Toll-like Receptor 4 Asp299Gly Polymorphism Research Articles

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.

Toll-Like Receptor 4 Gene Polymorphisms and Preeclampsia: Lack of Association in a Caucasian Population

Preeclampsia is a multifactorial disorder with genetic and environmental components. As Toll-like receptor 4 (TLR4) has an essential role in innate immune response, which is exaggeratedly activated in preeclampsia, our aim was to investigate whether two single nucleotide polymorphisms (SNPs) of the TLR4 gene--Asp299Gly (A896G) and Thr399Ile (C1196T)--are associated with preeclampsia in a Caucasian population from Hungary. In a case-control study, we analyzed blood samples from 180 preeclamptic patients and 172 normotensive, healthy pregnant women with the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method. The linkage disequilibrium (LD) profile of the TLR4 gene was investigated and tag SNPs were identified using data from the International HapMap Project. There were no significant differences in the genotype and allele frequencies of Asp299Gly and Thr399Ile polymorphisms between the two study groups. Additionally, no significant difference was found in the distribution of the estimated haplotypes created by the two polymorphisms between the preeclamptic and the control group. Furthermore, no significant differences were detected in the genotype, allele and haplotype frequencies of Asp299Gly and Thr399Ile TLR4 SNPs between patients with mild and severe preeclampsia, between patients with late and early onset of the disease, or between preeclamptic patients with and without fetal growth restriction. In conclusion, we did not find an association between TLR4 Asp299Gly and Thr399Ile gene polymorphisms and preeclampsia. As the Thr399Ile polymorphism is a highly informative tag SNP of the TLR4 gene, our results suggest that variations in this genomic region are not associated with preeclampsia. Nevertheless, further studies are required with determination of fetal TLR4 genotypes to explore the role of TLR4 gene polymorphisms in the risk of preeclampsia, especially in ethnically different populations.

Rarity of TLR4 Asp299Gly and Thr399Ile Polymorphisms in the Korean Population

PurposeActivation 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 MethodsIn 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).ResultsNo 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.ConclusionOur data and other Asian data suggest that a racial difference can be found in the frequency of the TLR4 polymorphism.

Common human Toll-like receptor 4 polymorphisms—Role in susceptibility to respiratory syncytial virus infection and functional immunological relevance

Evidence suggests that Toll-like receptor 4 (TLR4) contributes to immune recognition of respiratory syncytial virus (RSV). The TLR4 gene harbours a polymorphism–Asp299Gly–previously associated with reduced TLR4 signalling. To understand of how host genetic variation influences the outcome of RSV infection in children, we examined the association between the TLR4 299Gly allele and severe RSV disease. By genotyping 236 children with RSV infection and 219 healthy controls we found no association between the risk of severe RSV infection and Asp299Gly polymorphisms ( P > 0.05), and we demonstrate that the TLR4 Asp299Gly genotype does not influence susceptibility to either RSV serotype A or B ( P > 0.05). Finally, examining the functional impact of the TLR4 Asp299Gly polymorphism ( n = 58), we demonstrate that proinflammatory cytokine production following TLR4 activation was indistinguishable between homozygous (Asp/Asp) and heterozygous (Asp/Gly) subjects. We conclude that the Asp299Gly TLR4 polymorphism does not alter receptor function and does not influence the risk of severe RSV infection.

Lack of genetic association of the Toll-like receptor 4 (TLR4) Asp299Gly and Thr399Ile polymorphisms with spondylarthropathies in a Hungarian population

Bacteria have long been suggested as aetiological factors in the genetically susceptible host in spondylarthropathies, including ankylosing spondylitis (AS) and reactive arthritis (ReA). Variability of the Toll-like receptor 4 (TLR4) gene has been shown to play a role in the inflammatory response to certain bacterial infections. We investigated whether TLR4 Asp299Gly and Thr399Ile polymorphisms contribute to the genetic background of spondylarthropathies in a cohort of Hungarian patients with AS and ReA. DNA was obtained from patients with AS (n=138), ReA (n=91) and ethnically matched healthy controls (n=140). Genotyping was carried out by polymerase chain reaction-restriction fragment length polymorphism analysis and the results were confirmed by direct sequencing. No significant differences in allele or genotype frequencies were observed between controls and either the AS patients or the ReA patients. Clinical characteristics of these groups were unrelated to the presence of any of these polymorphisms. Toll-like receptor 4 Asp299Gly and Thr399Ile polymorphisms do not contribute to disease susceptibility in either AS or ReA. Functional abnormalities of the TLR4 signalling pathway suggested in spondylarthropathies seem not to be genetically determined by these two common polymorphisms.

