Occurrence Of Polymorphisms Within TLR4 And GR Genes In Chongqingnese Population And The Functional Significance Of The TLR4/11 367 Polymorphism

It has been demonstrated that the variations in genomic DNA sequence, i.e. gene polymorphisms, are important determinants for disease susceptibility, clinical phenotype diversity and different response to pharmacotherapies. Toll-like receptor 4 (TLR4) is the central signaling receptor for lipopolysaccharide (LPS) in mammals. Therefore, the difference in the expression of the TLR4 gene may be a determinant for susceptibility to sepsis. GR is a transcriptional factor activated by glucocorticoid and a regulator of the expression of various genes. It not only influences the stress and inflammation in traumatic patients, but also is associated with glucocorticoid resistance. Based on the current researches, this study was designed to investigate the occurrence of polymorphisms within the TLR4 and GR genes in Chongqingnese Han population and the functional significance of the G11 367C polymorphism, which is a novel variant we identified in the 3′untranslated region (3′UTR) of the TLR4 gene. Two hundred sixty-nine healthy volunteers were selected. The G11 367C, A+896G polymorphisms within the TLR4 gene and the ER22/23EK, N363S, BclⅠpolymorphisms within the GR gene were genotyped using single-tube bi-directional allele specific amplification and restriction fragment length polymorphism methods, respectively. TLR4 protein and mRNA expression on peripheral leukocytes and plasma tumor necrosis factorα(TNF-α) levels were determined by flow cytometry, real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Post-transcriptional effect of the G11 367C polymorphism was evaluated with dual-luciferase reporter assay system and real-time quantitative polymerase chain reaction. In addition, we compared the genotype distribution of the G11 367C and BclⅠpolymorphisms in relation to the development of multiple organ dysfunction and sepsis in patients with severe trauma (n=100, ISS 31.2±8.5). And we also compared the genotype distribution of the BclⅠpolymorphism in relation to the steroid-induced elevated intraocular pressure in patients with post-LASK. The purpose of this study was to provide basis for further investigation of genetic background of susceptibility to multigenic disease.The main results and conclusion were summarized as follows:1. The G11 367C polymorphism within the TLR4 gene 3′UTR and the BclⅠC to G polymorphism within the GR gene both are common allele in Chongqingnese Han population, with minor allele frequency of 13.01% and 23.50% respectively. These genotypes distribution were in agreement with the Hardy-Weinberg equilibrium (p=0.06 and 0.43). We did not observe genetic variation at position +896 within the TLR4 gene and at position ER22/23EK, N363S within the GR gene in the same population.2. In response to ex vivo LPS stimulation, TLR4 protein and mRNA expression on peripheral leukocytes was significantly higher in GG homozygous genotype for 11 367G allele compared with individuals heterozygous GC and homozygous CC. But there was no difference for TLR4 protein between heterozygous GC and homozygous CC.3. The plasma TNF-αlevels were significantly higher in GG homozygous genotype for 11 367G allele after LPS stimulation compared with individuals heterozygous GC and homozygous CC. But there was no difference between heterozygous GC and homozygous CC.4. We also found that the activity and mRNA expression of luciferase was significantly higher in HEK293 cells transfected with construct containing 11 367G allele than those transfected with construct containing 11 367C allele. This suggests that 11 367C can inhibit translation of the reporter gene mRNA significantly by inhibiting the expression level of the reporter gene.5. There were no significant differences in age, gender ratio and ISS scores between patients stratified according to the different genotypes of the TLR4 G11 367C polymorphism. MOD scores in trauma patients with 11 367G allele was significantly higher than 11 367C, suggested that trauma patients carrying 11 367C allele was less likely to have MOD. This association was not influenced by age, gender ratio and ISS. Even though statistically significant difference for the susceptibility to post-traumatic sepsis between the genotypes of 11 367 SNP was not detectable, patients with homozygous genotype for 11 367G allele showed highest incidence of sepsis. There were no significant differences in age, gender ratio, ISS scores, MOD scores and the susceptibility to post-traumatic sepsis between patients stratified according to the different genotypes of the BclⅠC to G polymorphism.6. In this study, we also found that BclⅠallelic frequency for the steroid-induced elevated intraocular pressure patients and control (G=0.19 and 0.22) was not significantly different.Together, the above results suggest that the TLR4/11 367 polymorphism may be a functional SNP, which could attenuate LPS-induced transmembrane signaling through alteration of post-transcriptional regulation of 3′UTR and target gene expression. The TLR4/11 367 polymorphism may be used as genetic biomarkers for the assessment of susceptibility to inflammatory diseases, such as sepsis and multiple organ dysfunctions secondary to severe trauma. But the functional significance of the BclⅠC to G variation within GR gene remains to be investigated.