CARD15 Mutations Research Articles

Mutation screening of the CARD15 gene in sarcoidosis

CARD15 was first identified as a susceptibility gene for Crohn's disease. More recently, CARD15 mutations were shown to be associated with the pediatric granulomatous inflammatory diseases, Blau syndrome and early-onset sarcoidosis (EOS). The aim of the present study was to evaluate whether CARD15 variants also play a role in patients with ordinary sarcoidosis other than EOS. We enrolled 135 Japanese sarcoidosis patients with uveitis as well as 95 healthy individuals and performed mutation analysis by direct sequencing of CARD15 exon 4. Direct DNA sequencing in the sarcoidosis patients showed eight CARD15 variants, including five novel mutations (13402C>T, 13543C>T, 13775C>A, 13937G>A, and 14079C>T). Compared with healthy individuals, CARD15 mutations are not common in the Japanese patients with sarcoidosis. Based on the results, we examined the clinical manifestations in patients with sarcoidosis according to their CARD15 mutations. Sarcoidosis patients with these mutations have no specific clinical features with regard to course of the disease or disease severity. Our results indicate that in general, CARD15 mutations may not contribute to the risk of sarcoidosis.

S1193 Phenotypic Associations of Crohn's Disease with Antibodies to Flagellins A4-Fla2, Fla-X, ASCA, pANCA and CARD15 Mutations in a Swiss Cohort

S1193 Phenotypic Associations of Crohn's Disease with Antibodies to Flagellins A4-Fla2, Fla-X, ASCA, pANCA and CARD15 Mutations in a Swiss Cohort

Genetic and environmental factors as predictors of disease severity and extent at time of diagnosis in an inception cohort of inflammatory bowel disease, Copenhagen County and City 2003–2005

Background and aims The etiology of the inflammatory bowel diseases (IBD), Crohn's disease (CD) and ulcerative colitis (UC) remains unknown. We aimed to investigate the influence of genetic, serological, and environmental factors on phenotypic presentation of IBD at diagnosis in a population-based Danish inception cohort from 2003–2005. Methods Three-hundred-forty-seven (62%) of 562 cohort patients were genotyped. ASCA and p/c-ANCA were determined and patients answered a questionnaire concerning environmental factors with possible influence on IBD. Results Fourteen percent of CD patients vs. 11% of controls were positive for common CARD15 mutation (ns), whereas more CD patients than healthy controls were homozygous for the OCTN-TC haplotype ( p = 0.03). ASCA was more common in CD (22%) than UC (14%) ( p = 0.045) and was related to age and localization of CD. p-ANCA was more frequent in UC ( p = 0.00001) but was related to pure colonic CD ( p = 0.0001). Sugar consumption was significantly higher in CD patients than in UC patients ( p = 0.0001) and more CD patients than UC patients had undergone appendectomy prior to IBD diagnosis ( p = 0.03). A possible relation between tonsillectomy and disease severity in CD, and a relation between use of oral contraception and disease localization of UC to rectum/left-sided colon were found. Conclusions In this cohort of unselected IBD patients we found a very low frequency of mutations in IBD susceptibility genes and observed a greater impact of ASCA and ANCA than of genetic factors on disease phenotypes. In addition, several environmental factors seemed to influence disease occurrence and disease presentation in both UC and especially CD.

The Role of CARD15 Mutations and Smoking in the Course of Crohn's Disease in a Mediterranean Area