No Evidence of Association between NOD2/CARD15 Gene Polymorphism and Atherosclerotic Events after Renal Transplantation

Stable renal transplant recipients (RTR) display high rates of atherosclerotic events (AE). Innate immunity and especially vascular inflammation play a role in the pathogenesis of atherosclerosis. It is illustrated both by an increased occurrence of postrenal transplant cardiovascular events in patients with elevated levels of C-reactive protein and by a correlation between posttransplant AE and Toll-like receptor-4 Asp299Gly polymorphism. Here, we analyze the influence NOD2/CARD15 gene polymorphism since NOD2 can modulate macrophage pro-inflammatory activity and macrophage is present in early atherosclerotic lesions. The incidence of single nucleotide polymorphism (SNP) in the three major polymorphic region of NOD2 gene (SNP8, SNP12 and SNP13) was assessed in 182 RTR and the correlation between such polymorphism and the development of AE was analyzed. No correlation was observed between NOD2 gene polymorphism and the occurrence of AE after renal transplantation. NOD2 gene polymorphism thus does not appear to influence cardiovascular complications in RTR.

Toll like Receptor 4 Is Involved in In VIvo Antiphospholipid-Mediated Thrombosis and Endothelial Cell Activation.

Background: Antiphospholipid antibodies (aPL) are associated with thrombosis and pregnancy loss in patients with Antiphospholipid Syndrome (APS). aPL and bacterial lipopolysaccharide (LPS) bind to and activate endothelial cells (EC) through NFκB and p38 MAPK pathways. Recent studies suggest that aPL might interact with toll-like receptor-4 (TLR-4), the receptor for LPS.Aim: to investigate the role of TLR-4 in antiphospholipid syndrome (APS).Methods: we examined: i) the aPL effects on thrombosis and EC activation in LPS non-responsive (LPS−/−) mice that display a spontaneous mutation of TLR-4 vs LPS-responsive (LPS+/+) mice displaying wild type TLR-4, ii) the prevalence of TLR-4 Asp299gly and Thr399Ile polymorphisms - both associated with decreased response to LPS - in 110 APS patients (with arterial and/or venous thrombosis) vs 220 controls (of same ethnic background). IgGs were purified from a patient with APS (IgG-APS) and from control serum (IgG-NHS). LPS −/− and LPS +/+ mice, in groups of nine, were treated with IgG-APS or with IgG-NHS twice intraperitoneally. Size of induced thrombi and # of leukocyte (WBC) adhering to endothelial cells in the microcirculation of endothelium of the cremaster muscle of mice (as a means to measure endothelial cell activation) were determined in vivo, seventy-two hours after the first injection. TLR-4 Asp299gly & Thr399Ile polymorphisms were evaluated by Allele-Specific PCR.Results: LPS +/+ mice treated with IgG-APS produced significantly larger thrombi when compared to mice treated with IgG-NHS (2166 ± 1419 μm2 vs 1176 ± 841 μm2) and significantly larger number of WBC adherence to ECs (4.5 ± 1.9 vs 2.2 ±1.1). Thrombus size and number of adhering WBC to ECs were significantly lower in LPS −/− mice treated with IgG-APS compared to LPS +/+ mice treated with IgG-APS [thrombus size: 779 ± 628 μm2 vs 2166 ± 1419 μm2 (p<0.05) and number of adherent WBC to EC: 1.0 ± 0.5 vs 4.5 1.9 (p<0.05)], respectively. The titer of anticardiolipin antibodies in the sera of mice injected with aPL was 48.2 ± 17.1 GPL (for LPS −/− mice) and 50.8 ± 11.2 GPL (for LPS +/+ mice), respectively (NS). A significant reduction in TLR-4 Asp 299gly & Thr399Ile polymorphisms was observed in APS patients (5%) compared to controls (11.4%) (p<0.05).Conclusions: These findings strongly suggest that TLR-4 is involved in aPL interaction with endothelial cells and mediates their pathogenic effects.

Effect of the functional toll-like receptor 4 Asp299Gly polymorphism on susceptibility to late-onset Alzheimer's disease

Experimental data have shown an upregulated expression of toll-like receptors, particularly toll-like receptor 4 (TLR4), in neurodegeneration. The Asp299Gly polymorphism of the TLR4 gene has been associated with an attenuated receptor signalling and a blunted inflammatory response. In the present study, we sought to determine whether this common genetic variant could influence susceptibility to late-onset Alzheimer's disease (LOAD) in an Italian population sample. A cohort of 277 LOAD patients and 300 cognitively healthy controls were genotyped for the TLR4 Asp299Gly polymorphism using restriction isotyping. The frequency of the minor 299Gly allele was significantly higher in the controls than in the LOAD cases (7.2% versus 3.1%, respectively, P = 0.003). Additionally, the frequency of the variant genotypes (Asp/Gly and Gly/Gly) was 13.0% in the controls and 5.4% in LOAD patients ( P = 0.002). After adjustment for age, gender, and the APOE ɛ4 carrier status, the odds ratio for the development of LOAD associated with the Asp/Gly and Gly/Gly versus Asp/Asp genotype was 0.37 (95% CI: 0.20–0.69, P = 0.002). Our data further support a role for innate immunity in neurodegeneration and give the first evidence that the TLR4 Asp299Gly variant may be protective toward the development of LOAD.

