Abstract

Recently, through a genome wide association study in Japanese knee osteoarthritis (OA) cases, a previously unknown gene, DVWA, was identified. The non-synonymous single nucleotide polymorphism (SNP) rs7639618 was subsequently found to be consistent and most significantly associated in Japanese and Han Chinese knee OA studies and functional relevant. Here, the association of the DVWA polymorphisms (rs7639618, rs11718863 and rs9864422) was genotyped in 1120 knee OA cases, 1482 hip OA cases and 2147 controls, all of white European descent from the Netherlands, the UK, Spain and Greece. Random effect DerSimonian and Laird meta-analyses were performed to assess the association in the different strata. To assess a more global effect, the original Japanese and Chinese data were included with the European. The meta-analyses provided evidence for global association of rs7639618 with knee OA with an odds ratio (OR) of 1.29, 95% confidence interval (CI) of 1.15–1.45 and a P-value of 2.70 × 10−5. This effect, however, showed moderate heterogeneity, and rs7639618 was not independently associated with knee OA in Europeans, with an OR of 1.16, 95% CI of 0.99–1.35 and a P-value of 0.063. Furthermore, no association was observed with hip OA in Europeans, with a P-value of 0.851. Our results suggest that there may be global relevance for the DVWA SNP rs7639618 among knee OA cases, however, the apparent lower effect size in combination with the higher risk allele frequency in the European samples highlights again the ethnic differences in effects of discovered OA susceptibility genes.

INTRODUCTION

Osteoarthritis (OA) is a prevalent, chronic degenerative joint disease that causes considerable disability among the elderly. There is no adequate therapy to reverse or slow down the degenerative process, consequently OA has a large socio-economic burden. OA is a complex disease reflected by phenotypic and genetic heterogeneity. Elucidation of common pathways that are involved in the onset and progression of the disease may assist in the development of new drug targets and in better management of this disabling condition in the future.

In the search for susceptibility loci for OA, it has become apparent that many loci show particular relevance to disease development at particular skeletal sites (1–4). Furthermore, OA susceptibility loci identified in Asian cohorts are often not as relevant to disease development in European cohorts, and vice versa (2,3,5–9), indicating ethnic differences in the OA genetic component. Meta-analysis, however, is known to provide an effective way to assess effect sizes in different independent studies while addressing the heterogeneity between them. Already, extensive meta-analysis has shown to be of value to explore the overall effects across ethnic groups and OA subtypes for the genes Asporin (ASPN) (8), growth and differentiation factor 5 (GDF5) (10) and endothelial differentiation gene 2 (EDG2) (7). Others await further replication (11).

Through a recent genome wide association (GWA) study in Japanese, a previously unknown gene, double von Willebrand factor domain A (DVWA), was found to harbour single nucleotide polymorphisms (SNPs) (rs7639618, rs11718863 and rs9864422) that showed consistent association with knee OA in Japanese and Chinese knee OA cohorts with the highest association for rs7639618 (12). It was further shown that the DVWA protein interacts with ß-tubulin and that the strength of binding is influenced by the alternate amino acids coded for by the two non-synonymous SNPs rs7639618 and rs11718863. In the current paper, meta-analyses were performed to explore replication of the Asian-associated DVWA SNPs in a large number of Europeans with knee OA (n = 1120) and European controls (n = 2147) from the Netherlands, the UK, Spain and Greece. An association analysis was also performed on 1482 Europeans with hip OA, such that the total number of European cases and controls genotyped was 4749. Finally, to assess the global effect across different ethnic groups, we included the original DVWA association data of the Japanese and Chinese knee OA cohorts.

RESULTS

The reported high linkage disequilibrium (LD) among the three SNPs in the DVWA gene was also observed in the European populations with an r2 = 1 between the DVWA SNPs rs7639618 and rs1171863 and r2 = 0.87 for SNPs rs7639618 and rs1171863 with rs9864422. Only data for rs7639618 are, therefore, presented here.

Table 1 shows allele and genotype counts for rs7639618 and allelic odds ratios (ORs) in cases and controls from each European and Asian cohort. All genotypes are in Hardy–Weinberg equilibrium (HWE) in the control samples. The frequency of the common predisposing G-allele differed considerably between Asian and European populations, being ∼50% in Asian controls and ∼85% in European controls. In the individual European cohorts, the G-allele showed modest significant predisposing association only in the UK cases (P = 0.018 for knee cases and P = 0.045 for hip cases).

Table 1.

