Analysis Of SNPs In The Promoter Of CD14 Gene And Their Relationship With LPS Response

As an important genetic factor, gene polymorphisms play a key role in disease susceptibility, clinical phenotype diversity and different response to pharmacotherapies. The CD14 receptor is a pattern recognition molecule in the innate immune response against microorganisms and other exogenous and endogenous stress factors. CD14 has specificity for LPS and other bacterial wall-derived components, and plays an important role in the activation of monocytes/macrophages by LPS, which induces overexpression of certain cytokines and inflammatory mediators that amplify and diversify the LPS signals. There were many studies about the CD14 gene polymophisms and their relationship with the difference of inflammatory disease susceptibility, clinical phenotype and response to pharmacyotherapies in different populations, but most studies focused on the analysis of -159 T/C polymorphism in the promoter of CD14 gene and it's correlation with many inflammatory diseases, there is little reports concerning other SNPs in CD14 gene and how the gene polymorphisms, including -159 T/C polymorphism, affect the expression of CD14 gene. In our study, the SNPs in the important function regions of CD14 gene in Chinese population was examined by direct sequencing firstly. Secondly, based on the bioinformatic analysis, the allelic and genotypic frequencies of SNPs in the promoter of CD14 gene in Chongqing Han population were further surveyed. Third, the associati of the SNPs with the level of peripheral plasma sCD14 before and after LPS stimulation was studied. Finally, the functional activity of the SNPs was investigated through gene transfection and Dual-Luciferase Reporter assay system. The main results and conclusions were summarized as follows:1. All important regions of CD14 gene in 27 samples from 10 representitive nationalities, such as Mongolian, Tibetan, Weiwuer, Dai, Bulang, Miao, Zhuang, Li, Chaoxuan and Han in China were directly sequenced, and four base mutations were foundin 5V flank and exon regions. The 557 C—>T transversion in exon region maybe only occurred in Chinese population. Bioinformatics analysis showed that, among the three SNPs in 5\flank region, the -1855 A/G polymorphism locates in the repeat region, which may have few important function, but the -1145 G/A and -159 T/C polymorphisms distribute in the pomoter region and are responsible for binding of several important nuclear transcription factors.2. The frequencies of-1145 G/A and -159 T/C polymorphisms were further conveyed in 298 healthy unrelated volunteers in Chongqing Han population with PCR-RFLP method. The results showed that both -1145 G/A and -159 T/C polymorphisms are high frequency SNPs, and the frequencies are 39.16% and 42.45% respectively. Linkage disequilibrium analysis showed that they are in strong LD, and the most common haplotypes are G-T and AC.3. Through assay of the level of peripheral plasma sCD14 before or after LPS stimulation with ELISA, the relationship between these SNPs and the expression of CD 14 was further examined. The results showed that, in Chongqing Han population, the -1145 G -*A or -159 T— C polymorphism maybe inhibit the expression of CD14, but the role is not affected by LPS stimulation. The -1145 and -159 SNP maybe affect the expression of CD14 and LPS response together.4. The functional activities of these polymorphisms were further investigated through construction of pGL3-CD14 promoter (-1392-+215) plasmids and Dual-Luciferase Reporter assay system. The results showed that, -1145 G/A cooperates with -159 T/C, regulate the transcription!of CD 14 gene, so that play an important role in the expression of CD 14, especially in LPJvresponse.