Associations between vitamin D receptor gene polymorphisms and osteoarthritis: an updated meta-analysis

RHEUMATOLOGY Rheumatology 2014;53:998 1008 doi:10.1093/rheumatology/ket418 Advance Access publication 4 February 2014 Meta-analysis Associations between vitamin D receptor gene polymorphisms and osteoarthritis: an updated meta-analysis Zhao-Hua Zhu 1,2, Xing-zhong Jin 2, Weiya Zhang 3, Mao Chen 4, Dong-Qing Ye 5, Yu Zhai 5, Fu-Long Dong 1, Cai-Liang Shen 1 and Changhai Ding 2,6 META- ANALYSIS Abstract Objective. Vitamin D receptor (VDR) gene polymorphisms may be associated with the risk of OA, however, evidence for this is controversial. This meta-analysis aims to confirm whether VDR gene polymorphisms are associated with OA. Methods. Meta-analyses on the association between OA and VDR ApaI, BsmI, TaqI and FokI polymorphisms were conducted using allele and homozygote contrast and contrasts in the recessive and dominant models. Stratification analyses by different demographic regions (Europe vs Asian) were also performed and pooled odds ratios (ORs) were obtained using the random effects model if the results were heterogeneous. Results. A total of 13 relevant studies involving OA patients (n = 2104) and controls (n = 2939) were included in the analysis. There were significant associations between VDR ApaI polymorphisms and OA in the Asian population (A vs a: OR = 1.16, 95% CI 1.02, 1.32, P = 0.025; AA vs Aa/aa: OR = 1.36, 95% CI 1.04, 1.77, P = 0.025; AA vs aa: OR = 1.35, 95% CI 1.00, 1.80, P = 0.047), but not in the whole population. There was also a statistically significant association between FokI polymorphism and OA (FF vs Ff/ff: OR = 0.65, 95% CI 0.44, 0.95, P = 0.024); however, this result was derived from only two studies. No significant associations were found between VDR TaqI and BsmI polymorphisms and OA. Conclusion. There are modest but statistically significant associations between VDR ApaI polymorphisms and the susceptibility of OA in the Asian population. Key words: vitamin D receptor, osteoarthritis, polymorphisms, meta-analysis. Introduction OA is the most common joint disease, characterized by joint pain and radiographic changes including osteophytes and joint space narrowing (JSN). The knees, hips 1 Orthopedics Department, First Affiliated Hospital of Anhui Medical University, Hefei, China, 2 Menzies Research Institute Tasmania, University of Tasmania, Hobart, Tasmania, Australia, 3 Academic Rheumatology, University of Nottingham, Nottingham, UK, 4 Division of Epidemiology, School of Public Health and Primary Care, Chinese University of Hong Kong, Hong Kong, 5 Department of Epidemiology and Biostatistics, School of Public Health, Anhui Medical University, Anhui, People s Republic of China and 6 Department of Epidemiology and Preventive Medicine, Monash University, Melbourne, NSW, Australia. Submitted 22 April 2013; revised version accepted 30 October 2013. Correspondence to: Cai-Liang Shen, Orthopedics Department, First Affiliated Hospital of Anhui Medical University, 218 Jixi Road, Hefei 230022, China. Email: shencailiang1616@163.com and hands are the synovial joints commonly affected by the disease. It is generally accepted that OA affects the whole organ, including articular cartilage, subchondral bone, meniscus, ligaments, muscle and synovium. Common risk factors of OA include age, gender, overweight/obesity [1], joint injury [2], occupation [3] and family history/aggregation of the disease [4, 5]. Several polymorphisms have been identified that may be associated with the disease, including vitamin D receptor (VDR), aggrecan (AGC), insulin-like growth factor-1 (IGF-1), estrogen receptor (ER), TGF-b, and collagen II, IX and XI [6, 7]. Among them, the VDR gene that is located on chromosome 12q12-q14 [8] has been reported most frequently. VDR gene polymorphisms have been associated with OA, especially with osteophytes [9]. They have been shown to be related to OA of the knee in Britons [10] as! The Author 2014. