Polymorphisms of the lipopolysaccharide-signaling complex in inflammatory bowel disease: association of a mutation in the Toll-like receptor 4 gene with ulcerative colitis

Genes encoding for receptors of the innate immune system are potential candidates for susceptibility to inflammatory bowel disease, e.g., mutations in the cytosolic receptor NOD2/CARD15 were associated with Crohn's disease. Herein, two mutations of the Toll-like receptor (TLR)-4 gene (Asp299Gly and Thr399Ile) resulting in impaired lipopolysaccharide signaling, the -159C/T promotor polymorphism of the CD14 gene, polymorphisms of the lipopolysaccharide binding protein gene and the bactericidal permeability increasing protein gene were evaluated in 102 patients with Crohn's disease, 98 patients with ulcerative colitis and 145 healthy controls. The allele and carrier frequencies for the Thr399Ile mutation in TLR4 gene were significantly increased in ulcerative colitis when compared to the controls (P = 0.014 and P = 0.018, respectively). None of the other five polymorphisms was associated with inflammatory bowel disease. In conclusion, a novel association between a functional polymorphism in TLR4 and ulcerative colitis is reported. This observation underscores the importance of impaired innate immunity in inflammatory bowel disease.     

Inducible and endothelial nitric oxide synthase genes polymorphism in inflammatory bowel disease

To assess the influence of inducible and endothelial nitric oxide synthase gene (NOS2A and NOS3) polymorphisms in susceptibility to Crohn's disease (CD) and ulcerative colitis (UC). A total of 505 inflammatory bowel disease (IBD) patients (221 with UC and 284 with CD) and 332 ethnically matched controls were studied. Patients and controls were genotyped by polymerase chain reaction -based techniques for a multiallelic (CCTTT)(n) repeat and biallelic marker (TAAA)(n) in the promoter region of the NOS2A gene and for a T/C polymorphism at position -786 in the promoter region and a polymorphism in exon 7(298Glu/Asp) of the NOS3 gene. There was not association between NOS2A and NOS3 genotypes, alleles or haplotypes frequencies with UC, CD and controls. Our data suggest that NOS2A and NOS3 polymorphisms do not play a major role in IBD predisposition.     

CD14 promoter polymorphism -159C>T is associated with susceptibility to chronic Chlamydia pneumoniae infection in peripheral blood monocytes

Chlamydia pneumoniae uses peripheral blood monocytes (PBMC) for systemic dissemination and has been linked to atherogenesis by inflammation mediated via TLR2/4 and CD14. We found 12.8% of 610 coronary artery disease (CAD) patients of Central European background to be chronically infected with C. pneumoniae based on the repeated detection of chlamydial DNA in PBMC. Among those the -159C>T CD14 promoter polymorphism was more frequent (OR 1.7, 95% CI 1.08-2.65, P=0.0224) than among C. pneumoniae-negative subjects matched for age and gender. The Arg753Gln TLR2 and Asp299Gly TLR4 polymorphisms were not related to chlamydial infection. Susceptibility for chronic chlamydial infection of PBMC in CAD patients appears associated with the CD14-159C>T promoter polymorphism encoding for enhanced CD14 expression.     

Toll-like receptor 4 (TLR4) gene polymorphisms in celiac disease

Toll-like receptors (TLRs) participate in the first line of immune defense through antigen pattern recognition, and ligands include exogenous and host-derived molecules. Coding variants in TLR4 have been associated with autoimmune diseases like ulcerative colitis, Crohn's disease, and rheumatoid arthritis. Our aim was to determine whether these polymorphisms are associated with celiac disease (CD). Two coding single nucleotide polymorphisms of TLR4 (Asp299Gly and Thr399Ile) were genotyped in 95 family trios with CD as well as in 186 patients and 186 unrelated controls. There were no differences in allele, genotype or haplotype distribution, or transmission between patient and control groups. Our results do not support association of these TLR4 variants with CD.     