To evaluate the role of CARD15 mutations and smoking in the main events of Crohn's disease (CD). A total of 182 patients with CD were included in a prospective study in order to evaluate the role of CARD15 mutations and smoking in the main outcomes of disease course: first operation and surgical recurrence. The following variables were evaluated in a univariable and multivariable analysis: age, sex, site of disease, pattern, smoking habit, extraintestinal manifestations, duration of disease, and CARD15 mutation. The Kaplan-Meier method for survival curves and Cox model for multivariable analysis were, respectively, used. A total of 110 patients were operated on and 32 were reoperated on. The 7-yr cumulative free rate of surgery was 42% (95% CI 34-51%). At multivariate analysis only stricturing and penetrating pattern were predictors of surgery (HR 1.7, 95% CI 1-2.8; HR 3.2, CI 1.8-5.5, respectively). The 7-yr cumulative free rate of reoperation was 75% (95% CI 0.52-0.88). At multivariable analysis in the model with any CARD15 mutation, only smoking habit at diagnosis (HR 3.6, 95% CI 1.4-9.1) was predictive of surgical recurrence. When single mutations were considered in the model smoking (HR 4.2, 95% CI 1.8-10.1) and L1007fs mutation (HR 2.9, 95% CI 1.1-7.3) were predictive of reoperation. In CD, smoking predicts recurrence after surgery. The role of CARD15 mutations in the clinical course of CD remains undefined.

Two-stage candidate gene study of chromosome 3p demonstrates an association between nonsynonymous variants in the MST1R gene and Crohnʼs disease

Genomewide linkage studies identified chromosome 3p21 as an IBD locus. Genomewide association studies have supported this locus and the Wellcome Trust Case Control Consortium (WTCCC) study narrowed it to a 0.6 Mb region. Our objectives were to perform a 2-stage candidate gene association study of the 3p locus and to identify linkage disequilibrium (LD) between significant single-nucleotide polymorphisms (SNPs) and an Oxfordshire subset (n = 282) of the WTCCC as well as the HapMap SNPs. A total of 197 SNPs in 53 genes from the 3p locus were genotyped on the Illumina platform in a screening cohort of 469 Crohn's disease (CD) patients and 461 controls. Significant associations were then genotyped on the iPLEX platform in the original as well as a second cohort of 139 CD patients, 670 ulcerative colitis (UC) patients, and 1131 controls. All cases and controls were Caucasian and from the Oxfordshire region of the UK. An intronic SNP rs1128535 in the TRAIP gene was associated with CD in the screening and validation cohorts (combined [n = 608] P = 0.0004 [corrected 0.002], odds ratio [OR] 0.77, 95% confidence interval [CI], 0.67-0.89]). No association was seen for UC. Epistasis was seen with the common CARD15 mutations (P = 0.00003 [corrected 0.0006], OR 0.48, 95% CI, 0.34-0.68). No LD was demonstrated with the WTCCC SNPs. Strong LD was demonstrated with 2 nonsynonymous HapMap SNPs in the MST1R gene in an adjacent LD block to the peak WTCCC association, suggesting a distinct association signal. The LD with these functional MST1R variants implicate this gene as having a possible role in CD pathogenesis.

Diverse effects of the CARD15 and IBD5 loci on clinical phenotype in 630 patients with Crohnʼs disease

Genetic variants at the CARD15 and IBD5 loci are strongly associated with Crohn's disease (CD), but evidence of the effect of these variants on the clinical expression of CD is conflicting and has often been hampered by small sample sizes. We studied 630 well-characterized patients to clarify the genotype/phenotype relationship in CD. Patients and healthy controls were genotyped for three common mutations in CARD15 and a marker of the IBD5 risk haplotype. Allele frequencies were compared between phenotypic subgroups using chi2 or Fisher's exact tests. Genotype/phenotype analysis was carried out using multinomial logistic regression modelling allowing for adjustment for correlated or confounding factors. The mean age at diagnosis was significantly lower in carriers of the CARD15 or IBD5 risk alleles. After correction for age and smoking, CARD15 mutations were strongly associated with both ileal disease (P=8.8 x 10(-6)) and stenotic disease (P=0.003), but the association with stenotic disease appeared to be due to a confounding effect with ileal disease. CARD15 mutations were also associated with the presence of granulomas (P=5.7 x 10(-5)), which remained significant after adjustment for age at diagnosis and disease location (P=0.0047). The IBD5 risk haplotype frequency was significantly elevated in cases with perianal disease (P=0.028) and axial spondyloarthropathy (P=0.012). Genetic variants at the CARD15 and IBD5 loci have diverse effects on clinical expression in CD. CARD15 mutations are significantly correlated with the presence of granulomas.

