| Pulmonary tuberculosis(p TB), caused by Mycobacterium tuberculosis, is one of the deadliest infectious diseases worldwideand both morbidity and mortality remain high. Tuberculosis(TB) causes more fatalities than any other single infectious disease and the incidence is increasing dramatically with the emergence of resistant strains in recent years. In 2012, an estimated 8.6 million people developed TB, among which approximately 66%(5.7 million) were newly diagnosed cases; 1.3 million died from the disease, including 320,000 deaths among HIV-positive patients. China is one of the 22 high-burden TB countries and the number of TB patients ranks second in the world.Although about one third of the world’s populationsis infected by Mycobacterium tuberculosis, only 10 percent of them will develop active TB. Many of these patients have significant risk factors, such as HIV infection, diabetes mellitus and otherimmunodeficiencies, while remaining risks may be attributable to other factors, includinginteractions of genetic andenvironmental conditions. It is accepted that hostgenetic factors play a role indetermining differential susceptibility to M.tuberculosis infection and disease outcome in humans.From 1983,a large number of efforts by linkage mapping were done to screen the human genome for TB susceptibility. Numerous candidate-based association studies of TB were performed to test the association of predicted functional single nucleotide polymorphisms(SNP) in candidate genes, including SLC11A1(NRAMP1), HLA, Toll-like receptor(TLR) genes, the IFN-γ and IL-12 signaling genes, etc. However, compared with other diseases, the genetic studies of TB susceptibilityare far less successful because of the complex confounding effect of environmental factors.Most reported TB-associated loci cannot be replicatedinother populations.Genome-wide association study(GWA study, or GWAS), also known as whole genome association study(WGA study, or WGAS) or common-variant association study(CVAS), is a genetic examination of testing the whole common variants in the same time for a large numberof individuals to see if any variant is associated with a trait. GWAS typically focus on associations between SNPs and major diseases. Recently, GWAS has been first used to delineate the genetic basis of TB in two large African cohorts, and two loci were found, which provides important clues to an early intervention and treatment of TB. China is itself the world’s second most number of TB patients, however, up to now, there has been no published TB GWAS specifically on Chinese Han population.On the other hand, among those whodevelop TB after exposure to M. tuberculosis, up to 70% are males. A male/female ratio of1.9±0.6 was documentedfor the worldwide case notificationrate andreached ashigh as 3 in somecountries. Evidence has also indicated that males are more prone to get severe forms of tuberculosis. Although socioeconomic and cultural factors might be related with under-notification inwomen, in particular in developing countries, a significant proportion of this sex-specific difference are also believed to be attributable to biological heterogeneity.Among the biological factors that mayaccount for the sex bias, sexual hormones, gender-related genetic background and metabolic characteristics aresuggested to play important roles.It appears reasonable to assume that host genetic variation, in particular of immune-related genes on the X chromosome might contribute to sex-specific differences in TB incidences.Part One. Genome-wide association study ofpulmonarytuberculosisObjectives: It is accepted that host genetic factors are crucial in determining susceptibility and resistance to pulmonary tuberculosis(TB). So far, significant genome-wide association(GWA) signals have been identified in case-control groups from Ghana and The Gambia, and the results were replicated in other Europeanethnic groups. The objective of this study was to comprehensively evaluate the associations of GWA study identified signals with p TB in Chinese Han population.Subjects and methods:To identify genetic markers for pulmonary tuberculosis(TB) in the Chinese Han population we conducted a genome-wide association(GWA)study of 500 TB cases and 600 controls by using the Affymetrix Genome-Wide Human SNP array 6.0. After rigorous quality control for phenotypes and cryptic relatednessas well as for SNP-wise quality, a total of 650,939 SNPs in 483 cases and 587 controls underwent statistical analyses. The top three MDS vectors calculated from the GWA study dataset were used as covariates to adjust for population structure in the logistic regression analyses. Next, we performed imputation with data derived from recent versions of the 1000 Genomes Project(Phase I integrated variant set, v3, March 2012). After standard quality-control of SNPs, we obtained 5,692,315 imputed SNPsfor subsequent analysis.To examinewhether there are common susceptibility loci in the Chinese Han and an African