Vitamin D receptor (VDR) gene FokI, BsmI, ApaI, and TaqI polymorphisms and osteoporosis risk: a meta-analysis

Osteoporosis is a disease of the bones in which the density of the bones decreases. The prevalence of this disease greatly varies in different populations of the world. Numerous studies have been investigated VDR gene polymorphisms as osteoporosis risk in different ethnic groups. In present meta-analysis, the aim is to find out the role of VDR gene polymorphisms (FokI, BsmI, ApaI, and TaqI) in osteoporosis risk. Suitable case-control studies for present meta-analysis were retrieved from four electronic databases. Open Meta-Analyst program was used for statistical analyses. Studies investigated BsmI (65 studies; 6880 cases/8049 controls), ApaI (31 studies; 3763 cases/3934 controls), FokI (18 studies; 1895 cases/1722 controls), and TaqI (26 studies; 2458 cases/2895 controls) polymorphisms that were included in the present meta-analysis. A significant association was found between the dominant model of FokI (ORff + Ffvs.FF = 1.19, 95% CI = 1.04–1.36, p = 0.01, I2 = 39.36%) in the overall analysis and recessive model of the Caucasian population of TaqI polymorphism (ORTT + Ttvs.tt = 1.35, 95% CI = 1.11–1.63, p = 0.002, I2 = 50.07%) with osteoporosis. On the other hand, no such effect is found in any other genetic models and in any other gene polymorphisms of the overall analyses or sub-group analyses. In conclusion, the authors found that the dominant model of FokI in the overall analysis and recessive model of TaqI in the Caucasian population are significantly associated with the development of osteoporosis.


Background
Bone is an active tissue that maintains itself by continuous formation and reabsorption [1]. Osteoporosis is a condition in which the density of the bone decreases due to the increased activity of the osteoclasts [2]. A great variance is observed in the prevalence of osteoporosis in different ethnic groups [3]. Age and gender are the two major contributing factors in the occurrence of osteoporosis. Worldwide, one out of three women over the age of 50 experiences osteoporotic fractures in comparison to one in five men of the same age group [4].
Genetic and environmental factors play a crucial role in the etiology of osteoporosis [5,6]. Calcium intake and exercise are the main risk factors for osteoporosis [5]. It is very well established that along with the environmental factors, individual genetics plays a key role in the development of osteoporosis, e.g., (i) low bone density is found in the female offspring of the osteoporotic women [7], (ii) male offspring of idiopathic osteoporotic men have low bone mineral density [8], and (iii) studies of female twins have shown heritability of bone mineral density (BMD) to be 57 to 92% [9,10].
Amongst all the genes studied in osteoporosis, the vitamin D receptor (VDR) gene polymorphism is the most important in the etiology of the disease [11,12]. VDR gene polymorphisms have been reported to be associated with the development of several bone diseases, multiple sclerosis, vitamin D-dependent rickets type II, and other complex diseases [13]. However, the mechanism by which the VDR gene influences bone mass has not been fully elucidated.
In human, VDR gene is found on the chromosome 12 (12q12-q14) with 11 exons and spans~75 kb genomic DNA. The most studied VDR gene polymorphisms are BsmI, ApaI, FokI, and TaqI. Although several studies between osteoporosis and VDR gene polymorphisms have been published, the results are contradictory [14,15]. This may be due to the differences in the designing of the studies, less number of samples, differences in ethnicities, or various other environmental factors. So, the aim of the present study was to find an association between VDR gene polymorphisms and osteoporosis risk.

Methods
Different databases (PubMed, Google Scholar, Springer-Link, and Science direct) were searched up to December 31, 2018, with the keywords "vitamin D receptor gene," "BsmI," "ApaI," "FokI," "TaqI," and "VDR," along with "osteoporosis." The retrieved studies were conducted between 1995 and 2018, and we examined all the retrieved papers thoroughly to determine their suitability for inclusion in the current meta-analysis.

Inclusion and exclusion criteria
Studies found suitable to be included in the present study should have (a) a case-control study and (b) reported the sample size and distribution of genotypes. Similarly, a study should be excluded if (a) the study was conducted on the animal model, (b) the study that has replication of data, (c) only cases were reported, and (d) book chapters or review articles.

Data extraction
From the selected articles, we extracted different information like (a) last name of the first author, (b) year of publication of the study, (c) country where the study was conducted, and (d) number of genotypes in different groups. We also checked whether the genotype distributions of control population of all the included studies were in agreement with Hardy-Weinberg equilibrium (HWE) by using the goodness of fit chi-squared test. All the data from the different papers were retrieved by the two authors (UY and PK) and if any discrimination was found, it was resolved by the consultation with the corresponding author.

