VDR gene ApaI polymorphism and risk of postmenopausal osteoporosis: a meta-analysis from 22 studies

Abstract Objective The ApaI polymorphism (G > T, rs7975232) of the vitamin D receptor (VDR) gene in the risk of postmenopausal osteoporosis has been widely researched, and the results have yielded conflicts. Therefore, we performed an updated pooled analysis to comprehensively assess the association between VDR ApaI polymorphism and postmenopausal osteoporosis risk. Methods We searched eligible studies about ApaI polymorphism and osteoporosis through the PubMed, Embase, CNKI and Wanfang databases; case–control studies containing available genotype frequencies of A/a were chosen. We used the odds ratio with 95% confidence interval to assess the strength of this association. Sensitivity analysis and publication bias assessment were performed. Trial sequential analysis (TSA) was performed to evaluate a sufficient sample. Results Twenty-two studies assessed the relationship between ApaI polymorphism and the risk of osteoporosis in postmenopausal women. The comprehensive analyses showed no significant association for ApaI polymorphism with postmenopausal osteoporosis in the overall population, equally valid for Asian and Caucasian subgroups with any genetic model. TSA still indicated the results were robust. Conclusion The present meta-analysis suggests that the VDR ApaI genotype may not affect the risk of postmenopausal osteoporosis in Asians and Caucasians.


Introduction
Osteoporosis is a progressive bone disease characterized by decreased bone mass and density, bone microstructure deterioration and altered bone proteins.it leads to an increased risk of bone fracture [1,2].about 30% of postmenopausal females have osteoporosis [3].Bone fractures caused by postmenopausal osteoporosis are extremely harmful and are one of the leading causes of disability and death in older female patients [4][5][6].it is generally accredited that osteoporosis is related to individual genetic differences, estrogen levels, nutritional status and lifestyle.it also can be induced by bone formation and bone resorption disorders caused by physical injury, diseases affecting bone metabolism or long-term use of hormone drugs [7].researchers recently found that osteoporosis is a multi-gene regulated, hereditary disease [8,9].Many factors affecting the relationships between gene polymorphism, bone mineral density (BMD) and fracture have been intensively investigated, such as type i collagen gene, calcitonin receptor gene, low-density lipoprotein receptor-related protein gene, cannabinoid receptor genes and so on [10][11][12].the vitamin D receptor (VDr) exerts an essential role in calcium homeostasis and bone metabolism by regulating osteocyte growth and differentiation, intestinal calcium absorption and parathyroid hormone secretion, and is one of the significant candidate genes to explore the genetic factors leading to osteoporosis [13][14][15].the VDR gene is located on chromosome 12 (12q13.1),longer than 100 kb, and more than 100 polymorphic sites are predicted [16,17].Apai (rs7975232) is one of the most representative single-nucleotide polymorphisms of the VDR gene [18].Apai is located near the 3′ end of the VDR gene and has been reported to be in strong linkage disequilibrium.this polymorphism does not change the amino acid sequence of the encoded protein but may affect gene expression by regulating messenger rna stability [19,20].it has previously been reported to be associated with the risk of osteoporosis.However, the conclusions were inconclusive.in 1992, Morrison et al. found that BMD and circulating osteocalcin levels might be affected by VDR Bsmi and Apai polymorphism [21]. in 1997, Vandevyver et al. reported for the first time that VDR Apai polymorphism was associated with BMD in postmenopausal females [5].Since then, epidemiological investigations have been widely reported regarding the assessment of Apai polymorphism and the susceptibility of postmenopausal osteoporosis.However, the relevant research results have been controversial. in an earlier survey of the chinese population, compared to individuals with the 'a' allele, those with the 'a' allele had higher BMD at the femur among postmenopausal Han women with primary osteoporosis and osteoporosis associated with type 2 diabetes [22].However, no significant correlation was observed between Apai polymorphism and BMD in another chinese postmenopausal female group [23].Different results also existed in those research studies on the european population [24,25].racial and regional differences or a limited sample size might cause this.Meta-analysis is a powerful statistical analysis tool to integrate and analyze the data of published articles, expected to make a more accurate and objective assessment of the related research results [26]. in recent years, several meta-analyses have explored the relationship between Apai polymorphism and the risk of osteoporosis, with inconclusive results.a recent study suggested that Apai rs7975232 polymorphism was significantly associated with the risk of type i primary osteoporosis, namely postmenopausal [27].Unfortunately, the authors did not further analyze whether there were racial differences in this positive association. in another study, although an ethnicity-based subgroup analysis of the association between postmenopausal osteoporosis and Apai polymorphisms was conducted [28], the study included perimenopausal and comorbid samples, which may have affected the accuracy of the results.therefore, it is necessary to perform an updated meta-analysis on the currently published eligible case-control studies to comprehensively assess the association of the Apai polymorphism with the risk of postmenopausal osteoporosis.

