Association between toll-like receptors/CD14 gene polymorphisms and inflammatory bowel disease in Korean population

The innate immune response in patients who develop inflammatory bowel disease (IBD) may be abnormal. However, the exact role of Toll-like receptors (TLRs) / CD14 gene in the pathogenesis of IBD has not been fully elucidated. We aimed to investigate the association between polymorphisms of TLR1, 2, 4, 6, and CD14 gene and susceptibility to IBD in Korean population. A total 144 patients of IBD (99 patients with ulcerative colitis, 45 patients with Crohn's disease) and 178 healthy controls were enrolled. Using a PCR-RFLP, we evaluated mutations of TLR1 (Arg80Thr), TLR2 (Arg753Gln and Arg677Trp), TLR4 (Asp299Gly and Thr399Ile), TLR6 (Ser249Pro) genes and the -159 C/T promoter polymorphism of CD14 gene. No TLR polymorphisms were detected in Korean subjects. T allele and TT genotype frequencies of CD14 gene were significantly higher in IBD patients than in healthy controls. In subgroup analysis, T allelic frequency was higher in pancolitis phenotype of ulcerative colitis. In Korean population, the promoter polymorphism at -159 C/T of the CD14 gene is positively associated with IBD, both ulcerative colitis and Crohn's disease.     

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.     

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.     

Rarity of TLR4 Asp299Gly and Thr399Ile polymorphisms in the Korean population

PURPOSE: Activation of the innate immune system and chronic low-grade inflammation are thought to be involved in the pathogenesis of atherosclerosis and also thought to be associated with type 2 diabetes and its complications. As a receptor for bacterial lipopolysaccharide and heat-shock proteins, Toll-like receptor 4 (TLR4) is one of the central regulators of the immune response. Recent studies have reported an association between TLR4 polymorphisms and diabetes and its complications in Caucasian populations. MATERIALS AND METHODS: In this study, we analyzed the association between TLR4 gene polymorphisms in patients with features of type 2 diabetes and healthy controls in Korea. Two polymorphisms of the TLR4 gene (Asp299Gly and Thr399Ile) were examined in 225 diabetic patients and 153 healthy controls using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and single-strand conformation polymorphism (SSCP). RESULTS: No Asp299Gly or Thr399Ile mutations were detected in any of the 378 subjects. Seven subjects from each group who had slightly different SSCP patterns were selected for sequencing, but we found no TLR4 polymorphisms on Exon3. The Asp299Gly and Thr399Ile TLR4 gene polymorphisms were absent in both groups, which was similar to the results for Japanese and Chinese Han subjects. CONCLUSION: Our data and other Asian data suggest that a racial difference can be found in the frequency of the TLR4 polymorphism.     

Toll-like receptor (TLR) 4 polymorphisms are associated with a chronic course of sarcoidosis

The aetiology of sarcoidosis, an inflammatory granulomatous multi-system disorder, is unclear. It is thought to be the product of an unknown exogenous antigenic stimulus and an endogenous genetic susceptibility. Toll-like receptors (TLR) are signal molecules essential for the cellular response to bacterial cell wall components. Lipopolysaccharide (LPS), for example, binds to TLR 4. Two different polymorphisms for the TLR4 gene (Asp299Gly and Thr399Ile) have been described recently. This leads to a change in the extracellular matrix function of TLR4 and to impaired LPS signal transduction. We genotyped a total of 141 Caucasian patients with sarcoidosis and 141 healthy unrelated controls for the Asp299Gly and Thr399Ile polymorphisms in the TLR4 gene. The mutations were identified with polymerase chain reaction followed by restriction fragment length polymorphism (RFLP) analysis. Among sarcoidosis patients the prevalence for each Asp299Gly and Thr399Ile mutant allele was 15.6% (22/141). In the control group the prevalence was 5.67% (8/141) (P = 0.07). In the subgroup of patients with acute sarcoidosis there was no difference in the control group (P = 0.93), but there was a highly significant association between patients with a chronic course of sarcoidosis and TLR4 gene polymorphisms (P = 0.01).     

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.     