Allele and genotypes for the DVWA SNP rs7639618 in cases and controls from both European and Asian OA studies

DVWA SNP rs7639618 Alleles (%)
 
Total OR (95% CI) P-valuea Genotypes
 
Total P-value HWE 
GG GA AA 
European populations (new data) 
 UK 
  Controls 1223 (83) 253 (17) 1476   504 215 19 738 0.487 
  TKR 635 (87) 97 (13) 732 1.35 (1.05–1.74) 0.018 275 85 366  
  THR 1904 (85) 328 (15) 2232 1.20 (1.00–1.44) 0.045 808 288 20 1116  
 Netherlands 
  Controls 1264 (86) 214 (14) 1478   538 188 13 739 0.459 
  GARP 634 (84) 118 (16) 752 0.91 (0.70–1.19) 0.447 263 108 376  
  GARP knee OA 232 (85) 42 (15) 274 0.94 (0.63–1.38) 0.735 98 36 137  
  GARP hip OA 165 (83) 33 (17) 198 0.85 (0.57–1.26) 0.414 67 31 99  
 Spain 
  Controls 448 (83) 94 (17) 542   189 70 12 271 0.103 
  TKR 414 (83) 84 (17) 498 1.03 (0.75–1.43) 0.8 171 72 249  
  THR 434 (81) 100 (19) 534 0.91 (0.67–1.24) 0.5 175 84 267  
 Greece 
  Controls 679 (85) 119 (15) 798   291 97 11 399 0.401 
  Knee OA 640 (87) 96 (13) 736 1.17 (0.87–1.56) 0.226 280 80 368  
Asian populationsb 
 Japan 
  Controls 651 (52) 607 (48) 1258   162 327 140 629 0.304 
  Disease cohort knee OA (set B) 799 (62) 483 (38) 1282 1.54 (1.32–1.81) 7.3 × 10−8 253 293 95 641  
 Japan 
  Controls resident cohort 554 (57) 412 (43) 966   166 222 95 483 0.184 
  Populations-based knee OA (set C) 305 (63) 179 (37) 484 1.27 (1.01–1.59) 0.038 99 107 36 242  
 China 
  Controls 404 (49) 422 (51) 826   106 192 115 413 0.156 
  Knee OA 477 (57) 357 (43) 834 1.40 (1.15–1.69) 7.2 × 10−4 145 187 85 417  
DVWA SNP rs7639618 Alleles (%)
 
Total OR (95% CI) P-valuea Genotypes
 
Total P-value HWE 
GG GA AA 
European populations (new data) 
 UK 
  Controls 1223 (83) 253 (17) 1476   504 215 19 738 0.487 
  TKR 635 (87) 97 (13) 732 1.35 (1.05–1.74) 0.018 275 85 366  
  THR 1904 (85) 328 (15) 2232 1.20 (1.00–1.44) 0.045 808 288 20 1116  
 Netherlands 
  Controls 1264 (86) 214 (14) 1478   538 188 13 739 0.459 
  GARP 634 (84) 118 (16) 752 0.91 (0.70–1.19) 0.447 263 108 376  
  GARP knee OA 232 (85) 42 (15) 274 0.94 (0.63–1.38) 0.735 98 36 137  
  GARP hip OA 165 (83) 33 (17) 198 0.85 (0.57–1.26) 0.414 67 31 99  
 Spain 
  Controls 448 (83) 94 (17) 542   189 70 12 271 0.103 
  TKR 414 (83) 84 (17) 498 1.03 (0.75–1.43) 0.8 171 72 249  
  THR 434 (81) 100 (19) 534 0.91 (0.67–1.24) 0.5 175 84 267  
 Greece 
  Controls 679 (85) 119 (15) 798   291 97 11 399 0.401 
  Knee OA 640 (87) 96 (13) 736 1.17 (0.87–1.56) 0.226 280 80 368  
Asian populationsb 
 Japan 
  Controls 651 (52) 607 (48) 1258   162 327 140 629 0.304 
  Disease cohort knee OA (set B) 799 (62) 483 (38) 1282 1.54 (1.32–1.81) 7.3 × 10−8 253 293 95 641  
 Japan 
  Controls resident cohort 554 (57) 412 (43) 966   166 222 95 483 0.184 
  Populations-based knee OA (set C) 305 (63) 179 (37) 484 1.27 (1.01–1.59) 0.038 99 107 36 242  
 China 
  Controls 404 (49) 422 (51) 826   106 192 115 413 0.156 
  Knee OA 477 (57) 357 (43) 834 1.40 (1.15–1.69) 7.2 × 10−4 145 187 85 417  

TKR, total knee replacement; THR, total hip replacement; GARP, Genetics OsteoArthritis and Progression.

aAllelic P-value.

bData of original manuscript (12).

