The Most Common Vitamin D Receptor Polymorphisms (ApaI, FokI, TaqI, BsmI, and BglI) in Children with Dental Caries: A Systematic Review and Meta-Analysis

Vitamin D participates in the calcification of enamel and dentin and the appropriate immune responses to oral microbial infections. We aimed to assess the association between the most common vitamin D receptor (VDR) polymorphisms (ApaI, FokI, TaqI, BsmI, and BglI) and the risk of dental caries in children. Methods: PubMed/MEDLINE, Cochrane Library, Web of Science, and Scopus databases were comprehensively searched until 19 January 2021. Meta-analysis with odds ratios as the effect estimate along with 95% confidence intervals and subgroup analysis were conducted using Review Manager 5.3 software. Publication bias and sensitivity analyses were conducted by Comprehensive Meta-Analysis, version 2.0 software. Results: Seventy-eight studies were retrieved from the databases, with nine studies included in the final analysis. Based on five genetic models, there was no association between ApaI (rs7975232), TaqI (rs731236), BsmI (rs1544410), FokI (rs2228570), and BglI (rs739837) polymorphisms and susceptibility to dental caries, except for the FokI (rs10735810) polymorphism. Conclusion: Among the VDR polymorphisms considered, an association was found between the FokI (rs10735810) polymorphism and the risk of dental caries, with a protective role of the f allele and ff genotype.


Eligibility Criteria and Study Selection
The inclusion criteria were: (1) case-control studies focusing on the association between VDR polymorphisms and the risk of dental caries; (2) studies reporting VDR polymorphisms (ApaI (rs7975232), FokI (rs10735810), TaqI (rs731236), BsmI (rs1544410), FokI (rs2228570), and BglI (rs739837)) in children (age < 18 years); (3) dental caries confirmed by clinical examinations; (4) studies reporting the frequencies of alleles or genotypes; and (5) a control group with no tooth decay. Reviews, conference papers, and studies with no control group or those among adults or reporting other polymorphisms of VDR were excluded. The data from published studies were retrieved independently by two authors (M.S. and R.S.) to retrieve the necessary information. In case of discrepancies between the data extracted by the two authors, a duplicate data extraction was performed by a third author (M.G.).

Quality Assessment
Three reviewers (M.S., A.K., and N.N.) independently assessed the quality of the selected studies by scoring them according to Table 2. We developed a quality assessment tool specifically for this study, which consisted of 7 criteria. The range of scores varies from 0 to 11, with higher scores indicating better study quality.

Statistical Analysis
The association between polymorphisms and dental caries susceptibility was calculated by odds ratios (ORs) with 95% confidence intervals (CIs) based on five genetic models (allele, homozygote, heterozygote, recessive, and dominant models). To calculate heterogeneity, a chi-square-based Q test and the I 2 statistic were used [27,28]. A p-value of > 0.10 and I 2 < 50% indicated that there was no heterogeneity between the studies. However, considering the diversity in the effect sizes and populations between the studies, we used a random effects model in all analyses. Subgroup analysis (based on ethnicity and genotyping method) and sensitivity analysis ("one study removed" and "cumulative analysis") were applied to find the effect of subgroups on the overall results and the stability of results, respectively. Funnel plots were used to determine publication bias. The p-value of (two-sided) < 0.05 was considered significant, but the size of the effect was also taken into consideration to determine the association between the polymorphism and dental caries. The forest plots and subgroup analysis were conducted by Review Manager 5.3 (RevMan 5.3) software, while publication bias and sensitivity analyses were performed using Comprehensive Meta-Analysis version 2.0 (CMA 2.0) software. The polymorphisms (ApaI (rs7975232), FokI (rs10735810), TaqI (rs731236), BsmI (rs1544410), FokI (rs2228570), and BglI (rs739837)) were demonstrated to not be in strong linkage disequilibrium (LD) with each other (r 2 < 1) using the LDlink online tool (https://ldlink.nci.nih.gov) (accessed on 6 November 2020) [29], and therefore all polymorphisms were included in the present meta-analysis.

