CARD15 Variants Research Articles

NOD2/CARD15 variants in Malaysian patients with sporadic colorectal cancer

Colorectal cancer (CRC) is one of the most common types of cancer in both developed and developing countries. This disease is triggered by and progresses via the sequential accumulation of multiple genetic alterations. In addition, the interaction between low-penetrance genes and environmental factors can also increase the risk of developing CRC. Since inflammatory bowel diseases (IBDs) are one of the predisposing factors for CRC, IBD-related genes might, to a certain extent, be associated with cancer initiation. The nucleotide oligomerization domain 2/caspase activating recruitment domain 15 gene (NOD2/CARD15) is the most well-established gene to be associated with increased susceptibility to Crohn's disease. Thus, various studies have been performed to investigate the potential contribution of this gene to CRC risk. In this study, we aimed to determine the frequency of the Arg702Trp, Gly908Arg, 3020insC, Pro268Ser, and JW1 variants of NOD2/CARD15, and to investigate their association with CRC susceptibility. A total of 130 CRC patients and 212 healthy controls were recruited for this study. Subsequently, real-time polymerase chain reaction with TaqMan was performed for the genotyping of these NOD2/ CARD15 variants. None of the NOD2/CARD15 variants was statistically associated to CRC susceptibility in our Malaysian population. Our findings were remarkably similar to those of other Asian cohorts, which indicated that these NOD2/CARD15 variants exhibit genetic heterogeneity between Caucasian and Asian populations.

Card15 mutations and gastric cancer in a Portuguese population

Background. CARD15 is involved in the innate immune response and mutations of this gene have been linked with increased risk of Crohn's disease and colorectal cancer. The relation between CARD15 mutations and gastric cancer (GC) remains controversial. Aims. To assess whether CARD15 mutations are risk factors for GC in Portugal and whether there are genotype–phenotype correlations in these patients. Methods. The 3 main CARD15 mutations (3020insC, R702W and G908R) were searched in 150 patients with GC and in 202 healthy controls. Results. Overall, CARD15 mutations were found in 28 patients (18.7%) and in 27 controls (13.4%) (p = 0.176). Individually, the incidence of 3020insC was significantly higher in patients than in controls (6.0% vs. 1.0%, p = 0.021). This polymorphism was linked with an increased risk for the intestinal-type of GC (p = 0.002), while no association was found with the diffuse and/or mixed types. Genotype frequencies for R702W (10.0% vs. 7.9%) and G908R (4.0% vs. 4.0%) were not statistically different between the two groups. Similarly, no significant associations were detected between these two polymorphisms and the different histological GC types. No correlations were observed between CARD15 mutations and family history, mean age at diagnosis or GC stage. Conclusions.The CARD15 3020insC variant is a risk factor for intestinal GC in Portugal. CARD15 variants are not correlated with age of diagnosis or family aggregation of the disease neither with the GC stage.

AB0119 Card15/nod2 mutations in turkish patients with ankylosing spondylitis

BackgroundIt has been shown that CARD15/NOD2 molecule has an important role in etiopathogenesis of many inflammatory diseases and provides susceptibility to diseases. CARD15/NOD2 is a cytoplasmic molecule, which recognizes some...

The Protease Genes Cyld and USP40 Are Associated With Crohn's Disease: Results From a European Consortium