Publication search
We searched electronic databases, including the PubMed, MeDline, eMBaSe, Weipu, cnKi and Wanfang databases, for all studies published up to august 2021 on the association between VDR Apai and postmenopausal osteoporosis risk.the search strategy included 'vitamin D receptor' or 'VDR' and 'osteoporosis' or 'fracture' in combination with 'genetic' or 'polymorphism' or 'variant' .

Criteria selection and data extraction
Studies containing rs7975232 associated with the risk of postmenopausal osteoporosis were included if they met all of the following criteria: the study had a case-control design; participants included postmenopausal women; the concrete number of genotypes; the study assessed the relationship between Apai and osteoporosis or fracture; and the study provided BMD values of the lumbar spine and femoral neck (BMD ≤ −2.5 standard deviations defined osteoporosis).review articles, case reports, comments, letters, conference reports and those that did not include concrete genotype data were excluded.all relevant studies were also searched and reviewed.if more than one article had been published using the same series of study subjects, we chose the one with the most participants.in this meta-analysis, 'postmenopausal' was identified primarily based on the Stages of reproductive aging Workshop (StraW)-10 of 2012 [29]; that is, 1 year after the last menstrual period.the perimenopausal sample in the studies was excluded (i.e.within 1 year of the last menstrual period) to avoid the effect of menopausal syndrome.the relevant information using a predesigned form included the first author, publication year, region, ethnicity, age, sample size, allele frequency of cases and controls, and genotyping method.

Quality assessment
We assessed the quality of these case-control studies using the newcastle-Ottawa Scale [30], including three aspects: selection method between the case group and control group; comparability between the case group and control group; and exposure evaluation method.two authors appraised quality independently.any different results were resolved by inviting a third author to award a consensus.Scores ranged from 0 (lowest) to 9 (highest).a study with a score ≥5 was regarded as a qualified study.

Statistical analysis
For the control group of each study, the observed genotype frequencies of the VDR Apai polymorphism were assessed for Hardy-Weinberg equilibrium (HWe) using the chi-squared goodness-of-fit test, and p < 0.05 was considered out of HWe.Odds ratios (Ors) and 95% confidence intervals (cis) accessed the strength of association between the Apai polymorphism and osteoporosis.the pooled Ors were performed for the allele model (a vs. a), dominant model (aa + aa vs. aa), recessive model (aa vs. aa + aa), heterozygote model (aa vs. aa) and homozygote model (aa vs. aa), respectively.a chi-square Q-test evaluated the heterogeneity assumption, and p < 0.05 indicated heterogeneity across studies.the summary Or estimate of each study was calculated by the fixed-effects model if there was no significant heterogeneity.Otherwise, the random-effects model was employed [31,32].the potential for publication bias was examined by Begg's test (funnel plot method) or egger's linear regression test [33], and two-sided p < 0.05 is considered statistical significance.the sensitivity analysis based on a one-by-one exclusion method was performed.We performed this meta-analysis using Stata software version 9.0 (Stata corporation, college Station, tX, USa). in addition, trial sequential analysis (tSa) was used to analyze the problem of random errors, such as false positives and negatives, in the repeated meta-analysis updates [34].We calculated the sample size needed to draw a definite conclusion by tSa 0.9.5.10 Beta software based on first type of error α = 0.05, second type of error β = 0.2 (power 80%) and a relative risk reduction of 20% [35,36].

Eligible studies
the study selection process for the meta-analysis is shown in Figure 1.Our meta-analysis identified 22 studies on the association between VDR Apai and osteoporosis, including 3642 osteoporosis cases and 3914 controls.Seventeen studies involved caucasians, and another five investigations involved asians.the main characteristics are presented in table 1. the newcastle-Ottawa Scale scores for the quality of case-control studies are presented in table 2, and all studies have scores ranging from 5 to 8. the observed genotype frequencies of the VDR Apai polymorphism in each control group were assessed by HWe, and six unequal studies were excluded (table 3).