Mutations in the gene for toll-like receptor 4 and multiple sclerosis

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system with heterogeneous pathological features, disease courses and genetical backgrounds. In this study we determined whether genetic variants of toll-like receptor (TLR) 4, which confer substantial differences in the inflammation elicited by bacterial lipopolysaccharide, are related to the development of MS. We found no differences in the frequencies of the cosegregating TLR4 Asp299Gly and Thr399Ile polymorphisms between Austrian MS patients (11.6%) and age-matched controls (13.7%). Furthermore, we could not detect any influence of these mutations on clinical parameters and serum levels of soluble adhesion molecules of MS patients. Our data indicate that these TLR4 polymorphisms have no influence on the incidence, progression and inflammatory parameters of MS.     

Single step high-throughput determination of Toll-like receptor 4 polymorphisms

BACKGROUND: Toll-like receptors are central components of host defence in humans, responsible for recognition of pathogen-associated molecular patterns and activation of innate immunity. Toll-like receptor 4 (TLR4) is activated by lipopolysaccharide (LPS) and other microbial components, thereby initiating the expression and release of pro-inflammatory cytokines. The common, frequently co-segregating allelic variants Asp299Gly and Thr399Ile have been related to susceptibility to gram-negative infections and sepsis and may be involved in the development of atherosclerosis. Identification of TLR4 Asp299Gly and Thr399Ile genotypes can be important for examination of genotype/phenotype relationships as well as for individual risk assessment of patients. METHODS: TLR4 Asp299Gly and Thr399Ile genotypes were detected by a single tube polymerase chain reaction (PCR), based on exonuclease degradation of dual labelled allele-specific oligonucleotides. The assay results were compared with conventional restriction fragment length polymorphism (RFLP) analysis. RESULTS: Genotypes of 345 individuals were determined simultaneously in a single PCR assay. Allele frequencies for our population were 6.8% for the TLR4 Asp299Gly polymorphism and 6.4% for the Thr399Ile polymorphism. Validation by RFLP analysis revealed a correct detection of all genotypes. CONCLUSIONS: We have developed a novel method for the detection of the TLR4 Asp299Gly and Thr399Ile mutations, permitting rapid genotyping which should be useful for large-scale population studies as well as applicable for routine clinical testing.     

Toll-like receptor 4 Asp299Gly and Thr399Ile polymorphisms in gastric cancer of intestinal and diffuse histotypes

In the present study we investigated the potential role of Toll-like receptor 4 (TLR-4) Asp299Gly and Thr399Ile polymorphisms as risk factors in the development of gastric cancer. TLR-4 Asp299Gly and Thr399Ile polymorphisms were investigated in 171 Italian patients with sporadic gastric cancer and in 151 controls. Unconditional regression (odds ratio and 95% confidence intervals) were used to investigate the association of the studied polymorphisms with gastric cancer. TLR-4 Thr399Ile polymorphism is linked with an increased susceptibility to gastric cancer (P = 0.023 and hazard ratio = 3.62). No significant association for TLR-4 Asp299Gly polymorphism was found. In the subgroup of patients with intestinal-type gastric cancer, a significant risk of gastric cancer was associated with TLR-4 Thr399Ile genotype (P = 0.006). Our results demonstrated that TLR-4 Thr399Ile polymorphism is linked with an increased susceptibility to gastric cancer. An increased risk for intestinal gastric cancer in carriers of the TLR4 Thr399Ile allele was observed. Future epidemiological studies should consider the possible interactions between proinflammatory genotypes (such as TLR and interleukin-1R polymorphisms) and other risk factors for cancer such as dietary habits and/or exposure to environmental carcinogens.     

The -295T-to-C promoter polymorphism of the IL-16 gene is associated with Crohn's disease

Recently, a T-to-C polymorphism at position -295 in the promoter region of the human interleukin-16 (IL-16) gene was reported. The expression of IL-16 is increased in inflammatory bowel disease, in particular in Crohn's disease. However, data concerning the IL-16 promoter polymorphism in inflammatory bowel disease are lacking. Thus, the current study aimed at the assessment of this polymorphism in Crohn's disease and ulcerative colitis. One hundred three patients with Crohn's disease, 100 patients with ulcerative colitis, and 120 healthy unrelated controls were genotyped for the promoter polymorphism. Furthermore, patients with Crohn's disease were stratified according to disease localization and the respective clinical phenotype (fistulizing, fibrostenotic, or inflammatory). The frequencies of the T allele (P < 0.01) and the TT genotype (P < 0.01) were significantly increased in patients with Crohn's disease compared to the controls, regardless of the disease phenotype or the site of intestinal involvement. An association with ulcerative colitis was not observed. Herein a new association between a promoter polymorphism of the IL-16 gene and Crohn's disease was observed and correlates with the previously described increased mucosal expression of IL-16 in inflammatory bowel disease.     

