Epistasis between Toll-like receptor-9 polymorphisms and variants in NOD2 and IL23R modulates susceptibility to Crohn's disease.

OBJECTIVES: Recent data suggest functional interactions between NOD2 and other receptors of the innate immune system modulating inflammatory responses. Here we analyzed the role of Toll-like receptor 9 (TLR-9) gene variants with respect to susceptibility to inflammatory bowel disease (IBD) and tested for genetic interactions with NOD2 and other susceptibility genes for Crohn's disease (CD). METHODS: The single-nucleotide polymorphisms (SNPs) -1237T/C (rs5743836) and 2848A/G (rs352140=p.Pro545Pro) in TLR9, the main CD-associated variants within the genes for NOD2, IL23R, ATG16L1, and variants in the IBD5 locus and in the DLG5 gene were assessed in 956 patients with IBD (606 CD and 350 ulcerative colitis) and in 792 healthy controls. The associations with disease susceptibility and phenotype, and epistatic gene-gene interactions, were analyzed. RESULTS: The TLR9 -1237T/C polymorphism showed significant interactions with NOD2 mutations. The frequency of -1237C was significantly higher in CD patients with at least one NOD2 mutation (P=0.004 vs. controls, odds ratio (OR) 1.60, 95% confidence interval (CI) (1.15-2.21)) and further increased in CD patients with two mutated NOD2 alleles (P=0.002 vs. controls, OR 2.37, 95% CI (1.35-4.15)). Significant gene-gene interactions were also observed for the TLR9 polymorphism -1237T/C with IL23R variants (most significantly with rs1004819, P=0.0007), with a particular high frequency of -1237C in CD patients carrying CD-protective IL23R variants. Epistatic interactions of the TLR9 -1237T/C SNP were also noted with the DLG5 113G/A variant (P=0.0007). CONCLUSIONS: Our results provide evidence for genetic interactions between polymorphisms in TLR9 and CD-associated variants in NOD2, IL23R, and DLG5, differentially modulating CD susceptibility.

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 Crohn's 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.

Allele 2 of the interleukin-1 receptor antagonist gene is associated with early gastric cancer.

PURPOSE: In advanced gastric cancer (tumor stages T2-T4), associations with polymorphisms of the interleukin-1 (IL-1) gene cluster have been made. In early-stage gastric cancer, which we defined as adenocarcinoma confined to the mucosa or submucosa (stage T1), the role of host genetic susceptibility remains to be determined. PATIENTS AND METHODS: Eighty-eight patients with early-stage gastric cancer (stage T1, 77 positive for Helicobacter pylori) and 145 controls were genotyped for polymorphisms in the IL-1 gene cluster and the tumor necrosis factor alpha (TNF-A) gene. Statistical analysis was performed using the chi2 test and the Fisher's exact test, respectively. RESULTS: The homozygous genotype IL-1RN*2/2 of the IL-RN gene was strongly associated with early-stage gastric cancer (P < .0001), whereas further associations with the IL-1 gene cluster were not observed. A weak association of the TNF-A-308A allele with the diffuse type of early-stage gastric cancer, and an association with a composite of two or three proinflammatory polymorphisms, which predispose to increased production of the proinflammatory cytokines IL-1beta and TNF-alpha, could also be demonstrated. CONCLUSION: The genotype IL-1RN*2/2 seems to be associated with early-stage gastric cancer. As opposed to advanced-stage gastric cancer, further proinflammatory cytokine polymorphisms were not associated independently, but might act in combination and mirror early steps of gastric carcinogenesis in hosts colonized by Helicobacter pylori. However, these findings await confirmation in future trials and should be underscored by gene expression studies.

The angiotensin I-converting enzyme gene insertion/deletion polymorphism is linked to early gastric cancer.

The insertion/deletion polymorphism of the angiotensin I-converting enzyme (ACE) gene has recently been linked to the pathogenesis and progression of human cancers. Using genomic DNA from 88 patients with early gastric cancer confined either to mucosa (pT(1a)) or submucosa (pT(1b)), we assessed the insertion (I) and deletion (D) polymorphism by PCR analysis and compared it with a large noncancer control population (n = 145). In the noncancer control group, the II genotype was observed in 33 (23%) individuals, whereas the ID and DD genotypes were found in 72 (50%) and 40 (27%) individuals, respectively. Interestingly, in the cancer group, we found the II genotype in six (7%) patients and the ID genotype in 46 (52%) individuals, whereas the DD genotype was observed in 36 (41%) individuals (P = 0.0034). Accordingly, the odds ratio for the II genotype was 0.20 [95% confidence interval (95% CI), 0.08-0.54; P = 0.009] and 0.55 for the ID/II genotype (95% CI, 0.31-0.96; P = 0.044) using the high-activity genotype DD as the reference category. No correlation was found among tumor type, tumor stage, the presence of Helicobacter pylori, and the ACE genotype. Our study provides further evidence that the ACE insertion/deletion gene polymorphism may be linked to the development of early gastric cancer.

