TNF-308 gene polymorphism and tuberculosis susceptibility: a meta-analysis involving 18 studies
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- Wang, Q., Zhan, P., Qiu, L. et al. Mol Biol Rep (2012) 39: 3393. doi:10.1007/s11033-011-1110-x
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A number of studies have investigated the association between TNF-308 (rs1800629 G/A) polymorphisms and the susceptibility towards tuberculosis (TB) in different populations. However, many of these studies provided inconsistent results. In this study, a systematic review and meta-analysis of the published studies was performed to gain a clearer understanding of this association. The PubMed, Embase, Web of Science and CNKI databases were searched for case–control studies published up to Jan 2011, we used no lower date limit. Data were extracted and pooled odds ratios (OR) with 95% confidence intervals (CI) were calculated. A total of 18 publications from 2001 to 2010, involving 2584 TB cases and 3817 controls were included. Overall, for the A allele carriers (G/A + A/A) vs. homozygote GG, the pooled OR was 1.03 (95% CI = 0.89–1.19; P = 0.912 for heterogeneity). For the allele A vs. allele G, the pooled OR was 1.07 (95% CI = 0.93–1.22; P = 0.013 for heterogeneity). In the stratified analysis by ethnicity, among Asians significant risk was found for allele A vs. allele G (OR = 1.22, 95% CI = 1.02–1.47; P = 0.152 for heterogeneity), no significant risks were found among Caucasians. This meta-analysis indicated that the TNF-308 polymorphism was not associated with the risk of TB in the total population, however the significant risk for TNF-308 A allele was found among Asians not Caucasians.
Tuberculosis (TB), primarily caused by Mycobacterium tuberculosis, continues to be an important public health problem despite the existence of national and international TB control programs. Recent data from the World Health Organization show that about 8–10 million new cases arise annually and eventually 2–3 million die of the disease every year [1, 2]. TB is an infectious agent that causes disease and death worldwide. It is estimated that one-third of the World’s population is infected with M. tuberculosis; however, only 10% of those infected ever develop the clinical disease . The central question that arises is whether TB patients are inherently susceptible to the disease or is the development of the disease caused by specific environmental factors. Clearly, environmental factors such as poor economic conditions, malnutrition, stress and overcrowding play a role in determining the susceptibility to TB in human populations. It is known that genetic and non-genetic factors of both the bacterium and the host have impacts on the immune response to M. tuberculosis. Several gene polymorphisms are described to be associated with susceptibility to human TB [4, 5].
The tumor necrosis factor (TNF)-α gene, which encodes the cytokine TNF-α, is located within the class III region of the MHC. TNF-α is an important cytokine in the pathogenesis of TB, because it plays a role in the formation and maintenance of granuloma . Serum level of TNF-α is significantly elevated in patients with advanced TB when compared with the levels of patients with a mild case of TB and control patients . In 1992, a genomic polymorphism resulting in substitution of the nucleotide adenine (A) for guanine (G) at position −308 was discovered within a regulatory region of the TNF-α locus (rs1800629 G/A) . The predominant homozygous allele, the heterozygous allele and the homozygous rare allele of the TNF-308 polymorphism are known as the homozygous wild-type genotype (G/G), the heterozygote (G/A) and the variant—homozygote (A/A), respectively. The presence of the A substitution leads to increases in the binding of nuclear factors and enhanced transcription of the gene [9, 10].
A relatively large number of studies have been published on the association of TNF-308 polymorphisms and the risk of TB, but the findings have been inconsistent. It is possible that a single study may have been to limited for the detection of a small effect of the polymorphisms on TB risk, especially when the sample size was relatively small. The previous meta-analysis  including ten studies, indicated no statistically significant association of TNF-308 polymorphisms with TB risk. When these meta-analyses were performed, the pooled sample size was relatively small. Since then, several additional studies with larger cohort populations have been reported. Simple differences in cohort populations and study design may also contribute to the disparities in the findings. Therefore, we performed a large and comprehensive meta-analysis including the most recent published studies to derive a more precise estimation of the phenotype association.
Materials and methods
Since our study was a meta-analysis based on published articles, we did not draft a statement of patient consent or seek the approval of internal review boards. The electronic databases of PubMed, Embase, Web of Science and CNKI (China National Knowledge Infrastructure) were searched for studies to include in the present meta-analysis, by using the terms “TNF”, “tumor necrosis factor”, “polymorphism” and “tuberculosis”. An upper date limit of Jan 01, 2011 was applied; we used no lower date limit. The search was carried out without restriction on language, but was limited to studies that had been conducted on human subjects. We also reviewed the Cochrane Library for relevant articles. The reference lists of reviews and retrieved articles were hand searched simultaneously. Only published studies with full text articles were included. When more than one of the same patient population was included in several publications, only the most recent or complete study was included in this meta-analysis.
The included studies met the following criteria: (1) evaluated TNF-308 polymorphism and TB risk; (2) case–control studies; (3) supplied the number of individual genotypes in TB cases and controls; and (4) indicated that the distribution of genotypes among controls were in Hardy–Weinberg equilibrium.
