Vitamin D receptor gene polymorphisms and susceptibility to urolithiasis: a meta-regression and meta-analysis

Background The currently available data with respect to the association between vitamin D receptor (VDR) gene polymorphism and risk to urolithiasis are inconclusive and inconsistent. Hence, an exhaustive meta-analysis can solve the discrepancies and provide a hint for upcoming investigations. Herein, a meta-analysis was carried out to attain a conclusive estimate of the association between VDR gene single nucleotide polymorphisms (SNPs) and urolithiasis risk. Methods The major databases, including ISI Web of science, Scopus, and PubMed/MEDLINE were searched systematically from until June 2020 to retrieve all relevant studies. Association between VDR gene polymorphisms, including FokI (rs2228570), TaqI (rs731236), BsmI (rs1544410), and ApaI (rs7975232), and urolithiasis risk was evaluated using pooled odds ratio (OR) and their corresponding 95% confidence interval (CI). Additionally, to seek for the potential source of heterogeneity, meta-regression analyses were exerted. Results Literature search led to finally finding of 33 studies evaluating the VDR gene SNPs and urolithiasis risk. It was observed that none of the four SNPs were significantly associated with urolithiasis predisposition. However, subgroup analysis confirmed higher risk of urolithiasis in East-Asian and Caucasian population with ApaI and TaqI gene polymorphism. The analyses of sensitivity acknowledged the results stability. Conclusion Although this meta-analysis did not support the association of FokI, TaqI, BsmI, and ApaI in the overall polled analysis, it suggests that ApaI and TaqI SNPs is associated with increased risk of urolithiasis in East-Asian and Caucasians populations.


Background
Urolithiasis is known as one of the prevalent diseases among urological disorders that has been associated with many complicated factors [1]. Urolithiasis is characterized by a high recurrence incidence, and its prevalence rate is 4-20% in developed countries, and the disease incidence continues to increase [2]. It is a multifactorial disorder, resulting from environmental influences, metabolic defects and genetic factors [3]. Numerous investigations recognized the importance of genes in this disorder.
Studies have shown that several genetic factors including single nucleotide polymorphisms (SNPs) in osteopontin (OPN), progestin and adiporeceptor 6 (PAQR6), calcium-sensing receptor (CaSR), and vitamin D receptor (VDR) are correlated with the risk of urinary calcium stone formation [4][5][6][7]. In spite of attribution of a genetic background in susceptibility to urolithiasis, little has been identified with respect to the relevant genetic loci for the disease. Two genome-wide association studies (GWASs) recognized four risk susceptibility genes, including CLDN14 in Europeans and Japanese [8,9], INMT-FAM188B-AQP1, RGS14-SLC34A1-PFN3-F12, and DGKH in Japanese [9]. That notwithstanding, these studies suggested further studies was needed to identify more risk loci as well as to recognize the molecular mechanisms attributed to the urinary calculi.
Broadly speaking, complex interactions of genetics and environmental factors, such as water intake, diet, urine pH, and infections have been associated with the etiopathogenesis of urolithiasis [10]. The underlying mechanisms of the development of calcium-containing stones, which are the most common type of kidney and bladder stones, have not fully been divulged [11]. Nowadays, the possibility of both free and fixed stones development has been suggested. The widely accepted explanation of the development of such stones relies on the increased solubility of the lithogenic elements in the urine [11]. Furthermore, it has been contemplated that the deposition of initial crystals occurs in the lumens of renal tubules [12,13]. However, recent observations imply that a development of Randall plaques in the renal papilla is the initial trigger of stone formation [14]. Such plaques are developed when calcium phosphate crystals are deposited in the basement of the thin loops of Henle and then extend into the urothelium. Calcium oxalate stones, which are responsible for almost 80% of all urinary stones, are developed after formation of calcium phosphate crystals. In fact, the binding of more calcium oxalate as well as matrix molecules present in the urine to the Randall plaques accelerates the formation of calcium oxalate stones [15].
Recent studies have demonstrated that receiving vitamin D supplements maybe put the individual at risk of developing kidney stones disease [16]. Moreover, vitamin D has an important role in calcium metabolism, such as absorption of calcium from intestine and its reabsorption in the kidneys. it through increasing the serum calcium levels could enhance the risk of urinary stone formation [17]. Vitamin D functions are dependent on the expression and nuclear activation of VDR [18]. Therefore, any alteration in the VDR may change the calcium metabolism, thus alter the urolithiasis risk. Taken together, studies have recommended that VDR play an essential role in the pathogenesis of urolithiasis [19].
The human VDR gene is placed on the chromosome 12q12-q14 that harbors more than 200 SNPs, among which FokI (or rs2228570), TaqI (or rs731236), BsmI (or rs1544410), and ApaI (or rs7975232) polymorphisms have been extensively investigated. VDR gene has at least five promoter regions, six untranslated exons, and eight protein-coding exons, which are alternatively spliced into BsmI, FokI, ApaI, and TaqI [20].. BsmI and ApaI are placed on the 9th intron of the 3′ terminal, TaqI is located on the 9th exon of the 3′ terminal, and FokI is established on the promoter of the 5′ terminal. Studies have reported that BsmI and TaqI SNPs are not involved in altering the protein structure of VDR; however, they have been suggested to play a role in the translation efficiency and stability of the corresponding mRNA [21]. Numerous studies have indicated the association of polymorphisms in the VDR gene with several human diseases [22,23].
A series of studies investigated the association between these polymorphisms of VDR gene and the risk of urolithiasis, but the findings have been conflicting . The inconsistent results were possibly because of clinical heterogeneity, small sample sizes, and low statistical power. In addition, previous meta-analyses [51][52][53] appeared to be out of date due to the availability of new data [45][46][47][48][49][50]. Therefore, we performed the most up to date meta-analysis with the aim of obtaining more accurate and updated results.

