma formed earlier this year is booked for study in August 2004.
Patient recruitment has been more difficult than initially anticipated. The time taken to recruit patients in the Crohn’s disease arm of this study is a consequence of the decline in number of patients who have had loop ileostomies formed for the management of refractory colonic Crohn’s disease. This reflects the increased use of anti-TNF monoclonal antibodies over this period. A further factor hindering recruitment has been the recent change in surgical practice to bury the distal limb of the defunctioning loop in the subcutaneous tissues, thereby limiting colonoscopic access. Recruitment of the control cohort has also been difficult, mainly because individuals without a history of inflammatory bowel disease have been reluctant to participate in a study which involves handling of fecal material. The delay in patient recruitment has necessitated extension of the initial arm of the study.
2. Patient events
Three of five Crohn’s disease patients demonstrated a rise in CRP after fecal installation suggestive of disease reactivation.
One control subject was withdrawn from the study because of unacceptable discomfort during colonoscopy. No other adverse events have been experienced.
3. Labeling and hybridization of the cRNA to Nimblegen microarray
RNA was prepared from peripheral blood monocytes and colonic biopsies from all participating individuals. Hybridization of cRNA to the Nimblegen microarray has been carried out for all samples collected up until June 2004. Where sufficient RNA has been available, experiments have been carried out in duplicate.
4. Genomic genotyping
DNA from all subjects has been genotyped for the three common Crohn’s disease associated CARD15 alleles. In addition, full medium resolution HLA types have been performed.
C. List of significant results (positive or negative).
We plan an interim analysis of the data in July 2004. Statistical assistance has been organized with experts at Nimblegen Inc. (Madison US) and Oxagen Ltd. (Abingdon UK). Final analyses will be available by December 2004.
D. Lay summary of this report.
Crohn’s disease comes in different forms. The aim of this study is to identify which genes are responsible for the type of Crohn’s disease that only affects the large bowel. We are particularly interested in the genes which are activated during the early stages of a disease flare. In order to do this, we have studied patients whose colon has been taken out of circuit by a “defunctioning ileostomy” as part of their treatment. Diverting the feces from the large bowel may result in disease remission, and disease is frequently reactivated following re-exposure to an individual’s own feces. To make sure that we are not identifying genes that normally activate in this situation, we have also studied patients who have had a defunctioning ileostomy for bowel cancer. We have studied the genetic changes that occur in the bowel itself (using tiny biopsies) and in circulating immune cells.
To date, we have successfully studied five patients with defunctioning ileostomy for colonic Crohn’s disease and four control subjects who have ileostomies for rectal cancer. Patient recruitment has been slow, but further patients are scheduled to be studied over the next three months. Once these results are available, we will analyze the data and we aim to present the results at the BMRP annual investigator meeting in February 2005.