Information was carefully extracted from all eligible publications independently by two authors according to the inclusion criteria listed above. Disagreement was resolved by discussion between the two authors. The following data were collected from each study: first author’s surname, year of publication, ethnicity (country of origin), source of controls, TB definition, total numbers of cases and controls, and numbers of cases and controls with G, A, G/G, G/A and A/A genotypes. Allele (G/A) distributions were derived from the genotype distributions. If data from any of the above categories were not reported in the primary study, items were treated as “NA, not applicable”. We did not contact the author of the primary study to request the information. Different ethnicities were categorized as Asian and Caucasian. We did not define any minimum number of patients as required to include a study in our meta-analysis.
Odds ratios (OR) with 95% confidence interval (CI) were used to assess the strength of the association between the TNF-308 polymorphism and the risk of TB. The OR of COPD associated with TNF-308 genotype, the A allele carriers (G/A + A/A) vs. G/G genotype, allele A vs. allele G were calculated, respectively. Subgroup analyses were carried out with respect to ethnicity. A heterogeneity assumption was checked by the chi-square-based Q-test . A P value greater than 0.10 for the Q-test indicated a lack of heterogeneity among studies, so that the pooled OR estimate of each study was calculated by the fixed-effects model (the Mantel–Haenszel method) . Otherwise, the random-effects model (the DerSimonian and Laird method) was used . One-way sensitivity analyses were performed to assess the stability of the results, namely, a single study in the meta-analysis was deleted each time to reflect the influence of the individual dataset on the pooled OR . An estimate of the potential publication bias was carried out by using the funnel plot, in which the standard error of log (OR) of each study was plotted against its log (OR). An asymmetric plot suggested a possible publication bias. Funnel plot asymmetry was assessed by the method of Egger’s linear regression test, which is a linear regression approach to measure the funnel plot asymmetry on the natural logarithm scale of the OR. The significance of the intercept was determined by the t-test, as suggested by Egger (P < 0.05 was considered representative of statistically significant publication bias) . All of the calculations were performed using STATA version 10.0 (Stata Corporation, College Station, TX).
Main data of studies included in the meta-analysis
Ethnicity (country of origin)
Number of cases/controls
Sharma et al. 
Fan et al. 
Yang et al. 
Merza et al. 
Trajkov et al. 
Wu et al. 
9 (GA + AA)
21 (GA + AA)
Kumar et al. 
Ates et al. 
Wang et al. 
7 (GA + AA)
93 (GA + AA)
Qu et al. 
Oh et al. 
Vejbaesya et al. 
Amirzargar et al. 
Oral et al. 
Correa et al. 
Fitness et al. 
Scola et al. 
Selvaraj et al. 
Main results of pooled OR with CI in the meta-analysis
Number of cases/controlsa
(G/A + A/A) vs. G/G
A vs. G (included 16 studies)
OR (95% CI)
OR (95% CI)
In the stratified analysis by ethnicity, no significant association was identified among Caucasians for both the A allele carriers vs. G/G (OR = 0.86, 95% CI = 0.68–1.09; P = 0.348 for heterogeneity) and allele A vs. allele G (OR = 0.87, 95% CI = 0.70–1.09; P = 0.033 for heterogeneity). However, among Asians significant risk was found for allele A vs. allele G (OR = 1.22, 95% CI = 1.02–1.47; P = 0.152 for heterogeneity), no significant association was identified among Asians for both the A allele carriers vs. G/G (OR = 1.15, 95% CI = 0.95–1.38; P = 0.329 for heterogeneity).
A single study involved in the meta-analysis was deleted each time to reflect the influence of the individual dataset on the pooled ORs, and the corresponding pooled ORs were not materially altered (data not shown).
Pulmonary TB, the most common clinical form of the disease, is a granulomatous disease of the lungs caused by M. tuberculosis. Clearly, environmental factors such as poor economic conditions, malnutrition, stress and overcrowding play a role in determining the susceptibility of humans towards TB. The genetic contribution of the host plays a significant role in determining susceptibility to developing the active form of the disease, the severity of infection and the health outcome of the patient [35, 36]. Many studies have focused on the candidate genes for TB susceptibility including those that are expressed in several cells from the innate or adaptive immune system such as Toll-like receptors, cytokines (TNF-α, TGF-β, IFN-γ, IL-1b, IL-1RA, IL-12, IL-10), nitric oxide synthase and vitamin D, both nuclear receptors and their carrier, and the vitamin D-binding protei [37, 38].
TNF-α is an essential cytokine for granuloma formation, previous studies have shown that mice deficient in TNF-α exhibit poorly formed granulomas with areas of extensive necrosis, resulting in widespread dissemination of M. tuberculosis and the rapid death of animals . Additionally, data of a recent study had revealed that TB could be reactivated by blocking effect of TNF-α . In the promoter region of the TNF-α gene, functional polymorphism at the locus −308 had been described . Generally, G to A substitution at position −308 represented a functional polymorphism which leads to different transcription rates in TNF-α production. In vitro, the allele A of TNF-308 polymorphism could lead to increased expression of the TNF-α gene [9, 10]. However, molecular and biologic mechanism of interaction between TNF-α gene polymorphism and risk of TB could not still be fully elucidated.