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Associations between VDR polymorphisms and OA well as osteophytosis of the lumbar in Australians [11] and Finns [12]. In contrast, other studies have reported no significant association between VDR gene polymorphisms and OA [13, 14]. This inconsistency may be partly attributable to the different populations, the environmental factors affecting vitamin D absorption and metabolism or the presence of confounding factors. A meta-analysis [15] including 10 studies (1591 cases and 1781 controls from Asian and European populations) reported in 2009 that there were no significant associations between the VDR TaqI, ApaI or BsmI and OA. The stratified analysis also showed no significant association between VDR and OA in either European or Asian populations. Since then there have been three new studies [16 18] that reported associations between VDR gene polymorphisms and OA. In this study we performed an updated meta-analysis aimed at investigating whether VDR gene polymorphisms are associated with OA. Materials and methods Literature search The keywords OA, osteoarthritis, VDR, vitamin D and polymorphism were used as searching terms for electronic databases including PubMed, Medline, Embase, the Cochrane Central Register of Controlled Trials (CENTRAL), Web of Science and the China National Knowledge Infrastructure database (CNKI). The search was done restricted to to English- and Chinese-language publications. The resultant literature was retrieved and the references were checked for other relevant publications. Only studies fully published up to March 2013 were included. When the literature contained duplicate publications, the most recent study was chosen. Study selection Two reviewers independently assessed the studies using pre-designed criteria. The inclusion criteria were (i) patients with OA diagnosed using ACR clinical OA criteria, imaging (e.g. Schneiderman or Kellgren Lawrence grade system) or total joint replacement due to primary OA; (ii) studies investigating the association between the VDR gene polymorphism and OA; (iii) a case control, cohort or cross-sectional design providing sufficient data to calculate an odds ratio (OR) and corresponding 95% CI;. (iv) the frequency of alleles or genotype distribution was available and (v) the genotype distribution of the control population met the Hardy Weinberg equilibrium (HWE) model. Interim analyses, studies with overlapping populations and comparative analyses of laboratory methods were excluded. Data extraction Two authors independently extracted the data from all eligible studies and discrepancies were resolved by discussion with consensus. The following information was collected: author, year of publication, country, OA sites, OA definition, genotype method and total number of cases and controls, and allele frequency of VDR single nucleotide polymorphisms (SNPs). If a consensus was not reached, a third author was involved and a final decision was made by a majority vote. Evaluation of study quality The methodological quality of these studies was evaluated by two independent reviewers with the modified Newcastle Ottawa Scale (NOS) [19], which describes three aspects of quality: selection, comparability and exposure of cases and controls. For cohort or crosssectional studies, the three aspects are selection, comparability and outcome measures. These three aspects consisted of four, two and three items, respectively, each of which was given 1 point, thus a study was awarded a maximum of 9 points if it fulfiled all nine items. A score >6 was defined as of high quality. Statistical analysis Data were processed with Stata 10.0 (StataCorp, College Station, TX, USA). A chi-square test was applied to determine whether the observed genotype frequencies in the controls were consistent with HWE. The heterogeneity of the included studies was tested using Cochran s Q statistics, and the magnitude of the heterogeneity was measured using the I 2 statistic (I 2 = 100% (Q df)/q). A study with an I 2 >25% was regarded as being without heterogeneity [20]. It was considered statistically significant when P-values were <0.1 from Cochran s Q tests [20]. The Mantel Haenszel method in the fixed-effects model was used if the result of the Q test was not significant [21]. Otherwise, we calculated the pooled OR and 95% CI using a random-effects model with the DerSimonian and Laird method [22]. Publication bias was detected by using Egger et al. s [23] and Begg and Mazumdar s [24] linear regression tests. Both of these were used to estimate funnel plot asymmetry, and P-values 40.05 were considered as statistically significant. Meta-analyses were performed on (i) allelic contrast, (ii) dominant model contrast, (iii) recessive model contrast and (iv) contrast of homozygotes. To evaluate demographic region-specific effects, we performed