Findings From the Manitoba IBD Risk Factor Study

What does the NOD-2 gene do?

It controls the production of the NOD-2 protein, a protein that has a role in cells responding to bacteria near their surface. If the NOD-2 protein is not working normally it is possible that cells in the gut will not be able to properly fend off potentially injurious bacteria.

 

Are there gene defects associated with IBD?

1. Brant S, Wang M-H, Rawsthorne P, Sargent M, Datta LW, Nouvet F, Shugart YY, Bernstein CN. A population-based case control study of CARD15 and other risk factors in Crohn’s disease and ulcerative colitis. American Journal of Gastroenterology 2007; 102: 313-323.

This was the first population based study to show the increased risk of having Crohn’s disease if harboring mutations in the NOD-2 gene. Approximately 15% of the general population of Manitoba has mutations in the NOD-2 gene while approximately 36% of those with Crohn’s disease have mutations in the NOD-2 gene. Having these mutations is a clue to the pathogenesis of Crohn’s disease but it is neither necessary nor sufficient to have these mutations in terms of getting Crohn’s disease (lots of healthy people have these mutations and lots of people with Crohn’s disease don’t have these mutations).

 

2. Okazaki T, Wang MH, Rawsthorne P, Sargent M, Datta LW, Shugart YY, Bernstein CN, Brant SR. Contributions of IBD5, IL23R, ATG16L1, and NOD2 to Crohn’s disease risk in a population-based case-control study: Evidence of gene-gene interactions. Inflammatory Bowel Diseases 2008; 14: 1528-1541.

This study conducted in collaboration with researchers at Johns Hopkins University assessed the DNA from subjects in the Risk Factor Study for other possible gene associations and did confirm that genes for other proteins can be mutated in IBD, although at a much lower likelihood than for NOD-2. IL-23R, for instance is a gene that codes for the protein IL-23R. This protein is a receptor that helps control the function of IL-23 a protein that is actively involved in inflammation.

With the mapping of the human genome a few years ago there are easier techniques available to scientists to determine what gene mutations may be associated with any chronic disease. This work is ongoing across the world but primarily amongst a National Institutes of Health (of the US) sponsored consortium of about 10 centres in North America and Europe that have pooled their resources. It is possible that within 5-10 years all the genes that have some association with IBD will be uncovered. While genetics is clearly important in determining if someone might be at risk, these and other studies have highlighted that there must be some important environmental component to developing IBD.

 

Is there an infectious cause of IBD?

For many years it has been suspected that a bug that causes a Crohn’s-like disease in cattle (Johne’s disease) may cause Crohn’s disease. This bug Mycobacterium paratuberculosis is difficult to find in humans. There have been a number of studies conducted in search of a possible association of Mycobacterium paratuberculosis with Crohn’s disease; some have been positive and some have been negative. This has become increasingly debated, but it is agreed by most involved in this research, that a major aspect lacking to advance our understanding in this area is a single, reproducible diagnostic test that can clinch the presence of Mycobacterium paratuberculosis with a high degree of certainty. Guessing that Mycobacterium paratuberculosis might be linked with Crohn’s disease has enormous implications for the beef and dairy industry of the entire world. So we should not guess. We should keep working towards understanding with certainty whether or not this infection is associated with Crohn’s disease.

 

1. Bernstein CN, Nayar G, Hamel A, Blanchard JF. A pursuit of animal borne infections in the mucosa of subjects with inflammatory bowel disease and population-based controls. Journal of Clinical Microbiology 2003; 41:4986-4990.

In this study the colon tissues of 25-30 subjects with each of Crohn’s disease, ulcerative colitis and healthy control volunteers were studied by the veterinarians of Manitoba (Agriculture Manitoba) to determine if the DNA of Mycobacterium paratuberculosis or other animal-borne infections was present in the colon. This study could not find any Mycobacterium paratuberculosis DNA in Crohn’s tissue, rarely in ulcerative colitis tissue and in about 25% of control tissue. This study concluded that there was no association between Mycobacterium paratuberculosis and Crohn’s disease.

 

2. Bernstein CN, Blanchard JF, Rawsthorne P, Collins MT. A population-based case control study of seroprevalence of Mycobacterium paratuberculosis in patients with Crohn’s disease and ulcerative colitisJournal of Clinical Microbiology 2004; 42: 1129-1135.

In this study conducted with researchers at the University of Wisconsin we aimed to determine if people with Crohn’s disease were more likely to have an antibody response in their blood to Mycobacterium paratuberculosis. Such a response might indicate that the infection has been present at some time. Nearly 1000 blood samples were studied for these antibodies and approximately 35% of Manitobans were positive for these antibodies. However, there was no difference in rate of antibody positivity among persons with any of Crohn’s disease, ulcerative colitis or healthy controls. We are still uncertain if having an antibody response to this bug even proves that the infection has been present.

