Association between Vitamin D receptor gene polymorphism and the risk of Multiple Sclerosis: Systematic review and meta-analysis

Background The association between the vitamin D receptor ( VDR ) gene polymorphism and the risk of Multiple sclerosis (MS) has been evaluated in several studiese. However, the findings were inconsistent and inconclusive. Methods All relevant studies reporting the association between the FokI (rs2228570) or/and TaqI (rs731236) or/and BsmI (rs1544410) or/and ApaI (rs7975232) polymorphisms of the VDR and susceptibility to multiple sclerosis published up to September 2019 were identified by comprehensive systematic database search in web of science, Scopus, and PubMed. Results A total of 30 case–control studies were included in this meta-analysis. The overall results suggested a significant association between TaqI gene polymorphism and MS risk under heterozygote contrast (OR = 1.27, 95%CI = 1.01–1.59, REM). Moreover, the pooled results of subgroup analysis decline presence of significant association under all defined genotype model. In subgroup analysis, BsmI gene polymorphism was associated with increased risk of MS under the recessive model in Asian population. In other hand, ApaI gene polymorphism was associated with decreased risk of MS under recessive and homozygote contrast (aa vs AA) models in Asian population. Conclusions This meta-analysis suggested a significant association between TaqI gene polymorphism and MS susceptibility. Furthermore, BsmI gene polymorphism was associated with an increased risk of MS in Asian population. In contrast, ApaI gene polymorphism was associated with a decreased risk of MS in Asian population. Future large scale studies on gene–environment and gene– gene interactions are required to estimate related risk factors and assist early diagnosis of patients at high risk for MS.


Abstract
Background The association between the vitamin D receptor ( VDR ) gene polymorphism and the risk of Multiple sclerosis (MS) has been evaluated in several studiese. However, the findings were inconsistent and inconclusive.

Background
Multiple sclerosis (MS) is a chronic, demyelinating disorder of the brain and spinal cord that mainly develops in young individuals [1]. Autoantibodies and reactive T cells against the myelin are recognized as implicit pathogenic function in the tissue damage and development of CNS inflammation [2]. The main etiology of the disease remains to be elusive, but it has been demonstrated genetic and environmental factors play important roles in susceptibility to the disease [3]. Vitamin D is a group of fat-soluble secosteroids that have functional and regulatory effects in the body. Vitamin D has been implicated in the development of the brain and spinal cord. Alternatively, the active form of vitamin D, 1,25-dihydroxyvitamin D has a wide anti-inflammatory and immunomodulatory properties [4,5]. Vitamin D exerts its immunomodulatory functions within the immune system by decreasing the presentation of major histocompatibility complex II (MHC-II) on T cells and monocytes. Vitamin D also reduces T cell proliferation and pro-inflammatory cytokine release [6]. The lower serum vitamin D levels compared to healthy controls have been reported in MS patients. Moreover, Vitamin D has positive effects on regulating MS risk development [7,8]. The effects of Vitamin D on the immune system are exerted by binding to the nuclear vitamin D receptor (VDR) [9]. Particular variants of the VDR gene are related to changes in vitamin D metabolism and function [10]. Taken together, these results suggested that VDR may play an important role in the pathogenesis of MS.
The human VDR gene is located on chromosome 12q12-14 and series of restriction fragment length polymorphisms (RFLP) in the human VDR gene have been reported, containing BsmI (rs1544410), ApaI (rs7975232), FokI (rs2228570), TaqI (rs731236) restriction sites [11]. ApaI, BsmI, and TaqI are localized near the 3′ end of the VDR gene in the intron between exons 8 and 9, and shown to be in strong linkage disequilibrium (LD) [12]. The 3′UTR of the VDR gene involved in the regulation of gene expression by regulation of mRNA stability and expression level [13]; Polymorphism FokI is located at the translation starting codon [14]. Therefore, we set out a meta-analysis of all eligible published case-control studies to obtain an exact evaluation of the association between MS and VDR gene polymorphisms.

Methods
The current meta-analysis was conducted according to the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) statement [25].

Literature review
All relevant studies reporting the association between the FokI (rs2228570) or/and TaqI (rs731236) or/and BsmI (rs1544410) or/and ApaI (rs7975232) polymorphisms of the Vitamin D receptor and susceptibility to multiple sclerosis published up to September 2019 were identified by comprehensive systematic electronic database search in web of science, Scopus, and PubMed. The following search terms were applied: (VDR" OR "vitamin D receptor") AND ("multiple sclerosis" OR "MS") AND ("polymorphisms" OR "single nucleotide" OR "polymorphism" OR "SNP" OR "variation" OR "mutation"). As a complementary approach, in order to detect additional relevant studies, manual references evaluation of the included studies was performed. In this meta-analysis, the strategy of the search was restricted solely to the English-language publications and human population.

Study selection
Two reviewers independently assessed titles and abstracts of all studies retrieved in the initial search. Articles not following the eligibility criteria were excluded by applying a hierarchical approach based on study design. The full-text examination was applied if we could not decide include or exclude based on titleand abstract. In particular conditions, if an author has published more than one study by the same case series, the most recently published study was included. Any disagreements were discussed and resolved by consensus.

Eligibility criteria
Studies considered eligible if met the following criteria: 1) all eligible case-control studies that evaluate the relationship between the VDR SNPs and the risk of MS as the main outcome; 2) sufficient data are available to extract or calculate odds ratios (ORs) and 95% confidence intervals; 3) contained genotype or allele distributions of case and healthy individuals for VDR gene polymorphism in the studies. The exclusion criteria were as follows: 1) studies which genotype or allelic frequency could not be extracted; 2) letter, case report, review, comment, book chapter, and abstract; 3) duplicated reports and studies with repetitive subjects. The application of these criteria recognized 30 casecontrol studies eligible for the meta-analysis.