subgroup analyses, categorized by European and Asian populations. Results Characteristics of eligible studies The selection process of eligible studies is depicted in Fig. 1. A total of 13 publications consisting of 2104 OA patients and 2939 controls met the inclusion criteria [10, 14, 16, 17, 18, 25 32]. Among these there were 7 comparisons for ApaI polymorphism, 11 comparisons for TaqI polymorphism, 6 comparisons for BsmI polymorphism and 3 comparisons for FokI polymorphism. There were seven studies on Europeans and six studies on Asians. Six studies were performed of the lumbar, three of the knee, four of the hip and two of the hand. One of these studies contained information about three different OA sites that were www.rheumatology.oxfordjournals.org 999

Zhao-Hua Zhu et al. FIG. 1Selection process of eligible studies 54 studies were identified after an initial search 29 excluded (11 review and meta; 5 animal studies; 7 vitro study; 6 relevant to other diseases) 25 studies were relevant to polymorphisms and OA treated independently [25]. Due to small sample size, we did not perform OA site-specific analyses. There were no repetitive studies. Characteristics of the included studies are presented in Table 1. All included studies were of high quality with an NOS score >6. Heterogeneity and publication bias Egger et al. s [23] and Begg and Mazumdar s linear regression tests [24] did not show any evidence of publication bias in the studies analysed. The distribution of the genotype in the control group of each study was consistent with HWE. The heterogeneity and publication bias of included studies on each gene polymorphism are presented in Table 2. Considering that significant heterogeneity was identified in overall and European studies of the BsmI gene, we performed a sensitivity analysis by repetitive calculation of the combined OR estimate and a heterogeneity test after removing one study at a time. It turned out that after omitting Aerssens et al. [14] from the overall studies, I 2 experienced the sharpest decline, from 52.1% to 0%. A similar trend was also found in the European population, where I 2 decreased from 65.5% to 0%. However, the results of association did not change after removing this study [14]. Similar sensitivity analysis processes were also performed involving the association tests of VDR TaqI and FokI genotypes, and we did not found any change in these results. Meta-analysis results 19 studies were left for more extraction 13 studies were totally in meta-analysis Table 2 summarizes the main results of this meta-analysis. The pooled ORs were computed for the allelic contrast, 6excluded(3relevant to other genes; 3 not case-control studies) 6 excluded (4 number of null and wild genotype could not be ascertained; 1 family study; 1 not in Hardy- Weinberg equilibrium) dominant model contrast, recessive model contrast and contrast of homozygotes. No statistically significant association was found between the genotype of BsmI and OA susceptibility. In the subgroup analysis, VDR BsmI polymorphisms were not associated with OA in Europeans or Asians. For the ApaI genotype, statistically significant associations were found among Asians between VDR ApaI polymorphism and OA susceptibility in the allelic contrast (A vs a: OR = 1.16, 95% CI 1.02, 1.32, P = 0.025) (Fig. 2), homozygotes contrast (AA vs aa: OR = 1.35, 95% CI 1.00, 1.80, P = 0.048) (Fig. 3), as well as recessive model contrast (AA vs Aa + aa: OR = 1.36, 95% CI 1.04, 1.77, P = 0.025) (Fig. 4). No significant association was found among Asians in the dominant model contrast (AA + Aa vs aa: OR = 1.15, 95% CI 0.69, 1.37, P = 0.12). In contrast, no statistically significant associations were found between VDR ApaI polymorphism and OA overall and in the European population (Table 2). With regard to the TaqI genotype, the meta-analyses of the allelic, recessive, dominant and homozygotes model contrast revealed no significant associations between VDR TaqI polymorphism and OA overall and in different ethnicities (Table 2). For the FokI genotype, a statistically significant association was noticed in the recessive model in the whole populations (FF vs Ff + ff: OR = 0.648, 95% CI 0.44, 0.95, P = 0.024). However, only two publications met the inclusion criteria [18, 29]. Allelic contrast analysis was available in one of the three studies involving the VDR FokI genotype [17]. 1000 www.rheumatology.oxfordjournals.org