The functional genetic variation in the PTPN22 gene has a negligible effect on the susceptibility to develop inflammatory bowel disease

The aim of this study was to assess the possible association between the protein tyrosine phosphatase non-receptor 22 (PTPN22) gene 1858C-->T (rs2476601, encoding R620W) polymorphism and inflammatory bowel disease (IBD). Our study population consisted of 1113 IBD [544 ulcerative colitis (UC) and 569 Crohn's disease (CD)] patients and 812 healthy subjects. All the individuals were of Spanish white origin. Genotyping of the PTPN22 gene 1858C-->T polymorphism was performed by real time polymerase chain reaction technology, using TaqMan 5'-allelic discrimination assay. The frequency of the PTPN22 1858T allele in healthy subjects was 6.2% compared with 6.7% in the UC patients and 5.1% in Crohn's patients. No statistically significant differences were observed when the PTPN22 1858C-->T allele and genotype distribution among CD patients, UC patients and healthy controls were compared. These results indicate that the PTPN22 1858C-->T polymorphism does not appear to play a major role in IBD predisposition in our population.     

Macrophage migration inhibitory factor, Toll-like receptor 4, and CD14 polymorphisms with altered expression levels in patients with ulcerative colitis

Ulcerative colitis is a multifactorial disease in which genetic factors play a major role. Functional mutations in the genes related to innate immune response exacerbate mucosal damage coupled with persistent inflammation. The cytokine macrophage migration inhibitory factor (MIF), CD14, and Toll-like receptor 4 (TLR4) are the central players with clearly defined roles in inflammation. The aim of this study was to investigate the association between MIF-173G > C, CD14-159C > T, and TLR4-299A > G polymorphisms and mononuclear cell expression in patients with ulcerative colitis (UC). Genotyping of MIF-173G > C, CD14-159C > T, and TLR4-299A > G polymorphisms was performed by amplification refractory mutation system-polymerase chain reaction and allele-specific amplification in 139 and 176 patients with UC and controls, respectively. Simultaneously, the expression levels of intracellular MIF, mCD14, and mTLR4 were determined in mononuclear cells using a flow cytometer. Polymorphisms in CD14-159C > T and TLR4-299A > G significantly affected mCD14 and mTLR4 expression levels and also increased susceptibility to UC. Although intracellular MIF expression levels differed among patient and control groups, the polymorphism in MIF 173G > C was not observed to be associated with a risk of UC.     

Toll-like receptor (TLR) 2 and 4 mutations in periodontal disease

Toll-like receptors (TLR) are signal molecules essential for the cellular response to bacterial cell wall components. Different functional effective polymorphisms for the TLR 4 gene (Asp299Gly; Thr399Ile) and for the TLR 2 gene (Arg677Trp, Arg753Gln) have recently been described that are associated with impaired lipopolysaccharide signal transduction. A total of 122 patients with chronic periodontal disease and 122 healthy unrelated controls were genotyped for the Asp299Gly and Thr399Ile polymorphism of the TLR 4 gene and the Arg677Trp and Arg753Gln mutation of the TLR 2 gene. The mutations were identified with polymerase chain reaction followed by restriction fragment length polymorphism (RFLP) analysis. The prevalence of the Asp299Gly and the Thr399Ile mutant allele was 4.1% (10/244) and 4.5% (11/244) among periodontitis patients. For the healthy controls the prevalence was 3.3% (8/244) for the Asp299Gly (P = 0.810) and 3.7% (9/244) for the Thr399Ile mutant allele (P = 0.819). The Arg753Gln mutant allele was found in 2.9% (7/244) of the periodontitis subjects as compared to 4.1% (10/244) in the control group (P = 0.622). The Arg677Trp mutant allele was not found in any of the study subjects. Unlike in ulcerative colitis there was not observed an association between chronic periodontitis and the various mutations of the TLR 2 and 4 gene.     

Gene expression in mononuclear cells from patients with inflammatory bowel disease

OBJECTIVES: Discovery of Nod2 as the inflammatory bowel disease 1 (IBD1) susceptibility gene has brought to light the significance of mononuclear cells in inflammatory bowel disease pathogenesis. The purpose of this study was to examine changes in gene expression in peripheral blood mononuclear cells in patients with untreated Crohn's disease (CD) and ulcerative colitis (UC) as compared to patients with other inflammatory gastrointestinal disorders and to healthy controls. METHODS: We used a 2400 gene cDNA glass slide array (MICROMAX) to examine gene expression in peripheral blood mononuclear cells from seven patients with Crohn's disease, five patients with ulcerative colitis, 10 patients with other inflammatory gastrointestinal disorders, and 22 age- and sex-matched controls. Results. Novel categories of genes differentially expressed in Crohn's disease and ulcerative colitis patients included genes regulating hematopoietic cell differentiation and leukemogenesis, lipid raft-associated signaling, the actin cytoskeleton, and vesicular trafficking. Conclusions: Altered gene expression in mononuclear cells may contribute to inflammatory bowel disease pathogenesis.     