Role of TLR4 C>1196T (Thr399Ile) and TLR4 A>896G (Asp299Gly) polymorphisms in a North Indian population with asthma: a case‐control study

Toll‐like receptor 4 (TLR4) is the most important TLR among the pattern recognition receptors which recognizes lipopolysaccharide of gram‐negative bacteria. They identify a highly conserved structure of microbes called pathogen‐associated molecular patterns and activate immune and inflammatory responses that have been shown to be involved in the pathogenesis of asthma. The role of TLR4 gene polymorphisms in asthma was detected in a total of 964 individuals, including 483 healthy controls and 481 asthma patients from a North Indian population. The genotyping was carried out using polymerase chain reaction–restriction fragment length polymorphism method. Statistical analysis revealed that the heterozygous genotype as well as the mutant (T) allele of the TLR4 C>1196T (Thr399Ile) polymorphism shows resistance towards asthma with OR = 0.70, 95% CI (0.49–0.99), P corrected value = 0.046 and OR = 0.72, 95% CI (0.52–0.98), P corrected value = 0.039, respectively. However, no association was found between the TLR4 A>896G (Asp299Gly) polymorphism and asthma patients (P > 0.05). This is the first study conducted in India conferring TLR4 (Thr399Ile) polymorphism resistance towards asthma, while lack of association was found between TLR4 (Asp299Gly) polymorphism and asthma in the studied North Indian population.     

TLR Polymorphisms in FMF: Association of TLR-2 (Arg753Gln) and TLR-4 (Asp299Gly, Thre399Ile) Polymorphisms and Myeloid Cell TLR-2 and TLR-4 Expression with the Development of Secondary Amyloidosis in FMF

Amyloidosis is the major complication of familial Mediterranean fever (FMF). Toll-like receptors (TLR) are involved in the activation of an innate immune system TLR-2 and TLR-4 recognize lipoteichoic acid and lipopolysaccharides (LPS), respectively. While TLR-2 Arg753Gln polymorphism upregulates, TLR-4 Asp299Gly and Thre399Ile polymorphisms downregulate inflammation. We investigated the effect of these polymorphisms on the development of amyloidosis in FMF patients. We also investigated myeloid cell TLR-2 and TLR-4 expressions in these patients. We studied 26 FMF patients and 13 FMF patients with amyloidosis. TLR-2 Arg753Gln and TLR-4 Asp299Gly and Thr399Ile polymorphisms were analyzed with the polymerase chain reaction-restriction fragment length polymorphism method. Myeloid cell baseline TLR-2 and TLR-4 and LPS-induced TLR-4 expressions were evaluated. The TLR-2 and TLR-4 polymorphism rate was compared with the results of 100 healthy subjects in our previous study. In addition, 13 healthy controls were enrolled for leukocyte TLR-2 and TLR-4 expressions. Serum amyloid A (SAA) levels were measured in these 13 control cases and in FMF patients during attack-free periods. The frequency of TLR-2 Arg753Gln, TLR-4 Asp299Gly, and Thr399Ile polymorphisms in healthy controls in our previous study were 1%, 3%, and 2%, respectively. The frequency of these polymorphisms were not different in FMF patients (with or without amyloidosis) compared to the control group. Likewise, myeloid cell TLR-2 and TLR-4 expressions were not different among the controls and FMF patients. However, LPS-induced TLR-4 expression in granulocytes was more prominent in FMF patients. There was no correlation between TLR-2 and TLR-4 expressions and SAA levels. Neither myeloid cell TLR-2 and TLR-4 expressions nor TLR-3 Arg753Gln, TLR-4 Asp299Gly, and Thr399Ile polymorphisms seem to affect the development of secondary amyloidosis in FMF patients in our study population.     

