Vitamin D Receptor ApaI Gene Polymorphism and Tuberculosis Susceptibility: A Meta-Analysis

Abstract

Aim: Vitamin D performs its actions through the vitamin D receptor (VDR), which acts as a transcriptional factor. Many case-control studies have been performed in the past to elucidate the association of the ApaI polymorphism of VDR gene and the risk of tuberculosis (TB). However, these studies have shown inconsistent and conflicting results. In the present study, a meta-analysis was performed to investigate the potential relationship between the VDR ApaI gene polymorphism and the risk of TB. Methodology: A quantitative synthesis was performed for the published studies based on the association between the VDR ApaI gene polymorphism and the risk of TB retrieved from PubMed (Medline) and EMBASE web databases. A meta-analysis was performed, and pooled odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated for all the genetic models. Results: We observed a decreased risk of TB in allelic contrast (a vs. A: p=0.009; OR=0.869, 95% CI=0.782 to 0.965), homozygous (aa vs. AA: p=0.006; OR=0.724, 95% CI=0.575 to 0.910), and heterozygous (aA vs. AA: p=0.698; OR=0.948, 95% CI=0.722 to 1.243) comparisons. Similarly, dominant (aa+Aa vs. AA: p=0.032; OR=0.842, 95% CI=0.720 to 0.985) and recessive (aa vs. AA+Aa: p=0.027; OR=0.796, 95% CI=0.650 to 0.975) models also demonstrated a decreased risk of TB, whereas a heterozygous genotype (Aa vs. AA: p=0.109; OR=0.873, 95% CI=0.740 to 1.030) did not indicate any association with the risk of TB. There was no evidence of publication bias and heterogeneity test. Conclusions: This meta-analysis suggests that ApaI polymorphism of the VDR gene is significantly associated with a decreased risk of TB. However, future larger studies with groups of populations are warranted to analyze this association.

Introduction

Tuberculosis (TB) is a major chronic infectious disease mainly caused by Mycobacterium tuberculosis, an intracellular pathogen that survives within macrophages. The disease is a major cause of death worldwide annually when compared with other infectious diseases (Global Tuberculosis Report, 2012). About one third of the world's population is thought to be affected with M. tuberculosis infection, but only a small fraction (5-15%) of the population develops active TB disease during their lifetime (Rosman and Oner-Eyupoglu, 1998). The observation indicates that there is an interindividual heterogeneity in host susceptibility to TB. Previous studies have reported that genetic variants may play an important role in TB susceptibility in different individuals (Nava-Aguilera et al., 2009). Thus, it is anticipated that the identification of host genetic factors for the susceptibility to TB would aid greatly in the development of therapeutic strategies and the control of this dreadful infectious disease.

Vitamin D is primarily metabolized to the intermediate compound 25-hydroxyvitamin D [25(OH)D] in the liver and subsequently binds to the intracellular receptors that regulate gene expression (Ogunkolade et al., 2006). The activated form of vitamin D exerts its cellular functions by modulating the transcription of target genes after binding to the nuclear vitamin D receptor (VDR) and is generally involved in macrophage activation and clearance of the bacteria inside the macrophage cells (Haussler et al., 1998; Deluca and Cantorna, 2001). Expression and nuclear activation of the VDR is essential for these activities of vitamin D. Several polymorphisms have been described in the 5′ regulatory region and 3′ untranslated region (UTR) of the VDR gene, among them ApaI (VDR 7975232 A>a), which is found in the intron 8 of the 3′UTR region, has shown an association with the stability of VDR mRNA along with other polymorphisms (Fang et al., 2005). Variant allele formation as a result of ApaI polymorphism may attenuate VDR functions and in turn susceptibility to TB may be enhanced by an imbalance in vitamin D status (Davies, 1995).

The significance of the role of the VDR gene and its immune response against TB provides evidence of vitamin D-related gene-environment interactions in the host response to TB (Bellamy, 2000). Several epidemiological studies have tried to decipher the possible association between the VDR ApaI gene polymorphism and TB susceptibility with inconsistent and contradictory results (Bornman et al., 2004; Selvaraj et al., 2004a, 2004b, 2008, 2009; Lombard et al., 2006; Babb et al., 2007; Olesen et al., 2007; Alagarasu et al., 2009; Vidyarani et al., 2009; Sharma et al., 2011). Inconsistency in the results across many of the studies could possibly be related to the ethnicity of the population, sample size, and individual studies that have low power to evaluate the overall effects. Thus, it is necessary to quantify and evaluate the true association of the VDR ApaI gene polymorphism and the risk of TB from all eligible studies with rigorous methods. In the present study, we have performed a meta-analysis, which is a powerful tool for exploring the risk factors associated with the genetic diseases, because it employs a quantitative method to combine the data drawn from individual studies where individual sample sizes are small with low statistical power, to provide reliable conclusions (Cohn and Becker, 2003; Areeshi et al., 2013). Here, we have done an updated meta-analysis, including the latest data to elucidate the association of the VDR ApaI gene polymorphism and TB risk.

