Thomas Griga

Polymorphisms in the DLG5 and OCTN cation transporter genes in Crohn’s disease

Background and aims: Recent data suggest identification of causal genetic variants for inflammatory bowel disease in the DLG5 gene and in the organic cation transporter (OCTN) cluster, both situated in previously described linkage regions. Patients and methods: The polymorphisms in DLG5 (113 G→A, 4136 C→A, and DLG5_e26), SLC22A4 (1672 C→T), and SLC22A5 (−207 G→C) were assessed in 625 patients with Crohn’s disease (CD), 363 patients with ulcerative colitis (UC), and 1012 healthy controls. Association with disease susceptibility, clinical phenotypes, and possible genetic interactions of these polymorphisms with disease associated CARD15/NOD2 mutations was analysed. Results: No significant association of DLG5 polymorphisms with CD or UC was observed. Homozygosity for the OCTN-TC haplotype was associated with an increased CD risk (OR = 1.65), which was even greater in the presence of CARD15 mutations. Genotype-phenotype analysis revealed that this association was particularly strong in patients with colonic disease. The TC haplotype was associated with non-fistulising non-fibrostenotic disease, an earlier age of disease onset, and reduced need for surgery. Conclusion: Our observations argue against a role of DLG5 polymorphisms in the susceptibility for inflammatory bowel disease, whereas the OCTN polymorphisms are associated with CD. However, due to the comparable weak association observed herein, extended linkage disequilibrium analyses of these variants with the IBD5 haplotype tagged single nucleotide polymorphims might be advisable before definitive conclusions about their causative role in CD can be drawn.

rs1004819 Is the Main Disease-Associated IL23R Variant in German Crohn's Disease Patients: Combined Analysis of IL23R, CARD15, and OCTN1/2 Variants

Background The IL23R gene has been identified as a susceptibility gene for inflammatory bowel disease (IBD) in the North American population. The aim of our study was to test this association in a large German IBD cohort and to elucidate potential interactions with other IBD genes as well as phenotypic consequences of IL23R variants. Methods Genomic DNA from 2670 Caucasian individuals including 833 patients with Crohns disease (CD), 456 patients with ulcerative colitis (UC), and 1381 healthy unrelated controls was analyzed for 10 IL23R SNPs. Genotyping included the NOD2 variants p.Arg702Trp, p.Gly908Arg, and p.Leu1007fsX1008 and polymorphisms in SLC22A4/OCTN1 (1672 C→T) and SLC22A5/OCTN2 (–207 G→C). Results All IL23R gene variants analyzed displayed highly significant associations with CD. The strongest association was found for the SNP rs1004819 [P = 1.92×10−11; OR 1.56; 95 % CI (1.37–1.78)]. 93.2% of the rs1004819 TT homozygous carriers as compared to 78% of CC wildtype carriers had ileal involvement [P = 0.004; OR 4.24; CI (1.46–12.34)]. The coding SNP rs11209026 (p.Arg381Gln) was protective for CD [P = 8.04×10−8; OR 0.43; CI (0.31–0.59)]. Similar, but weaker associations were found in UC. There was no evidence for epistasis between the IL23R gene and the CD susceptibility genes CARD15 and SLC22A4/5. Conclusion IL23R is an IBD susceptibility gene, but has no epistatic interaction with CARD15 and SLC22A4/5. rs1004819 is the major IL23R variant associated with CD in the German population, while the p.Arg381Gln IL23R variant is a protective marker for CD and UC.

The ATG16L1 gene variants rs2241879 and rs2241880 (T300A) are strongly associated with susceptibility to Crohn's disease in the German population.