Association of interleukin-18 gene single-nucleotide polymorphisms with susceptibility to inflammatory bowel disease

Interleukin-18 (IL-18) is believed to be one of the most important cytokines in the pathogenesis of inflammatory bowel disease (IBD). The aim of the study was to clarify the significance of single-nucleotide polymorphisms (SNPs) at the 5'-end of the IL-18 gene in the development of IBD. DNA was obtained from peripheral blood of 99 patients with ulcerative colitis (UC), 79 patients with Crohn's disease (CD), and 102 healthy controls. All participants were Japanese. SNPs at -656G/T, -607C/A, -137G/C, +113T/G, and +127C/T were determined by means of direct sequencing, and a genetic association with IBD was examined. The frequencies of the G allele at +113 and the T allele at +127 were significantly higher in patients with CD and UC compared with controls. The differences in allelic frequencies were more striking in patients with CD than in patients with UC, and at position +127 than at position +113. The haplotype estimation, according to the E-M algorithm, suggested that TACGT is closely associated with IBD, especially with CD. It was concluded that SNPs at the 5'-end of IL-18 gene might be closely related to the etiology of IBD.     

Microsatelite GT polymorphism in the toll-like receptor 2 is associated with colorectal cancer

Factors underlying genetic predisposition for development of sporadic colorectal cancer are largely unknown. The fact that this cancer is more common in patients suffering from inflammatory bowel disease raises the question of the relationship between chronic inflammation and cancer. Toll-like receptors 2 (TLR2) and 4 (TLR4) are critical in initiating innate immune response and inflammation toward various bacteria commonly found in the intestine. Recent evidence about the association of polymorphisms in these genes with ulcerative colitis and Crohn's disease, as well as other inflammatory conditions, was the basis for our investigation of their role in sporadic colorectal cancer. We assessed genotype and allele frequencies of TLR2 GT microsatelite polymorphism, TLR2 Arg753Gln, TLR4 Asp299Gly and TLR4 Thr399Ile polymorphisms in 89 colorectal cancer patients and 88 age- and sex-matched controls. The frequency of TLR2 GT microsatelite alleles with 20 and 21 GT repeats was decreased (p = 0.0044 and p = 0.001, respectively), while the frequency of the allele with 31 GT repeats was increased (p = 0.0147) in patients. The mutant allele Asp299Gly of TLR4 gene was slightly more frequent in colorectal cancer patients (p = 0.0269). In conclusion, we report an association of microsatelite GT polymorphisms of TLR2 gene and Asp299Gly polymorphism of the TLR4 gene with sporadic colorectal cancer among Croatians.     

Differential expression of vasoactive intestinal peptide receptor 1 expression in inflammatory bowel disease

There is conflicting evidence regarding the significance of vasoactive intestinal peptide (VIP) in inflammatory bowel disease (IBD). Involvement of the VIP receptor in IBD has not been reported. We examined the expression and localization of the VIP receptor in IBD. We determined the location of VIP receptor 1 (VIPR1) immunohistologically in surgically resected intestinal samples from 10 controls, 15 patients with ulcerative colitis, and 10 patients with Crohn's disease. A fluorescein-linked immunohistological study was performed using anti-VIPR1 antibody, with double-staining with antibodies to CD3, CD19, and CD68. Correlations with interleukin (IL)-4 and TNF-alpha expression were also investigated. Results showed that the number of VIPR1-positive cells was significantly increased in the inflammatory mucosa. VIPR1 was expressed in CD3-, CD19-, and CD68-positive cells. The proportion of VIPR1-positive cells among CD3-positive cells was significantly higher in the lamina propria of patients with ulcerative colitis than in those with Crohn's disease and the controls. The proportion of VIPR1-positive cells among CD68-positive cells was significantly higher in patients with ulcerative colitis and Crohn's disease than in the controls. A correlation between the numbers of VIPR1- and IL-4-positive cells was found in patients with ulcerative colitis, and between the numbers of VIPR1- and TNF-alpha-positive cells in patients with Crohn's disease. In conclusion, VIPR1 was widely expressed in infiltrating inflammatory cells, especially CD3- and CD68-positive cells in ulcerative colitis mucosa and CD68-positive cells in Crohn's disease mucosa. The differential expression of VIPR1 in ulcerative colitis and Crohn's disease mucosa suggests that the VIP system plays different roles in the pathogenesis of IBD.     

