Tuberculosis Immunity Related Gene Polymorphisms And Pulmonary Tuberculosis Susceptibility

Background: Tuberculosis, caused by the causative agent of mycobacterialtuberculosis (M.TB), was one of the earlier and the most influential diseases in thehistory of mankind due to its devastating effect on health and high mortality ratethroughout the world, which is responsible for more human deaths than any othersingle infectious disease and the incidence of the tuberculosis is increasing in recentyears. At present, there were more than500million people infected with M.TB, and5million TB cases and with an approximately1.3million annual incidence in China.According to the World Health Organization (WHO) report, China ranks the secondwith TB burden in the world, is one of the world's22TB high-burden countries.Studies have shown that one-third of the population will be infected under theexposure to the M.TB. Among the infected population, less than10%of those infectedwill develop to TB disease, suggesting that there is an inter-individual variation ingenetic susceptibility to tuberculosis in the general population.TB is a typical chronic intracellular infection, the innate and adaptive immuneresponse of host resistance to M.TB infection depends on the initial recognitionbetween cell surface pattern recognition receptors expressed on the T lymphocytes,macrophage and dendritic cells and components of M.TB, following activation ofantigen presentation, cascade reaction of downstream signaling pathways and theirregulatory genes such as cytokines, chemokines and so on, and thus remove or controlthe infection of pathogens. Because the genetic variation in genes involving in TBimmune response results in population differences in ability to response to theinfection and possible increased risk of individual pulmonary TB (pTB), which couldfurther affect the susceptibility of pTB as well as disease occurrence anddevelopment.Objectives: To investigation the relationship between gene polymorphisms andpTB susceptibility, we hypothesized that the functional SNPs in cytokines and itsreceptors, chemokine and pattern recognition receptors (PRRs) genes involved in TBimmune response are associated with pTB susceptibility individually or jointly andgene-gene interactions play important roles, the severity phenotype of pTB is genetically based, and closely correlated with the functional SNPs in some importantgenes.Methods: To test our hypothesis, we performed a case-control study, and totally115SNPs in20functional genes involved in TB immune response, includingcytokines and their receptors, chemokines and PRRs genes (INF-γ, TNF-α, IL-1β,IL-10, IL-12β, IL-12β1, IL-17, IL-23R, IL-6R, IL-6, CCL-1, CD-209, MCP-1,RANTES, SP-110, VDR, TLR2, TLR4, LXRs, MARCO and IRF5) were selectedusing the "sequence-based" and "map-based" selection approaches. SNPs genotypingwere performed by using PCR-RFLP, ARMS-PCR, Massarray or SNPstream assays,and the internal control and blinded re-testing were conducted by establishing qualitycontrol of the assays. In the case-control study, the peripheral blood was collectedfrom the new pTB cases confirmed by the clinical or laboratory tests from severalA-level hospitals. Healthy subjects who underwent the physical examination in thehospital were recruited during the study period and were matched for ethnic andgeographical origins to cases. A uniform health questionnaire was designed to conductepidemiological investigations for information of subjects. Microsoft excel was usedto establish the database after the repeated check and verification of all experimentaland questionnaire data, the SPSS17.0, SNPstats, Haploview and MDR software wasused to collect and analyze the data.Results:(1) Association analysis of cytokine and its receptor gene polymorphisms:SNPs-857C>T and-863A>C in promoter region of TNF-α gene were significantlyassociated with the susceptibility of pTB in Chinese Han population. Compared to TTgenotype of the-857C>T, subjects carrying CC genotype had a significantly0.68-folddecreased risk of pTB (OR=0.68,95%CI=0.53-0.86). While compared to AAgenotype of-863A>C, subjects carrying CC genotype had a significantly2.42-foldincreased risk of pTB (OR=2.42,95%CI=1.28-4.59), and also AA genotypeassociated with the severity degree of pTB "serious" level (OR=3.59,95%CI=1.41-9.11). In addition, we found that the genotype of carrying A allele of the-863A>C was associated with a significantly lower of serum TNF-α level than CCgenotype.