OBJECTIVES:We analyzed ATG16L1, a recently identified Crohns disease (CD) susceptibility gene, in a large cohort with inflammatory bowel disease (IBD) including potential interactions with other IBD genes as well as factors regulating its gene expression.METHODS:Genomic DNA from 2,890 Caucasians including 768 patients with CD, 507 patients with ulcerative colitis (UC), and 1,615 healthy controls was analyzed for 9 different ATG16L1 single nucleotide polymorphisms (SNPs). Genotyping included CARD15/NOD2 variants p.Arg702Trp, p.Gly908Arg, and p.Leu1007fsX1008 and polymorphisms in SLC22A4/OCTN1 (1672 C→T) and SLC22A5/OCTN2 (−207 G→C) as well as 10 CD-associated IL23R variants. The transcriptional regulation of ATG16L1 was studied in intestinal epithelial cells following stimulation with Toll-like receptor (TLR) ligands and proinflammatory cytokines and in a murine ileitis model and CD biopsies.RESULTS:All nine ATG16L1 gene variants analyzed displayed highly significant associations with CD demonstrating a CD-protective effect for the minor allele. The strongest associations were found for rs2241879 and the coding SNP rs2241880 (T300A); P = 3.6 × 10−6 and 3.7 × 10−6, respectively (OR 0.74, 95% CI 0.65–0.84 for both variants). The genotype-phenotype analysis revealed no significant associations. In UC, only rs6431660 was weakly disease-associated. There was no evidence for epistasis between the ATG16L1 gene and other susceptibility genes (IL23R, CARD15, SLC22A4/5). ATG16L1 mRNA expression was not upregulated in CD and murine ileitis, and was less than threefold increased in cells stimulated with proinflammatory cytokines and TLR ligands.CONCLUSION:ATG16L1 is a CD susceptibility gene without epistatic interaction with other CD susceptibility genes and is not upregulated in intestinal inflammation.

Novel Genetic Risk Markers for Ulcerative Colitis in the IL2/IL21 Region Are in Epistasis With IL23R and Suggest a Common Genetic Background for Ulcerative Colitis and Celiac Disease

OBJECTIVES:Recently, a genome-wide association study showed that single-nucleotide polymorphisms (SNPs) in the chromosome 4q27 region containing IL2 and IL21 are associated with celiac disease. Given the increased prevalence of inflammatory bowel disease (IBD) among celiac disease patients, we investigated the possible involvement of these SNPs in IBD.METHODS:Five SNPs strongly associated with celiac disease within the KIAA1109/TENR/IL2/IL21 linkage disequilibrium block on chromosome 4q27 and one coding SNP within the IL21 gene were analyzed in a large German IBD cohort. The study population comprised a total of 2,948 Caucasian individuals, including 1,461 IBD patients (ulcerative colitis (UC): n=514, Crohns disease (CD): n=947) and 1,487 healthy unrelated controls.RESULTS:Three of the five celiac disease risk markers had a protective effect on UC susceptibility, and this effect remained significant after correcting for multiple testing: rs6840978: P=0.0082, Pcorr=0.049, odds ratio (OR) 0.77, 95% confidence interval (CI) 0.63–0.93; rs6822844: P=0.0028, Pcorr=0.017, OR 0.73, 95% CI 0.59–0.90; rs13119723: P=0.0058, Pcorr=0.035, OR 0.75, 95% CI 0.61–0.92. A haplotype consisting of the six SNPs tested was markedly associated with UC susceptibility (P=0.0025, Pcorr=0.015, OR 0.72, 95% CI 0.58–0.89). Moreover, in UC, epistasis was observed between the IL23R SNP rs1004819 and three SNPs in the KIAA1109/TENR/IL2/IL21 block (rs13151961, rs13119723, and rs6822844).CONCLUSIONS:Similar to other autoimmune diseases such as celiac disease, rheumatoid arthritis, type 1 diabetes, Graves’ disease, and psoriatic arthritis, genetic variation in the chromosome 4q27 region predisposes to UC, suggesting a common genetic background for these diseases.

A polymorphism of the NFKBIA gene is associated with Crohn's disease patients lacking a predisposing allele of the CARD15 gene.