Methods
This study was performed in a stepwise process in accordance with the guidelines of the 2009 Preferred Reporting Items for Systematic Reviews and Metaanalyses (PRISMA) statement [54]. Besides, the current project does not contain any studies with human participants or animals performed by any of the authors. Registration in the International Prospective Register of Systematic Reviews (PROSPERO) was carried out.

Literature identification
A detailed systematic search was performed to identify candidate studies evaluating the associations between VDR gene polymorphisms and urolithiasis susceptibility (prior to June 2020). Three electronic databases, including Web of Science, MEDLINE, and Scopus were searched and for all of them, following combination of key words were used: ("urolithiasis" or "Kidney stone disease") AND ("VDR" OR "vitamin D receptor") AND ("polymorphisms" OR "SNP" OR "variation" OR "mutation"). Cross references within both original and review publications were done for additional pertinent studies. Original data were collected from English language and human population studies.

Inclusion/exclusion criteria
Studies included in quantitative analysis if met the following inclusion criteria: a) studies concerning the association between VDR gene polymorphisms and urolithiasis risk; b) Studies with case-control design; c) studies reporting sufficient data of genotype or allele frequency in order to calculate odds ratios (ORs) and 95% confidence intervals (CIs). On the other hand, duplicate data, case report, book chapter, review, letter, and abstracts were excluded.

Data extraction
All required data were extracted conforming to the standardized extraction checklist for the following data: the first author's name, journal and year of publication, country of origin, ethnicity, number of subjects in the case and control groups, mean or range of age, genotyping method, genotype counts in the case and control group. The extracted items were compared and any possible discrepancies were resolved by consensus.

Quality assessment
Methodological quality of eligible studies was evaluated by Newcastle-Ottawa Scale (NOS), a validated scale for non-randomized studies in meta-analysis. This scale consists of 3 parts with a total of 9 items. In this regards, studies with scores 0-3, 4-6 or 7-9 were of low, moderate, or high-quality, respectively [55].