Meta-analysis
the evaluations of the association between VDR gene Apai polymorphism and osteoporosis risk in postmenopausal women are summarized in table 4. the overall results suggested that there was no association between Apai polymorphism and the risk of postmenopausal osteoporosis in all genetic models (allele model: Or 0.943, 95% ci 0.804-1.105,p = 0.447; dominant model: Or 0.901, 95% ci 0.731-1.110,p = 0.328; recessive model: Or 0.947, 95% ci

Heterogeneity and sensitivity analyses
Several potential factors were excluded: literature quality, ethnicity, HWe and age.there was still significant heterogeneity in the overall comparisons and subgroup analyses.the sensitivity analysis based on a one-by-one exclusion method was performed.any single study could be omitted without any effect on the overall statistical significance, indicating that the results were stable.the results are shown in Figure 3. the regression meta-analysis was performed to explore further the source of heterogeneity, with the regional distribution as a covariable, and the heterogeneity still could not be reduced.We examined several studies with low consistency in sensitivity analyses individually.the heterogeneity was significantly lower (p > 0.1) after excluding three studies -Marozik et al. [24], Marozik et al. [37] and Wu et al. [38] -while the results of the meta-analysis had no effect and still supported no significant association between Apai polymorphisms and susceptibility to postmenopausal osteoporosis (see Supplementary material).

Publication bias
Publication bias was preliminarily examined by funnel plots and by egger's tests quantitatively.all graphical funnel plots of the included studies were symmetric, implying  that the publication bias was low in the overall meta-analysis.

Trial sequential analysis
We performed tSa to evaluate the accuracy of this meta-analysis.the analysis showed that the accumulated amount of the aa versus aa group had reached the required information size (n = 7926).Still, the Z curve did not cross the tSa boundary value and fell within the futility boundary, which means that our conclusions are robust with this sufficient evidence.the results are shown in Figure 4.

