Impact of Vitamin D Receptor Gene Polymorphism on Systemic Lupus Erythematosus Susceptibility: A Pooled Analysis.

Background: This study was designed to evaluate the influence of vitamin D receptor (VDR) gene polymorphisms on systemic lupus erythematosus (SLE) susceptibility. Methods: All eligible investigations were identified, the number of the various genotypes in the case and control groups were reviewed. A pooled analysis was performed using the Stata software. The study was carried out according to the Ethics Review Committee of The Third Xiangya Hospital, Central South University. Results: This meta-analysis included 19 studies. In our analysis, the VDR Apal polymorphism was correlated with SLE susceptibility in the overall population (AA vs. aa: odds ratio [OR] = 1.374, 95% confidence interval [CI]: 1.115-1.692, p = 0.003; AA + Aa vs. aa: OR = 1.342, 95% CI: 1.139-1.583, p < 0.01). The VDR Bsml and Apal polymorphisms were correlated with SLE susceptibility in Caucasian subjects (BB vs. Bb + bb: OR = 0.734, 95% CI: 0.593-0.909, p = 0.005; B vs. b: OR = 0.865, 95% CI: 0.760-0.983, p = 0.026; AA vs. aa: OR = 1.329, 95% CI: 1.016-1.740, p = 0.038). The VDR BsmI and FokI polymorphisms were correlated with SLE in African subjects (B vs. b: OR = 1.898, 95% CI: 1.458-2.470, p＜0.01; BB + Bb vs. bb: OR = 2.935, 95% CI: 1.944-4.430, p < 0.01; FF vs. Ff + ff: OR = 2.424, 95% CI: 1.673-3.512, p < 0.01; F vs. f: OR = 1.720, 95% CI: 1.417-2.087, p < 0.01; FF vs. ff: OR = 3.154, 95% CI: 2.083-4.774, p < 0.01; FF + Ff vs. ff: OR = 1.803, 95% CI: 1.363-2.384, p < 0.01). In addition, the VDR Apal polymorphism was correlated with SLE in female subjects (AA vs. aa: OR = 1.392, 95% CI: 1.049-1.849, p = 0.022) when stratified by gender. But there was no association between the VDR TaqI polymorphism and SLE susceptibility in our analysis. Conclusions: The VDR Apal polymorphism was associated with SLE susceptibility in general populations; in addition, Apal polymorphism was associated with SLE in female subjects. The VDR Bsml gene polymorphism was correlated with SLE susceptibility in Caucasian and African populations, whereas the VDR FokI polymorphism was correlated with SLE in African populations. But there was no association between the VDR TaqI polymorphism and SLE susceptibility in our analysis.


Introduction
Systemic lupus erythematosus (SLE) is an autoimmune disease that affacts our whole body. Many pathological processes are involved in the development of this disease, including immunoregulatory disorders, the emergence of autoantibodies and immune complexes that are resistent to autoantigens,leading to chronic damage in multiple systems 1 , which is easy to occur in women of childbearing age, and the peak age of diagnosis is 15-44 years old. The exact cause of SLE is currently unclear and is generally considered to be a result of a combination of host gene susceptibility and environmental factors 2 . The prevalence of first-degree relatives in SLE patients was 5.6%, the prevalence of fraternal twins was 2%-5%, while the prevalence of identical twins was 29%-57% 3 .
These genetic studies indicate that genetic susceptibility plays an important role in the occurance of SLE. With the progress of genome-wide sequencing, more than 40 loci associated with the susceptibility of SLE have been screened 4 . Among them, vitamin D receptor (VDR) gene, which is considered to be involved in the pathogenesis of SLE, is becoming a recent research hotspot.
As a novel immunomodulator ,vitamin D play an important role in regulating both innate immune responses and adaptive immune responses, such as mononuclear-macrophage chemotactic phagocytosis, immune tolerance, lymphocyte proliferation and differentiation, antibody secretion and other processes 5 . Vitamin D exerts its biological effects through binding with VDR, which is located on the 12q13-14, the VDR gene includes 11 exons and 8 introns 6 . Researchers have discovered over 470 SNPs in human VDR gene 7 . Although the VDR gene polymorphism does not cause an exact hereditary disease, it affects the susceptibility, severity, and response to treatment of various diseases 8,9 . The VDR gene polymorphisms associated with SLE mainly focuses on four loci of FokI (rs2228570), Bsml (rs1544410), ApaI (rs7975232) and Taql (rs731236). Researchers have reported that VDR polymorphism was correlated with SLE susceptibility, but due to factors such as heterogeneity, sample size, and environmental or regional differences in the study population, these results are always inconsistencies. In 2014, Zhou et al performed a meta-analysis including13 trials.They found that B and bb of BsmI, f and ff of FokI were correlated with SLE susceptibility in general populations and in Asian populations 10 , but not in Caucasian populations. Not long ago a study revealed that the VDR Bsml polymorphism was significantly correlated with SLE susceptibility in Chinese populations 11 , but not in Indian populations 12 . As the trial number of these analysis were not enough, we did this meta-analysis which included more trials to explore whether VDR polymorphism was correlated with SLE susceptibility.

Data retrieval and Quality evaluxation
We extracted the information below from included trials: first author's surname, publication date, study population, the ethnicity, the genetic locus of VDR, the method of gene detection, the number of cases and control groups, genotype distribution.
Consolidate the above information into a chart. These included studies were all case-control study.We evaluated the quality of these included literatures by Newcastle-Ottawa scale(NOS) quality score 14 .

