Analysis of the NOD2/CARD15 Gene in Patients Affected with the Aseptic Abscesses Syndrome with or without Inflammatory Bowel Disease

Objectives NOD2/CARD15 is a susceptibility gene for Crohn’s disease (CD). It is also involved, via different mutations, in the Blau syndrome. The syndrome of aseptic abscesses (AA) is characterized by visceral sterile collections of mature neutrophils that do not respond to antibiotics but regress quickly with corticosteroids. It is associated in two cases out of three with inflammatory bowel disease (IBD), and in particular with CD. We wanted to assess if changes on gene NOD2/CARD15 could contribute to the development of AA in patients with and without IBD. Methods Seventeen unrelated patients with AA from the French national register were genotyped for c.802C>T (p.Pro268Ser) and the three main CD associated variants, c.2104C>T (p.Arg702Trp), c.2722G>C (p.Gly908Arg) and c.3019_3020insC (p.Leu1007fsX1008), and 16 were screened for the 11 coding exons of NOD2/CARD15. Results The main variants associated with CD were found at a similar frequency in patients free of IBD and in those with CD. There was no significant difference in the main variants between patients with CD and those without IBD in our study and patients with CD and controls, respectively, from a large study of an ethnically similar population. No rare variant was found. A significant association between carriers of the silent variant c.1377 C>T and markers of severity of AA was observed. Conclusions These results suggest that the emergence of AA is not closely related to gene NOD2/CARD15. NOD2/CARD15 and other susceptibility genes might enhance the expression of AA as the result of a combination of polymorphisms. This work was supported by Grant PHRC 2004/CHU Clermont-Ferrand. Marc André is a recipient of a Contrat d’Interface Inserm-CHU Clermont-Ferrand.     

Update on the genetics of inflammatory bowel disease

Crohn’s disease and ulcerative colitis are related genetic disorders. Epidemiologic studies suggest that both disorders are caused by a complex interplay of genetic and environmental factors. Genetic linkage studies identify the general chromosomal locations of disease susceptibility genes, and a number of genetic linkages have been reported in inflammatory bowel disease (IBD). Most notable among these linkage regions has been the linkage in the pericentromeric region of chromosome 16, IBD1, among families multiply affected with Crohn’s disease. Recent studies have established that at least three coding region variants in the Nod2 gene are responsible for the linkage findings here, and Nod2 therefore represents the first definitively established gene contributing to the pathogenesis of IBD. The implications of these findings for advancing our understanding of Crohn’s disease are discussed.     

1007fs, G908R, R702W Mutations and P268S, IVS8+158 Polymorphisms of the CARD15 Gene in Turkish Inflammatory Bowel Disease Patients and Their Relationship with Disease-Related Surgery

Introduction CARD15 gene mutations may present different frequencies in populations and sometimes surgical interventions may become a necessary therapy for inflammatory bowel disease patients. Mutations of 1007fs, G908R, R702W and polymorphisms of P268S, IVS8⁺¹⁵⁸ of the CARD15 gene and their relation with disease-related surgery were investigated in Turkish inflammatory bowel disease patients in this study. Material and Method 1007fs, G908R, R702W mutations and P268S, IVS8⁺¹⁵⁸ polymorphisms of CARD15 gene were analyzed in 130 inflammatory bowel disease patients (67 Crohn’s disease, 63 ulcerative colitis) and 87 healthy controls. After obtaining DNA samples, genotyping was performed by polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) analysis. Results were evaluated by statistical analysis and accepted as significant if P < 0.05. Results R702W gene mutation was significantly lower in the inflammatory bowel disease group (1.5%) than the controls (4.8%) (P < 0.05). The overall allele frequency of mutations in the inflammatory bowel disease group (2.7%) was lower than in controls (6.6%) (P < 0.05). Disease-related surgery history was present in 20 Crohn’s and 25 ulcerative colitis patients; familial history was present in four Crohn’s and five ulcerative colitis patients. Statistically, no relationship was detected between disease-related surgeries and the investigated genetic tests. Conclusion In Turkish patients, no important relationship was detected between the investigated allele frequencies of the CARD15 gene and inflammatory bowel disease nor between disease-related surgeries and inflammatory bowel disease. Dedicated to the memory of the Turkish scientist Turgut Tukel MD. Thanks for his contributions and supports.     

