The Association Analysis Of CD14 And TLR2 Genes With Tuberculosis In The Han Chinese Population

Tuberculosis (TB) is a serious infectious disease caused by Mycobacterium tuberculosis (MTB). This chronic wasting disease was once called "white plague", leading the death rates among all infectious diseases around the world. Although about a third of the world's population is infected with Mycobacterium tuberculosis, only 5-15% of those develop clinical active TB during their lifetime. Some evidence suggests that certain genetic factors may involve in innate immunity and play important roles in susceptibility to TB. China has a much higher incidence of tuberculosis than the world average level. Whether the genetic factors increase the TB disease susceptibility in Chinese or not is still less known. The aim of the present study was to investigate whether genes are associated with the susceptibility to TB in a Chinese Han population.Monocyte/macrophage system plays a key role in the early identification of Mycobacterium tuberculosis and the incidence of tuberculosis. Therefore, we studied the monocyte/macrophage cell surface molecules-Toll-like receptors and their signaling pathway proteins in the extracellular which directly contact with the protein antigen of Mycobacterium tuberculosis in the frist stage. CD14, TLR2 and TLR4/MD-2 gene polymorphisms which concern with the susceptibility to TB were selected and identified as risk factors for TB in the Chinese Han population. Then further trials were done on the molecular basis of these high risk factors causing the increase of susceptibility to TB. Characteristic of this study was viewing molecular genetics in the host immune system to understand the pathogenesis of TB. It also provides a distinction between high-risk population tuberculosis, and a new idea in prevention and control of TB.Blood samples were obtained from 432 unrelated cases, diagnosed with TB. All patients were histological confirmed with criteria of the Chinese Ministry of Health on Tuberculosis. Control DNAs were obtained from 404 unaffected individuals of Han Chinese. According to references, suspicious TB-related gene polymorphisms were selected by using biological information database。In this study, four SNPs in the exon region of TLR2 gene, a microsatellite polymorphism in the intron regions of TLR2 gene; two SNPs in the exon region of TLR4 gene; all the 5 SNPs in the promoter region of CD 14 gene; and all the 6 SNPs in the promoter region of LY96 gene (encoding MD-2 protein) were selected. The polymorphisms were detected by polymerase chain reaction (PCR) method and followed by direct sequencing. Statistical analysis was as follows: The deviation from Hardy-Weinberg equilibrium (HWE) was examined in controls by the x2 test. Based on the logistic regression method, the case-control association of genotypes in five inheritance models (codominant, dominant, recessive, overdominant, log-additive) was tested for all the single SNPs. Our data showed that:①Different from foreign reports, the TLR2 Arg677Trp polymorphism, was not observed in either group. The TLR2 Arg753Gln polymorphism occur at very low frequency in the patients with TB (0.49%, compared with 0.49% in the control subjects, P= 0.094). No significant genetic association between 2 coding region SNPs (Asn199Asn and Ser450Ser) and TB were observed in the experiment (P> 0.05). No association in allelic polymorphism between control subjects and TB patients was found. However, the S/M genotype of the microsatellite polymorphism was more frequent in TB patients than in healthy controls (P= 0.01), while the S/L genotype was more popular in controls than in TB patients (P=0.007). The data suggests that the S/M genotype of the microsatellite (GT)n polymorphisms in intron 2 of the TLR2 gene may increase the susceptibility to tuberculosis in Chinese people, and the genotype S/L may act as a negative risk factor.②No Asp299Gly or Thr399Ile SNP in TLR4 was found from either the tuberculosis group or the healthy control group, indicating there was no significant association between these SNPs in the TLR4 gene and the susceptibility to TB.③The C allele of CD14 C-159T and G allele of CD14 C-159T and G allele of CD14 G-1145A were significantly associated with TB (P= 0.001 and P< 0.0001, respectively). Moreover, there was also significant association between the CC genotype of CD14 C-159T or GG genotype of CD14 G-1145A and TB (P= 0.027 and P x 0.001, respectively). Our results suggests that SNPs CD14 C-159T and G(-1145)A might be risk factors for the development of TB.