Association of polymorphisms in the interleukin-18 gene in patients with Crohn's disease depending on the CARD15/NOD2 genotype.

UNLABELLED: An increased expression of interleukin-18 (IL-18), a proinflammatory cytokine inducing interferon-gamma, has been found in Crohn's disease (CD). In the IL-18 gene, several partly functional relevant polymorphisms are known. This study sought to investigate associations of IL-18 polymorphisms in inflammatory bowel disease and CD according to CARD15/NOD2 mutation status and clinical phenotypes. METHODS: The IL-18 polymorphisms -607, -137, and the third position of codon 35 (c35/3) were genotyped in 210 patients with CD, 140 patients with ulcerative colitis, and 265 healthy controls using polymerase chain reaction and restriction fragment length polymorphism analysis. RESULTS: Frequencies of alleles and genotypes of the 3 polymorphisms and of the respective haplotypes and diplotypes displayed no significant differences between the whole groups of patients with CD and ulcerative colitis, respectively, compared with the controls. After stratification of patients with CD for CARD15/NOD2 status, significant associations of genotypes -137 CC (P = 0.018) and c35/3 CC (P = 0.010) and of the diplotype 2-2 (P = 0.018) were found in cases carrying CARD15/NOD2 mutations. Associations of genotypes -137 GG (P = 0.015) and c35/3 AA (P = 0.030) with colonic disease only in cases positive for CARD15/NOD2 mutations and of the genotype -607 AA (P = 0.007) with fistulas in cases negative for CARD15/NOD2 mutations were observed. CONCLUSIONS: In this study, significant differences of several genotypes and diplotypes within the IL-18 gene in CD depending on CARD15/NOD2 status have been found. In context with an increased expression of IL-18 in CD, it remains to be shown whether the expression of IL-18 is influenced by CARD15/NOD2 mutation status.

Recurrent severe gastrointestinal bleeding and malabsorption due to extensive habitual megacolon.

Dilatation of the colon and the rectum, which is not attributable to aganglionosis, is a rare finding and can be the result of intractable chronic constipation. We report a rare case of a 29-year-old male patient with impressive megacolon, in whom Hirschsprung's or Chagas disease was ruled out. In the present case, dilatation of the colon was most likely due to a behavioral disorder with habitual failure of defecation. Chronic stool retention led to a bizarre bulging of the large bowel with displacement of the other abdominal organs and severe occult blood loss. Because of two episodes of life-threatening gastrointestinal bleeding despite conventional treatment of constipation, a surgical approach for bowel restoration was necessary. Temporary loop ileostomy had to be performed for de-pressurization of the large bowel and the subsequent possibility for effective antegrade colonic lavage to remove impacted stools. Shortly after the operation, the patient was healthy and could easily manage the handling of the ileostomy. However, the course of the megacolon in this young adult cannot be predicted and the follow-up will have to reveal if regression of this extreme colonic distension with reestablishment of regular rectal perception will occur.

Prokinetic therapy: what can be measured by gastric scintigraphy?

OBJECTIVE: To evaluate whether gastric scintigraphy with quantitative analysis of gastric peristalsis may be a useful tool for documenting the effects of prokinetic therapy. METHODS: Gastric emptying was determined in eight patients with insulin dependent diabetes mellitus (IDDM) and nine patients with progressive systemic sclerosis (PSS) after ingestion of a semi-solid test meal. Fourier analysis of condensed images was used to evaluate contraction amplitudes of the entire stomach, as well as frequency and velocity of gastric contractions. Patients were studied twice, first under baseline conditions and then after 4 weeks of continuous oral prokinetic therapy with cisapride (10 mg t.i.d.). Findings were compared with data derived from 15 healthy controls. RESULTS: At baseline conditions IDDM and PSS patients exhibited reduced gastric peristalsis and delayed emptying compared to controls. After 4 weeks of prokinetic treatment, gastric contraction amplitudes increased significantly, especially in the gastric corpus and the proximal stomach, associated with a slight improvement of gastric emptying. Changes in frequency and velocity of gastric contractions during prokinetic treatment correlated with the improvement of gastric motility and emptying. CONCLUSIONS: Prokinetic therapy with cisapride stimulates gastric motility, and could be non-invasively monitored by scintigraphy. This radionuclide technique provides a number of relevant parameters of gastric function, such as gastric emptying, contraction amplitudes, frequency and velocity of gastric contractions, which give crucial insights into the mechanisms of action of prokinetic drugs.

