Macrophage migration inhibitory factor (MIF) -173G/C promoter polymorphism influences upper gastrointestinal tract involvement and disease activity in patients with Crohn's disease.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with increased expression in inflammatory bowel disease. The aim of the study was to analyze the role of the MIF -173G/C single nucleotide polymorphism in Crohn's disease (CD). METHODS: Using restriction fragment length polymorphism analysis, genomic DNA of 198 patients with CD and 159 unrelated controls was analyzed for the -173G/C SNP in the MIF promoter region. Colonic MIF mRNA expression was measured by quantitative polymerase chain reaction (PCR), serum MIF levels by enzyme-linked immunosorbent assay (ELISA). RESULTS: Thirty-six of the 146 G/G wildtype carriers (24.7%) but only 3 of the 45 G/C heterozygotes (6.7%) and only 1 of the C/C homozygotes (14.3%) were diagnosed with upper gastrointestinal tract involvement (P = 0.009, odds ratio [OR] = 0.22, 95% confidence interval [CI], 0.06-0.75 for wildtype versus hetero- and homozygous carriers). This result was confirmed in a second prospective study, in which all patients diagnosed with upper gastrointestinal involvement (n = 13) were G/G wildtype carriers (P = 0.01 versus controls). All patients (n = 12; 100%) with a Crohn's disease activity index (CDAI) > 300 were G/G wildtype carriers compared to only 65.6% wildtype carriers in the group with a CDAI < 150 (P = 0.016). MIF is expressed in the colonic mucosa of CD patients and intestinal epithelial cells but its mRNA expression does not correlate with the degree of inflammation and is not upregulated by proinflammatory cytokines. In CD, MIF serum levels are not influenced by the MIF -173G/C polymorphism. CONCLUSIONS: The MIF -173G/C polymorphism appears to be a factor contributing to a particular CD phenotype characterized by protection against upper gastrointestinal tract involvement and severe disease activity.

Homozygosity for the CARD15 frameshift mutation 1007fs is predictive of early onset of Crohn's disease with ileal stenosis, entero-enteral fistulas, and frequent need for surgical intervention with high risk of re-stenosis.

OBJECTIVE: The identification of CARD15 as a susceptibility gene for Crohn's disease (CD) offers new possibilities for patient classification and risk assessment. The purpose of this study was to carry out a CARD15 sequence analysis in a large single-center IBD cohort and to investigate the impact of different genotypes on disease phenotypes. MATERIAL AND METHODS: A total of 445 unrelated patients with IBD (68.1% CD, 28.5% ulcerative colitis (UC), 3.4% indeterminate colitis (IC)) were included in the study. Clinical data were recorded by detailed questionnaire and analysis of the charts. CARD15 variants (R702W, G908R, 1007fs (frameshift)) were identified by DNA sequence analysis. RESULTS: CARD15 variants were found in 142 inflammatory bowel disease (IBD) patients (31.9%) including 120 CD patients (39.6%). In CD, the presence of two CARD15 variants was associated with ileal disease (p=0.008 versus wild-type (wt); OR 4.04; 95% CI 1.36-11.96) and a fibrostenotic phenotype (p=0.002 versus wt; OR 5.47; 95% CI 1.61-18.58). Subgroup analysis of 19 patients (4.3%) homozygous for the CARD15 variant 1007fs (3020ins C) revealed an association with onset of CD at an early age (p=0.014 versus wt), ileal involvement (p=0.001), and intestinal stenoses in all patients (p=0.001) frequently requiring surgery (73.7%; p=0.093). Of these patients 78.6% developed re-stenoses after surgical resection; 52.6% of the homozygotes were diagnosed as having entero-enteral fistulas. CONCLUSIONS: Patients homozygous for the 1007fs mutation had an early disease onset with long-segment ileal stenoses and entero-enteral fistulas. They frequently needed surgical intervention and had a high risk of re-stenosis. Genotyping therefore appears to be an important diagnostic tool in identifying severely affected patients requiring individualized treatment strategies at an early stage of the disease.

Eight novel CARD15 variants detected by DNA sequence analysis of the CARD15 gene in 111 patients with inflammatory bowel disease.

We performed a limited DNA sequence analysis of the CARD15 gene in 89 patients with Crohn's disease (CD), 19 patients with ulcerative colitis (UC), and three patients with indeterminate colitis (IC), who were heterozygous carriers of one of the common CARD15 mutations [c.2104C>T (p.R702W), c.2722G>C (p.G908R), or c.3019_3020insC (p.Leu1007fsX1008)], the c.2462+10A>C variant, or of a new amino acid substitution in the 3'-end of exon 4. CARD15 exons 4, 5, 6, 8, and 11 were amplified by PCR and completely sequenced, thereby theoretically covering 73.9% of the described CARD15 variants and 96.6% of the mutated alleles. Using this approach, eight novel amino acid substitutions [c.1171C>T (p.R391C), c.1387C>G (p.P463A), c.2138G>A (p.R713H), c.2278C>T (p.R760C), c.2368C>T (p.R790W), c.2371C>T (p.R791W), c.2475C>G (p.N825K), and c.2546C>T (p.A849V)] were detected in six CD and two IC patients, and one UC patient. A severe disease phenotype was observed especially in patients who are compound-heterozygous for a common and a novel CARD15 mutation.

