Identification of NOD2/CARD15 mutations in Malaysian patients with Crohn's disease

OBJECTIVE: The NOD2/CARD15 gene has been identified as an important susceptibility gene for Crohn's disease (CD) but the three common disease predisposing mutations (DPM) found in developed countries have not been identified in Asian populations. The aim of our study was to look for the DPM in our multiracial population and to discover whether there were any differences in the three major ethnic groups; Malay, Chinese and Indian. METHODS: Blood samples from consecutive CD patients and healthy controls were obtained and analyzed for the three common mutations (R702W, G908R, 1007fs) but in addition to this, we also looked for the SNP5 and JW1 variants which are associated with CD in Ashkenazi Jews. A polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) technique was used to identify the mutations, which was confirmed by sequencing. The baseline socio‐demography and clinical characteristics of the CD patients were recorded. RESULTS: Overall 45 patients (three Malays, 15 Chinese, 26 Indians and one other) with confirmed CD and 300 controls were recruited. The three common DPM were not observed in either the CD patients or the controls. Neither the SNP5 nor the JW1 mutation was found in any of the controls. However, the SNP5 mutation was identified in six (13.3%) Indian CD patients and the JW1 mutation in eight CD patients who are different from those carrying the SNP5 mutation: one Malay (33.3%), two Chinese (13.3%), one other (Portuguese) and four Indians (15.4%). The presence of SNP5 was strongly associated with CD in the Indian population and that of JW1 was strongly associated with CD overall and in each of the major ethnic groups. There was a trend towards a younger age of onset and stricturing disease in patients carrying the JW1 mutation. CONCLUSION: These findings suggest the presence of novel DPM in the NOD2/CARD15 gene in Asian patients with CD.     

Clinical applications of NOD2/CARD15 mutations in Crohn's disease

The recent identification of the CARD15/NOD2 gene as a susceptibility locus for Crohn's disease represents an important step in the immunopathogenesis of inflammatory bowel disease. The gene explains about 20% of the genetic susceptibility CARD15 mutations are present in 30-50% of CD patients compared to 7-20% of healthy controls. The three risk alleles R702W, G908R and 1007fsInsC in NOD2 associated with susceptibility to Crohn's disease have demonstrated a remarkable amount of heterogeneity across ethnicities and populations, with regional variation across Europe. In non-Caucasian populations Crohn's disease continues to increase in incidence but this increase appears not to be a consequence of variation in NOD2. Genotype-phenotype analyses demonstrated an association of these mutations with ileum-specific disease and an increased incidence of the fibrostenotic phenotype. Although CARD15 variants do not predict response to the TNF alpha monoclonal antibodies, there are no data available on the possible influence of CARD15 mutations on response to other drugs. Screening for CARD15 mutations in order to identify high-risk individuals or to introduce an individualized disease management is therefore currently not recommended.     

Frequency analysis of NOD2 gene mutations in Korean patients with Crohn's disease]

BACKGROUND/AIMS: Several studies from Western populations have recently shown that three mutations in NOD2 gene (C2104T, G2722C, and 3020insC) are associated with susceptibility to Crohn's disease (CD). However, three mutations were shown not to be associated with CD in Japanese and Chinese population. Here, we have analyzed the frequency of three NOD2 mutations in Korean patients to determine whether the NOD2 mutations are associated with susceptibility to CD in Korean population. METHODS: Blood samples were obtained from 128 patients with CD, 47 patients with ulcerative colitis, 19 Behcet's colitis, and 200 healthy controls. DNA in the region of three NOD2 mutations was sequenced by single base extension method, and the frequency of mutations were analyzed. RESULTS: Among the subjects in our study groups, including patients with CD, ulcerative colitis, Behcet's colitis, and healthy controls, none had NOD2 mutations. CONCLUSIONS: Our results indicate that although three NOD2 mutations are associated with susceptibility to CD in Western populations, these might be rare and may not be associated with susceptibility to CD in Korean patients.     