Genetic advances in inflammatory bowel disease

Opinion statement The translation of basic science advances to tangible benefits in clinical practice remains a fundamental goal of biomedical research. Genetic approaches provide a unique opportunity to enhance bench-to-bedside translational efforts, with inflammatory bowel disease (IBD) being a model genetic disorder in several respects. The association of the NOD2 (CARD15) mutations to Crohnrss disease (CD) represents, in complex human disorders, one of the clearest cases of a definitive disease association. The NOD2 gene is located in the IBD1 genetic linkage region on chromosome 16 and functions as an intracellular pattern recognition receptor for components of bacterial peptidoglycan. CD-associated variants within NOD2 have a decreased capacity to appropriately signal with peptidoglycan and provide an important example of gene-environment interactions. Genetic variation in other innate immune receptors, notably Toll-like receptor (TLR)4, has been defined and may also play a role in CD pathogenesis. Replicated CD association in the IBD5 region on chromosome 5q has been reported, and candidate functional polymorphisms within the organic cation transporters OCTN1 (SLC22A4) and OCTN2 (SLC22A5) have been described. Confirmatory studies demonstrating altered OCTN activity and expression in primary human cells stratified on the IBD5 risk haplotype would provide important confirmatory support for disease contribution. A comprehensive understanding of IBD will involve integrating information from animal models, functional human polymorphisms, and expression studies from human IBD tissues. Genes and pathways implicated as contributing to IBD pathogenesis through multiple lines of evidence should be more intensively examined, including the tumor necrosis factor-α, MDR1, and peroxisome proliferator-activated receptor (PPAR)γ pathways. To attain full advantage of new genetic information, novel methods of classifying patients that go beyond phenotypic classifications presently utilized will be required. Genetic data will need to be integrated with novel biomarker development in IBD, including functional, expression (mRNA or protein), or biochemical indicators of physiologic/ disease processes and responses to therapies.     

The Relation Between NOD2/CARD15 Mutations and the Prevalence and Phenotypic Heterogeneity of Crohn’s Disease: Lessons from the Israeli Arab Crohn’s Disease Cohort

The prevalence of Crohn’s disease depends on geographic location and racial background. Arg702Trp, Gly908Arg, and Leu1007fsinsC mutations in the NOD2/CARD15 gene are associated with Crohn’s disease in Caucasians. The mutation rate among Israeli Jewish patients is 27%–41%. The prevalence of Crohn’s disease is much lower in the Israeli Arab compared to the Israeli Jewish population. We studied the NOD2/CARD15 mutation rate and disease phenotype (according to the Vienna classification) among the Israeli Arabs and compared them with those in an Israeli Jewish cohort. We recruited 66 Israeli Arab patients and 122 ethnically matched controls. Five patients (8.2%) and three controls (2.3%) carried one NOD2/CARD15 mutation. The phenotypic characteristics of the Arab and Jewish patients were very similar. We conclude that NOD2/CARD15 mutations do not contribute to Crohn’s susceptibility in the Israeli Arab population and suggest that NOD2/CARD15 mutations have an important effect on Crohn’s prevalence within a specific population but not on the phenotype. R. Safadi, MD, and R. Eliakim, MD, share senior authorship     

Investigation of association of the DLG5 gene with phenotypes of inflammatory bowel disease in the British population

Background and aims Mutations in the DLG5 gene are associated with an increased risk of inflammatory bowel disease (IBD) in some European populations. Initial investigation of a British IBD population showed evidence of association of one of three DLG5 variants, R30Q, in a family-based collection but not in a case-control cohort. We have now examined the association of the R30Q polymorphism in a large cohort of British IBD cases, tested for interaction between the DLG5 and CARD15 genes and assessed possible association of DLG5 with clinical features of Crohn’s disease (CD) and ulcerative colitis (UC). Materials and methods DLG5 R30Q and the CARD15 polymorphisms, Arg702Trp, Gly908Arg and Leu1007fs were genotyped in 1,148 IBD cases and 749 controls. DLG5 R30Q was also analysed in cases stratified by CARD15 genotype, disease subtype and smoking history. Results/findings No significant difference in frequencies of the R30Q variant was observed between IBD cases (9.9%) and controls (10.1%) or in cases analysed separately as CD and UC. There was also no significant difference in the frequency of R30Q between CD cases carrying risk-associated CARD15 alleles and those that did not. The frequency of R30Q was higher in CD cases with ileal disease than cases without (p=0.042) and higher in CD cases who had smoked than in nonsmokers (p=0.009). Interpretation/conclusion The R30Q variant in the DLG5 gene does not appear to be associated with an overall increase in the risk of disease in a British IBD cohort, but differences in its frequency in subgroups of CD patients warrant further investigation.     