Associations between VDR polymorphisms and OA TABLE 1 Characteristics of included studies in the meta-analysis Study author [Ref.] Country (ethnicity) Number of subjects (OA/control) Age, mean (S.D.), years OA site BMI, mean (S.D.), cases/ controls Smokers, %, cases/ controls OA definition Polymorphism Keen et al., 1997 [10] UK 351 (82/269) 58.3 (4.7) Knee 28.0 (4.0)/ 25.0 (3.6) 45/43 Radiographic OA (Kellgren Lawrence) TaqI Aerssens et al., 1998 [14] Belgium 314 (75/239) 70 (6) Hip 27 (4)/26 (4) NG Total joint replacement for OA BsmI Huang et al., 2000 [25] Japan 270 (134/ 136) Loughlin et al., 2000 [26] UK 740 (371/ 269) 63.8 (range 29 87) 65 (range 47 85) Granchi et al., 2002 [27] Italy 193 (143/50) 64 (range 22 88) Kawaguchi et al., 2002 [28] Noponen-Hietala et al., 2003 [29] Japan 205 (92/133) 22 (range 20 29) Finland 85 (29/56) 59 (range 42 75) Jordan et al., 2005 [30] UK 291 (182/ 109) Solovieva et al., 2006 [31] Finland 543 (160/ 383) Cheung et al., 2006 [32] China 579 (388/ 191) Hand, hip knee 24.3 (3.4)/ 23.6 (3.5) NG Radiographic OA (Kellgren Lawrence) BsmI, ApaI, TaqI Hip NG NG Total joint replacement for OA TaqI Hip NG NG Total joint replacement for OA BsmI Lumbar Lumbar 65.8 (3.0) Lumbar 56.3 (4.7) Hand 25.0 (3.8)/ 24.3 (3.5) 18 55 Lumbar Yuan et al. 2010 [16] China 462(178/284) 48.5 (13.1) Lumbar Eser et al., 2010 [17] Turkish 300 (150/ 150) Muraki et al., 2011 [18] Japan 787 (117/ 670) 20 30 Lumbar 65.6 (2.7) Knee Overall 27 (4.3) NG NG Lumbar disc degeneration (Schneiderman grade) NG NG Clinically and radiologically confirmed lumbar spinal stenosis NG NG A grade by Lane et al. [33] for either osteophyte score or space narrowing 21/23 Radiographic OA (Kellgren Lawrence scale 52) NG NG Lumbar disc degeneration (Schneiderman grade) NG NG CT examination (according to a four point scale) NG No smoking Lumbar disc degeneration (Schneiderman grade) NG Radiographic OA (Kellgren Lawrence scale 52) TaqI, ApaI TaqI, FokI BsmI ApaI, TaqI TaqI TaqI, ApaI FokI, TaqI FokI, ApaI Ref: reference; NG: not given. www.rheumatology.oxfordjournals.org 1001

Zhao-Hua Zhu et al. TABLE 2 Meta-analysis of associations between VDR ApaI, BsmI, TaqI and FokI polymorphisms and OA Polymorphism Population No. of studies Tests of association Tests of heterogeneity Begg and Mazumdar s [24] test for publication bias Egger et al. s [23] test for publication bias OR 95% CI P-value Model Q P-value I 2,% t P-value t P-value ApaI (A vs a) Overall 7 1.12 0.10, 1.24 0.060 F 3.94 0.685 0 0.39 0.712 0.12 0.914 European 1 0.96 0.74, 1.24 0.736 Asian 6 1.16 1.02, 1.32 0.024 F 2.27 0.810 0 0.20 0.848 1.77 0.327 AA vs Aa + aa Overall 7 1.23 0.99, 1.53 0.068 F 3.73 0.713 0 0.28 0.801 0.12 0.914 (recessive) European 1 0.99 0.67, 1.47 0.956 Asian 6 1.36 1.04, 1.77 0.025 F 2.06 0.841 0 2.21 0.327 1.77 0.327 AA + Aa vs aa Overall 7 1.11 0.94, 1.31 0.208 F 7.71 0.260 22.2 0.82 0.497 0.82 0.497 (dominant) European 1 0.88 0.55, 1.40 0.588 Asian 6 1.15 0.96, 1.37 0.122 F 6.63 0.250 24.6 0.59 0.659 0.70 0.611 (AA vs aa) Overall 7 1.22 0.95, 1.57 0.123 F 2.79 0.834 0 0.12 0.914 0.23 0.837 European 1 0.89 0.53, 1.52 0.680 Asian 6 1.35 1.00, 1.80 0.047 F 1.05 0.958 0 1.77 0.327 2.82 0.217 BsmI (B vs b) Overall 6 1.01 0.85, 1.20 0.944 F 4.58 0.469 0 0.26 0.840 0.26 0.840 European 3 1.00 0.81, 1.25 0.976 F 4.32 0.115 53.7 0.26 0.840 0.26 0.840 Asian 3 1.01 0.76, 1.35 0.940 F 0.26 0.878 0 NA NA NA NA BB vs Bb + bb Overall 6 1.23 0.57, 2.67 0.601 R 8.42 0.038 64.4 0.16 0.885 0.77 0.583 (recessive) European 3 1.17 0.50, 2.70 1.722 R 8.07 0.018 75.2 0.74 0.595 0.74 0.595 Asian 3 3.27 0.13, 8.09 0.469 F NA NA NA NA NA NA NA BB + Bb vs bb Overall 6 0.94 0.75, 1.19 0.593 F 1.11 0.953 0 0.17 0.875 0.08 0.951 (dominant) European 3 0.87 0.65, 1.22 0.423 F 0.57 0.753 0 0.08 0.951 0.05 0.970 Asian 3 1.00 0.74, 1.36 0.917 F 0.17 0.917 0 NA NA NA NA BB vs bb Overall 6 1.09 0.52, 2.28 0.811 R 6.27 0.099 52.1 0.24 0.834 0.41 0.904 European 3 1.00 0.46, 2.30 0.937 R 5.80 0.055 65.5 0.67 0.623 0.68 0.619 Asian 3 3.31 0.13, 8.21 0.465 F NA NA NA NA NA NA NA TaqI (T vs t) Overall 10 1.05 0.87, 1.27 0.583 R 19.30 0.023 53.4 1.18 0.273 1.18 0.273 European 4 1.05 0.91, 1.22 0.481 F 0.81 0.847 0 0.38 0.740 0.76 0.529 Asian 6 1.01 0.70, 1.47 0.957 R 17.83 0.003 72.0 4.67 0.210 4.89 0.320 (continued) 1002 www.rheumatology.oxfordjournals.org

