Vitamin D Receptor Gene FokI Polymorphism Contributes to Increasing the Risk of Tuberculosis: Evidence from a Meta-Analysis

Purpose In the present study, we explored the link between vitamin D receptor (VDR) FokI gene polymorphisms with tuberculosis(TB). Methods Based on a comprehensive search of PubMed, Embase, Web of Science, Elsevier Science Direct, Cochrane Library, CNKI, Wanfang, and Chongqing VIP databases, we searched case-control study on FokI gene polymor-phism and TB susceptivity. The Newcastle-Ottawa Scale(NOS) was used to evaluate the quality of the literature and extracted data, and R 4.0.5 software was used for the meta-analysis. Results: Among the 243 selected articles, 35 in the meta analysis. The meta-analysis showed that the FokI gene polymorphism allene gene model(f vs F, Odds ratio=1.22,95% condence interval:1.11-1.36); dominant model (ff+fF vs F, Odds ratio=1.29,95% condence interval:1.13-1.47); recessive model(ff vs fF+FF, Odds ratio=1.31,95% condence interval:1.09-1.56); codominant (ff vs FF, Odds ra-tio=1.48,95% condence interval:1.19-1.83); codominant(fF vs FF, Odds ratio=1.23,95% condence interval:1.09-1.39). The meta-analysis indicates a high level of heterogeneity between the VDR FokI gene polymorphism and TB and the race is a source of heterogeneity in the results. Conclusion The present update meta-analysis suggest that FokI gene polymorphism is signicantly associated with an increased risk of TB.


Introduction
Tuberculosis (TB) is an ancient disease for millennia and has always been a serious public health problem in history. Pulmonary tuberculosis (PTB) caused by Mycobacterium is one of the leading 10 causes of death globally currently. People are generally all susceptible to Mycobacterium, about 90% of adults could possibly developed TB [1]. The probability of developing TB disease is much higher among people infected with HIV and those with other risk factors of malnutrition, diabetes, smoking and alcohol consumption and so on. With the rapid development of molecular biology, the impact of host genetic variation in susceptibility of tuberculosis attracts much attention and regarding studies have shown it plays a critical role in increasing susceptibility to TB. At present, human leukocyte antigens (HLA) gene, natural resistance associated macrophage protein 1(NRAMP1) gene, vitamin D receptor (VDR) gene has become a focus of scholars at home and abroad.
The main role of vitamin D is regulating calcium and phosphorus metabolism. 1,25-Dihydroxyvitamin D3 is the most active metabolite of Vit D, and it has been proven to have an important role in regulating the immune system and humoral regulation. Macrophages play many essential roles in the immune responses through 1,25-Dihydroxyvitamin D3 activation. The active form of vitamin D3, 1,25-Dihydroxyvitamin D3 exerts Inhibition of growth of a tubercle bacillus in macro-phages. People usually study VDR to study role of Vit D. Recently, polymorphisms in the VDR gene, their polymorphism affects receptor activity and has been determined to be related to the pathogenesis of tuberculosis. Although multiple studies have con rmed the relationship with VDR gene polymorphism and TB, however, the ndings of these studies have been contradictory. How can we explain the relevant contradictory results?
Hence, we using meta-analysis to carry out comprehensive quantitative analysis and draw stable and reliable conclusions.

Literature search strategy
A literature search was carried out via computer and hand searches. We used vitamin D receptor or VDR or rs2228570 or FokI and tuberculosis and pulmonary search published articles in Chinese and English database. We use these databases: SinoMed data-base, CNKI, Wanfang, VIP, PubMed, Cochrane Library, Elsevier ScienceDirect, Embase, Web of Science and SpringLink.

Literature inclusion and exclusion criteria
The literature inclusion criteria: (1) type of study: case-control studies; (2) content of study: Fok gene polymorphisms and TB susceptibility; (3) research object: cases of inclusion were diagnosed TB, and exclusion autoimmune diseases, diseases of the endocrine system, long time use of adrenal cortical hormones or immunosuppressive drugs, and HIV infection [2,3]; (4) control of inclusion were healthy control group: X-ray chest showed no abnormalities, PPD test less than 5mm3; (5) the studies with enough data to calculate odds ratio(OR) and 95% con dence interval(CI) [4,5]; (6) there were no restrictions on race and gender, and aged ≥ 16years.
Literature exclusion criteria: (1) the studies with incomplete data or required data cannot be calculated OR and 95%CI; (2) there were reporting the clinical consequences; (3) repeated research; (4) controls were healthy subjects with no history of contact with TB patients.

Data extraction
All data were extraction independently by 2 investigators. The data were extraction included: the rst author's name, publication year, the area of research, case group and control group sample.