Statistical analysis
For evaluating the distribution of the genotype frequencies to see if it is deviated from Hardy-Weinberg equilibrium (HWE) in the control group, the χ2-test was employed [56]. The quality of association between VDR gene SNPs and urolithiasis risk was evaluated by the pooled OR and its corresponding 95% CI. Five different comparison model for FokI, TaqI, BsmI, and ApaI SNPs were as follow: dominant model, recessive model, allelic model, homozygote contrast, and heterozygote contrast. Presence of heterogeneity between included studies was estimated by Cochran's Q-statistic (P value< 0.10 was considered as statistically significant). Besides, to report quantitative heterogeneity, the I-squared (I2) test was used. The fixed-effected model was used if PQ statistic> 0.10 or I2 was< 50%; otherwise, the random-effected model was applied [57,58]. We assessed the predefined sources of heterogeneity among included studies by subgroup analysis and meta-regression analysis based on year of population, and genotyping method. The stability of our results was measured by sensitivity analysis. Additionally, sensitivity analysis was conducted in the presence of heterogeneity. Moreover, Begg's test, Egger's regression test and visual examination of the funnel plot were applied to measure publication bias (P value< 0.05 was considered as statistically significant) [59]. The data analyses were carried out using STATA (version 14.0; Stata Corporation, College Station, TX) and SPSS (version 23.0; SPSS, Inc. Chicago, IL).

Specifications of the included studies
The exact process of literature searches and study selection is depicted in the Fig. 1. Early literature search eventuated in identification of 207 records, 33 of which met the final inclusion criteria and included in quantitative analysis. Among 33 eligible studies, 20 studies investigated the FokI SNP, 22 studies TaqI SNP, 14 studies BsmI SNP and 16 studies ApaI SNP. The studies were published between 1999 and 2020 and had an overall good methodological quality with NOS scores ranging from 5 to 8. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and Taq-Man were used by majority of included studies as genotyping method. Tables 1 and 2 summarized the characteristics of the included studies.

Quantitative synthesis
As the reference categories for comparing, the FF genotype for FokI SNP, TT genotype for TaqI SNP, BB genotype for BsmI SNP, and AA genotype for ApaI were used.

Association of BsmI polymorphism and urolithiasis risk
Fourteen eligible publications with 3065 cases and 2915 controls were included and evaluated the association between BsmI polymorphism and urolithiasis risk. Among 14 studies, only five publications were carried out in Asia [27,44,62,66,68] and nine studies were in Europe [25, 29, 31-33, 36, 38, 46, 48, 65]. The statistical analysis demonstrated that there was no significant association between BsmI SNP and urolithiasis risk under any genetic models in both the overall population and the subgroup analysis.

Evaluation of the heterogeneity and publication bias
The results of publication bias test indicated that there was no evidence of publication bias for overall population and subgroup analysis of all FokI, TaqI, BsmI, and ApaI SNPs. Additionally, the shape of the funnel plot confirmed absence of publication bias. No heterogeneity in both the overall and subgroup analyses was detected except for FokI polymorphism (Fig. 3, Table 3).

Sensitivity analysis
Sensitivity analysis is an effective test to evaluate the influence of individual study on the pooled results. In the sensitivity analysis, each eligible study was sequentially removed to assess whether the individual data influence the pooled ORs. In this meta-analysis, the pooled results did not significantly affect by any single study in the dominant model for FokI, TaqI, BsmI and ApaI SNPs    Fig. 4), indicating that the combined results of our meta-analysis were statistically robust.

Meta-regression analyses
Potential sources of heterogeneity among included studies was estimated by meta-regression analyses ( Table 4). According that, the findings indicated that none of the expected heterogeneity parameter were the source of heterogeneity for the association between VDR gene polymorphism and the risk of urolithiasis (Fig. 5).