Background and aimsNuclear factor kappa-B (NFκB) plays a crucial role in diseases associated with dysregulated immune response. NFκB inhibitor α downregulates the activity of NFκB.Patients and methodsTo evaluate the contribution of the NFκB inhibitor α gene in Crohns disease single nucleotide polymorphisms in the 3′-UTR and at position −420 in the promoter were studied in 259 patients with Crohns disease genotyped for the variations of the CARD15 gene in comparison to 441 healthy controls. Additionally we screened the coding region of the NFκB inhibitor α gene for polymorphisms by SSCP analysis.ResultsIn comparison to controls the A allele and the AA genotype frequencies of the single nucleotide polymorphisms in the 3′-UTR were significantly increased only in Crohns disease patients without a variation in the CARD15 gene. Similarly, the difference between patients harboring no predisposing CARD15 alleles and patients harboring such a variation was significant.ConclusionThe findings indicate that the phenotype Crohns disease is to be substructured with respect to genetic susceptibility.

Budesonide for the treatment of collagenous colitis: first results of a pilot trial

OBJECTIVES:Collagenous colitis is a chronic watery diarrhea disorder characterized by a subepithelial collagen layer and a lymphoplasmacytic infiltration within the lamina propria. However, no standard treatment has been introduced by controlled clinical trials. Aim of the present pilot trial was to investigate the clinical effects of orally administered budesonide (3 mg t.i.d.) in 7 patients with collagenous colitis. In addition, the histomorphological changes after budesonide treatment were described in a group of 3 patients.METHODS:The study was performed as an open label pilot trial. Study end point was the clinical remission of collagenous colitis defined by stool frequency and stool consistency.RESULTS:The results indicate a rapid and sustained clinical response in all patients. Stool frequency significantly decreased (p < 0.001) from 10.43 ± 5.56 per day (4–20 per day) to 3.3 ± 1.2 (1–5 per day) after 10 days and to 1.86 ± 0.69 per day (1–3 per day) after 10 wk. Moreover stool consistency changed from watery (6 patients) or soft (1 patient) to soft (1 patient) or solid (6 patients). Clinical improvement was achieved within the first 10 days in all patients and maintained after dose reduction. In 3 patients no diarrhea recurred within 7, 12, or 15 months after treatment with budesonide was terminated. In these patients control biopsies were taken and showed a marked regression of both characteristics, the collagen band and the lymphoplasmacytic infiltration.CONCLUSIONS:With respect to the preliminary data from this pilot trial, budesonide with its high topical and low systemic effects seems to be of therapeutic clinical benefit in collagenous colitis. A therapeutic effect could be demonstrated for both therapeutic goals, the clinical response and morphological changes. Further studies on the effects of budesonide on mucosal collagen metabolism and long-term follow-up are warranted.

Role of the NFKB1 -94ins/delATTG promoter polymorphism in IBD and potential interactions with polymorphisms in the CARD15/NOD2, IKBL, and IL-1RN genes

Background: Recently, an association of the NFKB1 polymorphism −94ins/delATTG with ulcerative colitis (UC) has been reported. This 4‐bp insertion/deletion polymorphism is localized in the promoter region of the NFKB1 gene and appears to be functionally relevant. The aim of the present study was to confirm the association of the −94ins/delATTG (W/D) NFKB1 promoter polymorphism with UC in a population of German origin and to test for a potential association with Crohns disease (CD). Furthermore, potential interactions of the −94ins/delATTG polymorphism with the IKBL and the IL‐1RN genes should be determined. Materials and Methods: The study population comprised 630 patients with CD, 365 patients with UC, and 974 healthy controls. Genotyping was performed using polymerase chain reaction and restriction fragment length polymorphism analysis. For statistical evaluation, the chi‐square test and the Fisher exact test were used. Results: No significant association of the W/D NFKB1 polymorphism with CD or UC was detected. In addition, no significant interactions between the −94ins/delATTG NFKB1 polymorphism and polymorphisms within the IKBL and the IL‐1RN genes, respectively, were found in CD or UC. Also, no significant interactions of the NFKB1 polymorphism with mutations of the CARD15/NOD2 gene and with clinical phenotypes were detected in CD. Moreover, no associations of the NFKB1 polymorphism were found in UC depending on disease localization. Conclusions: The present study could not confirm the reported association of the −94ins/delATTG NFKB1 polymorphism with UC and also found no evidence for a role of this polymorphism in CD. The results do not give evidence for a role of this NFKB1 polymorphism in the pathogenesis of UC and CD.