Associations between VDR polymorphisms and OA TABLE 2 Continued Polymorphism Population No. of studies Tests of association Tests of heterogeneity Begg and Mazumdar s [24] test for publication bias Egger et al. s [23] test for publication bias OR 95% CI P-value Model Q P-value I 2,% t P-value t P-value TT vs Tt + tt Overall 10 1.01 0.81, 1.27 0.911 R 16.30 0.061 44.8 0.87 0.460 0.78 0.460 (recessive) European 4 0.97 0.769, 1.23 0.814 F 0.49 0.922 0 1.30 0.323 1.06 0.399 Asian 6 1.04 0.70, 1.54 0.430 R 15.39 0.009 67.5 1.43 0.225 1.48 0.214 TT + Tt vs tt Overall 9 1.16 0.91, 1.47 0.238 F 5.40 0.494 0 1.02 0.354 1.53 0.186 (dominant) European 4 1.19 0.93, 1.52 0.163 F 3.55 0.314 15.5 0.27 0.813 0.21 0.851 Asian 5 0.33 0.05, 2.02 0.230 F 0 0.998 0 0.03 0.978 0.51 0.701 TT vs tt Overall 9 1.04 0.77, 1.42 0.788 F 3.88 0.693 0 1.14 0.304 1.11 0.316 European 4 1.09 0.80, 1.50 0.592 F 2.30 0.512 0 0.49 0.674 0.30 0.794 Asian 5 0.33 0.05, 2.04 0.233 F 0 0.998 0 0.08 0.951 0.56 0.675 FokI (F vs f) Overall 3 0.89 0.61, 1.33 0.589 R 5.71 0.058 65.0 0.13 0.919 0.15 0.998 European 1 0.67 0.35, 1.29 0.233 NA NA NA NA NA NA NA NA Asian 2 0.97 0.60, 1.60 0.917 R 4.82 0.028 79.3 NA NA NA NA FF vs Ff + ff Overall 2 0.65 0.44, 0.95 0.024 F 0.13 0.714 0 0.28 0.825 NA NA (recessive) European 1 0.76 0.30, 1.90 0.553 NA NA NA NA NA NA NA NA Asian 1 0.74 0.52, 1.05 0.090 F 1.25 0.263 20.3 NA NA NA NA FF + Ff vs ff Overall 2 0.77 0.47, 1.27 0.308 F 1.43 0.232 29.9 6.20 0.102 NA NA (dominant) European 1 0.38 0.10, 1.36 0.135 NA NA NA NA NA NA NA NA Asian 1 0.88 0.51, 1.57 0.651 NA NA NA NA NA NA NA NA FF vs ff Overall 2 0.40 0.11, 1.43 0.158 R 3.06 0.080 67.3 2.53 0.240 NA NA European 1 0.18 0.05, 0.70 0.013 NA NA NA NA NA NA NA NA Asian 1 0.68 0.37, 1.24 0.204 NA NA NA NA NA NA NA NA NA: not available; R: random effects model; F: fixed effects model. www.rheumatology.oxfordjournals.org 1003

Zhao-Hua Zhu et al. FIG. 2OR (95% CI) for allelic contrast (A vs a) of the ApaI gene associated with OA FIG. 3OR (95% CI) for homozygotes contrast (AA vs aa) of the ApaI gene association with OA 1004 www.rheumatology.oxfordjournals.org

Associations between VDR polymorphisms and OA FIG. 4OR (95% CI) for recessive models contrast (AA vs Aa + aa) of the VDR ApaI gene association with OA Discussion In this meta-analysis we found that there were significant associations between VDR ApaI polymorphisms and OA in an Asian population but not in a European population. FokI polymorphism might also be associated with OA in the recessive model, but this was only derived from two publications. These results suggest that VDR gene polymorphisms may play roles in OA aetiology in the Asian population. Since restriction fragment length polymorphisms (RFLPs) of VDR genes were discovered in the early 1990s [34, 35], VDR genes have been reported to play a role in a wide range of common diseases, including OA [36], diabetes [37], cancer [38], tuberculosis [39] and cardiovascular diseases [40]. Strong associations between BMD and VDR gene polymorphisms have been reported in a number of studies [41, 42]. However, the association of VDR SNPs with development or progression of OA remain debatable. The contribution of BsmI SNP to hip, hand or knee OA, osteophytes and JSN was not significant, as observed in a study conducted by Baldwin et al. [13]. In contrast, Jordan et al. [30] highlighted an association of the B allele with the severity of osteophytes in patients with OA of the lumbar. In a UK study, subjects with VDR allele T of TaqI polymorphism had an increased risk of knee OA compared with those with the t allele [10], while other UK studies reported that TaqI polymorphism was not associated with knee OA, osteophytes and JSN [31, 43]. The frequency distribution of BsmI, ApaI and TaqI RFLPs was examined in Japanese female OA patients and no significant differences were observed in the frequency of each polymorphic VDR genotype between OA patients and controls [25]. Another recent study [18] found that the minor ff homozygous genotype was significantly associated with a greater prevalence of knee pain compared with the FF genotype for FokI polymorphism, while the associations of ApaI polymorphisms with knee pain were not significant. Uitterlinden et al. [44] reported that the VDR gene polymorphism was associated with osteophyte rather than JSN in knee OA patients. These suggest that further studies stratified by VDR genes and specific OA features are required. As a form of OA, disc degenerative diseases have been reported to have a close relationship with VDR allelic variation [45]. Kawaguchi et al. [28] investigated 205 Japanese volunteers between 20 and 29 years of age and reported that the Tt genotype of TaqI polymorphism was more frequently associated with the severity of disc degeneration than the TT genotype. In a study of 804 Chinese patients, Cheung [32] suggested that people www.rheumatology.oxfordjournals.org 1005