Discussion
In the current most recent meta-analysis, 33 casecontrol association studies evaluating the VDR gene SNPs and urolithiasis risk were analyzed. The results of pooled analysis revealed that none of the four SNPs in   attempted to disclose the possible association between VDR gene SNPs and urolithiasis risk; that notwithstanding, the findings still show discrepancies and a comprehensive meta-analysis seems to be required to shed insights on the unknown conundrums. As a result, we performed a meta-analysis to investigate the consequence of the common four SNPs in the VDR gene, namely FokI (rs2228570), TaqI (rs731236), BsmI (rs1544410), and ApaI (rs7975232) on the risk of urolithiasis. The discrepancies in outcome among the various ethnicities might be due to differences in geographic and ethnical diversity, and impression of ethnicity on the serum level of vitamin D as well as the VDR gene expression [69].
Reports have shown the role of environmental factors on the risk of different diseases. For example, seasonal differences may impress the serum level of vitamin D [70]. Among the pregnant women in south-eastern USA, season was indicated to be associated with vitamin D levels in non-Hispanic women [71]. Sun exposure has been shown to interact with functional variants of the VDR gene [72]. Additionally, sun exposure and the differences between high and low latitudes, it has been implied that people in high latitude regions experience lower levels of vitamin D, especially in those with darker skin (which is a natural barrier to the UV radiation) [73]. As a result, environmental stimuli may impress the functional variants of the VDR gene as well as serum levels of vitamin D and, hence, modify the risk of urolithiasis susceptibility, along with VDR genetic polymorphisms.
Vitamin D is a critical hormone and play a role in the metabolism of calcium. This vitamin implements its function by binding to the VDR. The genetic variations in the VDR gene have been shown to impress the interactions of the vitamin D/VDR, modulating the susceptibility risk for several pathologic conditions. FokI polymorphism can modulate the ATG start cordon in   [74,75]. Additionally, ApaI and TaqI SNPs have been shown to have potential to modify the mRNA transcription of VDR gene and can modulate the stability of VDR mRNA [21]. FokI SNP has been shown to have potential to modulate the function of transcription factors [76,77]. A recent meta-analysis by González-Castro in 2019 [78], including 23 studies (a total of 1536 cases/1767 controls for ApaI polymorphism, 1571 cases/ 1455 controls for BsmI polymorphism, 2145 cases/2280 controls for FokI polymorphism, and 2160 cases/2307 controls for TaqI polymorphism), indicated that BsmI polymorphism had a protective association with nephrolithiasis in the allelic and homozygous models. Moreover, both TaqI polymorphism and FokI polymorphism were associated with a decreased risk of nephrolithiasis in the heterozygous model. However, no association of ApaI polymorphism was detected with nephrolithiasis. However, our most recent update metaanalysis in 2020, by including 33 studies (a total of 2950 cases/3065 controls for ApaI polymorphism, 3065 cases/ 2915 controls for BsmI polymorphism, 3114 cases/3174 controls for FokI polymorphism, and 4188 cases/3955 controls for TaqI polymorphism), indicated that none of the VDR gene polymorphisms mentioned above were associated significantly with nephrolithiasis risk in the overall analysis except ApaI SNP. However, our subgroup analysis according to population stratification revealed that ApaI gene polymorphism increased risk of urolithiasis in East-Asian patients by the recessive, allelic and homozygous model and TaqI gene SNP in Caucasians population through the heterozygous model. On the other hand, a meta-analysis in 2014 with respect to the study of the associations between VDR gene SNPs and urolithiasis risk included 20 studies in the analysis [53]. They found that the TaqI polymorphism was associated with an increased risk of urolithiasis, whereas the ApaI, BsmI, and FokI polymorphisms did not show any significant association. Moreover, stratifying for ethnicity, a slightly increased risk was found among Asians as compared with Whites for TaqI SNP. On the other hand, our meta-analysis on 33 studies did not result in any strong significant association between all four SNPs and urolithiasis risk in the pooled overall comparison. However, subgroup analysis demonstrated a significant increased risk of urolithiasis in East-Asian and Caucasians populations in association with ApaI and TaqI genes polymorphism. In the current