Statistical analysis
We used stata (version 12.0,Stata Corp., College Station, TX, USA) to analyze data. We used gene counting method to calculate the frequency of the VDR polymorphism gene in each of the literatures. The four loci of VDR genes were studied (Meta-analysis of polymorphisms in Bsml, FokI, ApaI, and TaqI) and four different genetic models were

Publication bias and sensitivity analysis
Publication bias was tested by Egger's linear regression method (significance level at P < 0.05) and Begg's funnel plotting method. When there was publication bias, the results were adjusted by subtractive method 15 . Sensitivity analysis was performed after removing studies with the quality of the control group was poor and the control group did not meet the HWE test, and the results were observed to evaluate the stability of the results.

Literature search results and basic characteristics of inclusion studies
As was shown in Figure. 1, at first there were 93 studies that meet the search strategy, after elaborately screening, 29 papers were excluded and 64 articles were preserved, then 45 articles were excluded for all kinds of reasons and there were 19 trials preserved in the end 11,12,16-32 . Table 1 showed the basic characteristics and quality evaluation of the included studies.17 articles were written in English, while 2 were in Chinese, all of which were from 10 different countries. We included 2663 patients and 3252 controls. Table 1 revealed that the quality of these studies were moderate. Table 2 presented the number of various genotypes in both groups in each study.

The correlation between VDR BsmI polymorphism and SLE vulnerability
The forest plot in Figure 2 Figure 2 and Table 3). When stratified by race,  OR=1.898, 95% CI=1.458-2.470 P=0.000; P=0.000, Figure 2 and Table 3), However, no link was detected in Asian populations. When stratified by gender,no link was detected in female populations ( Table 3).

The link between VDR FokI Polymorphism and SLE vulnerability
As shown in Figure 3 11 Figure 3 and Table 3 Figure 3 and Table 3), while no link was found in Asian populations and in Caucasian populations. When stratified by gender, No link was found in female populations.

The correlation between VDR TaqI polymorphism and SLE vulnerability
As shown in Figure 5 11 Figure.5 and Table 3). No higher link was detected in any ethnicity or gender subgroup analysis.

Publication bias and sensitivity analysis
When the number of studies included was more than 6, we tested its publication bias. And it was found that VDR ApaI among overall populations had publication bias( AA+Aa vs aa genotype: P=0.029; AA vs aa genotype: P=0.017, Figure. 6). The results before and after adjustment by the clipping method were similar, indicating that this meta-analysis results are stable ( Table 4). The sensitivity analysis was performed after removing the study with poor quality and non-satisfaction of the HWE test. The difference between the OR value and the 95% CI change was not statistically significant, indicating a stable analytical result (Table 5).

Discussion
In this analysis, we included 2633 SLE patients and 3252 controls aiming to figure out whether the VDR gene polymorphism was correlated with SLE vulnerability. We found that the VDR BsmI, FokI, ApaI polymorphism was correlated with SLE vulnerability while VDR TaqI was not.
In the past,researchers have already done similar meta-analysis to explore whether VDR gene polymorphism was correlated with SLE vulnerability 10,33-35 . These pooled analysis showed that there was a link between VDR BsmI and FokI polymorphism and SLE susceptibility in Asian populations but not in Caucasian populations. In line with this, we found that VDR BsmI and FokI polymorphism could affect SLE susceptibility. However, our results also indicated that there was a link between VDR ApaI polymorphism and SLE susceptibility in Asian / Caucasian populations. In particular, for the first time we reported a link between VDR ApaI polymorphism and SLE susceptibility in female populations.
Compared with previous analysis,we included much more trials which might make our result more convincing.
The VDR is a 50,000-Da polypeptide 36 . VDR activation mediates a variety of "non-classical effects" that regulate cell growth, proliferation, apoptosis, and immune cell activation. It was reported that there was a correlation between VDR polymorphisms and many diseases like metabolic syndrome 37 , diabetes mellitus 38 , rheumatoid arthritis and rheumatoid- Some limitations in the context need to be pointed out. First, there were too few samples included in our analysis. When the study population is subgroup analyzed, the number of studies in some subgroups is small, which may lead to unreliable results. Second, heterogeneity is one of the foundations for assessing the reliability of meta-analysis results. Although our meta-analysis explains the source of partial heterogeneity through subgroup analysis, the heterogeneity between studies remains objective due to differences in study time, study area, and sample size. Last, due to limited data, we can only find differences between VDR polymorphisms and SLE susceptibility in different ethic populations and in female groups, while differences in other subgroups (e.g. male, adolescent or Juvenile SLE) require further study.

Conclusion
VDR ApaI polymorphism was associated with SLE susceptibility in general populations, in addition, ApaI polymorphism was associated with SLE in female subjects. VDR BsmI gene polymorphism were correlated with SLE susceptibility in Caucasian and African populations. While VDR FokI polymorphisms was correlated with SLE in African populations. But there was no association between VDR TaqI polymorphism and SLE susceptibility in our analysis.

Author Contributions
Na Liu, Shi-kun Yang and Ying-qiu Zhu analyzed the data for the manuscript and wrote the manuscript. Shi-kun Yang, Na Song, Wei-juan Zhang and Jin-ping Yang performed the literature search. Hao Zhang and Ming Gui edited the manuscript. Na Liu and Shi-kun Yang had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.  Tables   Table 1. Characteristics of studies included in the meta-analysis.   Figure 1 The study selection for eligible trials