Novel CARD15/NOD2 mutations in Finnish patients with Crohnʼs disease and their relation to phenotypic variation in vitro and in vivo

Three mutations (R702W, G908R, and 1007fs) of the CARD15/NOD2 gene associate with Crohn's disease (CD). Despite a strong linkage of CD to the inflammatory bowel disease (IBD) 1 region, only 16% of the Finnish CD patients carry 1 of these 3 mutations, pointing to the possibility of yet undetected founder mutations in the genetically isolated Finns. The aim of this study was to screen for CARD15 mutations in Finnish CD patients and to assess their functional consequences and relation to clinical phenotype. We performed CARD15 mutation screening in 240 CD probands. For functional studies, blood mononuclear cells were cultured alone or with muramyl dipeptide (MDP) and IL-8 levels were determined. We identified 30 different variants, including 12 new ones. Allele frequencies for the R702W, G908R, and 1007fs mutations were 3.3%, 0.4%, and 4.8%, respectively. The 1007fs variant was the only 1 associated significantly with CD. Five novel variants (R38M, W355X, P727L, W907R, R1019X) were found in 5 patients. The biochemical nature of these new mutations, data obtained by cross-species comparisons, as well as low IL-8 production favors their pathogenic role. All 5 patients with novel mutations presented a complicated form of ileal or ileocolonic disease. In conclusion, we identified 5 novel CARD15 mutations with an apparent pathophysiological role, but could not identify a putative Finnish founder mutation. It is still possible that regulatory mutations present in the flanking or intronic areas of the CARD15 gene contribute to the genetic susceptibility of CD. Homozygosity or compound heterozygosity for CARD15 gene mutations must be considered especially in complicated CD patients.

Las mutaciones del gen CARD15 presentan escasa relación con los fenotipos de la enfermedad de Crohn en Asturias

the association between the three common CARD15 gene mutations (R702W, G908R, L1007fs) and the genetic susceptibility to Crohn s disease (CD) have been confirmed by several studies, with some differences found, in relation to geographic areas and ethnic groups. To analyze the prevalence of CARD15 gen and its polymorphisms in patients with CD in Asturias and its possible correlation with the different genotypes of the disease. a total of 216 CD patients recruited from Asturias (North of Spain) and 86 ethnically matched healthy controls, were typed using Hybprobes on a LightCycler instrument for CARD15 mutations. Patients were subdivided according to Vienna classification. We have studied the frequency of these mutations in the different subgroups of CD patients and analyzed its contribution to the disease clinical characteristics and progression. carrier frequencies for CARD15 mutations in our CD patients were similar to controls (17.8 vs. 17.4%) respectively (NS). CD patients exhibited frequencies of 8.8, 3.0 and 6.0% for the R702, G908R and L1007fs polymorphisms respectively, whereas our control population had allele frequencies of 11.6, 2.3 and 3.5% for the three mutations respectively (NS). We did not find any relationship between CARD15 mutations and the different phenotypes of Crohn s disease, according to Vienna classification. in our CD population, other factors (i.e. environmental), in addition to genetics, must be mainly involved in the development of the disease.

IL23R R381Q and ATG16L1 T300A Are Strongly Associated With Crohn's Disease in a Study of New Zealand Caucasians With Inflammatory Bowel Disease