meta-analysis, thirteen more studies were added in comparison to the previous study, and did not support the previous finding in the overall analysis. The subgroup analyses were conducted based on the ethnicity to identify the potential impression of the genetic background on the association of VDR gene polymorphisms and urolithiasis. Our analysis resulted in identification of ApaI and TaqI polymorphism association with increased risk of urolithiasis in East-Asian and Caucasians populations. However, the previous meta-analysis identified the same association in only Asians [53]. These discrepancies may stem from diversities in the genetic backgrounds. Furthermore, given that solar UV radiation is involved in the process of vitamin D generation [79], the significant association of VDR gene TaqI SNP in Asians might be attributed to the partially higher amount of exposure to UVR [80]. Moreover, it has been implied that level of UV exposure may impress that the associations between VDR gene polymorphisms and disorders. In patients with non-Hodgkin lymphoma, it was reported that patients with CC genotype for TaqI SNP who experienced sun exposure less than 7 h per week exhibited higher risk of the disease in comparison to patients with TT genotype with the similar duration of sun exposure [81]. In addition, reports showed that the TaqI T allele was more common in prostate cancer patients in a southern European population compared with the controls [82]. Plus, in a British population, the association of FokI polymorphism was observed to be limited to cases with a high exposed to UV [83]. Other than that, gender has been known as also a major risk factor for urolithiasis risk. It was shown that the FokI polymorphism had significant differences in females but not males, implying to the role of gender on the function of VDR [44]. Nonetheless, lack of sufficient data hindered the subgroup analysis based on gender in the current meta-analysis, which need to be addressed in the further studies.
Data from GWASs as well as association studies in different ethnic groups have revealed that VDR gene polymorphisms play a role in altering the risk of urolithiasis  development. Although our analysis did not endorse the association of VDR gene BsmI, ApaI, FokI, and TaqI SNPs with susceptibility to urolithiasis, the gene can be of beneficial applications in populations with significant associations. Generally, the concept of personalized medicine has been widely accepted, implying to the consideration of genetic makeup of each patient in approaching with optimized medication. As a consequence, clarification of VDR gene polymorphisms contribution to the urolithiasis predisposition could be advantageous in clinics with respect to better diagnosis of subjects at risk as well as treatment with maximum efficacy. Despite we tried to perform the possibly well-suited analysis of the available data, a number of caveats and confining factors are related to this meta-analysis. First, our literature search was limited to only English-written papers, raising the chance of excluding of potentially worthwhile findings. Second, we could not analyze the role of age, gender, lifestyle, and other genetic variations, on the adjusted association of VDR gene SNPs and urolithiasis risk. Hence, additional works with respect to the gene-gene and gene-environment interactions is needed to approach with a more comprehensive estimation. Third, we noticed a significant heterogeneity among the studies for various comparisons, which may impress the perception of findings. Although we conducted subgroup analysis and weighted meta-regression in order to attenuate its effects. Finally, there were a number of VDR gene SNPs in the context of urolithiasis risk that could not be included in the meta-analysis due to lack of sufficient amount of data. Hence, it could barely implied that VDR gene could not convey a genetic risk factor for urolithiasis, merely regarding our findings.

Conclusion
In conclusion, the results of pooled analysis did not demonstrate any statistically significant association between all four SNPs and susceptibility to urolithiasis. However, subgroup analysis showed that the Recessive, allelic, and aa vs. AA model of ApaI and Tt vs. TT comparison of the TaqI gene polymorphism increased risk of urolithiasis in East-Asian and Caucasians population, respectively. Further genes should be evaluated to disclose the genetic mechanisms contributing to urolithiasis development. Moreover, the role of life style, age, and gender needs be considered in the stratification analyses for VDR gene SNPs and urolithiasis predisposition.