The interleukin-25 gene located in the inflammatory bowel disease (IBD) 4 region: no association with inflammatory bowel disease.

Genetic predisposition has been suggested to play an important role in the pathogenesis of inflammatory bowel diseases (IBDs). Linkage studies have identified a Crohn's disease susceptibility locus on chromosome 14 (14q11-12; IBD4). Interleukin-25 (IL-25) is a newly identified proinflammatory cytokine that has been shown to promote Th2 responses by inducing cytokines such as IL-4, IL-5 and IL-13. The IL-25 gene is located within this susceptibility region at 14q11.2. As IBDs are characterized by an imbalance of the Th1/Th2 cytokine response, we hypothesized that genetic alterations within the IL-25 gene might contribute to IBD. First, direct sequencing of the coding regions of the IL-25 gene in 40 patients with Crohn's disease or ulcerative colitis revealed only a newly reported polymorphism (c424C/A) in exon 2. Next, the frequency of this polymorphism was further investigated in 151 patients with Crohn's disease, 111 patients with ulcerative colitis, and 119 healthy controls to determine its clinical relevance. The genotypes of the c424C/A polymorphism did not reveal any significant differences between patients with Crohn's disease or ulcerative colitis and controls. Genoytype-phenotype relations in patients with Crohn's disease showed a comparable distribution of the c424C/A polymorphism in all subgroups of the Vienna classification. In summary, our data indicate that genetic alterations in the coding regions of the IL-25 gene are unlikely to play a role in IBDs, but the c424C/A polymorphism in the IL-25 gene should be investigated for a potential association with other chronic inflammatory and inherited disorders such as autoimmune diseases.     

Epistatic interaction between FCRL3 and MHC in Spanish patients with IBD

Inflammatory bowel disease (IBD), which comprises ulcerative colitis (UC) and Crohn's disease (CD), shows a multifactorial origin, with genetic and environmental factors involved. Although the genetic influence is clear for both diseases, the genetics involved is complex and epistatic interactions with other genes probably exist. The Fc receptor-like 3 gene (FCRL3) maps to the human chromosome 1q21-22 and certain single nucleotide polymorphisms (SNPs) in its promoter have been associated with some autoimmune diseases. Our aim was to study the role of two promoter SNPs of the FCRL3 gene (-169A>G, rs7528684 and -110C>T, rs11264799) in patients with IBD and their interaction with HLA-DRB1 and CARD15 in patients with UC and CD, respectively. A case-control study with 311 patients with CD, 324 patients with UC and 497 healthy controls was performed. All the individuals were White Spaniards. No significant associations were found between any FCRL3 SNP and CD or UC, but the stratification in patients with UC by human leukocyte antigen (HLA) showed a significant increase in heterozygosity at the FCRL3 locus, especially -169 AG (AG vs AA+GG, P= 0.0027, odds ratio = 3.6, 95% confidence interval 1.4-2.9), when HLA-DRB1*0103 carrier patients were compared with HLA-DRB1*0103 noncarriers. Our data suggest an epistatic interaction between genes in chromosomes 6p21 and 1q21-22, marked, respectively, by HLA-DRB1*0103 and FCRL3-169 AG.     

Lack of association of ACP1 gene with inflammatory bowel disease: a case-control study

The red cell acid phosphatease (ACP1) gene, which encodes a low molecular weight phosphotyrosine phosphatase (LMW-PTP), has been suggested as a common genetic factor of autoimmunity. In the present study, we aimed to investigate the possible influence of ACP1 polymorphisms in the susceptibility of inflammatory bowel disease (IBD). A total of 1271 IBD Spanish patients [720 Crohn's disease (CD) and 551 ulcerative colitis (UC)] and 1877 healthy subjects were included. Four single-nucleotide polymorphisms (SNPs), rs10167992, rs11553742, rs7576247 and rs3828329, were genotyped using TaqMan SNP genotyping assays. Common ACP1 alleles (i.e. ACP1*A, ACP1*B and ACP1*C) were determined by two of these SNPs. After the analysis, no evidence of association of the ACP1 genetic variants was found with CD or UC. Therefore, our results suggest that the ACP1 gene may not play a relevant role in the development of IBD.