Statistical analysis
Meta-analysis was done according to the method given in Rai et al. [16]. Briefly, statistical analysis of different vitamin D receptor gene polymorphisms and risk of osteoporosis were estimated by pooling the odds ratio (OR) with its corresponding 95% confidence intervals (CI). Heterogeneity was tested using Q statistics (a p value of less than 0.05 was considered significant). The I 2 statistics was also used to assess the discrepancy between studies. If the heterogeneity was higher (p value of Q test < 0.05 or I 2 > 50%) than the random effect model [17] that was applied, fixed effect model [18] was used. The heterogeneity may arise due to the differences in ethnicities or variation in study design or outcome. The funnel plot of precision by log odds ratio and standard error by log odds ratio was assessed for the possible publication bias, and if the funnel plot was found asymmetric, it denoted a publication bias [19]. The linear regression method of Egger was used to measure the asymmetry in the funnel plot [20], and a statistically significant publishing bias was considered to be a p value of < 0.05. The meta-analysis was conducted by Open Meta-Analyst program [21].

PRISMA (Preferred Reporting Items for Systematic
Reviews and Meta-Analyses) guideline was followed in the present meta-analysis. Flow chart of article selection was shown in Fig. 1 with specific reasons. Eighty-one studies were found to be eligible for

Eligible studies
For BsmI, a total of 65 studies with 6880 cases and 8049 controls were included in the meta-analysis .

Meta-analysis
Ethnicity was used for the sub-group analysis. Out of 65 studies, 37 belong to Caucasians, 22 were Asian, and 6 were of other origins. High heterogeneity was observed in all genetic models in all sub-groups. No significant association was found in any sub-group analyses in any genetic models (Table 1; Fig. 2). The ethnicity-based sub-group analyses were conducted. Out of 31 studies, 15 were Caucasians, 12 were Asians, and 4 were of other origin. High heterogeneity  Fig. 4). Studies were further analyzed by sub-group analysis on the basis of ethnicity. Out of 18, ten studies belong to Caucasians, five were Asians, and three were of other ethnicity. High heterogeneity was found in Asian and other studies; while in the Caucasian studies, low heterogeneity was observed. No significant association was found in any sub-group in any genetic model (Table 3; Fig. 4).

TaqI meta-analysis
High heterogeneity with insignificant association was found in the allele contrast model of TaqI polymorphism  (Table 4; Fig. 5).
The studies were further analyzed on the basis of ethnicity for sub-group analysis. Out of 26 studies, 17 belong to Caucasians, six were Asians, and three were of other ethnicity. High heterogeneity was observed in all groups, i.e., Asian, Caucasian, and other studies. Insignificant results were found in all the sub-groups of all the genetic models except for the recessive model of the Caucasian population (TT + Tt vs. tt) OR = 1.35, 95% CI = 1.11-1.63, p = 0.002 (Table 4; Fig. 5).

Sensitivity analysis
To conduct sensitivity analysis, all the studies deviated from the Hardy-Weinberg equilibrium (p < 0.05) were omitted. In BsmI, 21 studies [27, 30, 34, 38, 39, 44, 48- When these 21 studies were removed, heterogeneity was decreased in both the overall and in the sub-group meta-analyses except in the Asian studies.
In total of 18 FokI studies, control population in five studies [56,70,79,99,100] was not in HWE. When these studies were removed from the analysis, insignificant association was found in the main analysis (OR fvs.F = 1.12, 95% CI = 0.99-1.26, p = 0.05, I 2 = 46.48%), and no association was found in any sub-group. Removal of these studies decreases the heterogeneity both in the overall and in sub-group meta-analyses.
The control samples of nine ApaI studies [28,30,44,48,51,56,71,83,94] were not in HWE. Result of metaanalysis after removal of these nine studies showed no association between ApaI polymorphism and osteoporosis risk in the main/overall analysis (OR avs.A = 1.07, 95% CI = 0.90-1.27, p = 0.39, I 2 = 73.94%) and  [28,56,77,101] were deviated from the HWE. Results of meta-analysis of 22 studies (after elimination of 4 studies deviated from HWE) did not show any association between TaqI polymorphism and osteoporosis risk either in total studies (OR tvsT = 1.05, 95% CI = 0.85-1.29, p = 0.63, I 2 = 78.86%) or in any sub-group. Moreover, after removal of these 4 studies, there was an increase in the heterogeneities in overall and sub-group meta-analyses except the Asian population.

Publication bias
In all the genetic models in the overall and in sub-group meta-analyses for all polymorphisms, the funnel plots were symmetrical ( Fig. 6; Tables 1-4 (Tables 1-4).