NOD2 sequencing in Iranian children with Crohn's disease

Crohn's disease (CD) is a chronic inflammatory disease of the gastrointestinal tract. Although the exact etiology of disease is still unknown, mutations in the CARD15/NOD2 gene have been reported in association with CD in several studies. This study was performed to determine whether the CARD15/NOD2 gene confers susceptibility to Iranian pediatric patients with CD. All 12 coding exons of the CARD15/NOD2 gene were sequenced in 16 enrolled pediatric onset CD patients. Sequencing of the CARD15/NOD2 gene showed no mutation. However, two patients harbored polymorphisms within this gene. A heterozygous single nucleotide polymorphism rs3135500 C?>?Y in the exon 12.3 was detected in a 10-year-old girl with mild severity of CD and history of rectovaginal and perianal fistula, and multiple skin tags. The other 5-year-old boy with moderate to severe CD and a history of perianal fissures and oral candidiasis harbored heterozygous single nucleotide polymorphisms in exons 4.1 and 12.1. The results of the present study show that the CARD15/NOD2 mutations in Iranian patients with pediatric onset CD are not responsible for the pathogenesis of disease.     

Association of NOD2/CARD15 mutations with previous surgical procedures in Crohn's disease.

OBJECTIVE: The aim of this study is to assess the importance of NOD2/CARD15 gene mutations as prognostic factors for surgical indications in Crohn's disease. PATIENTS AND EXPERIMENTAL DESIGN: A total of 165 Crohn s disease patients were studied, considering previous surgery related to Crohn's disease. We analyzed for previous surgery in global procedures as well as separately for the two main surgical indications: ileal resection and fistula treatment. The need for appendectomy was also studied. All patients were genotyped for the three CARD15 mutations, and association studies were developed using Chi-square statistics and Fisher's exact test whenever appropriate. RESULTS: Carriers of the G908R or 1007fs mutation needed surgery more frequently, both for ileal resection and fistula repair. In contrast, appendectomy was not associated with CARD15 mutations. CONCLUSIONS: As previously reported in this population, the R702W mutation does influence parameters of disease or need of surgery. The need for Crohn's disease-related surgery is higher in carriers of the G908R or 1007fs CARD15 mutation in the Galician population. Nevertheless, the frequency of these mutations does not allow their use to predict the course of disease.     

NOD2/CARD15 mutations in Croatian patients with Crohn's disease: prevalence and genotype-phenotype relationship

BACKGROUND: Crohn's disease (CD) is a chronic inflammatory disorder of the gastrointestinal tract with variations in localization and behaviour. Mutations in the NOD2/CARD15 gene on chromosome 16q have been implicated in the pathogenesis of the disease and three main sequence variants, all single nucleotide polymorphisms (SNPs), have been identified in North American and European populations. AIMS AND METHODS: As no data exist in the Croatian population, we consecutively collected a cohort of 136 CD patients and 91 healthy controls to determine the prevalence of NOD2/CARD15 mutations and their association with phenotypic expression of the disease. All patients and controls were genotyped for Arg702Trp (Hugot SNP8), Gly908Arg (Hugot SNP12), and Leu1007fsinsC (Hugot SNP13) and allele frequencies were compared between the Crohn's patients and controls. The correlation of NOD2/CARD15 genotypes with the phenotypic expression of Crohn's disease was further assessed by logistic regression analysis. RESULTS: NOD2/CARD15 variants were found in 38/136 CD patients (27.9%) compared to 10/91 (10.9%) healthy controls (P = 0.0022). Allele frequencies in patients with CD were 13.97%, 4.4% and 11.76%, respectively, for SNP8, 12 and 13, compared to 5.49%, 1.12% and 4.40% in controls (P = 0.041, P = 0.162, P = 0.055). Six CD patients carried double mutations and, remarkably, we identified two homozygous mutants amongst the healthy control group. Surgery over the course of the disease and a younger age at onset of the disease were significantly more frequent in patients who were carriers of NOD2/CARD15 mutations. CONCLUSIONS: This report on NOD2/CARD15 mutations in Croatian patients with CD demonstrates that this gene is also implicated in susceptibility to CD in the Croatian population. Phenotypic association showed a younger age at diagnosis and a higher need for surgery in patients carrying NOD2/CARD15 mutations. However, the prevalence is somewhat lower compared to other reports, likely due to a more prominent colonic inflammation.     

