This M o n t h in Gastroenterology By Eugene B. Chang

Predicting Patterns of Crohn's Disease Through Genetic Markers

Last year, the first Crohn's disease gene, NOD2, was repor ted by 3 groups (Hugo J-P, et al. Nature 2001;411:599- 603; Ogura Y, et al. Nature 2001;411:603-606; Hampe J, Lancet 2001;357:1925-1928). NOD2 is located on chromosome 16 and encodes a protein that contains N-terminal caspase recrui tment domain (CARD) motifs, a nucleotide-binding domain, and a region of leucine-rich repeats toward its C-terminal domain (Figure 1). Although the function of NOD2 remains a mystery, several predictions have been made based on its putative protein structure. For instance, it has been proposed as a cytosolic receptor for endotoxin or l ipopolysaccharide (LPS) based on the presence of its LRR domain, as these motifs have also been found in ToU-like receptors that are bel ieved to be important sensors of the innate immune system. The finding that NOD2 is also highly expressed in innate immune cells such as monocytes and macrophages is consistent wi th this notion. Therefore, there was considerable exci tement that NOD2 mutations associated wi th Crohn's disease might explain the aberrations in immune activation, particularly by enteric flora, a major source of LPS and other bacterial products. However , this exc i tement was t empered by 2 key experimental observations. First, the LeulOO7fsinsC mutation, wh ich results in a frameshift mutation and truncation of NOD2 in the LRR region, is associated with an unanticipated impairment of LPS activation. Second, the other mutations associated wi th Crohn's disease are "silent," i.e., not resulting in any apparent functional or structural defects in the NOD2 protein. It has also been repor ted that the CARD motifs of NOD2 might implicate a role of this protein in programmed cell death, as they are structurally and functionally related to death domains of many apoptot ic proteins (Beutler B. Immuni ty 2001;15:5-14). Because apoptosis is essential for turning off an inflammatory response, loss of function associated wi th the LeulO07fsinsC mutation could result in uncontrol led prolifer- ation and activation of immune cells, leading to chronic inflammation and tissue destruction characteristic of Crohn's disease. The function(s) of NOD2 and its role in causing some types of Crohn's disease remains to be determined.

Despite the uncertainties of what NOD2 is, the studies by Cuthbert et al. and Abroad et al. appearing in this issue of GASTROENTEROLOGY underscore the immediate utility of genotypic information in the clinical arena. Both studies show that NOD2 mutations are highly associated wi th ileal Crohn's disease. In the study by Ahmad et al., 244 whi te Crohn's disease patients from a single center in the United Kingdom were rigorously phenotyped based on standard clinical, radiological, endoscopic, and histological critieria. Linkage disequilibrium mapping was performed on 340 polymorphisms in 24 HLA genes and 3 NOD2 polymorphisms. The NOD2 polymorphisms were strong predictors of ileal disease, but not associated with perianal or colonic disease. The relative risk was highest wi th the LeulOO7fsinsC mutation, which along wi th compound heterozygotes, also appeared associated wi th earlier and possibly more stricturing disease. Alleles on specific long-range HLA haplotypes also appeared to determine overall susceptibility. In the study by Cuthbert et al., 4 NOD2 polymorphisms were genotyped in a case-control cohor t of Crohn's disease, ulcerative colitis, and normal controls. As might be expected, NOD2 did not appear to be a significant risk factor for ulcerative colitis. Although there was a 3-fold increased risk in heterozygotes wi th the NOD2 mutation, monozygotes, or compound heterozygotes showed a greater than 200-fold risk of disease compared wi th controls. Finally, like the o ther study, there was a much higher incidence of ileal disease in patients wi th NOD2 mutations.

Much more work needs to be done in developing a molecular classification of Crohn's disease. This will require not only a greater understanding of NOD2 function, further refinements of patient stratification, but also identification of other causative or susceptibility genes associated wi th Crohn's disease. Ultimately, this genetic information will become an important part of our approach, management, and t reatment of these diseases.

See pages 854 and 867

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Figure 1. Functional regions of the NOD2/CARD15 protein. Arrows indicate sites of 3 mutations associated with Crohn's disease.

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Figure 2. Kaplan-Meier estimates of (A) patient survival and (B) allograff survival after liver transplan- tation in HCV- and non~tCV~nfected patients.

