Genetic Susceptibility Of The TLR1、 TLR2 And TLR6 Genes Of Tuberculosis Patients In Yunnan Province

Mycobacterium tuberculosis(MTB)infection leads to a seriously public health problem.Approximately 1/3 population has be infected by M.tuberculosis,but only5%-10% of which developed into active tuberculosis(TB).Although BCG vaccine could protect the infants from MTB infection,the ratio of MTB infection is still high.That the epidemic ratio of MTB showed difference among various populations suggested host genetic factors might be one of the influenced factors for MTB infection,disease progression,and treatment effect.Toll-like Receptors(TLR)belong to pattern recognition receptor family,which active downstream pathway by recognizing pathogen-associated molecular pattern or injury-related molecular pattern.Then,cytokines are synthesized and released to inducing inflammatory reaction.The patterns of MTB could be recognized by TLRs,and the Myeloid Differentiation factor 88 gene(My D88)is active to induce transcription of cytokines and further lead to immune response.218 TB patients and 240 general controls were collected from Yunnan Province.Seven Single nucleotide polymorphisms(SNP)in the TLR1,TLR2,and TLR6 genes were genotyped by using Snap Shot method.The genotype and allele frequencies of rs5743596,rs5743557,rs11938228,and rs3775073 showed significant difference between TB patients and controls.The genotype CC of rs5743596 and genotype AA of rs11938228 played risk factors in TB infection,but genotype TT of rs5743596 and genotype TT of rs5743557 were the protective factor for TB infection in Yunnan population.Furthermore,the allele C,C,A,and C of rs5743596,rs5743557,rs11938228,and rs3775073 were the risk factor for TB infection,respectively.Total 39 and 46 haplotypes were constructed in TB patients and controls.Two haplotypes(TCCTACT and TCCTATC)showed higher frequencies in TB patients.After dividing TB patients into five subgroups,including 65 cases of secondary pulmonary tuberculosis,60 cases of secondary pulmonary tuberculosis with pleural tuberculosis;29 cases of secondary pulmonary tuberculosis complicated with bronchial tuberculosis,33 cases of pleural tuberculosis and 17 cases of other types of tuberculosis.,we also identified genotype or allele frequencies showed significant difference among them.Two alleles of rs4833095 led to missense mutation of the TLR1 gene,plasmids containing two alleles were constructed.The MTB proteins ESAT6 and CFP10 were constructed and expressed to infect cells.The results showed that the TNFα expressing level increased at 24 h after TLR1 plasmids transfection,but no significant difference was found between two kinds of TLR1 plasmids containing two alleles.The SNPs rs5743596 and rs5743557 located in the promotor region of the TLR1 gene.Thus,RNA structures and dual-luciferase reporter assay were performed to verify their function.The results showed that two alleles of rs5743557 could lead to change of RNA structure.However,no structure change was identified by two alleles of rs5743596.The firefly expressing level showed significant difference between two alleles of each of these two SNPs.the plasmid containing allele C of rs5743596 and allele T of rs5743557 statistically increased the firefly expressing levels.The SNP rs11938228 located in the intron region of the TLR2 gene,but no RNA structure change was found between two alleles of this SNP.Taken together,the association between variants of seven SNPs in the TLR1,TLR2,and TLR6 genes and TB infection were identified in Yunnan population.The results provide theoretical basis for individualized treatment of TB patients.