Background: Proteases and protease inhibitors affect several components that contribute to mucosal barrier integrity, and therefore may be important players in the pathophysiology of Inflammatory Bowel Disease (IBD). Aim: We aimed to elucidate if and which proteases/ protease inhibitors are involved in IBD pathogenesis. Methods: In a first step, a systematic review was performed of all relevant published genetic studies in Crohn's disease (CD) and ulcerative colitis (UC), to prioritize all known proteases/protease inhibitors based on the amount of available genetic evidence for association with IBD. In a second step, the topranked genes for CD and UC were followed up in a genetic association study. A total of 185 haplotype tagging SNPs in 23 genes were genotyped in an exploratory cohort of 650 CD (CD1), 721 UC (UC1), and 542 healthy controls (HC1). Replication of SNPs with puncorrected<0.1 was performed in 4 independent cohorts: CD2 (n=634), UC2 (n=528), HC2 (n=900); CD3 (n=377), UC3 (n=290), HC3(n=354); CD4 (n=432), UC4 (n=432); and CD5 (n=227), UC5 (n=141). Cases and controls were compared according to the additive genetic model. False Discovery Rate (FDR) correction was applied to correct for multiple testing (R 2.9.1). Results: All SNPs showing significant associations (pFDR<0.05) in the combined cohort [CD1-5 (n=2320) vs HC1-3 (n=1796), and UC1-5 (n=2112) vs HC1-3] are shown in Table 1. Strongest evidence was found in CD for CYLD, located 9kb downstream of CARD15, with 2 risk increasing and 2 protective SNPs. To test whether the signal seen in CYLD is driven by thewell-established CARD15 association, logistic regressionwas performed. CARD15 data were available in 1135 CD and 674 HC. The final model included both CARD15 and CYLD, pointing to independent signals from both genes (pCYLDrs12324931<0.001, OR=2.1[1.6-2.8]; pCARD15rs2066845<0.001, OR=2.9[1.8-4.8]; pCARD15rs2066847<0.001, OR= 3.7[2.5-5.5]). Patients without CARD15 variants showed significant association with rs12324931 (p=0.005, OR=2.9[1.3-6.6]). Another interesting outcome is the strong signal seen in CD for USP40, located near ATG16L1. Logistic regression analysis in CD1 (versus HC1), showed that rs10929178 and rs12472244 (USP40), and rs2241880 (ATG16L1) are independently associated with risk for CD (p=0.009, OR[0.6-0.9]; p=0.05, OR[0.8-1.0]; and p=0.009, OR[0.6-0.9] respectively). Conclusions: We provide strong evidence for association of CYLD on 16q12.1 in CD patients, which is independent from CARD15. CYLD, a cytoplasmic deubiquitinating enzyme, is a key negative regulator of NF-κB, and has been shown to be significantly down-regulated in the intestine of IBD patients. Moreover, we identified several variants in other protease/protease inhibitor genes which are implicated in either CD (USP40, a ubiquitin-specific peptidase), UC (DAG1, MST1, PSMB8), or both (APEH, USP3). Table 1

Linking Crohn's Disease and Ankylosing Spondylitis: It's All about Genes!

Linking Crohn's Disease and Ankylosing Spondylitis: It's All about Genes!

CARD15 Gene Variants and Risk of Reoperation in Crohn's Disease Patients

Several studies have investigated, with conflicting results, the risk factors for reoperation in Crohn's disease (CD) patients. CARD15 gene variants have been identified as a major genetic risk factor for CD patients and associated with ileal disease, stenosis, and risk of surgery. However, data regarding the association between these variants and the need for reoperation are very few and conflicting. This study evaluated the risk factors of reoperation, including CARD15 gene variants. A total of 253 consecutive CD patients, recruited in four Italian tertiary-care inflammatory bowel disease (IBD) referral centers, who had submitted to surgery for CD, were included in the study. Clinical characteristics of CD patients, time and main indications for surgery, type of operation, postoperative therapy, and time to second surgery were recorded. CARD15 gene variants were determined by DNA sequencing analysis in each center. Factors related to surgical recurrence, including CARD15 variants, were estimated by Cox proportional hazard regression. In all, 89 patients (35.1%) showed at least one surgical recurrence. Reoperation was significantly correlated with stenosis as indications at initial surgery only. CARD15 variants were found in 36.0% of patients, but did not correlate significantly with the demographic and clinical characteristics of the patients, rate of first surgical recurrence, and time to second operation. CARD15 variants did not significantly affect the reoperation rate, irrespective of indications for surgery. Reoperation for CD is correlated with stenosis at initial surgery, but not with CARD15 gene variants. This finding does not justify more aggressive prophylactic therapy on the basis of CARD15 genotype.

