Toll-like receptor 4 gene polymorphisms and susceptibility to juvenile idiopathic arthritis.
ABSTRACT To determine if polymorphisms within the Toll-like receptor 4 (TLR4) gene are associated and linked with juvenile idiopathic arthritis (JIA). To investigate any possible gene-gene (epistatic) interaction between TLR4 and macrophage migration inhibitory factor (MIF) gene polymorphisms.
313 simplex families (each containing one affected JIA proband) were genotyped. Two known functionally important single nucleotide polymorphisms (SNPs) within the TLR4 gene (Asp299Gly and Thr399Ile) were typed by SNaPshot ddNTP primer extension and capillary electrophoresis. Single point and multipoint transmission disequilibrium tests (TDT) were carried out through the extended TDT and TDT phase packages for the two TLR4 SNPs. Epistatic interaction between TLR4 haplotypes and the previously JIA associated MIF CATT(7)-MIF-173*C promoter haplotype was investigated by chi(2) test and unconditional logistic regression in Stata version 7.
No distortion from random inheritance was observed by single point analysis for TLR4 Asp299Gly (p = 0.89) or TLR4 Thr399Ile (p = 0.40). Similarly, no distortion in transmission was seen when the TLR4 haplotypes were studied (p = 0.54). Additionally, no evidence for gene-gene interaction between TLR4 polymorphisms and the previously associated MIF gene polymorphisms was found (p = 0.40).
No linkage or association was seen for Asp299Gly or Thr399Ile SNPs of TLR4 with JIA susceptibility. No evidence of an epistatic interaction between these TLR4 polymorphisms and MIF polymorphisms was found.
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ABSTRACT: Tuberculosis (TB) still remains the leading cause of mortality due to bacterial pathogen. The only currently available vaccine against TB, Bacille Calmette-Guérin (BCG) is at best credited with a 50% overall protective efficacy. Skin testing with purified protein derivative (PPD) from Mycobacterium tuberculosis is the method of detecting BCG-induced cell mediated immunity, in vivo. In the previous study we found that approximately 60% young volunteers with no history of TB, who had been subjected to neonatal BCG vaccination and revaccination(s) at school age, developed delayed type hypersensitivity (DTH) to tuberculin. The remaining volunteers were persistently tuberculin negative. Moreover, we found a significant association between BCG driven development of DTH to PPD and the polymorphism within the CD14 C/T(-159) gene for macrophage receptor recognising mycobacterial compounds. It has suggested that the CD14 gene variants may play a role in the appearance and persistence of DTH to PPD in BCG vaccinated subjects. In order to extend our study on a possible role of CD14 in BCG driven DTH response to PPD, we measured the expression of mCD14 on macrophages, stimulated or not stimulated with mycobacterial antigens, and the serum levels of sCD14. Considering the importance of CD14 - TLR2/TLR4 interactions in macrophage signalling, we determined the polymorphism of TLR2 and TLR4 genes as well as macrophage expression of TLR2 for the volunteers with and without skin reactivity to PPD. We observed a subtle but significant decrease in CD14 density on adherent monocytes from tuberculin positive versus tuberculin negative volunteers. However, we found no difference in CD14 density on monocytes enriched in CD14+ cells using anti-CD14 mAb coupled to magnetic beads. A significant increase in CD14 density was observed on macrophages stimulated with PPD and LPS but not with live BCG bacilli. However, this increase as well as serum levels of soluble sCD14 were similar in the volunteers with and without skin reactions to PPD. Thus, our suggestion on the role of CD14 in the generation of DTH to tuberculin in BCG vaccinated subjects should be further explored. The most important CD14 co-receptors are Toll-like receptors (TLRs) which activate nuclear factors for the production of inflammatory cytokines. However, we could see no association between the polymorphisms of TLR4 (Asp299Gly and Thr399Ile) and TLR2 genes (Arg753Gln and Arg677Trp) and skin responses to PPD. Also, the TLR2 density was similar on monocytes from tuberculin negative and tuberculin positive volunteers.Folia Histochemica et Cytobiologica 02/2008; 46(3):353-9. · 0.81 Impact Factor
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ABSTRACT: Toll-like receptors (TLRs) are a family of transmembrane pattern recognition receptors (PRR) that play a key role in innate and adaptive immunity by recognizing structural components unique to bacteria, fungi and viruses. TLR4 is the most studied of the TLRs, and its primary exogenous ligand is lipopolysaccharide, a component of Gram-negative bacterial walls. In the absence of exogenous microbes, endogenous ligands including damage-associated molecular pattern molecules from damaged matrix and injured cells can also activate TLR4 signaling. In humans, single nucleotide polymorphisms of the TLR4 gene have an effect on its signal transduction and on associated risks of specific diseases, including cirrhosis. In liver, TLR4 is expressed by all parenchymal and non-parenchymal cell types, and contributes to tissue damage caused by a variety of etiologies. Intact TLR4 signaling was identified in hepatic stellate cells (HSCs), the major fibrogenic cell type in injured liver, and mediates key responses including an inflammatory phenotype, fibrogenesis and anti-apoptotic properties. Further clarification of the function and endogenous ligands of TLR4 signaling in HSCs and other liver cells could uncover novel mechanisms of fibrogenesis and facilitate the development of therapeutic strategies.Fibrogenesis & Tissue Repair 10/2010; 3:21.
Article: Quadruple-allele dipstick test for simultaneous visual genotyping of A896G (Asp299Gly) and C1196T (Thr399Ile) polymorphisms in the toll-like receptor-4 gene.[show abstract] [hide abstract]
ABSTRACT: Toll-like receptor-4 (TLR4) is a central regulators of innate immune response as it interacts with bacterial lipopolysaccharide (LPS) and also with endogenous molecules, such as heat-shock proteins and fibrinogen. Two common single nucleotide polymorphisms, A896G (Asp299Gly) and C1196T (Thr399Ile), have been found in the exon 3 of human TLR4 gene, which lead to structure alteration of the extracellular domain of TLR4 thereby influencing the receptor ability for recognition and ligand binding. We propose a simple, rapid and reliable method for the simultaneous detection of the two SNPs in TLR4 gene that involves: (a) exponential amplification of the genomic region that spans the two SNPs, (b) quadruple primer extension (PEXT) reaction using two allele-specific primers per SNP, and (c) a simple-to-perform dipstick test that allows visual and simultaneous detection of the four alleles within minutes without the need for specialized instrumentation. The method was applied to the simultaneous detection of the two SNPs in 90 samples of general Greek population and the results showed 100% concordance with those obtained by direct sequencing. The entire assay, starting from genomic DNA, can be run in less than 1.5h. The dipstick test eliminates multiple incubation and washing steps that are common in microtiter well-based assays and does not require highly trained personnel. Because of these advantages, it is suitable for the routine clinical laboratory or even for point-of-care testing.Clinica chimica acta; international journal of clinical chemistry 10/2011; 412(21-22):1968-72. · 2.54 Impact Factor