 

3. Bernstein CN, Wang MH, Sargent M, Brant SR, Collins MT. Testing the interaction between NOD-2 status and serological response to Mycobacterium paratuberculosis in IBD. Journal of Clinical Microbiology 2007; 45: 968-71.

Because of the high rate of positive antibody responses in Manitobans, we considered whether having a NOD-2 gene mutation and having the antibody response might be more likely to identify subjects with Crohn’s disease. In other words, if one has a NOD-2 gene mutation and then gets a Mycobacterium paratuberculosis infection, will that increase their risk for Crohn’s disease. This study did not find any link between having a NOD-2 gene mutation and being positive in the blood by antibody testing for Mycobacterium paratuberculosis.

We, and others, will continue to conduct studies on Mycobacterium paratuberculosis to determine if there is any association with Crohn’s disease. Even if not associated with Crohn’s disease, it is important to understand whether having any Mycobacterium paratuberculosis in beef or milk we consume could be harmful to humans in any way.

 

Could the bug that causes stomach ulcers (H. pylori), cause IBD?

1. Streutker C, Bernstein CN, Chan VL, Riddell RH, Croitoru K. PCR analysis for species-specific Ribosomal DNA detects Helicobacter species DNA in intestinal tissues from patients with inflammatory bowel disease. Journal of Clinical Microbiology 2004; 42: 660-664.

H pylori is a bug that resides in the stomach and can cause stomach ulcers or even stomach cancers. Finding the association between H. pylori and ulcers revolutionized the way ulcers were treated. Those associated with H pylori are treated with antibiotics to eradicate the H pylori. Together with researchers at McMaster University we studied colon tissue samples from subjects with Crohn’s disease, ulcerative colitis and healthy controls to determine if the DNA for H. pylori could be found. We did find an increase in H. pylori DNA in ulcerative colitis tissue. However, the implication of this finding is unknown. Treating patients with antibiotics does not seem to cure ulcerative colitis, although this has not been fully studied. There are groups who are very likely to be infected with H. pylori (such as the First Nations of Manitoba) yet they have low rates of ulcerative colitis.

 

2. Bernstein CN, Sargent M, Leslie WD. Serum osteoprotegerin is increased in Crohn’s disease: A population based case control study. Inflammatory Bowel Diseases 2005; 11: 325-330.

Using the blood from our IBD patients and healthy controls in our Risk Factor Study we also explored the expression of certain bone-related proteins. We found changes in one particular protein, osteoprotegerin, in Crohn’s disease. This protein is associated with maintaining bone mineralization but interestingly has also been associated with colitis in animal models of IBD. In fact administration of this protein may prove to ultimately be a therapy of some forms of IBD.

 

Could measles, mumps, or rubella be associated with IBD?

1. Bernstein CN, Rawsthorne P, Blanchard JF. A population-based case control study of measles, mumps and rubella and inflammatory bowel disease. Inflammatory Bowel Diseases 2007; 13: 759-762.

In the 1990s there was an interest mostly from the UK in exploring the association of either measles infection or the measles vaccine and the development of Crohn’s disease. Since measles infection rates were plummeting in the developed world and Crohn’s disease rates were rising there was a hypothesis put forward that there was an association with measles vaccine (protecting against getting measles) and developing Crohn’s disease. A number of studies refuted this possibility. The measles vaccine is typically administered together with mumps and rubella vaccine. After getting the infections of these viruses or the vaccines to them people will mount an antibody response in their blood. In this study we explored whether Crohn’s disease or ulcerative colitis patients were more likely to have a positive antibody response to these viruses compared to healthy controls. The only positive finding of this study was that patients with Crohn’s disease were less likely to have an antibody response to rubella (German measles). This suggests that possibly having a rubella infection or having gotten the rubella vaccine was in some way protective against getting Crohn’s disease. These are indeed confusing results. But we certainly did not find an association with measles infection or vaccine (or the other viruses) and having IBD.

 

Could bugs residing within the bowel be the problem?

1. Eckburg P, Bik EM, Bernstein CN, Purdom E, Dethlefsen L, Sargent M, Gill SR, Nelson K, Relman DA. Diversity of the human intestinal microbial flora. Science 2005; 308: 1635-1638.

In this landmark study we collected colon tissue from 3 healthy controls and researchers at Stanford University tediously and methodically looked for the DNA for every possible bacteria that could be residing in the human bowel. It was discovered that up to 60% of species of bacteria found had never been known before; and that regardless of where in the colon the tissue was from, there was a similar pattern of bacteria present. However there were marked differences of colon bacteria types between different people. The colonic bacteria of each individual is so unique that it is likely as unique as an individual’s fingerprint. Understanding what the bacteria are of the normal human colon helps to understand what may be present in disease states like IBD.