Role of ATG16L1 Thr300Ala polymorphism in inflammatory bowel disease: A Study in the Spanish population and a meta-analysis

Thr300Ala polymorphism in ATG16L1 was reported as a susceptibility factor to Crohn's disease (CD). Inconsistently replicated associations with ulcerative colitis (UC) and specifically with ileal CD were also reported. Our aims were: to replicate the ATG16L1 Thr300Ala association with inflammatory bowel disease (IBD) in the Spanish population, to perform a meta-analysis to determine the risk conferred to the different IBD subgroups, and to test for the interaction with CARD15 or IL23R risk loci. Thr300Ala (rs2241880) single nucleotide polymorphism (SNP) was genotyped in 712 IBD patients and 745 controls by TaqMan technology. Genetic frequencies were compared with chi-square tests. Our findings were pooled in a meta-analysis. In Spain, we observed an association of rs2241880 with CD (P = 0.008; odds ratio [OR, 95% confidence interval, CI] = 1.28 [1.06-1.54]), but not with UC. No significant differences emerged when patients were stratified by clinical features. Similarly, the meta-analysis demonstrated a significant association only with CD (P < 10(-4); OR [95% CI] = 1.33 [1.28-1.38]). A significant difference between ileal CD patients and controls was observed, but heterogeneity was found in comparisons involving colonic CD patients and definite conclusions cannot be drawn. No interaction between rs2241880 and the established CARD15 or IL23R susceptibility variants was observed. The Thr300Ala polymorphism is associated with CD, regardless of the CARD15 or IL23R status, but not with UC. Stratification by clinical phenotypes did not show definitive results because of the existing heterogeneity among studies.

Increased Immune Reactivity Predicts Aggressive Complicating Crohn's Disease in Children

The ability to identify children with CD who are at highest risk for rapid progression from uncomplicated to complicated phenotypes would be invaluable in guiding initial therapy. The aims of this study were to determine whether immune responses and/or CARD15 variants are associated with complicated disease phenotypes and predict disease progression. Sera were collected from 796 pediatric CD cases and tested for anti-Cbir1 (flagellin), anti-outer membrane protein C, anti-Saccharomyces cerevisiae, and perinuclear antineutrophil cytoplasmic antibody by using enzyme-linked immunosorbent assay. Genotyping (Taqman MGB) was performed for 3 CARD15 variants (single nucleotide polymorphisms 8, 12, and 13). Associations between immune responses (antibody sum and quartile sum score, CARD15, and clinical phenotype were evaluated. Thirty-two percent of patients developed at least 1 disease complication within a median of 32 months, and 18% underwent surgery. The frequency of internal penetrating, stricturing, and surgery significantly increased (P trend < .0001 for all 3 outcomes) with increasing antibody sum and quartile sum score. Nine percent of seropositive groups had internal penetrating/stricturing versus 2.9% in the seronegative group (P = .01). Twelve percent of seropositive groups underwent surgery versus 2% in the seronegative group (P = .0001). The highest antibody sum group (3) and quartile sum score group (4) demonstrated the most rapid disease progression (P < .0001). Increased hazard ratio was observed for antibody sum group 3 (7.8; confidence interval, 2.2-28.7), P < .002 and quartile sum score group 4 (11.0; confidence interval, 1.5-83.0, P < .02). The rate of complicated CD increases in children as the number and magnitude of immune reactivity increase. Disease progression is significantly faster in children expressing immune reactivity.

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.

M2047 The CARD15 Variants Rs2066843 and Rs2076756 Are New Independent Crohn's Disease Susceptibility Genes Associated with Severe Penetrating Disease Phenotype and Frequent Need for Surgery

M2047 The CARD15 Variants Rs2066843 and Rs2076756 Are New Independent Crohn's Disease Susceptibility Genes Associated with Severe Penetrating Disease Phenotype and Frequent Need for Surgery

CARD15 and IL23R influences Crohnʼs disease susceptibility but not disease phenotype in a Brazilian population