Discussion
this meta-analysis estimated associations between Apai polymorphism in the VDR gene and the risk of postmenopausal osteoporosis.the integrated analysis results demonstrated that Apai polymorphism was not significantly associated with the risk of postmenopausal osteoporosis in the overall population, and further subgroup analyses by ethnic groups showed a similar result.
Vitamin D plays a crucial role in calcium and phosphate homeostasis and skeletal metabolism, and its function is regulated by the VDR gene [39].the VDR gene has been widely studied because of its essential role in affecting susceptibility to osteoporosis by regulating bone metabolism and homeostasis [13,21,40].current studies on VDR gene polymorphism mainly correspond to the restriction sites of Bsmi, Apai, Taqi and Foki. the polymorphic loci of different regions had different effects on VDR gene expression.Bsmi and Apai were located in the eighth intron near the 3′ end, and their polymorphisms did not affect the amino acid sequence of VDR.Taqi is located at exon 9, a synonym mutation, and does not change the amino acid sequence.Foki is located at the 5′-end transcription initiation site, and its polymorphism can lead to changes in amino acid sequence length.current studies believe the polymorphic sites in the 5′-'end promoter region can affect the mrna expression pattern and expression level. in comparison, the polymorphic sites in the 3′-end untranslated region can affect mrna stability and protein translation efficiency.Such effects are related to the cell type, differentiation stage and activity state [41].gtex [42], eQtls [43] and other databases are used to analyze how these polymorphic sites affect mrna expression [44]. in recent years, several meta-analyses have focused on VDR gene Apai polymorphism and susceptibility to osteoporosis, but the results were inconsistent.in a pooled analysis of chinese individuals by Wang et al., no evidence of the relationship between the VDR Apai polymorphism and osteoporosis risk was observed with any genetic model [45].Yet another study by Ji et al. found that the vertebral fracture cases had a significantly higher frequency of the aa genotype of Apai in caucasians [18].
regional and racial differences are likely reasons for the different results.there are significant differences in VDR gene Apai allele frequencies in diverse populations (p < 0.001) [46].according to HapMap28 [47], gg homozygotes account for approximately 37% of caucasians compared to fewer than 10% of asians, suggesting that ethnic differences must be considered when assessing the prevalence in specific populations.in addition, in several studies addressing geographical trends in the incidence of osteoporotic fractures, changes in dietary structure [48], lifestyle [49] and environment [50] have been associated with BMD and fracture incidence [51].the Mediterranean diet is effective in preventing osteoporosis [52].the chinese diet, which includes a high amount of fruit, vegetables and soy, has also been associated with a lower fracture risk [53]. in contrast, traditional Western dietary patterns, refined and high in fat and carbohydrates, are more likely to lead to progressive bone loss [54], and the incidence of osteoporotic fractures is generally higher in europe and the USa [51]. in norway, older people at higher altitudes and inland from the coast are at higher risk of hip fracture than those in coastal areas, especially among older women, which may be related to temperature and climate change at different latitudes [55,56].low vitamin D synthesis (inadequate UVB exposure) also increases the risk of fracture in older adults [57].
We conducted this updated meta-analysis to more accurately assess the effect of VDR gene Apai polymorphism on the risk for osteoporosis in postmenopausal women.a total of 22 studies with 3642 osteoporosis cases and 3914 controls were included in this meta-analysis.to our knowledge, this is the latest updated meta-analysis for this polymorphic site.We found no significant correlations between VDR Apai and postmenopausal osteoporosis susceptibility in overall populations, and the same was valid for subgroup analysis.Several studies support our conclusions.Shen et al. found that the VDR Apai polymorphism was not associated with the risk of premenopausal osteoporosis for caucasians or asians after analyzing 14 case-control studies [58], and the earlier analysis by Zintzaras et al. involved seven studies that were consistent with ours [59].through sensitivity analysis and publication bias detection, the results of our meta-analysis were accurate and credible.considering that calcium is an essential nutrient for bone health, the guidelines also tip a balanced daily calcium intake as a dietary measure to reduce fracture risk [60].We focused on checking the details of the participants' nutritional habits in each study and determined that there was no difference in calcium intake between the case and control groups.
this meta-analysis covered almost all published eligible studies' original data and should be a valuable supplement to the related studies.However, there are still some limitations to this study.First, some confounding factors have yet to be analyzed, such as smoking, alcohol and other variables.Second, in subgroup analyses, studies focused on the chinese population may not have enough statistical power to assess the association between Apai and postmenopausal osteoporosis in asian women.Future studies with sizeable, diverse population composition will help validate our findings.in conclusion, our results support no genetic association between VDR Apai polymorphism and osteoporosis in asian and caucasian postmenopausal women.For a more accurate evaluation of the relationship between VDR gene polymorphism and postmenopausal osteoporosis, a multicenter, large-sample study is required, and the synergistic effects of various factors such as diet, environment, exercise and so on should be considered.
Potential conflict of interest no potential conflict of interest was reported by the authors.

Figure 1 .
Figure 1.Flow chart indicating the search results.
a p-Value for heterogeneity test.random-effects model was used when p < 0.05.Ci, confidence interval; Or, odds ratio; VDR, vitamin D receptor.Forest plots of all selected studies on the association between vitamin D receptor (VDR) Apai polymorphism and the risk of osteoporosis in the (a) allele model, (B) dominant model, (C) recessive model, (D) heterozygote model and (e) homozygote model.Ci, confidence interval; Or, odds ratio.

Figure 3 .
Figure 3. Sensitivity analysis of the association between vitamin D receptor (VDR) Apai polymorphism and postmenopausal osteoporosis risk.results were computed by omitting each study (left column) in turn.meta-analysis fixed-effects estimates (exponential form) were used.Bars, 95% confidence interval.

Figure 4 .
Figure 4. trial sequential analysis of the association between the vitamin D receptor (VDR) gene Apai polymorphism allele model and susceptibility to postmenopausal osteoporosis.the required information size was calculated based on a two-sided α = 5%, β = 15% (power 80%) and a relative risk reduction of 20%.m, mutation; tSa, trial sequential analysis; W, wildtype.

Table 1 .
main characteristics of studies included in the meta-analysis.

Table 3 .
Distribution of VDR Apai genotypes for osteoporosis cases and controls: Apai rs7975232 (G > t).

Table 2 .
results of quality assessment by the Newcastle-Ottawa Scale for case-control studies.

Table 4 .
Ors and 95% Cis for osteoporosis and the VDR Apai polymorphism under different genetic models.