Final Progress Report
Proposal No. IBD-0044
Principal Investigator: Herbert J. Van Kruiningen, DVM, Ph.D., M.D.
Applicant Organization: The University of Connecticut (Storrs, U.S.A.)
Project Title: Epidemiology of Familial Crohn's Disease
Period of Award: December 1, 2002 – March 31, 2006
Project Aims
1. To complete an ongoing epidemiological study of factors relating to the onset of Crohn’s disease in multiplex, simplex, and control families in northern Belgium.
2. To seek viruses in Crohn’s disease tissues in support of leads developed from the interviews of multiplex families.
Accomplishments Towards Meeting Those Aims
During 2003 the PI returned to Belgium for three weeks to interview additional control families. The questionnaire previously used was administered by the PI and the same nurse Marie Joossens. Four control families, matched appropriately with multiplex families, were interviewed, a total of 24 subjects. That brought the number of control families to ten, and it was the opinion of the epidemiologist co-investigator in this study, Robert Vlietinck, that we analyze the assembled data for 21 multiplex families and 10 controls.
Over the ensuing two years, three meetings were held, during which the PI and Robert Vlietinck analyzed the data, meeting for four to five days on each occasion. The data had been entered into SAS and 1,200 pages of data were analyzed. Twenty-one families with three or more first-degree relatives were studied together with 10 matched control families. Simplex families, i.e., families in which one case of Crohn’s disease occurred, were not included because of insufficient numbers of participants. Within those that were studied, there were 74 patients with CD, 84 unaffected family members and 59 controls. Data from a 176 item questionnaire were analyzed, including questions about first symptoms, childhood vaccinations and disease, food items, potable water supplies, social activities, travel, pets, and home and surrounding environment. Possible factors linked to disease were evaluated using univariate and multivariate logistic regression. Our first manuscript was published in April 2005, in which we reported that patients had more appendicitis during adolescence, ate less oats, rye and bran than controls, and consumed more unpasteurized cheese. Patients drank significantly less tap water and more well water than controls. Clustering of cases in time occurred in 13 of the 21 affected families. The drinking of well water and the absence of household pets were defined as important risk factors for familial Crohn’s disease.
A second manuscript describing pedigrees, temporal relationships among cases and family histories has been submitted to the Journal of Clinical Gastroenterology and is currently under review. Several new cases of inflammatory bowel disease, four with Crohn’s disease and one with ulcerative colitis have been recognized within the original 21 families and are being followed by Belgian colleagues.
A third manuscript is currently in revision and reports a “Search for Evidence of Recurring or Persistent Viruses in Crohn’s Disease.” In this study tissue blocks obtained from surgical specimens from patients and a control population were analyzed by PCR or RT-PCR, for EBV, CMV, HSV 1, HSV 2, HHV 8, pestiviruses, and enteroviruses. Additionally, seven sets of serum samples, including pre-operative and post-operative samples, from CD patients were analyzed serologically for antibodies to EBV. The tests revealed evidence of EBV nucleic acid in tissues of 11 patients from a total of 70 tested (15.7%) and in tissues of 3 of 41 control subjects (7.3%). Evidence of pestivirus was found in one CD patient, while one patient and one control were positive for CMV. No HSV 1 or 2, HHV 8 or enteroviruses were found. The serologic tests revealed that 5 of 7 CD patients had antibodies against the early protein, the capsid protein and the EBV nuclear antigen (EBNA). The titers were not significantly altered post-surgically. None of the patients had antibodies of the IgM isotype.
Significant Results
1. Families with familial Crohn’s disease inhabited homes surrounded by intensively tilled agricultural land.
2. During childhood and adolescent years multiplex families had an inordinate frequency of enterovirus infections, including hand, foot and mouth disease and hepatitis A.
3. There was evidence that CD patients had an extraordinary frequency of diseases acquired by the oral route or by fecal-oral transmission.
4. Multiplex families had less than usual daily contact with a bird, a dog or a cat in the home during childhood.
5. Patients with CD ate oats, rye and bran less frequently than the control populations.
6. Consumption of unpastuerized cheeses and raw pork constitute risk factors.
7. There was no evidence that patients with familial Crohn’s disease differ from patients with sporadic disease.
8. Smoking and appendicitis were recognized with high frequency in multiplex families.
9. Firstborns within multiplex families were affected more frequently than later-born siblings.
10. Within sibships an affected sibling was consecutive in birth order with another affected sibling 36% of the time.
11. Clustering of cases in time occurred in 13 of the 21 affected families.
12. Molecular tests revealed evidence of EBV in 15.7% of patients and in 7.3% of controls.
13. No HSV 1 or 2, HHV 8 or enteroviruses were found.
14. Evidence of pestiviruses and CMV was rare.
15. Five of seven CD patients had various antibodies against EBV. None of the patients had antibodies of the IgM isotype.
Lay Summary
Environmental factors are believed to trigger the onset of Crohn’s disease in genetically susceptible individuals. The aim was to assess environmental and familial factors that might be etiologically related to Crohn’s disease. Twenty-one families with three or more affected first-degree relatives were studied, together with 10 matched control families. There were 74 patients with Crohn’s disease, 84 unaffected family members, and 59 controls. Family members were interviewed together at the parental home. A 176 item questionnaire delved into first symptoms, childhood vaccinations and diseases, food items, potable water supplies, social activities, travel, pets, and home and surrounding environment. Questions were directed specifically for two time frames, childhood until age 20, and a 10-year epoch before the onset of first symptoms within a family. There were significantly more smokers in the patients and their relatives than in controls. Patients had more appendicitis, hand, foot and mouth disease and hepatitis A during adolescence, ate less oats, rye and bran than controls, and consumed more unpasteurized cheese. Patients drank significantly less tap water and more well water than controls. Families that ultimately came to contain familial CD reported less than expected daily contact with household pets during childhood. Clustering of cases in time occurred in 13 of the 21 affected families. Our data suggest that the drinking of well water represents an important risk factor for Crohn’s disease. Close daily contact with a cat or dog in the home may have a protective effect.