The genetics of inflammatory bowel disease

The inflammatory bowel diseases (IBD) comprise complex genetic disorders, with multiple contributing genes. Linkage studies have implicated several genomic regions as likely containing IBD susceptibility genes, with some observed uniquely in Crohn's disease (CD) or ulcerative colitis (UC), and others common to both disorders. The best replicated linkage region, IBD1, on chromosome 16q contains the CD susceptibility gene, NOD2/CARD15. NOD2/CARD15 is expressed in peripheral blood monocytes and is structurally related to the plant R proteins, which mediate host resistance to microbial pathogens. Three major coding region polymorphisms within NOD2/CARD15 have been highly associated with CD among patients of European descent. Having one copy of the risk alleles confers a 2-4-fold risk for developing CD, whereas double-dose carriage increases the risk 20-40-fold. All 3 major CD variants exhibit a deficit in NF-kappaB activation in response to bacterial components. Carriage of NOD2/CARD15 risk alleles is associated with ileal location, earlier disease onset, and stricturing phenotype. Other IBD genomic regions include IBD2 on chromosome 12q (observed more in UC), and IBD3, containing the major histocompatibility complex region. A short genomic region has been associated with CD on chromosome 5q, but the precise contributing gene is as yet unidentified. The characterization of additional IBD susceptibility genes could potentially lead to the identification of novel therapeutic agents for IBD, make possible a molecular reclassification of disease, and increase understanding of the contribution of environmental factors (notably, tobacco and the intestinal microbial milieu) to intestinal inflammation.     

<Emphasis Type="Italic">CARD15</Emphasis> Genotype-Phenotype Relationships in a Small Inflammatory Bowel Disease Population with Severe Disease Affection Status

Inflammatory bowel disease (IBD; MIM# 266600) is subdivided on the basis of clinical findings as either Crohn’s disease (CD), ulcerative colitis (UC), or indeterminate colitis (IC). Three previously described mutations within the IBD susceptibility gene CARD15 (R702W, G908R, 1007fs) increase susceptibility to CD with a terminal ileal and/or ileocolonic location and fibrostenosing behavior. We undertook an association study using 477 unrelated IBD patients (248 CD, 172 UC, 57 IC) and 104 population controls to determine whether these previously described associations could be replicated in a small, accurately phenotyped cohort. Case-control and family-based approaches were employed to analyze CARD15 mutant allele and haplotype data. Analyses were initially performed in unstratified IBD cohorts. The R702W mutant allele was associated with CD on case-control analysis (q=0.036, P=.004), and 1007fs with CD on pedigree disequilibrium testing (P=.020). All 3 CARD15 mutations increased susceptibility to a variety of CD subphenotypic manifestations, including early-onset CD in individuals with a family history of IBD, and CD complicated by extraintestinal disease. We also present evidence to suggest that R702W may predispose to a more generalized form of CD. Additionally, we confirm that CARD15 mutations are associated with terminal ileal/ileocolonic, and to a lesser extent, fibrostenosing CD.     

The pathogenesis of inflammatory bowel disease: translational implications for clinicians

Research in the pathogenesis of inflammatory bowel disease (IBD) has dramatically broadened our understanding of these complex disorders. These clinical manifestations result from a dysregulated immune response in the presence of luminal bacteria. Recent identification of mutations in the NOD2 gene, a protein involved in the sensing of bacteria, offers genetic support for the model of perturbed host-microbial interactions in Crohn’s disease. Several immunologic pathways have been identified that play a role in maintaining gut immune homeostasis. Abnormal expression of proinflammatory, deleterious cytokines such as tumor necrosis factor-’a and interferon-γ results in direct and indirect tissue damage. The search for specific causative microbial agents in IBD continues to be intense. This paper describes the advances in our understanding of IBD pathogenesis, with an emphasis on how this information is translated into patient care. The next stage of research will take advantage of such molecular biologic techniques to identify new pathogenetic mechanisms and targets for therapy tailored to individual patients.     