The leukocyte count predicts the efficacy of treatment with azathioprine in inflammatory bowel disease.

INTRODUCTION: Azathioprine has variable efficacy in inflammatory bowel disease. Previous studies suggested that either neutropenia, an increase in the mean corpuscular volume, the assessment of thiopurine methyl-transferase activity or erythrocyte 6-thioguanine values might predict the treatment response. However, due to the conflicting results of the preceding studies there are yet no established laboratory values which allow an estimation of the clinical response. PATIENTS AND METHODS: 45 patients with Crohn's disease and 39 patients with ulcerative colitis were enrolled in this retrospective evaluation. After a minimum of six months therapy with azathioprine patients in remission were compared with those who did not achieve a stable remission with respect to the number of leucocytes, lymphocytes, neutrophil granulocytes and the mean corpuscular volume. RESULTS: Patients who went into remission during treatment with azathioprine displayed significantly lower leukocyte counts if compared to patients who were not in remission (p = 0.004 in Crohn's disease and 0.003 in ulcerative colitis). A similar tendency was also observed with respect to the granulocyte count (p = 0.007 in Crohn's disease and 0.004 in ulcerative colitis). The mean corpuscular volume did not correlate with the response to purine analogues. DISCUSSION: The absolute leukocyte count and the percentage of granulocytes seem to predict the response to purine analogues in inflammatory bowel disease and possibly offers a feasible and cost effective diagnostic tool for the assessment of therapeutic efficacy. Subsequent preferably prospective studies should aim to define the optimal cut-off value for the leukocyte count.

Crohn's disease: an immunodeficiency?

Autoimmunity appears to be a key factor in Crohn's disease as it develops in a genetically susceptible host if the immunological tolerance towards bacterial antigens within the gastrointestinal tract is abrogated. The resulting excessive immunological activity leads to a chronic sometimes transmural inflammatory process within the bowel wall. However, several lines of evidence are compatible with an immunodeficiency preceding these processes: humoral or cellular immune defects can predispose to inflammatory bowel disease. An increased bacterial adherence at the intestinal mucosa, which is possibly attributable to impaired expression of defensins was observed in Crohn's disease. Furthermore, the 3020insC mutation of the NOD2/CARD15 gene which is associated with Crohn's disease results in impaired cytokine transcription. Lastly, therapeutic approaches such as the use of antibiotic therapy or granulocyte macrophage colony stimulating factor are in line with the concept of an immunodeficiency being a crucial element in Crohn's disease.

NOD2/CARD15 genotype influences MDP-induced cytokine release and basal IL-12p40 levels in primary isolated peripheral blood monocytes.

BACKGROUND: The functional link between mutations in NOD2 and Crohn's disease (CD) has not been entirely elucidated. The 1007fs mutation results in loss of NF-kappaB activation in response to muramyl dipeptide (MDP) but has also been linked to an increased IL-1beta processing and IL-12 release. METHODS: We investigated the basal and MDP-triggered mRNA expression and protein release for TNF-alpha, IL-10, IL-1beta, and IL-12p40 in peripheral blood monocytes from 40 CD patients and 15 healthy individuals with different NOD2 genotypes. RESULTS: Monocytes from individuals with 2 mutated NOD2 alleles (homozygous and compound-heterozygous individuals) displayed an impaired release of TNF-alpha and IL-10 but also of IL-1beta and IL-12p40 in response to MDP. In contrast to other NOD2 variants, the presence of at least 1 1007fs allele in double-mutated individuals completely abrogated NOD2 receptor function. Interestingly, monocytes from CD patients with 2 mutated NOD2 alleles displayed significantly higher basal levels of IL-12p40 in cell culture supernatants compared to wildtype CD patients and control individuals (P = 0.002 and P = 0.008, respectively). This was regardless of the IL23R genotype and was not mirrored by increased IL-12p40 plasma levels in these individuals. CONCLUSIONS: The CD-associated NOD2 variants lead, in a dose- and mutation-dependent manner, to an impaired release of TNF-alpha, IL-10, IL-1beta, and IL-12p40 in response to MDP. The finding of increased basal levels for IL-12p40-related cytokines in monocytes with 2 mutated NOD2 alleles is likely to set a new link between NOD2 mutations and the inflammatory mechanisms underlying CD.

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 Crohn's 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 x 10(-6) and 3.7 x 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.

The 14-bp deletion polymorphism in the HLA-G gene displays significant differences between ulcerative colitis and Crohn's disease and is associated with ileocecal resection in Crohn's disease.