NOD2/CARD15 mutations and presence of granulomas in pediatric and adult Crohn's disease

OBJECTIVES: The etiology and mechanism leading to granuloma formation in patients with Crohn's disease (CD) are presently unknown. The first susceptibility gene to be identified as a risk factor for CD is the NOD2/CARD15 gene on Chromosome 16. Mutations in NOD2 could affect the intracellular response to bacterial products and may eventually lead to granuloma formation. The association between NOD2 and granulomas has not been previously explored. We evaluated a possible association between NOD2 mutations and granuloma formation, and compared the prevalence of granulomas in both pediatric and adult cohorts. METHODS: Patients were consecutively recruited through pediatric gastroenterology and adult gastroenterology programs. Patients were eligible if CD was confirmed, and they had undergone full colonoscopy with biopsy and/or surgical resection. Patients underwent genotyping for NOD2 disease-associated mutations. RESULTS: A total of 230 patients were enrolled into the study, of whom 169 patients met all inclusion/exclusion criteria (Group 1, 77 patients [age range 1-16 years]; Group 2, 92 patients [age range 17-68 years]). Surgical resection was performed more often in adults (P < 0.005), and gastroscopy was performed more frequently in children (P < 0.001). Granulomas were found in 34% of the patients studied. The prevalence of granulomas did not differ by age, age group, or gender. A disease-associated NOD2 mutation was found in 37.8% of patients. Granulomas were found in 39% of patients with NOD2 mutations compared with 31% of those without NOD 2 mutations (difference was not significant). In addition, no difference was noted for the specific mutations. CONCLUSIONS: We did not find any correlation between NOD2 mutations and granuloma formation. The cause of granulomas in CD remains elusive.     

Association of NOD2/CARD15 mutations on Crohn's disease phenotype in an Italian population

AIMS: To confirm the prevalence of NOD2/CARD15 mutations in Italian inflammatory bowel disease patients and to define the role of the different mutations on Crohn's disease phenotype. PATIENTS AND METHODS: A total of 177 patients with Crohn's disease and 92 patients with ulcerative colitis and 164 control participants were investigated for the presence of Arg702Trp, Gly908Arg and Leu1007fsinsC NOD2/CARD15 mutations. Allele frequencies in Crohn's disease and ulcerative colitis patients were compared with those observed in the control population. Genotype-phenotype correlations with the major clinical features were also established and estimated risks (odds ratio with 95% confidence interval) for the mutations were calculated by logistic regression and multiple correspondent analysis. RESULTS: Gly908Arg and Leu1007fsinsC mutations were significantly more frequent in Crohn's disease patients compared with healthy controls (P<0.01 and <0.003 respectively). Indeed, using a logistic regression model adding terms for age (differently distributed between cases and controls) and sex, a significantly increased risk of having Crohn's disease compared with healthy controls was found for all NOD2 mutations: Leu1007fsinsC (odds ratio=7.35; 95% confidence interval: 1.73-31.3), Gly908Arg (odds ratio=5.70; 95% confidence interval: 1.37-23.7) and Arg702Trp (odds ratio=2.45; 95% confidence interval: 1.10-5.47). As far as the genotype-phenotype correlations are concerned, by multivariate conditional logistic regression methods, we found a significant association between Gly908Arg mutations and familial history of inflammatory bowel disease, between Leu1007fsinsC mutations and appendectomy and between Arg702Trp mutations and fibrostenotic phenotype of Crohn's disease. A nonsignificant association between Arg702Trp variants and ileal disease was also found (odds ratio=8, 95% confidence interval: 0.99-64.9). CONCLUSIONS: The results of the study confirm a significant association of CARD15 gene mutations in our Italian Crohn's disease population and the impact of different NOD2/CARD15 mutations on specific disease phenotypes.     