Discussion
The vitamin D receptors are the members of the nuclear hormone receptor (NR1I) family and expressed in different organs like the intestine, thyroid, and kidney in humans [103]. It is primarily responsible for the endocrine action of vitamin D that regulates calcium homeostasis and reduces the risk of osteoporosis. VDR is translocated from the cytoplasm to the nucleus when activated by binding of its ligand 1α,25-dihydroxyvitamin D 3 (1, 25(OH) 2 D 3 ) [104]. Several studies have documented that the onset of osteoporosis is caused by VDR gene polymorphisms [81]. VDR gene polymorphisms are also associated with other diseases like breast cancer [105], diabetes [106], myocardial infarction [107], and metabolic syndrome and inflammation [108].
Meta-analysis is a well-established statistical tool used for combining the data of small sample-sized individual studies. Meta-analysis increases the power of the study and decreases type I and II errors. During the past two decades, a number of meta-analyses were published which assessed the polymorphism of small effect genes as risk factor for different diseases and disorders, e.g., Down syndrome [16], neural tube defects [109], Glucose 6-phosphate dehydrogenase deficiency [110], depression [111], schizophrenia [112], Alzheimer [113], breast cancer [114], colorectal cancer [115], esophageal cancer [116], and prostate cancer [117]. During literature search, we identified seven metaanalyses [15,[118][119][120][121][122][123] investigating the relationship between VDR gene polymorphisms and osteoporosis.
BsmI, ApaI, FokI, and TaqI polymorphisms were included in seven, four, two, and two meta-analyses respectively. BsmI polymorphism studies were included in all seven meta-analyses. In six meta-analyses, no significant association was found between osteoporosis susceptibility and BsmI polymorphism [15,[118][119][120][121][122]. Zhang et al [123] conducted a meta-analysis of the risk of osteoporosis in postmenopausal women with 36 studies including 7192 subjects and found a marginally significant association (OR bvs.B = 1.2; CI = 1.00-1.46; p = 0.052). In all the meta-analyses, a low between study heterogeneity was found in all the studies except the study conducted by Yu et al [120]. ApaI polymorphism was included in four meta-analyses [118,120,122,123]. Zintzaras et al [118], Yu et al [120], Wang et al [122], and Zhang et al [123] included seven, six, three, and eighteen studies, respectively, in their meta-analyses, and all four studies reported no association between ApaI polymorphism and osteoporosis risk. Zintzaras et al [118] and Zhang et al [123] conducted meta-analyses of three and 18 studies of FokI polymorphism, and no significant association was found between FokI polymorphism and osteoporosis. Both groups [118,123] also conducted meta-analyses of TaqI polymorphism studies and again reported no association between TaqI polymorphism and osteoporosis susceptibility.
In the present meta-analysis, four common VDR gene polymorphisms (BsmI, ApaI, FokI, and TaqI) were included. A total of 65 (14929 samples), 31 (7697 samples), 18 (3617 samples), and 26 (5353 samples) studies for BsmI, ApaI, FokI, and TaqI polymorphisms, respectively, were included. We found a significant association in the dominant model of FokI polymorphism (ff + Ff vs. FF OR = 1.19, 95% CI = 1.04-1.36, p = 0.01) with low heterogeneity (I 2 = 39.36). No association was found in sub-group analysis on the basis of ethnicity in any genetic model except in the Caucasian population in the recessive model of TaqI polymorphism (TT + Tt vs. tt OR = 1.35, 95% CI = 1.11-1.63, p = 0.002) with moderate heterogeneity (I 2 = 50.07). The frequency of different VDR gene polymorphisms varies in different ethnic/regional populations. Due to this, the effect of these polymorphisms might vary from population to population.
The present meta-analysis has few demerits like (i) used crude odds ratio, (ii) only genetic polymorphisms considered, and other factors such as environmental factors or food habits that are not included which might have important roles in the etiology of osteoporosis. With these limitations, the present study has some strength like (i) this is the largest meta-analysis conducted both in number of included studies and number of sample size (81 studies; 19268 samples) and (ii) included all common VDR polymorphisms (BsmI, ApaI, FokI, and TaqI).

Conclusion
In conclusion, we found that the dominant model of FokI polymorphism is associated with osteoporosis, and also the recessive model of TaqI polymorphism is a risk factor for the osteoporosis in the Caucasian population. The other polymorphisms (BsmI and ApaI) have no role  in the osteoporosis in total or in the stratified populations. In addition, it has been suggested that different gene-gene and gene-environment interactions should also be considered in future case-control studies, which could clarify the genetics of osteoporosis. Acknowledgements Upendra Yadav is highly grateful to the VBS Purvanchal University, Jaunpur, for providing financial assistance to him in the form of PDF.
Authors' contributions UP and PK have retrived articles , both have extracted data from the included studies, and VR and UY written the manuscript. All author(s) have read and approved the manuscript.

Funding
There was no funding for this review.

Availability of data and materials
The data and materials will be available with the corresponding author upon reasonable request.

Ethics approval and consent to participate
The article does not contain any studies with human or animal subjects performed by any of the authors.

Consent for publication
Not applicable.