Recently, separate genome-wide association analyses have identified nonsynonymous SNPs in IL23R and ATG16L1 (rs11209026; c1142G>A, R381Q, and rs2241880; c1338A>G, T300A, respectively) as strong candidate susceptibility factors for Crohn's disease (CD) in whites. The aim of our study was to test whether these SNPs are associated with CD in a population-based cohort of New Zealand Caucasian inflammatory bowel disease (IBD) patients. Allele frequencies of rs11209026 and rs2241880 were determined in 496 CD patients, 466 ulcerative colitis (UC) patients, and 591 controls. Distribution of the relevant alleles was compared between controls and IBD patients. rs11209026 and rs2241880 genotype distributions were examined both within IBD clinical subphenotypes and CARD15 genotypes. rs11209026 and rs2241880 were both associated with CD (P valuers11209026=0.0026, OR 0.54, 95% CI 0.36-0.81; P valuers2241880=0.0001, OR 1.41, 95% CI 1.18-1.67). In addition, there was evidence for association of rs11209026 with UC (P value=0.037, OR 0.66, 95% CI 0.45-0.98). No significant association was observed between IL23R genotype or ATG16L1 genotype and IBD subphenotypes. IL23R was associated with CD and UC only in the absence of CARD15 mutations, whereas ATG16L1 was associated with CD in the presence and absence of CARD15 mutations. We replicated the previously reported associations between CD and rs11209026 and rs2241880, confirming that IL23R and ATG16L1 are susceptibility loci for CD in the New Zealand population. We also provide further evidence for association of rs11209026 with UC and a report of an additive effect between IL23R and CARD15 genotypes in CD.

Estimating risks of common complex diseases across genetic and environmental factors: the example of Crohn disease

Background:Progress has been made in identifying mutations that confer susceptibility to complex diseases, with the prospect that these genetic risks might be used in determining individual disease risk.Aim:To use Crohn...

Hemorragia digestiva baja masiva en pacientes con enfermedad de Crohn

We aimed at evaluating the frequency of acute severe bleeding in CD and its potential association to some risk factors, including clinical features of CD, environmental factors, and genetic alterations. 174 consecutive patients with CD (103 female (59%) and 71 men (41%), with a mean age of 37 years) were included. We analyzed all major acute lower gastrointestinal (GI) hemorrhage related to CD. Potential risk factors like smoking, site of disease, and presence of gene mutations in CARD15, TLR-4, and CD14 were also analyzed. Three patients (1.7%) suffered from severe acute lower GI bleeding. All patients required surgery to resolve their hemorrhage, and this indication represented 3.4% of all surgical procedures related to CD. All three patients were young ( < 25 years) and suffered ileal CD with inflammatory pattern (L1-B1 in the Vienna Classification). No relationship was found between acute bleeding and any of the potential risk factors evaluated. Acute severe GI bleeding is a rare, but severe complication in CD patients, and presents mainly in patients with inflammatory ileal disease. An association of endoscopy and arteriography is necessary for diagnosis. Urgent surgery is usually required in these patients.

CARD15 mutations in Dutch familial and sporadic inflammatory bowel disease and an overview of European studies

The single nucleotide variations R702W, G908R and L1007fs in the CARD15 gene have been found to be independently associated with Crohn's disease. The aim of this study was to evaluate the prevalence of these gene variations in Dutch multiple inflammatory bowel disease-affected families, in sporadic inflammatory bowel disease patients and in healthy controls. Dutch Caucasians from multiple inflammatory bowel disease-affected families were recruited, including 78 probands with Crohn's disease, 34 probands with ulcerative colitis and 71 inflammatory bowel disease-affected and 100 non-affected family members. In addition, 45 sporadic inflammatory bowel disease patients (36 Crohn's disease and nine ulcerative colitis), and 77 unrelated healthy controls were included. Genomic DNA was isolated to determine CARD15 R702W, G908R and L1007fs. For these mutations, we evaluated disease susceptibility and correlation with inflammatory bowel disease phenotypes. In all included unrelated inflammatory bowel disease-affected probands, the R702W, G908R and L1007fs allele frequencies were 8.8, 6.1 and 11.0%, respectively, for Crohn's disease, and 4.7, 0 and 2.3% for ulcerative colitis. In controls, the allele frequencies were 5.9, 0.7 and 1.9%, respectively. G908R and L1007fs were associated with Crohn's disease (P=0.006 and 0.001, respectively). Compound heterozygotes for any of the three mutations were 11 (9.2%) in Crohn's disease patients, but none in ulcerative colitis patients nor controls. Carriage of CARD15 mutations was not associated with familial disease (P>or=0.38). Inflammatory bowel disease-affected family members of Crohn's disease probands carrying L1007fs, however, were carriers significantly more often than expected (P<0.001). In Crohn's disease patients, a significant trend was found between carriage of at least one CARD15 mutation and between carriage of L1007fs and behaviour of disease, including more carriers with stricturing and even more with penetrating disease (P=0.006 and 0.017, respectively). In the Dutch population, CARD15 G908R and L1007fs are associated with Crohn's disease. Although no difference was found between sporadic and familial cases, in L1007fs-positive multiple affected families the inflammatory bowel disease-affected relatives are more likely than expected to carry this mutation. In Crohn's disease, carriage of at least one CARD15 mutation is associated with a more complicated disease behaviour.