Study Selection
Seventy-eight studies were retrieved from the databases (Figure 1). After removing and excluding duplicate and irrelevant records, 14 full texts were evaluated for eligibility. Then, five full-text articles were excluded for different reasons: one article was a systematic review, one article had no control group, one article reported other VDR polymorphisms, and two articles reported VDR polymorphisms in adults. At last, nine studies were included in the qualitative and quantitative analysis.  Flowchart of the study selection. * One article was a systematic review. One article had no control group. One article reported other vitamin D receptor (VDR) polymorphisms. Two articles reported VDR polymorphisms in adults.

Quality Assessment
The seven criteria used for quality assessment are shown in Table 2. The maximum possible score was 11, while the minimum was 0. Table 3 shows the characteristics of nine studies included in the meta-analysis [21,[30][31][32][33][34][35][36][37]. Out of nine studies, three each were reported from China [21,36,37] and Brazil [31,33,35], and one each from Turkey [32], Czech Republic [34], and India [30]. There were three studies each on Caucasian, Asian, and mixed ethnic participants. The source of the control was population-based/school-based in all studies. Abbreviations: PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism.

Meta-Analysis
There was no association between the TaqI (rs731236) polymorphism and susceptibility to dental caries based on the five genetic models (Table 7). 76%], respectively. Although the effect estimates for homozygote, heterozygote, and recessive models were >1, these estimates were derived from only one study each, and wider confidence intervals indicate that the sample sizes in these studies were very small. These findings indicate that there was no association between the BsmI (rs1544410) polymorphism and susceptibility to dental caries.    Table 9 demonstrates that there was no association between the FokI (rs2228570) polymorphism and susceptibility to dental caries and there was a lack of heterogeneity between the studies (I 2 = 0%) in all five genetic models. The odds ratio for most of these models was closer to 1, with narrow confidence intervals indicating no association.  (Table 10). There was no association between the BglI (rs739837) polymorphism and susceptibility to dental caries. Table 10. The results from meta-analysis of the association between BglI (rs739837) polymorphism and dental caries risk based on five genetic models.

Subgroup Analysis
As there was an adequate number of studies on the TaqI (rs731236) polymorphism, subgroup analyses in relation to ethnicity and genotyping were conducted (Table 11). The overall effect still remained insignificant with none of the subgroups demonstrating any association between the TaqI (rs731236) polymorphism and susceptibility to dental caries across the five genetic models. Abbreviations: OR, odds ratio; CI, confidence interval.

Sensitivity Analysis
We conducted "cumulative analysis" and "one study removed" analyses to evaluate the stability of the findings related to six polymorphisms. The results show that the results were consistent/stable for the six polymorphisms. Additionally, for the TaqI (rs731236) polymorphism, we removed two studies [34,36] reporting an HWE deviation in the control group and found that the pooled ORs still remained the same.

Publication Bias
The funnel plots ( Figure 2) and p > 0.05 for both Egger's and Begg's tests demonstrate a lack of publication bias with regard to all six polymorphisms considered in this review.