The Role of Toll-like Receptor 4 Asp299Gly and Thr399Ile Polymorphisms and CARD15/NOD2 Mutations in the Susceptibility and Phenotype of Crohnʼs Disease

We investigated the influence of 2 common Toll-like receptor 4 (TLR4) polymorphisms on susceptibility and disease characteristics of Crohn's disease (CD). 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. 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). The TLR4 Asp299Gly polymorphism is a risk factor for CD. TLR4 and CARD15/NOD2 mutations may contribute to distinct disease phenotypes.

Relationship between In Vitro Lipopolysaccharide-Induced Cytokine Response in Whole Blood, Angiographic In-Stent Restenosis, and Toll-Like Receptor 4 Gene Polymorphisms

In coronary in-stent restenosis (ISR), a substantial contribution of inflammation is assumed. We evaluated the association between polymorphisms in the Toll-like receptor 4 (TLR4) gene and cytokine response after lipopolysaccharide (LPS) challenge and the development of ISR. Patients were included after successful elective stent placement in a native coronary artery and were scheduled for follow-up angiography after 6 months. Quantitative coronary analysis was performed off-line. Patient whole blood was challenged with LPS for 24 h. Baseline and stimulated concentrations of interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha, and IL-10 were assessed by ELISA. Two cosegregating single-nucleotide polymorphisms in the TLR4 gene (Asp299Gly and Thr399Ile) were analyzed by allele-specific PCR amplification of genomic DNA. A total of 236 consecutive patients were included, and 40 (17%) developed ISR. Median baseline and stimulated cytokine concentrations did not differ between patients with and without ISR. In multivariate analysis, male sex, unstable angina, hypertension, and chronic total occlusion were predictors of ISR. TLR4 genotypes were not associated with baseline or stimulated cytokine concentrations or with angiographic variables at follow-up. In vitro cytokine response to LPS challenge is not increased in patients with ISR. Functionality of the TLR4 Asp299Gly polymorphism could not be demonstrated in this setting, and this polymorphism was not associated with angiographic outcome, calling into question its role in the progression of neointimal tissue growth.

TLR4 Asp299Gly and Thr399Ile polymorphisms are very rare in the Chinese population

Endotoxin is an important component of bio-aerosols that contribute to airway inflammation and airflow obstruction. Toll-like receptor 4 (TLR4) mediates the host response to bacterial lipopolysaccharide (LPS), and the Asp299Gly and Thr399Ile polymorphisms have been associated with the development of respiratory diseases. We hypothesized that TLR4 polymorphisms may be associated with a decline of lung function in cotton workers who were exposed continuously to endotoxin. We investigated these two polymorphisms in our 20-year longitudinal cohort of Han Chinese cotton and silk textile workers. The two polymorphisms were genotyped using TaqMan and PCR-restriction fragment length polymorphism methods. No homozygous or heterozygous variant genotypes of the Asp299Gly and Thr339Ile polymorphisms were detected in 491 samples of this population. Our results suggest that the TLR4 Asp299Gly and Thr339Ile polymorphisms are very rare in the Chinese population.

TLR4 Asp299Gly and Thr399Ile polymorphisms are very rare in the Chinese population.

Endotoxin is an important component of bio-aerosols that contribute to airway inflammation and airflow obstruction. Toll-like receptor 4 (TLR4) mediates the host response to bacterial lipopolysaccharide (LPS), and the Asp299Gly and Thr399Ile polymorphisms have been associated with the development of respiratory diseases. We hypothesized that TLR4 polymorphisms may be associated with a decline of lung function in cotton workers who were exposed continuously to endotoxin. We investigated these two polymorphisms in our 20-year longitudinal cohort of Han Chinese cotton and silk textile workers. The two polymorphisms were genotyped using TaqMan and PCR-restriction fragment length polymorphism methods. No homozygous or heterozygous variant genotypes of the Asp299Gly and Thr339Ile polymorphisms were detected in 491 samples of this population. Our results suggest that the TLR4 Asp299Gly and Thr339Ile polymorphisms are very rare in the Chinese population.

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.

TLR4 Asp299Gly polymorphism is not associated with coronary artery stenosis

Inflammation and innate immunity may play a role in the pathogenesis of atherosclerosis. The Asp299Gly polymorphism in the toll-like receptor 4 ( TLR4) gene reduces responsiveness to lipopolysaccharide and has been associated with reduced incidence and slower progression of carotid atherosclerosis. We analyzed this polymorphism in relation to susceptibility to and severity of coronary artery disease. Methods: 1400 participants (mean age: 63 years, 31% female) in the Southampton Atherosclerosis Study were genotyped for the TLR4 Asp299Gly polymorphism using the tetra-primer PCR method. Results: There was no significant difference between the frequencies of the Asp/Gly or Gly/Gly genotypes combined, compared to the Asp/Asp genotype, in patients with 0, 1, 2 or 3 coronary arteries with >50% stenosis ( χ 3 d. f. 2 =0.4, P=0.94). No associations were observed between genotype groups and cardiac risk factors ( P>0.05). Conclusion: The findings of this study do not support the hypothesis that the TLR4 Asp299Gly polymorphism influences predisposition to and progression of coronary artery disease.