HLA-G is a non-classical MHC class Ib molecule predominantly expressed in cytotrophoblasts and under pathological conditions also in chronically inflamed and in malignant tissues. Recently an increased expression of HLA-G was found in ulcerative colitis (UC), but not in Crohn's disease (CD). The HLA-G gene is located in IBD3, a linkage region for inflammatory bowel disease (IBD). A 14-bp deletion polymorphism (Del+/Del-) within exon 8 of the HLA-G gene might influence transcription activity and is therefore of potential functional relevance. To investigate whether the 14-bp deletion polymorphism is associated with IBD, 371 patients with CD, 257 patients with UC and 739 controls were genotyped. The heterozygous genotype (P = 0.031) and the Del+ phenotype (P = 0.038) were significantly increased, whereas the homozygous Del- phenotype (P = 0.038) was significantly decreased in UC when compared with CD. Thus, the 14-bp deletion polymorphism within the HLA-G gene displayed significant differences between UC and CD. Moreover, a significant increase of the Del+ allele (P = 0.002) and the Del+/Del+ genotype (P = 0.013) and a consecutive decrease of the Del-/- genotype (P = 0.024) were observed in those CD cases positive for ileocecal resection. Thus, a potential effect of the HLA-G gene in IBD may affect both UC and CD. Other polymorphisms linked to the 14-bp deletion polymorphism might also contribute to immunopathogenesis. As there are several partly functional polymorphisms within the promoter region potentially influencing HLA-G expression, further studies in IBD are necessary in the context of differential expression of HLA-G between UC and CD.

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 Crohn's 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 = 1.92x10(-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 = 8.04x10(-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.

Genetic variants and the risk of Crohn's disease: what does it mean for future disease management?

Genetic research in inflammatory bowel disease, especially in Crohn's disease, has made significant progress during recent years. There have been > 10 total genome scans that have been performed, and susceptibility loci on several chromosomes have been identified. Together with candidate gene studies, these scans have led to the identification of several susceptibility genes, with CARD15 being the most important. These genetic data have already provided important insights into the pathophysiology of inflammatory bowel disease and are stimulating future research. On the other hand, genotype-phenotype associations have illustrated the heterogenic nature of the disease. Although the clinical application of this knowledge is so far limited, there is significant optimism that an individual management of patients based on genetic data will be possible in the near future.

A polymorphism of the bactericidal/permeability increasing protein (BPI) gene is associated with Crohn's disease.

BACKGROUND: The bactericidal/permeability increasing protein (BPI) is involved in the elimination of gram-negative bacteria. A functionally relevant single nucleotide polymorphism of the BPI gene causes an amino acid exchange (Glu216Lys). STUDY: To evaluate whether this single nucleotide polymorphism contributes to the predisposition to inflammatory bowel disease, we compared the allele frequencies of 265 patients with Crohn's disease, 207 patients with ulcerative colitis, and 608 healthy controls. RESULTS: The Glu/Glu genotype frequency was decreased significantly in Crohn's disease patients as compared with controls (P < 0.027). No differences were obvious in patients with ulcerative colitis. CONCLUSIONS: Failure of the innate intestinal immune system could be involved in the pathogenesis of Crohn's disease via reduced/impaired defense against gram-negative bacteria.

Polymorphisms of the interleukin-18 gene in periodontitis patients.

BACKGROUND: Interleukin (IL)-18 regulates the expression of the proinflammatory cytokine interferon (IFN)-gamma. The present study sought to test the putative involvement of six different IL-18 gene polymorphisms in pre-disposition to destructive periodontal disease. METHODS: A total of 123 patients with periodontitis and 121 healthy controls were genotyped for six IL-18 polymorphisms at position -656, -607, -137, +113, +127 and codon 35/3. Genotyping has been performed by PCR and restriction fragment length polymorphism analysis. The frequencies of alleles and genotypes as well of haplotypes within both study groups were compared using the Pearson Chi-square test at a level of significance of 5% (p<0.05). RESULTS: Coseggregation was found to be 100% for the two polymorphisms at position -656 and -607 as well as for the polymorphisms at position -137, +113, and +127. The distribution of genotypes for the IL-18 gene polymorphism at position -656/-607 (p=0.854), at position -137/+113/+127 (p=0.320), and at codon 35/3 (p=0.481) was not significantly different among periodontitis patients if compared with healthy control subjects. The distribution of haplotype combinations for the -607 and -137 polymorphism also showed not significant difference between the both study groups (p=0.545). CONCLUSION: Herein the six different IL-18 gene polymorphisms were not associated with destructive periodontal disease.