In 2001, Selvaraj et al.  demonstrated an association between the TNF-308 polymorphisms and TB risk. Subsequently, many investigators have sought to implicate polymorphisms in TNF in the pathogenesis of TB. To date, the results of candidate gene case–control studies have been inconsistent. Some studies have found positive associations between the polymorphisms of TNF-308 and TB risk, while others have not [17, 21, 24–27, 29, 30, 32]. Several factors may be influencing these differences. First, if another variant in or near the TNF gene was one of the causal variants, the true association could easily be missed. Different linkage disequilibrium patterns with the functional variant may lead to variable results in different populations. It is feasible that the TNF gene variant is playing a role in cooperation with other gene variants exhibiting more limited biology. Second, several TB association studies have shown different results in different races, suggesting racial differences may be associated with genetic risk. Finally, small sample size and specific environmental exposures, such as industrial dust, are also confounding factors for diseases with a strong gene-environmental interaction, and may help explain the inconsistencies among the observational studies.
In the present meta-analysis, we have combined 18 eligible studies with 2584 cases and 3817 controls to evaluate the association between the TNF-308 polymorphisms and TB risk. We found no association between TNF-308 gene polymorphisms and TB in the total population. In the stratified analysis by ethnicity, among Asians significant risk was found for allele A vs. allele G (OR = 1.22, 95% CI = 1.02–1.47), no significant association was identified among Caucasians for allele A vs. allele G, suggesting that there could be an interaction between TNF-308 allele A polymorphisms and TB among Asians not Caucasians. Our results were also an indirect validation that the functionality of TNF-308 polymorphisms which leads to different transcription rates in TNF-α production [9, 10].
Among Asians significant risk was found for allele A vs. allele G (OR = 1.22, 95% CI = 1.02–1.47), however no significant association was identified for the A allele carriers vs. G/G (OR = 1.15, 95% CI = 0.95–1.38). The two publications [23, 25] only supplied the number of (GA + AA) genotype and the allele (G/A) distributions were not available. The number of studies for genotypes and alleles was different, for alleles the number of included studies was 16 not 18. The two publications [23, 25] from Asian missing the allele (G/A) distributions may explain the inconsistent results between genotypes and alleles.
The previous meta-analysis by Pacheco et al.  that showed no association between TNF-308 polymorphism and TB risk. That analysis included ten published studies, and only four Asian population studies. Pacheco et al.  analyzed the genotype of the TNF-308 polymorphism associated with TB, but did not examine the association with the allele of TNF-308 gene and TB. In addition, that meta-analysis did not include the subgroup analysis which considered populations of different ethnicity. We have improved upon that previous meta-analysis; in particular, by including more Asian studies and using a generally more comprehensive search strategy. We also explored heterogeneity and potential publication bias in accordance with published guidelines.
Meta-analysis has been recognized as an effective method to solve a wide variety of clinical questions by summarizing and reviewing the previously published quantitative research. By using meta-analysis, a multitude of genetic polymorphisms have been associated with specific disease states. Monocyte chemoattractant protein-1 promoter-2518 , Fc receptor-like 3 C169T , STAT4  and FcgammaRIIa-R/H131  polymorphisms were associated with systemic lupus erythematosus risk. TGFBR-1 , COX-2 , APE1 , ERCC2/XPD  and CYP2E1  polymorphisms have been proven to be associated with cancer risk by meta-analysis. There are several limitations inherent to meta-analysis that should be considered when interpreting these results. First, heterogeneity is a potential problem when interpreting any results obtained by meta-analyses. We minimized this likelihood by performing a careful search for published studies, using explicit criteria for study inclusion, precise data extraction and strict data analysis. However, some pooled ORs were obtained from heterogeneous studies. Second, only published studies were included in this meta-analysis. The presence of publication bias indicates that non-significant or negative findings may be unpublished. Last, the studies included in the present meta-analysis are case–control studies, not randomized population-based surveys, and may be biased by problems of stratification. It is possible that controls were not recruited from exactly the same genetic population as the TB patients.
Despite these limitations, the present meta-analysis suggests that the TNF-308 polymorphisms examined were not associated with the risk of TB in the total population, however the significant risk for TNF-308 A allele was found among Asians not Caucasians. Further case–control studies based on larger sample sizes regarding multiple SNPs and haplotypes are still required in future studies. Moreover, gene–gene and gene–environment interactions should also be considered in the analysis.
This study was supported by a grant from the Major Program of Nanjing Medical Science and Technique Development Foundation in 2007 (Molecular Predictor of Personalized Therapy for Chinese Patients with Non-small Cell Lung Cancer) (Lk-Yu).
Conflict of interest
The authors declare there are no conflicts of interest in this research.