Although many genetic variants are identified in association with Crohn's disease (CD), CARD15, IL23R, and ATG16L1 association with CD have been firmly confirmed in Caucasians of European ancestry. The prevalence of CD is rapidly rising in Brazil, where European ancestry is firmly admixed with natives and Africans, resulting in a heterogeneous population. We investigated the contribution of CARD15, IL23R, and ATG16L1 with CD risk in a heterogeneous Brazilian population. Genotyping for CARD15 (R702W, G908R, 3020insC), IL23R (rs1004819, rs7517847, rs11209026, rs10889677, rs1495965), and ATG16L1 (rs2241880) was performed in 187 children and adults with CD and 255 healthy ethnically matched controls. Clinical records were systematically reviewed and detailed phenotypic information was obtained. At least 1 CARD15 risk allele was present in 30% of the CD patients compared with 10% of controls. Variants of CARD15 (3020insC and R702W) and IL23R (rs1004819, rs11209026, and rs1088967) were associated with CD. However, no genotype-phenotype correlations were found among the Brazilian CD population with CARD15 or IL23R variants. No significant association was achieved with ATG16L1. CARD15 and IL23R confer susceptibility to CD in the Brazilian population. However, the presence of these variants did not influence disease phenotype. Further research should be focused on larger sample sizes with population admixture analysis to better understand the risks and genotype-phenotype correlation in populations like Brazil where the prevalence of CD is rapidly rising.

OCTN and CARD15 gene polymorphism in Chinese patients with inflammatory bowel disease

To investigate the single nucleotide polymorphism (SNPs) distribution of NOD2/CARD15 (R702W, G908R), OCTN1 1672C/T and OCTN2-207G/C in Chinese patients with inflammatory bowel disease (IBD). A total of 61 patients with Crohn's disease (CD), 151 patients with ulcerative colitis (UC), and 200 unrelated healthy controls were genotyped. Genotyping was performed by sequence specific primer polymerase chain reaction (PCR-SSP) or by restriction fragment length polymorphism (PCR-RFLP) analysis. Among the subjects in our study groups, including patients with CD, UC and healthy controls, none had OCTN and CARD15 variants and very rare IBD family history was found in our patients with the percentage of 0 (0/61 with CD) and 1.3% (2/151 with UC). Our results indicate that although OCTN or CARD15 variation is associated with susceptibility to IBD in Western populations, these might be rare and may not be associated with susceptibility to IBD in Chinese patients.

Association Between Genetic Variants in theIL-23RGene and Early-Onset Crohn's Disease: Results From a Case-Control and Family-Based Study Among Canadian Children

Interleukin (IL)-23 is a key regulator of inflammation and influences the activities of T-helper 17 (Th-17) lymphocytes. Recent reports indicate that variants in the gene coding for its receptor (IL-23R) are strongly associated with Crohn's disease (CD). We investigated whether DNA variants in the IL-23R gene determine susceptibility for CD in Canadian children. A case-control and case-parent trio design was implemented at three pediatric centers across Canada. Cases of CD (</=20 yr) along with their parents and controls were recruited. DNA samples were collected and genotyped for 10 single nucleotide polymorphisms (SNPs) in the IL-23R gene and three common SNPs in the CARD15 gene. Transmission disequilibrium-based tests were applied to the case-parent data and logistic regression models to the case-control data to study the association between the SNPs and CD. A total of 259 CD cases, 139 controls, and 232 families (167 trios and 65 dyads) were studied. The mean age at diagnosis was 13.3 yr (range 2.6-20 yr). The majority of the patients were Caucasian. Case-control analysis revealed significant associations with three SNPs (rs1004819, rs7517847, and rs11209026 [R381Q]) and borderline nonsignificant associations with three other SNPs (rs10489629, rs10889697, and rs11465804) in the IL-23R gene. Having any CARD15 variant was associated with a significant risk for CD (P < 0.0001). Analyses of case-parent data confirmed the findings from the case-control analysis including significant associations with the R381Q SNP (P= 0.002). The common variant in this SNP conferred risk for CD. These associations were largely independent of the CARD15 gene. Our findings confirm recently reported genome-wide associations between the IL-23R gene and CD. They suggest that the gene is also associated with pediatric-onset CD among Canadian children.