Polymorphisms of toll-like receptor 2 and 4 genes in rheumatoid arthritis and systemic lupus erythematosus

Human toll-like receptors (TLRs) participate in the innate response and signal the activation of adaptive immunity. Therefore, these TLRs may be important in autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). We investigated, by using a polymerase chain reaction restriction-fragment length polymorphism method, the possible association between the polymorphisms of TLR2 (Arg677Trp and Arg753Gln) and TLR4 (Asp299Gly and Thr399Ile) genes with the susceptibility or severity of RA and SLE. Our study population consisted of 122 patients with SLE, 224 patients with RA, and a control group of 199 healthy individuals. The TLR2 polymorphisms were very rare in our population; no individual carrying the TLR2-Arg677Trp polymorphism was observed, whereas the TLR2-Arg753Gln polymorphism was present in only 1% of the total population. We found no statistically significant differences in the TLR4-Asp299Gly and the TLR4-Thr399Ile genotype or allele distribution between SLE patients, RA patients, and control individuals. Similarly, no association was found with any of the demographic and clinical parameters tested either in RA or in SLE patients. In conclusion, a case-control study was used to analyze, for the first time, the influence of TLR2 and TLR4 gene polymorphism on the predisposition and clinical characteristics of SLE and RA but provided no evidence for association of TLR2 or TLR4 gene polymorphism with either disease in the population under study.     

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.     

Association of Helicobacter pylori infection with Toll‐like receptor‐4 Thr399Ile polymorphism increased the risk of peptic ulcer development in North of Iran

Toll‐like receptor‐4 (TLR4) polymorphisms may influence host immune response against Helicobacter pylori (H. pylori). This study aimed to investigate whether TLR4 polymorphisms are associated with H. pylori susceptibility and risk of peptic ulcer development or not. The TLR4 + 3725 G/C polymorphism was studied using polymerase chain reaction with confronting two‐pair primers (PCR–CTPP). In addition, TLR4 Asp299Gly and Thr399Ile polymorphisms were evaluated by PCR‐restriction fragment length polymorphism (RFLP). There was no significant difference in TLR4 + 3725 G/C and Asp299Gly genotype frequencies between non‐peptic ulcer (NPUD) and peptic ulcer (PUD) individuals in the context of peptic ulcer development and susceptibility to infection with H. pylori. Nevertheless, a significant association with increased risk for PUD development was observed for polymorphism TLR4 Thr399Ile [odds ratio (OR) = 4.2; 95% confidence interval (CI) = 1.35–13.26; p = 0.01]. Correspondingly, TLR4 Thr399Ile polymorphism was associated with H. pylori susceptibility (OR = 0.27; 95% CI = 0.08–0.88; p = 0.04). In addition, TLR4 Thr399Ile polymorphism increased 4.2‐fold, the risk of peptic ulcer development in individuals infected by H. pylori carrying CT + TT genotype. Our results showed that TLR4 Thr399Ile polymorphism along with H. pylori infection may play critical roles in peptic ulcer development in North of Iran.     

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.     

Polymorphisms of the ICAM-1 Gene Are Associated with Inflammatory Bowel Disease, Regardless of the p-ANCA Status

The intercellular adhesion molecule-1 (ICAM-1) is of paramount importance for the initiation and propagation of various inflammatory conditions. An increased frequency of allele R241 of the ICAM-1 gene was previously described in p-ANCA-negative as compared to p-ANCA-positive ulcerative colitis and vice versa in Crohn's disease. One hundred sixteen healthy unrelated controls, 121 patients with ulcerative colitis, and 96 patients with Crohn's disease were genotyped for two polymorphisms of the ICAM-1 gene (R/G241, exon 4; and K/E469, exon 6), employing dot-blot hybridization and stratified according to their p-ANCA status. When compared with the control group the frequency of the allele R241 (P = 0.024) and the heterozygous genotype R/G241, P = 0.032) were significantly increased in ulcerative colitis, whereas the homozygous genotype G/G241 was found less frequently (P = 0.022). The heterozygous genotype K/E469 was observed less frequently (P = 0.001 and 0.037, resp.) than the homozygous genotype E/E469, which was more frequent in Crohn's disease and ulcerative colitis (P = 0.002 and 0.012, respectively). Further significant differences concerning the allele or genotype distribution were not observed. After stratification for the p-ANCA status significant differences concerning the frequencies of both the R241 and the E469 alleles were not detected when p-ANCA-positive inflammatory bowel disease and p-ANCA-negative inflammatory bowel disease were compared. Ulcerative colitis and Crohn's disease are associated with polymorphisms of the ICAM-1 gene, which might therefore represent a functional candidate gene. However, the observed associations are independent of the p-ANCA status.