NOD2/CARD15, NOD1/CARD4, and ICAM-1 gene polymorphisms in Turkish patients with inflammatory bowel disease

Purpose The genetic susceptibility of people with certain NOD2/CARD15, NOD1/CARD4, and ICAM-1 gene variants to inflammatory bowel disease is still under investigation. The aim of this study was to investigate polymorphisms in the NOD2/CARD15 (R702W, G908R, and 3020insC), NOD1/CARD4 (E266K, D372N), and ICAM-1 (G241R, K469E) genes, which are known to be associated with inflammation, in Turkish patients with inflammatory bowel disease and healthy control groups. Methods The genotypes of 70 patients with endoscopically and histopathologically diagnosed Crohn's disease (38 men, 32 women; mean age, 38.8 ± 1.3), 120 patients with ulcerative colitis (67 men, 53 women; mean age, 41.7 ± 1.3) and 106 healthy control subjects (37 men, 69 women; mean age, 35.7 ± 1.4), who stated that they had never had any prior bowel disease history, were compared. A polymerase chain reaction-restriction fragment length polymorphism analysis was performed for two variants of the ICAM-1 gene, the three main variants of the NOD2/CARD15 gene, and the E266K variant of the NOD1/CARD4 gene, and DNA sequencing was used for the D372N polymorphism of the NOD1/CARD4 gene. Results In this study, the three previously described Crohn's disease-predisposing variants of the NOD2/CARD15 gene and the polymorphisms examined in the NOD1/CARD4 and ICAM-1 genes were not found to be associated with ulcerative colitis or Crohn's disease. Conclusions These findings suggest that the polymorphisms observed in the NOD2/CARD15, NOD1/CARD4, and ICAM-1 genes are not genetic susceptibility factors for Crohn's disease or ulcerative colitis in Turkey.     

IGR2096a_1 T and IGR2198a_1 C alleles on IBD5 locus of chromosome 5q31 region confer risk for Crohn’s disease in Hungarian patients

Background and aims  We investigated the possible association of IBD with C1672T of SLC22A4 and G-207C of SLC22A5 alleles, and with the novel IGR2096a_1 (rs12521868) and IGR2198a_1 (rs11739135) susceptibility loci, all located on IBD5 locus of chromosome 5q31. Materials and methods  DNA of 217 Crohn’s disease, 252 ulcerative colitis, and 290 control patients were analyzed by polymerase chain reaction/restriction fragment length polymorphism methods. Results  Neither the C1672T and G-207C alleles, nor the TC haplotype were found to be risk factors. By contrast, the minor allele frequencies of IGR2096a_1 T (47.2%) and IGR2198a_1 C (45.9%) were increased in Crohn’s disease compared with the controls (38.2% and 37.7%, respectively; p < 0.05); multivariate regression analysis revealed a risk nature for Crohn’s disease (OR = 1.748, 95% CI 1.186–2.574; p = 0.007 for T allele, OR = 1.646, 95% CI 1.119–2.423, p = 0.011 for C allele of IGRs). Conclusion  The data suggest a special haplotype arrangement of susceptibility genes at the IBD5 locus in Hungarians, which nation differs historically from the surrounding Caucasian ethnicities in its origin.     

Association of <Emphasis Type="Italic">SLC22A4/5</Emphasis> Polymorphisms with Steroid Responsiveness of Inflammatory Bowel Disease in Japan

Purpose We investigated the association between steroid responsiveness and single nucleotide polymorphisms of SLC22A4/A5 located within inflammatory bowel disease 5 locus. Our goal is personalized steroid therapy adjusted to match individual variations in drug responsiveness in each inflammatory bowel disease patient. Methods Unrelated Japanese cohorts of 94 patients with Crohn’s, 94 patients with ulcerative colitis, and 257 healthy control subjects were consecutively enrolled in this study. Genotyping and haplotype analysis focusing on steroid responsiveness was performed by using 15 single nucleotide polymorphisms. Results The G allele of −368T > G in SLC22A5, in which strong linkage disequilibrium was observed and the limited diversity of three haplotypes was estimated, was significantly associated with steroid resistance in Japanese patients with Crohn’s disease (P = 0.016). Haplotype analysis between −446C > T and −368T > G in the SLC22A5 promoter region showed that the CG allele appeared to be a risk haplotype for steroid resistance (CG: odds ratio, 4.13; 95 percent confidence interval, 1.41–12.1; P = 0.016). Conclusions This extensive linkage disequilibrium may form a general risk haplotype for steroid resistance in Crohn’s disease in Japanese. Further analyses of the pharmacogenomics of steroid responsiveness are warranted to achieve the goal of individualized steroid therapy against inflammatory bowel disease. Supported by a grant-in-aid from the Ministry of Health, Labour and Welfare (K.I.), Japan. Address of correspondence: Yoshiaki Arimura, M.D., First Department of Internal Medicine, Sapporo Medical University, S-1, W-16, Chuo-ku, Sapporo, 060-8543, Japan. E-mail: arimura@sapmed.ac.jp     