European Mathematical Genetics Meeting, Heidelberg, Germany, 12th–13th April 2007

European Mathematical Genetics Meeting, Heidelberg, Germany, 12^th–13^th April 2007

This Month in Gastroenterology

This Month in Gastroenterology

Maladie de Crohn et génétique : connaissances actuelles

Crohn's disease as ulcerative colitis are the main chronic inflammatory bowel diseases (IBD) in the developed countries of the world with an estimated prevalence of 1/250. Morbidity of these diseases are important and some patients live with a considerable symptom burden despite medical treatment. In 2001, mutations in the gene CARD15 (also named NOD2) have been identified and involved in susceptibility for Crohn's disease. Theses findings have played an important role to elucidate the leading role of innate immune response in the bowel and in understanding the pathogenesis of IBD and have helped to identify potential targets for therapeutic intervention. This paper summarizes the important knowledge acquisitions in the physiopathology of Crohn's disease. It underlines the important environmental, immunologic and genetic components leading to this multifactorial disease.

CARD15 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.

Common NOD2/CARD15 variants are not associated with susceptibility or the clinicopathologic characteristics of sporadic colorectal cancer in Hungarian patients.

BackgroundEpidemiological observations suggest that cancer arises from chronically inflamed tissues. Inflammatory bowel disease (IBD) is a typical example as patients with longstanding IBD are at an increased risk for developing colorectal cancer (CRC) and mutations of the NOD2/CARD15 gene increase the risk for Crohn's disease (CD). Recently, NOD2/CARD15 has been associated with a risk for CRC in some studies, which stemmed from ethnically diverse populations. Our aim was to identify common NOD2/CARD15 mutations in Hungarian patients with sporadic CRC.MethodsA total of 194 sporadic CRC patients (m/f: 108/86, age at diagnosis of CRC: 63.2 ± 9.1 years old) and 200 healthy subjects were included. DNA was screened for SNP8, SNP12 and SNP13 NOD2/CARD15 mutations by denaturing-HPLC and confirmed by direct sequencing.ResultsNOD2/CARD15 mutations were found in 28 patients (14.4%) and in 23 controls (11.5%, p = NS). Allele frequencies for SNP8/R702W (1.8% vs. 1.5%) SNP12/G908R (1.8% vs. 1.8%) and SNP13/3020insC (3.6% vs. 2.5%) were also not statistically different between patients and controls. The clinicopathologic characteristics of CRC patients with or without NOD2/CARD15 mutations were not significantly different.ConclusionOur results suggest that common NOD2/CARD15 mutations alone do not contribute to CRC risk in the Hungarian population.

Mutations in CARD15 and smoking confer susceptibility to Crohn's disease in the Danish population

Objective. Three CAspase Recruitment Domain (CARD15) mutations have shown to predispose to Crohn's disease in Caucasian populations. The aim of this study was to investigate the mutation frequency in patients with inflammatory bowel disease and in healthy controls in Denmark. Material and methods. Genotyping of the three common CARD15 mutations was carried out on 388 patients with Crohn's disease, 565 patients with ulcerative colitis and 796 healthy controls using real-time PCR. Allele and genotype frequencies in the three groups were compared. A possible additive effect of smoking on CARD15 mutations was also examined. Results. Carrying at least one CARD15 mutation was significantly more common in patients with Crohn's disease compared with healthy controls (21% versus 10%; p <0.001). A gene–dosage effect was observed (ORadj.smoking 22.2; p<0.001 for carrying two CARD15 mutations versus ORadj.smoking 1.8; p=0.01 for carrying one CARD15 mutation). The 1007insC protein truncating mutation was the major contributing mutation. Ileal involvement was more common in Crohn's disease patients with CARD15 mutations as opposed to patients without CARD15 mutations (ORadj.smoking 3.6; p<0.001). Smoking was independently associated with Crohn's disease (OR 1.8; p<0.001), but no multiplicative effect of smoking on CARD15 genotypes was found. Conclusions. In the Danish population, CARD15 mutations were found to be associated with Crohn's disease, hence supporting the hypothesis of a genetic component contributing to the disease. Further research for other genes possibly involved in Crohn's disease may result in the use of genetic testing for diagnosis or treatment of Crohn's disease in the future.