Increased expression of the chemokine fractalkine in Crohn's disease and association of the fractalkine receptor T280M polymorphism with a fibrostenosing disease Phenotype.

BACKGROUND: The fractalkine receptor CX3CR1 has been shown to be involved in inflammation and immune response. Recently, two polymorphisms of CX3CR1 (V249I and T280M) were reported. AIMS: Our aim was to analyze fractalkine expression and the role of CX3CR1 polymorphisms in Crohn's disease (CD). METHODS: We determined fractalkine mRNA expression in the intestinal epithelial cell (IEC) line SW480 after stimulation with proinflammatory cytokines as well as in inflamed (n = 14) and noninflamed (n = 14) CD lesions by quantitative PCR. By restriction fragment length polymorphism analysis, genomic DNA from 206 patients with CD and 211 unrelated controls was analyzed for the two single nucleotide polymorphisms in the CX3CR1 gene, which result in the V249I and T280M substitutions. RESULTS: All proinflammatory stimuli (TNF-alpha, IL-1beta, LPS) significantly increased fractalkine mRNA expression in IEC. There was also a significant increase in fractalkine mRNA expression in inflamed lesions of CD patients when compared to noninflamed colonic mucosa (p = 0.02). Intestinal fractalkine mRNA levels correlated highly with IL-8 mRNA expression levels (r = 0.931). However, there was no difference in the V249I and T280M genotype frequencies between CD patients and the control group. In the CD group, 33.0% were heterozygous and 8.3% homozygous for the V249I polymorphism, while 23.3% were heterozygous and 4.4% homozygous for the T280M polymorphism. All T280M homozygotes were diagnosed of intestinal stenosis (p = 0.03 vs wildtype and heterozygous carriers) and had significantly more often ileocolonic involvement more often than patients with wildtype and heterozygous genotypes (p = 0.01). These associations were independent of the CARD15 genotype status. CONCLUSIONS: The expression of the chemokine fractalkine is upregulated by proinflammatory cytokines and enhanced in inflamed CD lesions. The CX3CR1 T280M polymorphism appears to influence CD phenotype and localization.

The role of Toll-like receptor 4 Asp299Gly and Thr399Ile polymorphisms and CARD15/NOD2 mutations in the susceptibility and phenotype of Crohn's disease.

BACKGROUND: We investigated the influence of 2 common Toll-like receptor 4 (TLR4) polymorphisms on susceptibility and disease characteristics of Crohn's disease (CD). METHODS: Genomic DNA from 204 patients with CD and 199 unrelated controls was analyzed for the presence of 2 single nucleotide polymorphisms in the TLR4 gene, resulting in the amino acid substitutions Asp299Gly and Thr399Ile. In addition, the carrier status for the 3 common CD-associated CARD15/NOD2 gene mutations, Arg702Trp, Gly908Arg, and 1007fs, was determined. The frequency of the different genotypes was compared, and a detailed genotype-phenotype correlation was performed. RESULTS: An almost 2-fold increase in the frequency of the TLR4 Asp299Gly phenotype was observed in patients with CD (14.2%) compared with healthy controls (7.5%, P = 0.038, odds ratio = 2.03). The prevalence of a stricturing phenotype was increased in patients heterozygous for 1 of the TLR4 polymorphisms studied (Asp299Gly, 34.5%; Thr399Ile, 36.7%) compared with patients with wild-type TLR4 (17.1% and 16.7%; P = 0.04 and 0.02, respectively). The presence of the Asp299Gly polymorphism in the absence of CARD15/NOD2 mutations was a particularly strong predictor of the stricturing disease phenotype that was present in 47.4% of the patients with Asp299Gly+/NOD2- compared with 10.1% of the patients with the Asp299Gly-/NOD2+ status (P = 0.0009; P = 0.0004 for Thr399Ile+/NOD2- versus Thr399Ile-/NOD2+). In contrast, there was a trend toward a higher prevalence of the penetrating phenotype in the TLR4-/NOD2+ group (71.6%) compared with the TLR4+/NOD2- group (47.4%, P = 0.059). CONCLUSIONS: The TLR4 Asp299Gly polymorphism is a risk factor for CD. TLR4 and CARD15/NOD2 mutations may contribute to distinct disease phenotypes.