Linking genetic susceptibility to Crohnʼs disease with Th17 cell function: IL-22 serum levels are increased in Crohnʼs disease and correlate with disease activity and IL23R genotype status

We analyzed the influence of Crohn's disease (CD)-associated IL23R gene variants on IL-22 that is expressed in IL-23R+ Th17 cells. IL-22 serum levels were measured in 242 CD patients and in 31 healthy controls. Subanalyses included serum levels of IL-6, TNF-alpha, IL-17A, IL-17F, C-reactive protein (CRP), and leukocyte count. In all patients, genotyping for 10 CD-associated IL23R single nucleotide polymorphisms (SNPs) and the 3 main CD-associated CARD15 variants was performed. There was a highly significant increase in IL-22 serum expression in CD patients compared to healthy controls (P = 2.53 x 10(-9)). IL-22 serum levels correlated with disease activity: IL-22 levels in patients with a Crohn's disease activity index (CDAI) >150 were significantly higher than in patients with a CDAI <150 (P = 0.001), while TNF-alpha and IL-6 were not significantly different between these 2 groups. Analyzing the effect of 10 IL23R variants on IL-22 serum levels, we demonstrated that the quotients of mean IL-22 serum levels of carriers of the minor allele to the mean serum IL-22 in wildtype carriers correlated highly with the corresponding CD susceptibility risk for each gene variant (r = 0.807). The IL-22 levels in carriers of CD risk-increasing IL23R variants were significantly higher than in carriers of CD risk-decreasing IL23R variants (P = 0.008). The Th17 cytokine IL-22 is expressed at high levels in CD and correlates with disease activity, offering a better separation between active and inactive CD than IL-6 and TNF-alpha. IL23R genotypes influence IL-22 serum expression, linking genetic CD susceptibility to Th17 cell function for the first time.

Role of NOD2/CARD15 in coronary heart disease.

Background:Bacterial DNA has been repeatedly detected in atheromatous lesions of coronary heart disease (CHD) patients. Phylogenetic signatures in the atheroma lesions that are similar to those of bacterial biofilms on human barrier organs, including the respiratory or gastrointestinal tract, raise the question of a defective barrier function in CHD. NOD2 plays a major role in defense against bacterial invasion. Genetic variation in the CARD15 gene, which encodes NOD2, was previously shown to result in a barrier defect that causes chronic inflammatory disorders (e.g. Crohn disease). In the present study, we investigated the possible involvement of NOD2/CARD15 in the pathology of CHD by i) analyzing the local expression of NOD2 in atherectomy versus healthy tissue (n = 5 each) using histochemical immunofluorescence and ii) by testing the three major functional CARD15 variants (R702W, G908R and 1007fs) for association with early-onset CHD in 900 German patients and 632 healthy controls.Results:In atherectomy tissue of CHD patients, NOD2 was detected in inflammatory cells at the luminal sides of the lesions. However, the allele and genotype frequencies of the three major CARD15 polymorphisms did not differ between CHD patients and controls.Conclusion:The NOD2 up-regulation in atheroma lesions indicates an involvement of this protein in the pathology of CHD. Although NOD2 could be important in local immune response mechanisms, none of the analyzed CARD15 variants seem to play a significant role in the etiology of CHD.

A study in three European IBD cohorts confirms that the ATG16L1 c.898A > G (p.Thr300Ala) variant is a susceptibility factor for Crohn’s disease

