Association of NOD2 (CARD 15) genotype with clinical course of Crohn's disease: a cohort study

doi:10.1016/S0140-6736(02)08590-2

The Lancet

Volume 359, Issue 9318, 11 May 2002, Pages 1661-1665

https://doi.org/10.1016/S0140-6736(02)08590-2 Get rights and content

Summary

Background

Crohn's disease is a heterogeneous disorder for which NOD2 (CARD 15) has been identified as a susceptibility gene. We investigate the relation between NOD2 genotype and phenotypic characteristics of patients with Crohn's disease.

Methods

Hypotheses about the relation between NOD2 genotype and Crohn's disease phenotype were generated retrospectively from a group of 446 German patients with this disorder. Positive findings (p<0·10) were verified in prospectively established cohorts of 106 German and 55 Norwegian patients with Crohn's disease. All patients were genotyped for the main coding mutations in NOD2, denoted SNP8, SNP12, and SNP13, with Taqman technology.

Findings

In the retrospective cohort, six clinical characteristics showed noteworthy haplotype association: fistulising, ileal, left colonic and right colonic disease, stenosis, and resection. In the German prospective cohort, these haplotype associations could be replicated for ileal (p=0·006) and right colonic disease (p≤0·001). A similar trend was noted in the Norwegian patients.

Interpretation

We recorded a distinct relation between NOD2 genotype and phenotype of Crohn's disease. Test strategies with NOD2 variations to predict the clinical course of Crohn's disease could lead to the development of new therapeutic paradigms.

Introduction

Crohn's disease is a chronic intestinal inflammatory disorder that is characterised by a dysregulated mucosal immune response. Results of epidemiological and genetic linkage studies1, 2, 3 suggest that there is a genetic component that causes this disorder, and the pericentromeric region of chromosome 16 has proved the most probable location of this component. Results of positional cloning and candidate-gene analysis of chromosome 16 have identified nucleotide oligomerisation domain (nod) 2 as a gene linked to Crohn's disease.4, 5, 6 Recently, the nomenclature of NOD2 has been changed to caspase activating recruitment domain (card) 15. NOD2 has been shown to have a role in the cause of Crohn's disease in four independent and ethnically different groups of patients.4, 5, 6

The association of high risk of Crohn's disease with NOD2 mutations suggests a potential diagnostic applicability. Furthermore, no patient with ulcerative colitis who is homozygous for SNP13—the most important truncating NOD2 mutation—has so far been identified, which suggests that the range of mutations of NOD2 might facilitate differential diagnosis of Crohn's disease and ulcerative colitis.⁴

Crohn's disease has a range of clinical manifestations, and its heterogeneity has been highlighted by results of several epidemiological studies aimed at identification of subgroups or clusters of phenotypes of this disorder.7, 8, 9, 10 Age at diagnosis, location (terminal ileum, colon, ileocolon, upper gastrointestinal tract), and development of complications (stricturing, penetrating) are useful variables for clinical classification.⁷ Such classification schemes would, however, benefit further from associated genetic factors, since clinical classifications can change over time.¹¹

Phenotype clustering in families and in homozygotic twins12, 13 suggests that genetic factors affect clinical outcome in Crohn's disease. Since mutations in NOD2 are an important factor in cause of Crohn's disease, investigation of the range of mutations of NOD2 for possible genotype-phenotype relations seems worthwhile.

NOD2 is a member of the NOD gene family, and has a role in mediation of inflammatory response to bacterial antigens. Therefore, NOD2-related clinical manifestations can be assumed to show the pathophysiological effect of intestinal flora—eg, ileocolonic or fistulising disease.

Investigation of the genotype-phenotype relation in patients with Crohn's disease is methodologically challenging, since many clinical characteristics have to be assessed. We have therefore used a two-stage study design, in which a set of hypotheses was first generated by exploratory analysis of a retrospectively characterised group of patients. Hypotheses emerging as potentially important were then validated in a second, prospectively established, verification cohort.

We investigated the importance of the three major functional variants of NOD2—SNP8, SNP12, and SNP13—on the clinical course of Crohn's disease. Presence of stenoses, fistulae, arthritis, other extraintestinal complications, and resections, relapse frequency, and location of disease (ileal, right colon, left colon) were examined.

Section snippets

Study populations

We analysed two groups of patients. Patients in all cohorts were recruited consecutively. For the retrospective group, we invited—through the German Crohn's and Colitis Foundation—about 9000 people to take part in the study, and we received over 6000 responses. Of these, we included the first 446 patients with Crohn's disease who fulfilled the criteria listed below. Patients were eligible for the study if inflammatory bowel disease had been diagnosed with standard clinical criteria and by

Genotyping

We prepared genomic DNA from 10 mL fresh or frozen blood samples with the blood Gigakit (Invitec, Berlin, Germany). We assayed mutations with Taqman (Applied Biosystems, Foster City, CA, USA) as described.¹⁸ In brief, genomic DNA was arrayed and dried on 96-well and 384-well plates. Taqman PCR was set up with Genesis pipetting robots (Tecan, Männedorf, Switzerland). We amplified samples with ABI9700 PCR machines (Applied Biosystems), and measured fluorescence with ABI7700 and ABI7900

Statistical analysis

We tested qualitative clinical characteristics for significant SNP haplotype association with a log-likelihood ratio test. We estimated haplotype frequencies of the three NOD2 SNPs in patients with or without the specific characteristic, with an expectation maximisation algorithm as implemented in the HAPMAX computer program.²¹ Twice the log-likelihood ratio between the two data models that do or do not distinguish between phenotypic categories roughly follows a X² distribution, with k-1

Role of the funding source

The sponsors of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.

Results

446 patients were recruited for the retrospective sample, and 161 for the prospective group. Both groups had a closely similar distribution of sex and age at diagnosis (table 1).

Haplotype frequencies for SNP8, SNP12, and SNP13 were estimated for controls and patients (table 2). These frequencies differed significantly between the two geographic populations, both in controls (p^r=0·006) and in patients (p^t≤·001). Therefore, possible associations between genotypes and clinical variables were

Discussion

We have shown that haplotypes with SNP13 are associated with ileal disease (table 4) whereas, at least in German patients, all three SNPs seem to be associated with increased risk for right-colonic disease (table 5). These results suggest that NOD2 genotype has an effect on the clinical presentation of Crohn's disease. No association with general markers of disease severity (as assessed by relapse frequency, age of onset, and need for resections) was noted. This absence of association

GLOSSARY

caspase activating recruitment domain (card): Caspases are important enzymes in the apoptosis process.
haplotype: A specfic set of alleles present at two or more linked loci on a chromosome.
nfkb: Nuclear factor kappa B, a transcription factor that regulates expression of most inflammation genes.
nucleotide oligomerisation domain (nod): A structural characteristic of the NOD gene family. Members of the family include APAF1, CED4, NOD1, NOD2, and some cytosolic products of plant disease-resistance genes.

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Mechanisms of DiseaseAssociation of NOD2 (CARD 15) genotype with clinical course of Crohn's disease: a cohort study

Summary

Background

Methods

Findings

Interpretation

Introduction

Section snippets

Study populations

Genotyping

Statistical analysis

Role of the funding source

Results

Discussion

GLOSSARY

Am J Hum Genet

Gastroenterology

Lancet

Mol Med Today

Gastroenterology

Am J Hum Genet

Lancet

J Biol Chem

J Biol Chem

Mapping of a susceptibility locus for Crohn's disease on chromosome 16

Nature

A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease

Nature

Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease

Nature

Mechanisms of Disease
Association of NOD2 (CARD 15) genotype with clinical course of Crohn's disease: a cohort study