The prevalence of genetic and serologic markers in an unselected European population-based cohort of IBD patients

The aetiology of inflammatory bowel disease (IBD) is unknown, but it has become evident that genetic factors are involved in disease susceptibility. Studies have suggested a north-south gradient in the incidence of IBD, raising the question whether this difference is caused by genetic heterogeneity. We aimed to investigate the prevalence of polymorphisms in CARD15 and TLR4 and occurrence of anti-Saccharomyces cerevisiae (ASCA) and antineutrophil cytoplasmic antibodies (pANCA) in a European population-based IBD cohort. Individuals from the incident cohort were genotyped for three mutations in CARD15 and the Asp299gly mutation in TLR4. Levels of ASCA and pANCA were assessed. Disease location and behaviour at time of diagnosis was obtained from patient files. Overall CARD15 mutation rate was 23.9% for CD and 9.6% for UC patients (P < 0.001). Mutations were less present in the Scandinavian countries (12.1%) versus the rest of Europe (32.8%) (P < 0.001). Overall population attributable risk was 11.2%. TLR4 mutation rate was 7.6% in CD, 6.7% in UC patients and 12.3% in healthy controls (HC), highest among South European CD patients and HC. ASCA was seen in 28.5% of CD patients with no north-south difference, and was associated with complicated disease. pANCA was most common in North European UC patients and not associated with disease phenotype. The prevalence of mutations in CARD15 varied across Europe, and was not correlated to the incidence of CD. There was no association between mutations in TLR4 and IBD. The prevalence of ASCA was relatively low; however related to severe CD.

Association of interleukin-1 receptor-associated kinase M (IRAK-M) and inflammatory bowel diseases

Objective. Inflammatory bowel diseases (IBD) have a complex genetic background. The interleukin receptor associated kinase-M (IRAK-M) is a NF-κB-mediated, negative regulator of Toll-like receptor (TLR) signaling. A functional mutation in a negative regulator might induce impaired endotoxin tolerance and increased inflammatory responses. IRAK-M is situated on chromosome 12q14, a susceptibility locus for IBD, which makes it a good candidate gene. The objective of the study was to analyze a large cohort of IBD patients for the association between IBD and IRAK-M. Material and methods. A total of 542 patients with IBD (309 Crohn's disease (CD), 233 ulcerative colitis (UC)) and 305 controls were studied. Two single nucleotide polymorphisms (V147I and V270I) and six microsatellite markers were evaluated using association analysis and the haplotype sharing statistic. Results were stratified for CARD15 mutations R702W, G908R and 1007fsinsC. Results. No significant differences in IRAK-M allele frequencies were observed between IBD, UC, CD or subgroups of CD or UC and controls. Five out of 36 UC patients (13.9%) with an IBD-associated CARD15 mutation were carriers versus 2/167 (1.2%) in non-carriers (OR 13.1, 95% CI 1.0–164.5). No interaction was observed for CD. Conclusions. No evidence was found to suggest an association between IBD, CD, UC or subsets of CD and UC and IRAK-M. However, an interaction was found between IRAK-M and CARD15 in UC patients. In CARD15 mutant patients, the production of IRAK-M upon stimulation might be impaired. Further studies are needed to determine whether an impaired negative regulation of the TLR-signaling pathway might be partly responsible for the development of IBD.