NOD2 exonic variations in Iranian Crohn's disease patients

Purpose

The NOD2 gene is known to have a strong association with Crohn??s disease, but different trends were reported in occurrence of NOD2 variants in distinct ethnicities. The aim of this study was to assess all exonic sequences of the NOD2 gene in Iranian Crohn's disease patients and healthy controls to identify any existing variation and evaluate their association with Crohn's disease.

Methods

A total of 90 non-related Crohn's disease patients and 120 sex- and age-matched healthy controls of Iranian origin were enrolled in this study. The participants were referred to a tertiary center in a 2-year period (2006?C2008). The exonic regions of the NOD2 gene were amplified by polymerase chain reaction and evaluated by direct sequencing.

Results

A total of 21 sequence variations were identified among all exonic regions of the NOD2 gene, of which eight had an allele frequency of more than 5%. Eight new mutations (one in exon 2 and seven in exon 4) were observed. The three main variants (R702W, G908R, and 1007fs) showed allele frequencies of 13.3%, 2.2%, and 1.7%, respectively. Three new variations (P371T, A794P, and Q908H) and R702W mutation were significantly more frequent in Crohn's disease patients compared to controls.

Conclusions

Eight novel mutations were identified in the NOD2 exons, but the pathophysiological importance of these variants remains unclear. Iranian patients with their different genetic reservoirs may demonstrate some novel characteristics for disease susceptibility.     

Lack of common NOD2 variants in Japanese patients with Crohn's disease

BACKGROUND & AIMS: Previous studies have linked Crohn's disease (CD) to the pericentromeric region of chromosome 16 (IBD1). Three independent studies of Western populations have recently shown that 3 variants of NOD2, a gene located at 16q12, are associated with susceptibility to CD. Here, we have evaluated the 3 NOD2 variants in Japanese patients to determine whether the gene is also associated with susceptibility to CD in a non-Western population. METHODS: Blood samples were obtained from 350 patients with CD, 272 patients with ulcerative colitis, and 292 healthy controls at 3 hospitals in Japan. DNA was sequenced in the region of the 3 NOD2 variants (C2104T in exon 4, G2722C in exon 8, and 3020insC in exon 11) by genomic polymerase chain reaction followed by direct sequencing. RESULTS: Among the subjects in our 3 study groups, including patients with CD, patients with ulcerative colitis, and healthy controls, none had common NOD2 variants that have been associated with CD in white patients. CONCLUSIONS: These results indicate that genetic variation, which may predispose some human populations to CD, may not be present in other populations and specifically that common variants in NOD2 found in white patients with CD are not associated with CD in the Japanese population.     

Toll-like receptor 4 and NOD2／CARD15 mutations in Hungarian patients with Crohn＇s disease： Phenotype-genotype correlations