Genetic and pathogenetic insights into inflammatory bowel disease

Crohn’s disease (CD) and ulcerative colitis (UC) are chronic inflammatory disorders of the gastrointestinal tract that share clinical and pathologic characteristics. The most credible hypothesis is that CD and UC result from an inappropriate and exaggerated mucosal immune response to normal constituents of the mucosal microflora that is in part genetically determined. However, there is reason to believe that the main pathologic processes in these two diseases are distinct. For example, the CARD15/NOD2 gene has been identified as a susceptibility gene for CD but not for UC. Moreover, the study of patients and mouse models of inflammatory bowel disease has clearly shown that, in CD, the tissue-damaging inflammatory reaction is driven by interleukin-12-activated Th1 cells, whereas a humoral response predominates in UC.     

Current concept on the pathogenesis of inflammatory bowel disease-crosstalk between genetic and microbial factors： Pathogenic bacteria and altered bacterial sensing or changes in mucosal integrity take ＂toll＂ ？

The pathogenesis of inflammatory bowel disease(IBD)is only partially understood.Various environmentaland host(e.g.genetic-,epithelial-,immune and non-immune)factors are involved.It is a multifactorialpolygenic disease with probable genetic heterogeneity.Some genes are associated with IBD itself,while othersincrease the risk of ulcerative colitis(UC)or Crohn's disease(CD)or are associated with disease locationand/or behaviour.This review addresses recent advancesin the genetics of IBD.The article discusses the currentinformation on the crosstalk between microbial andgenetic factors(e.g.NOD2/CARD15,SLC22A46A5 andDLG5).The genetic data acquired in recent years help inunderstanding the pathogenesis of IBD and can identify anumber of potential targets for therapeutic intervention.In the future,genetics may help more accurately diagnoseand predict disease course in IBD.     

Disease Behavior in Children with Crohn’s Disease: The Effect of Disease Duration, Ethnicity, Genotype, and Phenotype

Background Disease behavior in Crohn’s disease (CD) may be modified by disease location and genotype. Disease behavior may change over time, and thus analysis requires follow-up. To date, there have been few pediatric studies that have evaluated the association between disease behavior and genotype with prolonged follow-up. The aim of our study was to evaluate the effect of genotype, phenotype, and ethnicity on disease behavior in pediatric CD. Methods Evaluation of 128 pediatric CD was followed by analysis of 232 pediatric and adult-onset CD patients. Inclusion required at least 2 years of follow-up. Phenotype, ethnicity, and disease duration were recorded. Patients were genotyped for polymorphisms in the NOD2/CARD15 gene. Results Colonic involvement was more frequent in younger patients. Pediatric disease at end of follow-up was classified as inflammatory (78%), penetrating (7%), and stricturing (17%). Duration of follow-up (mean 4.9 pediatric and 6.4 years mixed) was associated with more stricturing and penetrating disease. There was no association between mean age of onset and NOD2/CARD15, or either of these with disease behavior. These observations were replicated in the mixed cohort. Sephardic Jewish origin was inversely correlated with inflammatory behavior (P = 0.006), independent of NOD2/CARD15 genotype. Conclusions Duration of disease and ethnicity, irrespective of NOD2/CARD15 genotype and age of onset, were the only predictors for penetrating or stricturing disease. Ron Shaoul, Esther Leshinsky-Silver, and Arie Levine contributed equally in this endeavor.     