Zhao-Hua Zhu et al. younger than 40 years of age with at least one t allele were approximately six times more susceptible to disc degenerative diseases than those without a t allele. Videman et al. [12] reported that the MRI signal intensity of intervertebral discs in men with the ff and Ff genotypes was 9.3% and 4.3%, respectively, lower than the intensity in men with the FF genotype (P = 0.006). The summary scores of bulging, intensity and disc height were 4.0% and 6.9% worse in men with Ff and ff genotypes, respectively, than those in men with the FF genotype (P = 0.029). Although dozens of association studies relating VDR gene polymorphisms to OA have been published, results are controversial [14, 25, 46]. This inconsistency may be due to factors such as small sample sizes, confounding factors, clinical heterogeneity and multiple pathological mechanisms of OA. As a powerful statistical method, meta-analysis provides a quantitative approach to combine data from independent studies as well as to examine and explain the heterogeneity [47]. In 2009 Lee et al. [15] carried out a meta-analysis to examine the association of VDR gene polymorphisms with OA. However, this metaanalysis did not find evidence of significant associations between VDR TaqI, BsmI and ApaI polymorphisms and OA. Stratification by ethnicity also reported no significant associations between VDR gene polymorphisms and OA among European or Asian patients. In our study we updated the previous systematic review with new studies on the association between OA and the four RFLPs for ApaI, BsmI, TaqI and FokI. The updated meta-analysis included a total of 13 studies (2104 patients) and demonstrated that there was a significant association between the ApaI gene and OA in the Asian population for the recessive model, allelic contrast and homozygotes contrast. No evidence of significant associations was revealed between VDR TaqI or BsmI polymorphisms and OA susceptibility. With regard to the FokI gene, there was a statistically significant association reflected by the pooled result of two included literatures. However, because of the limited data available in the literature, whether the recessive model of FokI genotypes is associated with OA remains inconclusive. One of the included studies contained data regarding three different OA groups (knee, hip and hand) that were treated independently [25]. The repetitive use of data from this study (46 had polyarticular OA) may affect the final results, however, we performed a sensitivity analysis showing it does not change the final results after removing this article or only using data from one group. Some potential limitations of this meta-analysis should be mentioned. First, all included literature was searched based on Chinese and English language, thus language bias may exist. Second, OA appears to be more prevalent in females and its incidence increases with age [48, 49]. The mechanisms underlying OA at different sites could be different. Due to the limited data, we were unable to perform further stratified analyses for the association for specific sites of OA and further adjustment for confounding factors such as gender, BMI, smoking and so on. Third, the number of studies of FokI gene polymorphisms was small. There was insufficient statistical power to explore the real relationship between VDR FokI polymorphisms and OA. Fourth, as the demographic region subgroup analyses were restricted to Europeans and Asians, the results are applicable to only these ethnic groups. In spite of these potential limitations, our meta-analysis combined the most recently published data [16 18] regarding the association between VDR gene polymorphisms and OA. The literature numbers and sample sizes included in the current study were larger than in the previous meta-analysis [15], which significantly increased the statistical power of the pooled results. Besides, the studies included in our meta-analysis were of relatively high quality since all of their NOS scores were >6. In conclusion, this meta-analysis