Materials and Methods

Literature search strategy

We performed a PubMed, Medline, and EMBASE web database search covering all research articles published with a combination of the following key words, that is, “VDR, Vitamin D gene (polymorphism OR mutation OR variant) AND tuberculosis susceptibility or TB” (last updated on October 2013). We evaluated potentially relevant genetic association studies by examining their titles and abstracts and procured the most relevant publication matching with the eligible criteria for a closer examination. Besides the online database search, the references listed in the selected research articles were also screened for other potential articles that may have been missed in the initial search.

Inclusion and exclusion criteria

To minimize heterogeneity and facilitate the proper interpretation of this study, published articles included in the current meta-analysis had to meet all the following criteria, that is (1) they must have appraised the association between VDR ApaI gene polymorphism and TB risk, (2) employed a case-control study design, (3) clearly described confirmed TB patients and TB-free controls, (4) have available genotype frequency in the “cases” and in the “controls,” (5) been published in the English language, (6) data collection and analysis methodology should be statistically acceptable. In addition to above, when the case-control study was included in more than one research article using the same case series, we selected the research study that incorporated the largest number of individuals. The major reasons for study exclusion were data overlapping, case-only studies, review articles, repeated literature, and genotype frequencies or sample size or case-control not reported.

Data extraction and quality assessment

For each retrieved publication, the methodological quality assessment and data extraction were independently abstracted in duplicate by two independent investigators using a standard procedure. Data collection form was used to confirm the accuracy of the collected data by strictly following the inclusion criteria as stated above. The major characteristic abstracted from the retrieved publications included the name of first author, publication year, the country of origin, source of cases and controls, number of cases and controls, study type, and genotype frequencies. Cases related with disagreement/discrepancy on any item of the data from the selected studies were fully debated with investigators to achieve a final consensus.

Statistical analysis

To appraise the association between the VDR ApaI gene polymorphism and TB risk, pooled odds ratios (ORs) and their corresponding 95% confidence intervals (CIs) were estimated. Heterogeneity assumption was examined by the chi-square-based Q-test (Wu and Li, 1999). Heterogeneity was considered significant at p-value<0.05. The data from single comparison were combined using a fixed-effect model (Mantel and Haenszel, 1959) when no heterogeneity was obtained. Otherwise, the random-effect model (DerSimonian and Laird, 1986) was used for the pooling purpose. Moreover, I² statistics was employed to quantify interstudy variability and larger values suggested an increasing degree of heterogeneity (Higgins and Thompson, 2003). Hardy-Weinberg equilibrium (HWE) in the controls was calculated by the chi-square test. Funnel plot asymmetry was measured by the Egger regression test, which is a type of linear regression approach to measure the funnel plot asymmetry on the natural logarithm scale of the OR. The significance of the intercept was measured by the t-test (p-value<0.05 was considered a representation of statistically significant publication bias) (Egger et al., 1997). A comparative evaluation of “meta-analysis” programs was done by using uniform resource locator www.meta-analysis.com/pages/comparisons.html. The Comprehensive Meta-Analysis (CMA) V2 software program (Biostat) was utilized to perform all statistical analysis involved in this meta-analysis study.

Results

Characteristics of the published studies

A total of 11 articles were finally selected after the literature search from the PubMed (Medline) and EMBASE web databases. All retrieved articles were inspected carefully by reading their titles and abstracts, and the full texts for the potentially relevant publications were further checked for their suitability for this meta-analysis (Fig. 1). Research articles either showing VDR polymorphism to predict survival in TB patients or considering VDR variants as indicators for response to therapy were excluded straightaway. Similarly, studies investigating the levels of VDR mRNA or protein expression or relevant review articles were also excluded from this meta-analysis. We included only case-control or cohort design studies stating the frequency of all three genotypes. Besides the database search, the supporting references available in the retrieved articles were also checked for other potential studies. After careful screening and following the inclusion and exclusion criteria, 11 eligible original published studies were finally considered for this study (Table 1). Distribution of genotypes, HWE p-values in the controls, and susceptibility toward TB have been shown in Table 2.

FIG. 1.

Flow diagram of study selection process (inclusion and exclusion criteria of retrieved studies).

Table 1.