AIM: To determine common NOD2/CARD15 mutations and TLR4 D299G polymorphism in Hungarian patients with CD. METHODS: A total of 527 unrelated patients with CD (male/female: 265/262, age: 37.1 (SD 7.6) years) and 200 healthy subjects were included. DNA was screened for possible NOD2/CARD15 mutations by denaturing high-performance liquid chromatography (confirmed by direct sequencing). TLR4 D299G was tested by PCR-RFLP. RESULTS: NOD2/CARD15 mutations were found in 185 patients (35.1%) and in 33 controls (16.5%,P<0.0001). SNP8/R702W (10.8% vs 6%, P= 0.02), SNP13/3020insC (19.4% vs 5%, P<0.0001) and exon4 R703C (2.1% vs 0%, P= 0.02) mutations were more frequent in CD, while the frequency of SNP12/G908R was not increased. The frequency of TLR4 D299G was not different (CD: 9.9% vs controls: 12.0%). Variant NOD2/CARD15 allele was associated with an increased risk for CD (ORhet=1.71, 95%CI=1.12-2.6, P= 0.0001, ORtwo-risk alleles = 25.2, 95%CI =4.37- ,P<0.0001), early disease onset (carrier: 26.4 years vs non-carrier: 29.8 years, P=0.0006), ileal disease (81.9% (?) 69.5%, OR = 1.99, 95%CI = 1.29-3.08, P= 0.02, presence of NOD2/CARD15 and TLR4: 86.7% vs 64.8%), stricturing behavior (OR = 1.69,95%CI = 1.13-2.55, P= 0.026) and increased need for resection (OR=1.71, 95%CI: 1.13-2.62, P= 0.01), but not with duration, extra-intestinal manifestations, familial disease or smoking. TLR4 exhibited a modifier effect: age of onset in wt/TLR4 D299G carriers: 27.4 years vs NOD2mut/TLR D299G: 23 years (P = 0.06), in NOD2mut/wt: 26.7 years. CONCLUSION: These results confirm that variant NOD2/ CARD15 (R702W, R703C and 3020insC) alleles are associated with earlier disease onset, ileal disease, stricturing disease behavior in Hungarian CD patients. In contrast, although the frequency of TLR4 D299G polymorphism was not different from controls, NOD2/TLR4 mutation carriers tended to present at earlier age.     

Association of NOD2/CARD15 variants with Crohn's disease in a Greek population

OBJECTIVE: Single nucleotide polymorphisms in the NOD2/CARD15 gene have recently been shown to be associated with Crohn's disease (CD), but whether this susceptibility extends to all ethnic groups and geographic areas remains unknown. The aim of the present study was to evaluate the NOD2/CARD15 mutations in Greek patients with CD. METHODS: Individuals were genotyped for three NOD2/CARD15 mutations: R702W, G908R and L1007fsinsC. Blood samples were obtained from 120 patients with CD, 85 patients with ulcerative colitis, and 100 unrelated healthy controls. RESULTS: Mutations in NOD2/CARD15 were observed with significantly greater frequency in CD patients (98/120, 81.7%) than in ulcerative colitis patients (40/85, 47%) (P < 0.0001) or in healthy individuals (21/100, 21%) (P < 0.0001). For CD patients, compared with controls, the odds were increased for carriage of the R702W (odds ratio, 12.25) and less for the G908R (odds ratio, 5.2) and L1007fsinsC (odds ratio, 3.9) mutations. The age of onset of CD was lower in Greek mutation carriers as compared with non-carriers of Greek origin (28.2 +/- 14.6 years versus 34 +/- 12.3 years, respectively; P = 0.036). Additionally, the frequency of NOD2/CARD15 mutations was increased in ileitis or ileocolitis compared with non-ileal disease. CONCLUSIONS: The NOD2/CARD15 mutations are risk factors for CD in Greece, they appear to predict an earlier age of onset and are associated particularly with ileitis or ileocolitis.     

Crohn's disease in four members of a family, two of whom are dizygotic twins

Familial occurrence of Crohn's disease (CD) is well known, but the disease is rarely reported to occur in dizygotic twins. We present an additional case of dizygotic twins, both of whom developed CD, from a family in which two other members are affected. The 16-year-old son contracted the disease 2 years before his 50-year-old father, and 13 years before his twin sister. Another sister was affected 6 years after the onset of the disease in the propositus. HLA haplotyping of the three children matched the Mendelian ratio. The multiple occurrence of CD in blood relatives, especially in siblings, emphasizes the importance of genetic factors in the development of this disease. This family history, however, could point to psychic influences promoting the occurrence of CD on the basis of a polygenic disease susceptibility.