Data extraction
Two reviewers independently extracted all data according to standardized extraction form for the following data: the author's name, journal and year of publication, country of origin, ethnicity, number of case and control for each gender separately, mean or range of age, genotyping method, total sample size of cases and controls, and the number of cases and controls for each genotype. For quality assessment of the included publications, the Newcastle-Ottawa Scale (NOS) was applied [26]. Studies with scores 0-3, 4-6 or 7-9 were of low, moderate or high-quality, respectively.

Statistical analysis
Deviation from Hardy-Weinberg equilibrium (HWE) for distribution of the allele frequencies was analyzed using Chi-Square test in the control group. Sensitivity analysis was conducted to evaluate the stability of the results by removing the studies not in HWE.The strength of association between the FokI and/or TaqI

Study characteristics
The primary search yielded a total of 636 publications from web of science, Scopus, and PubMed databases. After the removal of duplicates and title abstract review, only 76 studies remained for full-text examination. Eventually, 30 studies have met inclusion criteria and included for quantitative synthesis. The search workflow is shown in Figure 1.
Study characteristics are summarized in Table 1 The studies were published between 1999 and 2019. Taq-Man and PCR-RFLP genotyping method were used by most studies.

Quantitative synthesis
The distributions of FokI, TaqI, BsmI and ApaI genotypes of the included studies are shown in Table 2. FF for FokI SNP, TT for TaqI SNP, BB for BsmI SNP and AA for ApaI were used as the reference category. The heterogeneities in the comparisons (I 2 _50%, fixed-effects models; I 2 >50%, random-effects models) ascertain the application of Fixed-effects or random-effects models.

Meta-analysis for FokI (rs2228570) polymorphism and MS
Overall 16 case-control studies with 3057 cases and 2852 controls were analyzed for assessment of FokI gene polymorphism and MS risk. Of 16 studies, 9 studies carried out in Europe continent [19,22,[32][33][34][35][36][37][38] 4 studies in Asia continent [16,17,39,40] One study in America continent [41] and finally 2 studies in Australia [15,42] (Table 1). No significant association was observed between FokI gene polymorphism and MS risk across all genetic models. Additionally, subgroup analysis based on geographical location was performed which the pooled results rejected any association between FokI gene polymorphism and risk of MS in European and Asian population. Since there was only one study for American, and two studies for Australian populations, these studies were excluded from the subgroup analysis. The results of pooled ORs, heterogeneity tests and publication bias tests for different analysis models are shown in Table 3. (Supplementary file Figure 1 and 2).
The results of pooled ORs, heterogeneity tests and publication bias tests for different analysis models are shown in Table 3.

Evaluation of heterogeneity
Significant heterogeneity existed for FokI, TaqI, BsmI and ApaI gene polymorphism in all of the genetic models. Furthermore, in subgroup analysis, there was significant heterogeneity for studies were carried out in Asian and European countries (Table 3).

Publication bias
Publication bias was estimated by using funnel plot, Begg's and Egger's tests. No evidence of Publication bias was seen for all four SNP and subgroup analysis under all genetic models. Additionally, the shape of the funnel plot appeared to be symmetrical which demonstrated that there was no significant publication bias (Figure 4).

Sensitivity analysis
Sensitivity analysis was conducted after sequentially removing each eligible study. This approach is to enumerate as an inevitable step for analyzing multiple criteria. The significance of the pooled ORs was not affected by any single study in the dominant model for FokI,TaqI,BsmI and ApaI SNPs ( Figure 5), indicating that our results were statistically robust.

Discussion
The VDR gene, as a pleiotropic gene, has been associated wiht several diseases. In the previous studies, the relationship between VDR gene SNPs and autoimmune disorders was evaluated in several meta-analyses. The study of Feng et al.. [55] described that TaqI or BsmI gene polymorphism in the VDR gene was significantly connected with autoimmune thyroid diseases. Mao et al. [56] represented that the BsmI B allele may act as a risk factor for the onset of systemic lupus erythematosus (SLE) among Asians and overall populations and also the FokI FF genotype act as a potential risk factor for SLE predisposition in Asians. Furthermore, Tizaoui et al. [57] showed that the VDR TaqI and FokI gene polymorphisms may increase the risk of rheumatoid arthritis (RA) in European population . And finally, Wang et al.. [58] reported that the ApaI and BsmI gene polymorphisms were related with elevated susceptibility to type 1 diabetes (T1D) in Asian population. Collectively, it could be assumed that VDR gene polymorphisms act as a potential risk factor in the development or progression of autoimmune disorders.
Although four meta-analyses have been conducted over the course of past 10 years to evaluate the association between the VDR gene polymorphisms and MS, these findings were inconclusive due to the variations of the literature and selected databases. Hence, for resolving these inconsistencies, and to decrease the heterogeneity and the probability of random errors, we set out an updated meta-analysis. In this meta-analysis, 30 studies met the inclusion criteria and included quantitative synthesis. No evidence of publication bias for all four SNP in subgroup analysis and overall populations under five genetic models was observed. Regarding the essential role of genetic factors in the pathogenesis of MS, we categorized our results according to ethnicity. Our meta-analysis revealed that

Ethics approval and consent to participate
This study has been approved by ethic committee of Tehran University of Medical Sciences.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sector.

Supplementary Files
This is a list of supplementary files associated with the primary manuscript. Click to download.