suggests that VDR ApaI polymorphisms may be associated with OA and the association may be population dependent, currently only observed in the Asian population. Further studies with high-quality and large sample sizes in different populations are warranted. Rheumatology key messages. There are statistically significant associations between vitamin D receptor ApaI gene polymorphisms and the risk of OA in the Asian population. Acknowledgements The authors wish to thank Amir Sabah Hassan for his kind linguistic support. Disclosure statement: The authors have declared no conflicts of interest. References 1 Muthuri SG, Hui M, Doherty M et al. What if we prevent obesity? Risk reduction in knee osteoarthritis estimated through a meta-analysis of observational studies. Arthritis Care Res 2011;63:982 90. 2 Muthuri SG, McWilliams DF, Doherty M et al. History of knee injuries and knee osteoarthritis: a meta-analysis of observational studies. Osteoarthritis Cartilage 2011;19: 1286 93. 3 McWilliams DF, Leeb BF, Muthuri SG et al. Occupational risk factors for osteoarthritis of the knee: a meta-analysis. Osteoarthritis Cartilage 2011;19:829 39. 4 Zhang W, Doherty M. How important are genetic factors in osteoarthritis? Contributions from family studies. J Rheumatol 2005;32:1139 42. 5 Zhang W, McWilliams DF, Ingham SL et al. Nottingham knee osteoarthritis risk prediction models. Ann Rheum Dis 2011;70:1599 604. 6 Spector TD, MacGregor AJ. Risk factors for osteoarthritis: genetics. Osteoarthritis Cartilage 2004;12(Suppl A): S39 44. 1006 www.rheumatology.oxfordjournals.org

Associations between VDR polymorphisms and OA 7 Ramonda R, Lorenzin M, Modesti V et al. Serological markers of erosive hand osteoarthritis. Eur J Intern Med 2013;24:11 5. 8 Miyamoto K, Kesterson RA, Yamamoto H et al. Structural organization of the human vitamin D receptor chromosomal gene and its promoter. Mol Endocrinol 1997;11: 1165 79. 9 Uitterlinden AG, Burger H, Huang Q et al. Vitamin D receptor genotype is associated with osteoarthritis. J Clin Invest 1997;100:259 63. 10 Keen RW, Hart DJ, Lanchbury JS et al. Association of early osteoarthritis of the knee with a Taq polymorphism of the vitamin D receptor gene. Arthritis Rheum 1997;40: 1444 9. 11 Jones G, White C, Sambrook P et al. Allelic variation in the vitamin D receptor, lifestyle factors and lumbar spinal degenerative disease. Ann Rheum Dis 1998;57:94 9. 12 Videman T, Leppävuori J, Kaprio J et al. Intragenic polymorphisms of the vitamin D receptor gene associated with intervertebral disc degeneration. Spine 1998;23:2477 85. 13 Baldwin CT, Cupples LA, Joost O et al. Absence of linkage or association for osteoarthritis with the vitamin D receptor/type II collagen locus: the Framingham Osteoarthritis Study. J Rheumatol 2002;29:161 5. 14 Aerssens J, Dequeker J, Peeters J et al. Lack of association between osteoarthritis of the hip and gene polymorphisms of VDR, COL1A1, and COL2A1 in postmenopausal women. J Arthritis Rheum 1998;41: 1956 50. 15 Lee YH, Woo JH, Choi SJ et al. Vitamin D receptor TaqI, BsmI and ApaI polymorphisms and osteoarthritis susceptibility: a meta-analysis. Joint Bone Spine 2009;76: 156 61. 16 Yuan HY, Tang Y, Liang YX et al. Matrix metalloproteinase-3 and vitamin D receptor genetic polymorphisms, and their interactions with occupational exposure in lumbar disc degeneration. J Occup Health 2010;52:23 30. 17 Eser B, Cora T, Eser O et al. Association of the polymorphisms of vitamin D receptor and aggrecan genes with degenerative disc disease. Genet Test Mol Biomarkers 2010;14:313 7. 18 Muraki S, Dennison E, Jameson K et al. Association of vitamin D status with knee pain and radiographic knee osteoarthritis. Osteoarthritis Cartilage 2011;19: 1301 6. 19 Wells GA, Shea B, O Connell D et al. The Newcastle Ottawa Scale (NOS) for assessing the quality of nonrandomized studies in meta-analyses. http://www. ohri.ca/programs/clinical_epidemiology/oxford (2 March 2012, date last accessed). 20 Higgins JP, Thompson SG. Quantifying heterogeneity in a meta-analysis. Stat Med 2002;21:1539 58. 21 Mantel N, Haenszel W. Statistical aspect of the analysis of data from retrospective studies of disease. J Natl Cancer Inst 1959;22:719 48. 22 DerSimonian R, Laird N. Meta-analysis in clinical trials. Control Clin Trials 1986;7:177 88. 23 Egger M, Davey Smith G, Schneider M et al. Bias in metaanalysis detected by a simple, graphical test. BMJ 1997; 315:629 34. 