Association of chronic non-bacterial osteomyelitis with Crohn’s disease but not with CARD15 gene variants

Chronic non-bacterial osteomyelitis (CNO) is an inflammatory, non-infectious disorder of the skeletal system with unknown etiology. Besides bone-inflammation, patients may present with inflammatory involvement of other tissues. Chronic recurrent multifocal osteomyelitis (CRMO) is the most severe form of CNO. We describe the occurrence of Crohn’s disease (CD) in four patients, previously diagnosed with CRMO. Mutations in CARD15, encoding the NOD2 protein, have recently been found in patients with CD. Based on the occurence of CNO and CD in these four and several reported patients, we hypothesized that CD and CRMO might share a common autoinflammatory process. Thus, we searched for CD associated CARD15 gene variants R702W, G908R and 1007fs in 29 CNO patients, 4 of them additionally diagnosed with CD. In the latter one out of the four showed compound heterozygosity for the gene variants R702W and 1007fs. The allele frequency in the 25 patients diagnosed with CNO but not CD was not different from that already reported in healthy people (R702W 4.0%, G908R 2.0%, 1007fs 2.0%). The occurrence of non-bacterial bone inflammation and granulomatous intestinal inflammation seems to represent an extended phenotype of CD, which partly might be explained by potential disease causing mutations in CARD15. However, CNO without intestinal inflammation is not associated with common CARD15 gene variants. Therefore, other variants of genes coding for proteins involved in innate immunity and inflammation might predispose for the occurrence of CNO.     

NOD2/CARD15基因R702W、G908R及L1007fs多态性与广西壮族人群炎症性肠病的相关性

目的:探讨我国广西壮族人群NOD2/CARD15基因R702W、G908R及L1007fs的遗传多态性与炎症性肠病的相关性.方法:分别收集2007-02/2010-10在广西地区无亲缘关系的壮族(n=70)和汉族(n=76)IBD患者及壮族(n=80)和汉族(n=84)正常对照者的肠黏膜组织.采用酚氯仿法提取各组织样本DNA,采用聚合酶链反应-限制性片段长度多态性分析(PCR-RFLP)方法对NOD2/CARD15基因R702W、G908R及L1007fs进行检测,统计基因型及等位基因频率,分析上述3个多态性位点与广西壮族人群炎症性肠病的相关性.结果:广西壮族和汉族IBD患者与正常对照者均未发现NOD2/CARD15基因R702W、G908R及L1007fs突变型基因型,所有多态性位点上的基因型全部为野生型纯合子,其基因型频率和等位基因频率分布在IBD患者和正常对照者中差异无统计学意义(P>0.05).结论:NOD2/CARD15基因R702W、G908R及L1007fs多态性与广西壮族人群炎症性肠病无明显相关性.     

Contribution of the NOD1/CARD4 insertion/deletion polymorphism +32656 to inflammatory bowel disease in Northern Europe

BACKGROUND: NOD1/CARD4 and NOD2/CARD15 are both intracellular pattern-recognition receptors. The NOD1/CARD4 gene lies within a previously described inflammatory bowel disease (IBD) locus (7p14). An association has been suggested between the NOD1/CARD4+32656 deletion*1 variant of a complex deletion*1/insertion*2 polymorphism and IBD in 1 recent study in Europe. Our aim was to assess the influence of NOD1/CARD4+32656 on disease susceptibility and phenotype in the Scottish and Swedish IBD populations. METHODS: A total of 3,962 individuals (1,791 IBD patients, 522 parents, 1,649 healthy controls) from 2 independent populations (Scotland and Sweden) were genotyped for NOD1/CARD4+32656 A/C by TaqMan and direct sequencing. Case-control, Transmission Disequilibrium Testing (TDT) and detailed genotype-phenotype (Montreal) analyses were performed. The case-control analysis had 80% power to detect an effect size of odds ratio (OR) 1.21 for IBD. RESULTS: In case-control analyses in Scottish and Swedish patients, none of the genotypes studied in IBD, Crohn's disease (CD) or ulcerative colitis (UC), differed significantly from controls (deletion*1 allelic frequency 73.9%, 73.6%, 73.9%, and 73.6%, respectively: all P > 0.8). No epistatic interaction with NOD2/CARD15 was seen for CD susceptibility. TDT analysis in our Scottish early onset cohort was negative. CONCLUSIONS: This variant allele of NOD1/CARD4+32656 is not associated with a strong effect on susceptibility to IBD in children and adults in Northern Europe. A gene-wide haplotype-based approach may be preferable to analysis of individual variants to assess the contribution of the NOD1/CARD4 gene to IBD.