Main Characteristics of Included Studies Summarized for the Meta-Analysis

Author	Year	Country of origin	Study design	Genotyping method	Cases	Controls	Source of genotyping
Sharma et al.	2011	India	HB, PB	PCR-RFLP	76	461	Blood
Alagarasu et al.	2009	India	HB	PCR-RFLP	111	146	Blood
Selvaraj et al.	2009	India	HB	PCR-RFLP	65	60	Blood
Vidyarani et al.	2009	India	HB	PCR-RFLP	40	49	Blood
Selvaraj et al.	2008	India	HB	PCR-RFLP	51	60	Blood
Olesen et al.	2007	West Africa	HB	TaqMan	320	345	Blood
Babb et al.	2007	South Africa	HB	PCR-RFLP	249	352	Blood
Lombard et al.	2006	South Africa	HB	PCR-RFLP	104	117	Blood
Bornman et al.	2004	West Africa	PB	PCR-RFLP	106	634	Blood
Selvaraj et al.	2004a	India	HB	PCR-RFLP	46	64	Blood
Selvaraj et al.	2004b	India	HB	PCR-RFLP	64	103	Blood

HB, hospital based; PB, population based; PCR-RFLP, polymerase chain reaction-restriction fragment length polymorphism.

Table 2.

Distribution of Gene Polymorphism of Studies Included in the Meta-Analysis

	Control				Case
	Genotype			Minor allele	Genotype			Minor allele	HWE
Author (year)	AA	Aa	aa	MAF	AA	Aa	aa	MAF	p-Value
Sharma et al. (2011)	239	175	47	0.29	41	28	7	0.27	0.080
Alagarasu et al. (2009)	44	81	21	0.42	48	46	17	0.36	0.095
Selvaraj et al. (2009)	23	25	12	0.40	25	29	11	0.39	0.022
Vidyarani et al. (2009)	14	25	10	0.45	17	16	7	0.37	0.848
Selvaraj et al. (2008)	16	31	13	0.47	24	19	8	0.34	0.780
Olesen et al. (2007)	70	166	101	0.54	83	166	67	0.47	0.906
Babb et al. (2007)	116	173	63	0.42	101	108	40	0.37	0.914
Lombard et al. (2006)	29	84	4	0.39	17	86	1	0.42	0.001
Bornman et al. (2004)	266	292	76	0.35	41	53	12	0.36	0.762
Selvaraj et al. (2004a)	22	34	8	0.39	13	25	6	0.42	0.353
Selvaraj et al. (2004b)	39	49	15	0.38	20	35	9	0.41	0.950

HWE, Hardy-Weinberg equilibrium; MAF, minor allele frequency.

Publication bias

The Begg funnel plot and the Egger test were carried out to appraise the publication bias among the selected studies for the meta-analysis. The appearance of the shape of funnel plots was symmetrical in all genetic models. The Egger test was performed to provide the statistical evidence of funnel plot. The findings showed a lack of publication bias among all comparison models (Table 3).

Table 3.

Statistics to Test the Publication Bias and Heterogeneity in the Meta-Analysis

	Egger's regression analysis			Heterogeneity analysis
Comparisons	Intercept	95% Confidence interval	p-Value	Q-Value	p_{heterogeneity}	I² (%)	Model used for the meta-analysis
a vs. A	0.69	−1.51 to 2.90	0.49	10.87	0.36	8.08	Fixed
aa vs. AA	0.36	−0.95 to 1.69	0.54	5.60	0.84	<0.0001	Fixed
Aa vs. AA	0.30	−2.69 to 3.30	0.84	17.38	0.06	42.48	Fixed
aa+Aa vs. AA	0.59	−2.32 to 3.52	0.65	17.07	0.07	41.44	Fixed
aa vs. AA+Aa	0.28	−0.81 to 1.37	0.57	4.43	0.92	<0.0001	Fixed

Test of heterogeneity

To test heterogeneity among the selected studies, the Q-test and I² statistics were employed. No significant heterogeneity was detected in any of the models, that is, allele (a vs. A), homozygous (aa vs. AA), heterozygous (aA vs. AA), dominant (aa+Aa vs. AA), and recessive (aa vs. AA+Aa) genotype models, which were included for this meta-analysis. Therefore, the fixed-effect model was applied to synthesize the data (Table 3).

Quantitative synthesis

We pooled all 11 studies together, which resulted in 1232 confirmed TB cases and 2391 controls, for the assessment of overall association between the VDR ApaI polymorphism and the risk of TB. The pooled ORs from overall studies indicated a decreased risk between VDR ApaI gene polymorphism and TB risk in allelic contrast (a vs. A: p=0.009; OR=0.869, 95% CI=0.782 to 0.965), homozygous (aa vs. AA: p=0.006; OR=0.724, 95% CI=0.575 to 0.910), and heterozygous (aA vs. AA: p=0.698; OR=0.948, 95% CI=0.722 to 1.243) comparisons. Likewise, dominant (aa+Aa vs. AA: p=0.032; OR=0.842, 95% CI=0.720 to 0.985) and recessive (aa vs. AA+Aa: p=0.027; OR=0.796, 95% CI=0.650 to 0.975) models also showed a decreased TB risk. Heterozygous genotype (Aa vs. AA: p=0.109; OR=0.873, 95% CI=0.740 to 1.030) failed to show any association with the risk of TB (Figs. 2 and 3).

