Analysis Of SNPs In TLR4 Promoter And Their Clinical Relevance In Patients With Major Trauma

Clinical epidemiology study has shown that sepsis is an important cause of death of patients with major trauma, and LPS plays a central role in the pathogenesis of sepsis. We carried out a great quantity of exploratory study on LPS receptors in the past ten years, reseach results demonstrate that TLR4/CD14/MD-2 complex is the crucial pattern recognition receptor of LPS, and CD14, MD-2 gene polymorphisms are associated with susceptivity to sepsis and prognosis of patients with major trauma. TLR4 is also a key molecule of pattern recognition receptor in LPS passthway, though its polymorphisms in the coding sequence and 3′untranslated region (UTR) are well associated with the susceptibility to sepsis, however the distribution of its promoter polymorphisms in China population, and their clinical relevance in patients with major trauma is still unclear.In this study, we selected the potential SNPs in TLR4 gene promoter, which maybe influence TLR4 gene expression through bioinformatics analysis. Then, we genotyped the selected SNPs in Chongqing Han population by method of restriction fragment length polymorphism. To study the function of the SNPs, we constructed different allele promoter plasmids and measured TLR4 promoter activity by Dual-Luciferase Reporter assay system. We also examined different genotype carriers'TLR4 protein levels and the significance of their whole-blood leukocytes response to LPS stimulation. Finally, we investigated the possible functional significance by observing the association with the prognosis of patients with major trauma. The main results and conclusions were summarized as follows:1. Bioinformatics analysis shows that there are twelve SNPs in the upstream 3000 bp of TLR4 promoter. Among them, -2242T→C, -1892G→A, and -1837 A→G can change the type or numbers of binding transcription factors, which indicats that the three SNPs may affect the transcription activities of TLR4 promoter.2. Genotyping 379 cases of Chongqing Han healthy population shows that -2242T→C, -1892G→A, and -475 A→G all exist in Chinese population, with the frequencies 43.27% , 27.70%, and 42.75% respectively.3. TLR4 gene promoter (-2331 ~ +14), and the other vectors containing a single mutant genotype of -2242 C, -1892 A, or -1837 G, a double mutant genotype of -2242 C, -1892 A, or -2242 C, -1837 G, or -1892 A, -1837 G, and a combined mutant genotype of -2242 C, -1892 A, and -1837 G are constructed through PCR and site-directed mutagenesis technique. Luciferase Reporter assay shows that -2242 C promoter has a higher activity than -2242 T promoter, it implys that–2242 T→C base variation can enhance the activity of TLR4 promoter. There are no significant difference between -1892 A promoter and -1837 G promoter activity. Fluorescence-activated cell sorter (FACS) shows -2242 CC carriers have higher TLR4 protein expression levels than -2242 TT carriers, which indicates that -2242 T→C may enhance TLR4 gene expression through changing promoter activity.4. Measuring LPS stimulated whole-blood leukocytes, we found that -2242 CC carriers have the highest TNFα, IL-6, and IL-8 levels and -2242 TT carriers have the least. It indicates that -2242 T→C can significantly enhance reactive potency to LPS. Results show that -2242 SNP may have an important significance in sepsis assessment.5. In view of the functional effect of the–2242 SNP shown by the above results, we further investigated the clinical relevance of this SNP in 153 patients with major trauma. The result shows that patients who possessed the–2242 C allele were more likely to experience complications with organ dysfunction and sepsis after major trauma. So the TLR4/–2242 polymorphism may be an important functional variant which might be used as a relevant risk estimate for sepsis and other complications in trauma patients.