(2) Association analysis of chemokine gene polymorphisms: Genotypefrequencies of rs159291, rs159294and rs210837of gene clusters located in the CCL1and rs2107538in RANTES gene were significantly difference between cases andcontrols (p<0.05). Logistic regression analysis revealed that subjects carrying AAgenotype had a significantly0.71-fold decreased risk of pTB (OR=0.71, 95%CI=0.56-0.92) compared with carrying GG genotype of rs159291. For thers159294and rs210837, compared with carrying the AA genotype, subjects carryingTT or GG genotype of rs159294or rs210837will increase the susceptibility of pTB(OR=1.17,95%CI=1.01-1.35; OR=1.59,95%CI=1.07-2.36, respectively). To evaluatethe combined effect of the polymorphisms on pTB susceptibility, totally eighthaplotypes of frequency greater than1%among the three blocks were constructed forCCL1gene, of which only the AT haplotype in the third block containing rs159291and rs159294was significantly associated with an increased risk of pTB compared toGT haplotype (OR=1.16,95%CI=1.00-1.35).(3) Association analysis of patterrecognition receptor gene polymorphisms: Among53SNPs scatted across thesegenes, the allele and genotype of rs729302in IRF5gene, rs17009726and rs6761637in MARCO gene, and rs7975232in VDR gene were significantly difference betweenpTB case and controls (p<0.05). In the multivariate logistic regression analysis,compared with AA and CA genotypes of rs729302in IRF5gene, subjects with CCgenotype were significantly associated with a decreased risk of pTB in dominantmodel (OR=0.71,95%CI=0.54-0.93) and the frequencies of haplotype2(CG) presentin block1(rs729302and rs4728142) displayed a significant difference between twogroups (OR=0.83,95%CI=0.72-0.96), indicating an association between IRF5geneticvariants and pTB; Conversely, subjects carrying of the G and C allele had a1.65and1.24fold increased risk of pTB for rs17009726(OR=1.65,95%CI=1.32-2.05) andrs6761637(OR=1.24,95%CI=1.02-1.52) respectively in MARCO gene and both GChaplotype involved in block2and TGCC haplotype in block3were associated withan increased pTB risk after100000permutations analysis using haploview (OR=1.62,95%CI=1.31-2.00; OR=1.31,95%CI=1.06-1.60, respectively). We also found thatsubjects carrying A allele of rs797523(ApaΙ) in VDR gene was significantlyassociated with pTB (OR=0.82,95%CI=0.69-0.98), and A allele was correlated withhigh serum concentrations of25-hydroxyvitamin D (25-OHD), indicating thatrs797523(ApaΙ) play a protective role for pTB through a positive regulator of vitaminD expression.(4) Gene-gene interaction analysis: There was an interaction betweenrs2107538in RANTES and rs3091324in CCL1and the individual who wereclassified as "high-risk" genotype with an1.4-fold increased risk of pTB (OR=1.40,95%CI=1.17-1.67) according to MDR reduces dimensionality of multilocus genotypeinformation to a one-dimensional factor with two levels-"high-risk" and "low-risk"genotypes. Also, the gene-gene interaction among rs7975232in VDR, rs2077344in MARCO, rs7656411in TLR2and rs729302in IRF5genes were identified and therisk of individuals with "high-risk" genotype suffering from pTB will been increasedby1.36fold (OR=2.36,95%CI=1.95-2.84) according to the two classification.Conclusions: Our study found that polymorphisms in TNF-α, CCL1, RANTES,IRF5, MARCO and VDR genes were significantly associated with pTB susceptibilityindividually or jointly with other SNPs, and also TNF-α and VDR polymorphismswere respectively correlated with its serum expression levels. The results of our studycontribute to the knowledge on pTB susceptibility mechanism and possible multiplegene-gene interactions and provide a "proof-of-principle" approach for molecularepidemiological studies in pTB susceptibility. The identified risk genotypes orhaplotypes in our study could be used, if validated, as molecular markers to improveour ability to define high-risk populations who may be susceptible to pTB in theChinese population.