FIG. 2.

Forest plot of overall tuberculosis (TB) risk associated with vitamin D receptor (VDR) ApaI gene polymorphism. The squares and horizontal lines correspond to the study-specific odds ratio (OR) and 95% confidence interval (CI).

FIG. 3.

Forest plot of overall TB risk associated with VDR ApaI gene polymorphism. The squares and horizontal lines correspond to the study-specific OR and 95% CI.

Discussion

The VDR influences both innate and adaptive immunity. Innate immunity genes are important in modulating the host susceptibility to TB because the first line of defense against M. tuberculosis involves the identification and uptake of the bacterium by macrophages and dendritic cells (Rockett et al., 1998). The significance of polymorphic variant genotypes of the VDR gene has been emphasized on the susceptibility or resistance to various infectious diseases, including TB (Hill, 2001). Genes involved in protective immunity are under great selective pressure, showing higher variability than other genes (Cooke and Hill, 2001). It has been reported that genotype frequencies of VDR polymorphisms differ between populations and may contribute to inconsistent associations with disease development (Nava-Aguilera et al., 2009). As we know that TB is one of the most common infectious diseases with a high morbidity and mortality. A well-established genetic marker surely would have a significant influence in screening and prevention of TB. Epidemiological research studies performed in different ethnicity and geographical populations showed inconsistent and conflicting results because they were underpowered and it was difficult to conclude exactly by examining the alleles separately. For the said reason of conflicting results, this meta-analysis was performed to explore whether the VDR ApaI polymorphism has any role in the development of TB. Pooled ORs generated from large sample size and population can enhance the statistical power, and combining data from various studies has the advantage of reducing random errors (Ioannidis et al., 2008).

Although previous published meta-analysis by Gao et al. (2010) has reported no association between the ApaI polymorphism of VDR gene and the risk of TB, in this study, the associations for the allele, the dominant model, and the recessive model were examined, and it was found that ApaI polymorphism of VDR gene was associated with a decreased risk of TB compared with wild-type AA genotype in the overall analysis. One of the possible explanations is that VDR gene has more than 62 polymorphisms distributed throughout the gene, and it is possible that the analyzed variant do not influence individually owing to the linkage disequilibrium with other VDR polymorphisms. Moreover, the publication bias was tested, and we found that there was no publication bias for overall population and the results for overall population might be more robust to some extent.

Furthermore, the VDR gene has gained special attention because early VDR studies in mouse models reported that vitamin D-deficient mice are increasingly sensitive to autoimmune diseases (Bouillon et al., 2008). However, susceptibility toward TB is polygenic, and multiple candidate genes are likely to be involved in the process of active disease development (Bellamy, 2006). Due to the multifactorial nature of TB infection and complex nature of the immune system (Möller and Hoal, 2010), VDR genetic polymorphism cannot be solely responsible for the predisposition of TB.

In this study, no significant heterogeneity was found between selected studies in the test of heterogeneity, indicating that our present combined analysis is unbiased. However, the findings from our current analysis should still be interpreted with full caution. First, we only included the studies published in the English language and those abstracted and indexed by the selected electronic databases were included for data analysis; it is possible that some pertinent studies published in other languages and indexed in other electronic databases may have missed. Second, the abstracted data were not stratified by other factors, for example, HIV status or severity of the TB infection, and these results are based on unadjusted parameters. Third, we did not test for gene and environment interactions because of the insufficient data available in the published reports.

Conclusion

In conclusion, this meta-analysis systematically analyzed the association between the VDR ApaI gene polymorphism and the risk of TB. The outcomes of our study suggested that ApaI polymorphism of VDR gene is associated with reduced risk of TB. Considering the predictive values, future large designed case-control studies with environmental factors in different populations are necessary to further evaluate the role of VDR ApaI polymorphism in TB susceptibility and to validate this association with this complex disease.

Footnotes

Acknowledgments

We acknowledge the software-related support provided by the Institute of Life Sciences, Bhubaneswar, India, and Medical Microbiology Unit of EMS Department, College of Nursing & Allied Health, Jazan University, Jazan, Saudi Arabia.

Author Disclosure Statement

No competing financial interests exist.

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