Discussion
The present meta-analysis evaluated the association between VDR polymorphisms (ApaI (rs7975232), FokI (rs10735810), TaqI (rs731236), BsmI (rs1544410), FokI (rs2228570), and BglI (rs739837)) and the risk of dental caries in children. None of the polymorphisms were associated with the risk of dental caries, except for the FokI (rs10735810) polymorphism, with the f allele and ff genotype of this polymorphism having a protective role in dental caries occurrence.
The role of genetic factors in the risk of dental caries is still largely unknown despite numerous studies. Dental caries is a multifactorial disease caused by interactions between environmental factors, behavioral factors, several genetic factors, and gene-environment interactions [31]. Advances in transcriptional research have provided a variety of data on the interaction between VDR and other transcriptionally active proteins, demonstrating the potential of VDR to exert a wide range of biological reactions [38]. Vitamin D is known as a modulator of calcium homeostasis and plays an important role in regulating electrolytes and blood pressure. Evidence has shown that the most active metabolite of this vitamin can regulate the immune response and also has anti-inflammatory activity [39]. VDR gene polymorphisms have been shown to be strongly related to mineral density [32,40,41] and a meta-analysis [42] confirmed this. Although results from individual studies remain inconsistent, a meta-analysis of controlled clinical trials showed that early vitamin D supplementation could reduce the risk of dental caries by 47-54% [20]. Although the mechanism of action is unknown, VDR gene polymorphisms could modulate the effect of vitamin D supplementation. For instance, one study found some VDR polymorphisms to modify the association of vitamin D supplementation with the risk of a specific type of cancer [43]. The role of VDR polymorphisms in modifying the effect of vitamin D supplementation on dental caries needs further exploration.
VDR plays an important role in regulating the expression of genes associated with the immune response, calcium homeostasis, and cell differentiation and proliferation [18]. The distribution of VDR polymorphisms could show different patterns based on ethnicities and age [44][45][46][47]. Research has shown ethnic differences in vitamin D status and their correlation to hormonal homeostasis and bone phenotype, as well as the influence of environmental factors such as lifestyle, diet, and sun exposure [17,18]. However, we could not find any differences based on ethnicities in this meta-analysis.
Our meta-analysis showed a protective role of the FokI (rs10735810) polymorphism on dental caries. This might be due to its interactions with co-transcription factors [18] and its location ( Figure 3) [18,48]. riety of data on the interaction between VDR and other transcriptionally active proteins, demonstrating the potential of VDR to exert a wide range of biological reactions [38]. Vitamin D is known as a modulator of calcium homeostasis and plays an important role in regulating electrolytes and blood pressure. Evidence has shown that the most active metabolite of this vitamin can regulate the immune response and also has anti-inflammatory activity [39]. VDR gene polymorphisms have been shown to be strongly related to mineral density [32,40,41] and a meta-analysis [42] confirmed this. Although results from individual studies remain inconsistent, a meta-analysis of controlled clinical trials showed that early vitamin D supplementation could reduce the risk of dental caries by 47-54% [20]. Although the mechanism of action is unknown, VDR gene polymorphisms could modulate the effect of vitamin D supplementation. For instance, one study found some VDR polymorphisms to modify the association of vitamin D supplementation with the risk of a specific type of cancer [43]. The role of VDR polymorphisms in modifying the effect of vitamin D supplementation on dental caries needs further exploration.
VDR plays an important role in regulating the expression of genes associated with the immune response, calcium homeostasis, and cell differentiation and proliferation [18]. The distribution of VDR polymorphisms could show different patterns based on ethnicities and age [44][45][46][47]. Research has shown ethnic differences in vitamin D status and their correlation to hormonal homeostasis and bone phenotype, as well as the influence of environmental factors such as lifestyle, diet, and sun exposure [17,18]. However, we could not find any differences based on ethnicities in this meta-analysis.
Our meta-analysis showed a protective role of the FokI (rs10735810) polymorphism on dental caries. This might be due to its interactions with co-transcription factors [18] and its location ( Figure 3) [18,48]. The meta-analysis has several limitations and strengths. Limitations include the presence of fewer published reports on this topic hindering the performance of any meta-regression analysis, studies with small sample sizes, and clinical and statistical heterogeneity between the studies. Some studies included in the meta-analysis did not match The meta-analysis has several limitations and strengths. Limitations include the presence of fewer published reports on this topic hindering the performance of any metaregression analysis, studies with small sample sizes, and clinical and statistical heterogeneity between the studies. Some studies included in the meta-analysis did not match cases with controls, used genotyping methods different from other studies, and had controls with a deviation of the HWE. It also needs mentioning that we could not conduct any analysis to adjust the effect of multiple testing or multiplicity within the included studies. Despite the limitations, this review demonstrates several strengths in the form of the lack of publication bias, the suitable quality of all the included studies, and the use a population-based source for recruiting controls in all the studies. More studies on larger sample sizes and different ethnicities will help to explore the influence of different VDR polymorphisms on the risk of dental caries.

Conclusions
Out of the six VDR polymorphisms explored in this meta-analysis, an association was only observed between the FokI (rs10735810) polymorphism and the risk of dental caries, with the f allele and ff genotype demonstrating a protective role in the occurrence of dental caries.