④No genetic variation was observed at positions-538 and-475 of LY96 gene. For the orther SNPs-1625,-1201,-1174 and-1064, no significant differences between patients and controls were observed in allele, haplotype or genotype distribution (P> 0.05). Our results suggest that polymorphisms in the LY96 promoter region are not associated with TB, and may not play a major role in susceptibility to TB in a Chinese population.Pairwise Linkage Disequilibrium (LD) was calculated for the cases and controls in Han Chinese population. Constructed the haplotype blocks based on the results of LD analyses.The association analysis of the haplotypes with TB was similar to that of genotypes of single SNP by logistic regression. The significance of all these tests was 0.05.①We found strong LD (D'> 0.75) between some pairs of the markers, such as Arg753Gln, Asn199Asn and Ser450Ser in the TLR2 gene, C-1625G,C-1201G,G-1174T和A-1064G in the LY96 gene. Positions G-1145A and C-159T in the CD14 gene were also found to be in tight linkage disequilibrium (D'= 0.6685).②Therefore, we constructed haplotype blocks consisting of some nearby SNPs which were in strong LD. And the association between these haplotypes and the susceptibility of TB was also analysised. Our data showed that the haplotypes constructed by Arg753Gln/Asn199Asn/Ser450Ser and C-1625G/C-1201G/G-1174T/A-1064G had no significant association with TB. However, haplotype-specific association analysis revealed that the G-1145A/C-159T haplotype block showed significant association with TB (P< 0.0001).③In addition, no significant linkage was found in SNPs between different genes and no significant association was found in haplotype constructed by SNPs between different genes.In order to evaluate the influence of TB-related gene polymorphisms on gene expression in vivo, Flow cytometry and enzyme-linked immunosorbent assay were used to detect target gene expression in blood mononuclear cell surface and serum.①The expression of TLR2 was lower in healthy volunteers possessing the S/M genotype compared to those with the S/L genotype (P= 0.002) or with the M/M genotype (P= 0.038). The expression of TLR2 was lower in TB patients with the S/M genotype compared to patients with the S/L genotype (P= 0.001) or with the M/M genotype (P= 0.016).②The levels of sCD14 in the sera of TB patients were also higher than those in the control subjects (mean level of concentration, P< 0.001). In neither the patients with pulmonary TB nor the control subjects was there any association between the G-1145A and C-159T genotype and the levels of mCD14 and sCD14 (P> 0.05). However, the TA and CG haplotype of the CD14 gene had much higher expression level than CA haplotype in both PBMCs and serum (P< 0.05). In additon, before the patients with TB started anti-TB treatment, the levels of mCD14 on monocytes were higher than those in the control subjects (P< 0.001, respectively).For the haplotypes which showed highly significant correlation between CD 14 C-159T/G-1145A loci haplotypes and susceptibility to tuberculosis (such as CG; OR= 13.7, P<0.0001), we further constructed the CD14 promoter containing such haplotypes in vitro. The expression plasmids were transfected to U937 cells. By using luciferase reporter gene expression, haplotypes affect the function of CD14 promoter were analysised. The results showed that:pGL3-TA and pGL3-CG can be effective in stimulating the expression of fluorescent reporter gene, whose fluorescence intensity was significantly higher than pGL3-CA (respectively, P= 0.013 and P= 0.0015). Tips CD 14 gene promoter which contain TA, CG haplotypes was significantly higher than that contain CA haplotype. The data fully supported the in vivo test results.Conclusion:(1) This is the first time to study the association between 4 SNPs in the exon region of TLR2 gene, a microsatellite polymorphism in the intron 2 regions of TLR2 gene in Han Chinese population. Different with foreign groups, there were no significant genetic association between 4 SNPs and susceptibility to TB in Han Chinese population. However, significant genetic association between the microsatellite polymorphism in the intron 2 regions of TLR2 gene and TB was observed. (2) This is the first time to study the association between 2 SNPs in the exon region of TLR4 gene in Han Chinese population. Different with foreign groups, there were no significant genetic association between 2 SNPs and susceptibility to TB in Han Chinese population. (3) This is the first time to study the association between 6 SNPs in the promoter region of LY96 gene worldwide. Our results suggest that polymorphisms in the LY96 promoter region are not associated with TB, and may not play a major role in susceptibility to TB. (4) Our results suggest that the CD14 G(-1145)A polymorphism might be a new risk factor for TB. This SNP has strong linkage with CD14 C-159T, and the haplotype constructed by these 2 SNPs has extremly significant association with susceptibility to TB (P< 0.0001). Further in vivo (clinical samples) and the in vitro test (gene transfer) results showed that, CD14 Gene SNP loci haplotypes influenced promoter strength, the expression of CD 14 levels of TB patients, and ultimately the TB susceptibility.