Hepatitis C Infection Impairs Patient and AIIograft Survival After Liver Transplantation One of the major indications for or thotopic liver transplantation in the United States is cirrhosis secondary to hepatitis C (HCV) infection, alone or in combina- tion wi th alcohol abuse. Infection of the aUograft by HCV is Inevitable, resulting In a high incidence of hepatitis (75%- 80%) and significant development of fibrosis or cirrhosis (10%-21%) at 5 years. Fibrosing cholestatic hepatitis occurring In up to 10% of HCV-infected patients also accelerates allograft failure and death. Despite the development of these adverse events, several single center, or mul- t icenter studies have been unable to show a significantly higher mortality in HCV-infected liver transplant patients compared with recipients wi thout HCV. Because these data were largely derived from small cohor t studies, Forman et al. used data from the United Network of Organ Sharing that included over 11,000 patients w h o underwent close to 12,000 liver transplants be tween 1992 and 1998. This large database provided a more powerful analysis to detect a survival differ- ence. An increased rate of death and allograft failure was found in HCV-posit ive liver transplant patients compared with non-HCV recipients, even after adjusting for potential confounding variables including donor, recipient, and transplant center characteristics (see Kaplan-Meier curves). The HCV effect was also more p ronounced in female recipients. Based on these nationally representative data, the findings indicate a greater risk of death and allograft failure in HCV-positive recipients. In light of the shortage of donor organs and increasing numbers of patients wi th HCV infection, it is essential that approaches to improve survival of patients and allografts in these patients be developed.

See page 889

Identifying Causes of 6-Mercaptopurine Resistance in Patients With Inflammatory Bowel Diseases by Measuring 6-MP Metabolic Profiles Azathioprine (AZA) and 6-mercaptopurine (6-MP) are immunomodulators that have been useful in the treatment of active and quiescent inflammatory bowel disease in both pediatric and adult patients. However, nearly half of patients with active Crohn's disease and a third with quiescent disease did not respond to or cannot take these medications because of adverse effects. Many of these treatment faihlres result from problems with optimal dosing, either dose-dependent, idiosyncratic reactions, or inadequate levels to achieve a therapeutic response. In this regard, individual variations in 6-MP/AZA metal> olism likely account for differences in response and tolerance to thiopudne. 6-MP and ASA are both inactive prodrugs that are metabolized to several active metabolltes, including 6-thioguanine nucleotides (6-TGN) and 6-methylmercapto ribonudeotides (6-MMPR). Optimal 6-TGN red blood cell levels are significantly and independently associated with clinical remission. However, 6-MMPR appears to be cytotoxic, especially associated with hepatotoxicity. However, there are 2 other metabolic pathways, 1 catalyzed by xanthine oxidase (XO), and the other by thiopurine methyltransferase (TPMT), the latter thought to be the more important. The frequency distribution of TPMT activity in the general population is trimodal: 0.3% having low-to-absent activity, 11% intermediate activity, and 89% with normal to high-enzyme activity. Those with low activity are more likely to develop drug-related toxicity caused by rapid conversion of 6-MP/AZA to active metabo- lites. However, patients inheriting high TPMT activity may require higher than standard doses of thiopurines to achieve optimal 6-TGN levels.

The study by Duhinsky et al. was undertaken to determine if dose escalation in patients failing therapy because of subtherapeutic dosing optimizes 6-TGN levels and therapeutic efficacy. Therapeutic efficacy and adverse events were followed in 51 inflammatory bowel disease patients and 6-TGN and TPMT activities were measured blindly to clinical information. Interestingly, the study identified a novel subgroup of 6-MP/AZA-resistant IBD patients that were unable to achieve optimal 6-TGN production because of shunting toward potentially toxic 6-MMPR levels on dose escalation. Although preliminary, these data suggest the 6-MP metabolic profiling may be useful in o p ' ~ thiopurine therapy and identifying causes of treatment resistance or toxicity.

See page 904

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6-TGN Figure 3. 6-MP metabolism. Oral AZA is rapidly converted to 6-MP by a nonenzymatic process. Initial 6-MP transformations occur along com- peting catabolic (XO or TPMT) and anabolic (HPRT) enzymatic pathways. Once formed, 6-thiosine 5' monophosphate (6-TIMP) may be transformed either into 6-TGN by the rate-limit- ing inosine monophosphate dehydrogenase (IMPDH) and guanosine monophosphate syn- thetase (GMPS) enzymatic pathways or methyl- ated into 6-MMPR.

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