population,we combined our dataset from China with a previous Ghanaian GWA study dataset(1,359 cases, 1,952 controls; 793,694 SNPsgenotyped; 5,426,380 imputed SNPs). The 1537 most promising variants were then replicated in two additional groups, an additional Chinese Han groupof 1,272 cases and 1,733 controls and a study group from Mongolia,comprising of 121 cases and 231 controls.Results and Conclusions:The combined analysis of the present GWA study in Chinese Han and the two replication groups identified SNP rs932347 T at 1q32.2which was associated with resistance to TB(Padditive = 1.45 10-9; OR = 0.69; 95% CI = 0.61-0.78).As the frequency of SNPrs932347 was 1.5% in the Ghanaian study group compared to 17% in the Chinese Han group, it was not found to be associated in the Ghanaian group. As Mycobacterium africanum is not a cause of TB in Asia, we performed a stratified analysis with the Ghanaian study group, involving only cases infected by non-M. africanum strains. Although not significant, the effect direction of SNP rs932347 T, indicated by an OR of 0.84 in the African group, was identical. SNPs rs4331426 and rs2057178, the susceptibility loci in the Ghanaiancase-control group, were not associated with susceptibility or resistance, respectively, in the Chinese Han population. Again, the differences of allele frequencies of rs4331426 G and rs2057178 A, which were 4% and 5% in the Chinese Han and 45% and 32% in the Ghanaian groups, respectively, provide an explanation. In conclusion, we identified the first TB resistance locus in the Chinese Han population through a genome-wide association study.Part Two. A case-control study for polymorphism of X-linked geneassociated with pulmonary tuberculosis Objectives: Among those developing tuberculosis(TB) after exposure to Mycobacterium tuberculosis, approximately 70% are males. Host genetic variation, particularlyimmune-related genes on the X chromosome,might contribute to sex-specific differences in TB incidences. To study whether X-linked gene variation is associated with sex-specific presentation of pulmonary TB(p TB), three SNPs of the TLR8, CD40 LG and IRAK1 genes on the X chromosome were genotyped.Subjects and methods: A total of 1011 cases and 1105 controls of the Chinese Han population were recruited. Among them,923 cases and 1033 controls whofulfilled the inclusion criteria were subjected to further analyses..The p TB patientswere enrolled from the Hebei Chest Hospital and Shijiazhuang Fifth Hospital from March 2007 to August 2009. Healthy individuals who took part in routine physical examinations at the Beijing Electronic Hospitalwere recruited as controls.Five ml full venous EDTA anticoagulated blood samples were collected from all cases and controls after written informed consent was obtained. The study protocol was approved by the Ethics Committee of the Hebei Chest Hospital, Shijiazhuang Fifth Hospital, and the Beijing Electronic Hospital.Frequencies of the variants were analyzed independently as well as in their combinations. Three types of multiple logistic regression analysis(combined analysis of males and females with sex as a covariate, a male-only analysis, and a female-only analysis), applying codominant and recessive models, were used to calculate the effect that alleles and genotypes exerted on the disease status after controlling for age, smoking status and BCG vaccination. In addition, a logistic regression analysis(recessive model)with an interaction term between genetic variants and sex was performed after controlling for age, sex, smoking status and BCG vaccination.Results and conclusions:In the recessive model of multiple logistic regression with the interaction term of the three SNPs and sex, only the interaction of CD40 LG rs3092923CT + CC or C/(-) and sex had a significant effect(Pcorr. =0.0213, ORmale, 95% CImale = 0.47 [0.32-0.70]). This significance of the interaction term was attained after excluding the effectsof age, smoking and BCG vaccination status, suggesting that CD40 LG rs3092923 might play a role in sex-specific differences in p TB susceptibility.For TLR8 rs3764880 AG + AA or A/(-)and IRAK1 rs3027898 CA + AA or A/(-), the P value of the interaction item between genetic variants and sex was not significant(P =0.5008 and 0.9213, respectively). CD40LGrs3092923 and its combined effects with the other two SNPswere associated with an increased risk of p TB only in males. In males, the rs3092923 genotype C/(-) conferred relative protection(OR = 0.52, 95%CI = 0.35-0.78, Pcorr. = 0.0045) and the combined effects of three SNPsincreased gradually as the number of risk alleles increased(OR = 2.58, 2.83,and 2.96 for one, two, and three risk alleles, respectively). For the remaining SNPs, significance was obtained only for the AA genotypeof IRAK1rs3027898 in the combined and female-only analysis. Our results indicate a role of a CD40 LG variant and its combined effectswith distinct TLR8 and IRAK1 variants in susceptibility to p TB inmales. |