The IL‐10 gene is not involved in the predisposition to inflammatory bowel disease

Although genetic predisposition for inflammatory bowel disease (IBD) is well established, little is known about the accountable genes. The pathogenesis of IBD is characterized by an imbalanced activation of Th1‐ and Th2‐lymphocytes. IL‐10 represents an anti‐inflammatory cytokine which downregulates the production of Th1‐derived cytokines. To evaluate the role of the IL‐10 gene in IBD, two polymorphisms in the promoter region (G/A at position —1082 and C/A at position —592) were genotyped in 142 patients with Crohns disease (CD), 104 patients with ulcerative colitis (UC), and 400 healthy controls. Significant differences were not apparent, neither in the allele frequencies of either polymorphism, nor in the haplotype frequencies. Screening of the coding region of the IL‐10 gene by polymerase chain reaction — single strand conformation polymorphism (PCR‐SSCP) analysis revealed a rare sequence variation in exon 1 leading to an amino acid exchange (G→A; G15R) in two patients with CD and five healthy controls. Therefore, polymorphisms of the IL‐10 gene are not demonstrably involved in the predisposition of IBD in our cohorts of patients.

The polymorphism at position -174 of the IL-6 gene is not associated with inflammatory bowel disease.

Objective Inflammatory bowel diseases (IBD) are multifactorial disorders, characterized by failure to limit the inflammatory response to luminal antigens. Genetic factors play an important role in the pathogenesis, but little is known about the accountable genes. Increased secretion of pro-inflammatory cytokines appears crucial in the initiation of the inflammatory response. Methods To evaluate the role of the IL-6 gene in IBD, a functionally relevant polymorphism in the promoter region (G/C at position −174) has been genotyped in 169 patients with Crohns disease (CD), 133 patients with ulcerative colitis (UC) and 440 healthy controls by using restriction fragment length polymorphism (RFLP) analysis. Results No significant differences were apparent in the allele, genotype and carrier frequencies between patients and controls. Conclusion High secretion of IL-6 does not seem to play a major role in the genetic predisposition to IBD.

Collagenous colitis: implications for the role of vascular endothelial growth factor in repair mechanisms.

Objectives Collagenous colitis is a chronic inflammatory bowel disease with a band-like subepithelial deposition of immature extracellular matrix. Because the extracellular matrix deposition is potentially reversible, an imbalance between fibrogenesis and fibrolysis with reduced matrix degradation has been suspected. Vascular endothelial growth factor plays a central role in extracellular matrix degradation. Therefore, we investigated the expression of vascular endothelial growth factor in the colonic mucosa of patients with collagenous colitis before and after long-term treatment with oral budesonide. Method A quantitative immunohistochemical method was used to measure the amount of immunoreactive vascular endothelial growth factor, tenascin and leucocyte common antigen within the epithelium and the lamina propria of colonic biopsies by area morphometry. Results Strong immunostaining for vascular endothelial growth factor within the epithelium and the lamina propria, and for tenascin, was seen in patients with collagenous colitis compared with normal controls. The enhanced immunostaining for vascular endothelial growth factor within the lamina propria was accompanied by the accumulation of leucocytes, detected by staining for leucocyte common antigen. After long-term treatment with oral budesonide, the amount of immunostaining for leucocyte-derived vascular endothelial growth factor within the lamina propria decreased significantly to normal levels. In contrast, staining for vascular endothelial growth factor within the epithelium remained significantly increased. Conclusions Our data suggest an important role of vascular endothelial growth factor in counteracting the local imbalance of fibrogenesis and fibrolysis, leading to an accumulation of immature subepithelial matrix in collagenous colitis.