24 Begg CB, Mazumdar M. Operating characteristics of a rank correlation test for publication bias. Biometrics 1994; 50:1088 101. 25 Huang J, Ushiyama T, Inoue K et al. Vitamin D receptor gene polymorphisms and osteoarthritis of the hand, hip, and knee: a case-control study in Japan. Rheumatology 2000;39:79 84. 26 Loughlin J, Sinsheimer JS, Mustafa Z et al. Association analysis of the vitamin D receptor gene, the type I collagen gen COL1A1, and the estrogen receptor gene in idiopathic osteoarthritis. J Rheumatol 2000;273:779 84. 27 Granchi D, Stea S, Sudanese A et al. Association of two gene polymorphisms with osteoarthritis secondary to hip dysplasia. Clin Orthop Relat Res 2002;(403):108 17. 28 Kawaguchi Y, Kanamori M, Ishihara H et al. The association of lumbar disc disease with vitamin D receptor gene polymorphism. J Bone Joint Surg Am 2002;84-A:2022 8. 29 Noponen-Hietala N, Kyllönen E, Männikkö M et al. Sequence variations in the collagen IX and XI genes are associated with degenerative lumbar spinal stenosis. Ann Rheum 2003;62:1208 14. 30 Jordan KM, Syddall H, Dennison EM et al. Birthweight, vitamin D receptor gene polymorphism, and risk of lumbar osteoarthritis. J Rheumatol 2005;32:678 83. 31 Solovieva S, Hirvonen A, Siivola P et al. Vitamin D receptor gene polymorphisms and susceptibility of hand osteoarthritis in Finnish women. Arthritis Res Ther 2006;8:R20. 32 Cheung KM, Chan D, Karppinen J et al. Association of the Taq I allele in vitamin D receptor with degenerative disc disease and disc bulge in a Chinese population. Spine 2006;31:1143 8. 33 Lane NE, Nevitt MC, Genant H et al. Reliability of new indices of radiographic osteoarthritis of the hand and hip and lumbar disc degeneration. J Rheumatol 1993; 20: 1911 8. 34 Faraco JH, Morrison NA, Baker A et al. ApaI dimorphism at the human vitamin D receptor gene locus. Nucleic Acids Res 1989;17:21 50. 35 Morrison NA, Yeoman R, Kelly PJ et al. Contribution of trans-acting factor alleles to normal physiological variability: Vitamin D receptor gene polymorphism and circulating osteocalcin. Proc Natl Acad Sci USA 1992;89: 6665 9. 36 Cicuttini FM, Spector TD. Genetics of osteoarthritis. Ann Rheum Dis 1996;55:665 7. 37 Wang Y, Deb DK, Zhang Z et al. Vitamin D receptor signaling in podocytes protects against diabetic nephropathy. J Am Soc Nephrol 2012;23:1977 86. 38 Yin M, Wei S, Wei Q. Vitamin D receptor (VDR) gene polymorphisms and prostate cancer risk: a metaanalysis of 36 published studies. Int J Clin Exp Med 2009;2: 159 75. 39 Gao L, Tao Y, Zhang L et al. Vitamin D receptor genetic polymorphisms and tuberculosis: updated systematic review and meta-analysis. Int J Tuberc Lung Dis 2010;14: 15 23. 40 Cozzolino M, Stucchi A, Rizzo MA et al. Vitamin D receptor activation and prevention of arterial ageing. Nutr Metab Cardiovasc Dis 2012;22:547 52. www.rheumatology.oxfordjournals.org 1007

Zhao-Hua Zhu et al. 41 Morrison NA, Qi JC, Tokiata A et al. Prediction of bone density from vitamin D receptor alleles. Nature 1994;367: 284 7. 42 Melhus H, Kindmark A, Amer S et al. Vitamin D receptor genotypes in osteoporosis. Lancet 1994;344:949 50. 43 Valdes AM, Hart DJ, Jones KA et al. Association study of candidate genes for the prevalence and progression of knee osteoarthritis. Arthritis Rheum 2004;50:2497 507. 44 Uitterlinden AG, Burger H, van Duijn CM et al. Adjacent genes, for COL2A1 and the vitamin D receptor, are associated with separate features of radiographic osteoarthritis of the knee. Arthritis Rheum 2000;43:1456 64. 45 Jones G, White C, Sambrook P et al. Allelic variation in the vitamin D receptor, lifestyle factors and lumbar spinal degenerative disease. Ann Rheum Dis 1998;57:94 9. 46 Baldwin CT, Cupples LA, Joost O et al. Absence of linkage or association for osteoarthritis with the vitamin D receptor/type II collagen locus: the Framingham osteoarthritis study. J Rheumatol 2002;29:161 5. 47 Chen B, Cao L, Zhou Y et al. Glutathione S-transferase T1 (GSTT1) gene polymorphism and gastric cancer susceptibility: a meta-analysis of epidemiologic studies. Dig Dis Sci 2010;55:1831 8. 48 Loughlin J, Mustafa Z, Irven C et al. Stratification analysis of an osteoarthritis genome screen-suggestive linkage to chromosomes 4, 6, and 16. Am J Hum Genet 1999;65: 1795 8. 49 Kaprio J, Kujala UM, Peltonen L et al. Genetic liability to osteoarthritis may be greater in women than men [letter]. BMJ 1996;313:232. 1008 www.rheumatology.oxfordjournals.org