Background and aimsA recent study reported that a nonsynonymous SNP rs2241880 (c.898A>G, p.Thr300Ala) within ATG16L1 confers susceptibility to Crohn's disease (CD). We analyzed ATG16L1 c.898A>G in three independent European inflammatory bowel disease (IBD) cohorts from Germany, Hungary and the Netherlands. MethodsIn total, we included 910 European IBD patients and compared the ATG16L1 c.898A>G genotype frequency with 707 ethnically matched healthy controls. We included patients from 3 populations originating from Germany (CD n=310; ulcerative colitis [UC] n=179), Hungary (CD n=147; UC n=117), and the Netherlands (CD n=157). Subtyping analysis was performed in respect to CARD15 alterations and clinical characteristics. ResultsWe found a highly significant association of c.898A>G to CD. The association was significant (p=0.0005) for the total CD cohort but also for the individual populations from Germany (p=0.02) and Netherlands (p=0.02) whereas in the Hungarian CD patients a clear trend was observed (p=0.19; OR 1.227, 95% CI 0.910; 1.654). No association was found between c.898A>G and UC. No statistical interactions were observed between ATG16L1 c.898A>G and CARD15 variants. Furthermore no association to a CD subphenotype was detected. ConclusionsWe confirm that ATG16L1 variant c898A>G confers a risk variant for CD but is not associated with a distinct CD phenotype.

Heterozygosity for IL23R p.Arg381Gln confers a protective effect not only against Crohn’s disease but also ulcerative colitis

A recent study reported that a non-synonymous single nucleotide polymorphism (rs11209026, p.Arg381Gln) located in the IL23R gene is a protective marker for inflammatory bowel disease. To analyse the frequency of p.Arg381Gln in three independent European inflammatory bowel disease cohorts and to evaluate how this variant influences disease behaviour. We assessed a European cohort of 919 inflammatory bowel disease patients and compared the IL23R p.Arg381Gln genotype frequency with 845 healthy controls. Inflammatory bowel disease patients originated from Germany [Crohn's disease (CD): n = 318; ulcerative colitis (UC): n = 178], Hungary (CD: n = 148; UC: n = 118) and the Netherlands (CD: n = 157). Ethnically matched controls were included. We performed subtyping analysis in respect to CARD15 alterations and clinical characteristics. The frequency of the glutamine allele of p.Arg381Gln was significantly lower in inflammatory bowel disease patients compared with controls in a pooled analysis of all three cohorts (P < 0.000001) as well as in the individual cohorts (Germany: P = 0.001, Hungary: P = 0.02 and the Netherlands: P = 0.0002). The p.Arg381Gln genotype distribution was similar between CD and UC. We did not observe either statistical interactions between p.Arg381Gln and CARD15 variants or any significant associations between p.Arg381Gln genotype and subphenotypes. The p.Arg381Gln IL23R variant confers a protective effect against both CD and UC, but does not determine disease phenotype.

Mutations in pattern recognition receptor genes modulate seroreactivity to microbial antigens in patients with inflammatory bowel disease

Background and aims:A number of antibodies against microbial epitopes or self-antigens have been associated with Crohn’s disease. The development of antibodies reflects a loss of tolerance to intestinal bacteria that...

CARD15 variants predict small-bowel stenosis in Crohn's disease

CARD15 variants predict small-bowel stenosis in Crohn's disease

Adalimumab in patients with Crohn’s disease – safety and efficacy in an open‐label single centre study

To evaluate the efficacy and safety of adalimumab, a human antitumour necrosis factor-alpha antibody, in induction and maintenance of remission in patients with Crohn's disease either refractory or intolerant to infliximab in a single centre cohort. Sixteen Crohn's disease patients received 160 mg adalimumab subcutaneously in week 0, followed by 80 mg every other week. Clinical response was assessed based on Crohn's disease activity index and laboratory parameters (leukocyte count, C-reactive protein). In all patients genotyping for CARD15 variants and the +1059G/C polymorphism in the C-reactive protein gene was performed. In 10 of 16 patients (63%) treated with adalimumab, remission (CDAI score <150) was induced for at least 8 weeks independent of CARD15 or +1059G/C CRP status. In six of these 10 patients ongoing remission is observed now for more than 24 weeks. Adalimumab significantly decreased C-reactive protein serum levels and Crohn's disease activity index. There was one serious complication (fungal pneumonia). Six patients intermittently developed minor dermatological problems resolving after topical therapy. Otherwise, treatment was generally well tolerated. Adalimumab can induce and maintain remission in patients with moderate to severe Crohn's disease intolerant or refractory to infliximab. Further experience from larger cohorts is required to evaluate dose regimen and safety profiles in Crohn's disease therapy.