Literature quality evaluation
We evaluated the literature with 2 investigators using Newcastle-Ottawa Scale(NOS). The scale includes a total of 8 items including the selection, comparability and exposure. The highest score is 9 points, and the score more than 7 points as high-quality literature[6].

Statistical analysis
In this study, we considered the f allele is a gene that increases risk. Therefore, we use the ve models: allele contrast(f vs. F), dominant model(ff + fF vs. FF), recessive model(ff vs. fF + FF), co-dominant model(ff vs. FF, fF vs. FF) to calculate OR and 95%CI. The het-erogeneity between studies was assessed with a based Q test and I2 statistics. If there is no signi cant heterogeneity (I2 < 50%, Q test P > 0.05), select the xed effect model; otherwise, choose random Effect model. Sensitivity analysis to verify the stability and reliability of meta-analysis results [4,5,7]. The Begg rank correlation method was used to statistically assess publication bias. All the statistical tests were conducted with R software(version 4.0.5), and the statistical signi cance was de ned as p < 0.05. All p values were two-sided.

Characteristics of inclusion studies
Two hundred and forty-three studies were identi ed after database and manual literature searches. According to the inclusion and exclusion criteria, 35 relevant studies  were considered in this metaanalysis. A ow chart of the study selection process is shown in Fig. 1. And detailed characteristics of the enrolled studies were listed in Table 1.

Characteristics of inclusion studies
We pooled all 35 studies together for the assessment of the relationship between the VDR FokI polymorphism and TB risk. In this study used four models for meta-analysis.  OR = odds ratio; CI = con dence interval; R = random effect model.

Sensitivity analysis
In the sensitivity analysis, each study was deleted one at a time to evaluate the im-pact of each individual data set on the combined OR. Results showed no signi cant dif-ferences in the corresponding combined ORs, suggesting the stability of this me-ta-analysis ( Supplementary Fig. 2).

Subgroup meta-analysis
In order to assess the possible impact of different ethnic groups on the overall esti-mate, we will divide the analysis into three subgroups according to ethnicity, namely Asian ethnic group, African ethnic group and Caucasian ethnic group. The results of the comparison of the ve models are shown in Table 3 and Supplementary Fig. 3.

Publication bias
The meta-analysis results of the four models were through Begg's rank correlation test and showed no evidence con rmed of publication bias(p 0.05). The results showed in Table 4.

Discussion
Tuberculosis is one of the major causes of tuberculosis morbidity and mortality, and the VDR gene may play an important role in regulating host susceptibility to tuberculosis due to the potential role of VDR in tuberculosis morbidity and mortality. Many scholars believe that the de ciency of vitamin D3 in the human body will increase the suscepti-bility to tuberculosis, and the change of the VDR gene will have a certain impact on its cytological function. However, many studies have produced contradictory association data between VDR FokI gene polymorphism and tuberculosis risk. This shows that TB infection situation will be affected by environmental and genetic factors interact.
We conducted a meta-analysis based on 35 literatures that met the inclusion and exclusion criteria as of November 2020, and con rmed that VDR FokI gene polymor-phism was associated with tuberculosis. We found that the f allele was a risk factor for pulmonary tuberculosis in the allele model, dominant, recessive, and co-dominant model. This meta-analysis found that f gene mutations in Asian and Caucasian populations were associated with tuberculosis in different models. However,an insigni cant association was found in Africans for all comparison models. To some extent, this nding re ects the existence of ethnic differences, suggesting that this polymorphism may play a multi-functional role in the pathogenesis of tuberculosis, or interact with other genetic and en-vironmental factors. Previous studies, including the WHO TB report, have shown that yellow people are more likely to develop TB than blacks and whites.
The present study has some advantages over the previous ones. First of all, this is an updated metaanalysis, adding many new research results compared with the two me-ta-analyses in 2015.We use different models to compare. Second, we evaluated the quality of each literature included in this study.
Thirdly, we carried out sensitivity analysis on the results of this study, and the results were all stable. However, there are some limitations to consider in this study, and I should pay attention to the potential publication bias when interpreting the results, although no signi cant publication bias was found in this study. Second, patient heterogeneity and potential confounders may have distorted the analysis. Third, VDR polymorphism may be related to the clinical characteristics of PTB. Due to the lack of raw data from the authors, we performed subgroup analysis by eth-nicity, while we did not stratify or analyze other factors, such as gender or clinical and environmental variables, but the limited data available did not allow us to study this correlation.

Conclusions
In summary, this meta-analysis shows that VDR FokI polymorphism is associated with PTB susceptibility, suggesting that this polymorphism may play an important role in the risk of PTB. There are differences in the degree of susceptibility among different populations. Asian and Caucasian populations are more susceptible to f allele mutations, but no correlation has been found in African populations.  Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) ow chart of study identi cation process