Meta-analyses of the allelic effect

Meta-analysis was first conducted on the pooled European knee OA cases with the allelic model following the initial finding from the Asian populations (Fig. 1 and Table 2). No significant association with the G-allele was detected, with an OR of 1.16 [95% confidence interval (CI): 0.99–1.35] and a P-value of 0.063, and there was no heterogeneity, with a P-value of 0.374 (I2 = 3.6). We subsequently carried out a meta-analysis of the allelic association for the pooled European and Asian knee OA cases, which generated a highly significant P-value of 2.7 × 10−5, with a random summary OR of 1.29 (95% CI: 1.15–1.45) (Fig. 1 and Table 2). Although not significant in this analyses, the heterogeneity observed was moderately high (P = 0.121, I2 = 40.5). Sensitivity of this global effect was subsequently tested by removing the discovery sample (the Japanese disease cohort, set B) with the analysis focusing on the remaining six knee OA case groups (the replication studies). The association of the G-allele of rs7639618 with knee OA among the replication studies was significant with a P-value of 2 × 10−4 and a summary OR of 1.24 (95% CI: 1.11–1.38) with a much lower measure of heterogeneity (P = 0.339, I2 = 11.9%; Table 2). There was no association of the G-allele in the European hip OA cases (P = 0.851; Table 2).

Figure 1.

DerSimonian–Laird OR meta-analysis (random effects) of the association between DVWA SNP rs7639618 and OA as measured by allele frequency data. Black squares (OR) and 95% CI (bar) are shown for each study. The pooled ORs and their 95% CIs are represented by the unshaded diamonds. Summary ORs are given for each ethnic group as well as the combination of the groups. The left-hand column lists the individual studies, each one defined by country of origin.

Figure 1.

DerSimonian–Laird OR meta-analysis (random effects) of the association between DVWA SNP rs7639618 and OA as measured by allele frequency data. Black squares (OR) and 95% CI (bar) are shown for each study. The pooled ORs and their 95% CIs are represented by the unshaded diamonds. Summary ORs are given for each ethnic group as well as the combination of the groups. The left-hand column lists the individual studies, each one defined by country of origin.

Table 2.

Random-effects meta-analysis of four European and three Asian studies for the allelic association of DVWA SNP rs7639618 with knee and hip OA

Summary (number of studies) Allele model
 
OR (95% CI) P-value for the test of combined OR P-value for the test of heterogeneity (I2%) 
Knee OA 
 All studies combined (7)a 1.29 (1.15–1.45) 2.7 × 10−5 0.121 (40.5) 
 Replication studies (6)b 1.24 (1.11–1.38) 2 × 10−4 0.339 (11.9) 
 European (4)c 1.16 (0.99–1.35) 0.063 0.374 (3.6) 
 Asian (3)d 1.43 (1.28–1.59) 1.50 × 10−10 0.358 (2.8) 
Hip OA 
 All studies combined (3)e 1.02 (0.81–1.29) 0.851 0.140 (49.1) 
Summary (number of studies) Allele model
 
OR (95% CI) P-value for the test of combined OR P-value for the test of heterogeneity (I2%) 
Knee OA 
 All studies combined (7)a 1.29 (1.15–1.45) 2.7 × 10−5 0.121 (40.5) 
 Replication studies (6)b 1.24 (1.11–1.38) 2 × 10−4 0.339 (11.9) 
 European (4)c 1.16 (0.99–1.35) 0.063 0.374 (3.6) 
 Asian (3)d 1.43 (1.28–1.59) 1.50 × 10−10 0.358 (2.8) 
Hip OA 
 All studies combined (3)e 1.02 (0.81–1.29) 0.851 0.140 (49.1) 

Heterogeneity was evaluated using the Cochran Q test and I2 values (%).

aAll knee studies combined consist of total knee replacement (TKR) UK, the GARP knee OA, TKR Spain, knee OA Greece, disease cohort knee (set B) Japan, population cohort knee (set C) and knee OA China.

bAll knee studies combined excluding the discovery sample (Japanese disease cohort knee, set B).

cEuropean knee studies consist of UK TKR, the GARP knee OA, Spain TKR and Greece knee OA.

dDisease cohort knee Japan (set B), population cohort knee Japan (set C) and knee OA China.

eEuropean hip OA studies consisting of UK and Spanish total hip replacements and GARP hip OA cases.

DISCUSSION

A compelling association of two common functionally relevant non-synonymous SNPs (rs7639618 and rs1171863), located in the novel gene DVWA, was observed in knee OA cases from Japan and China opening up a novel etiological mechanism for OA (12). In the current study, meta-analyses were performed to assess whether these allelic associations were also independently relevant to Europeans and to assess the global effect by analysing the European and original Asian samples together.

A moderately significant association was observed in the UK sample alone, but when the four European studies were analysed together, the association in the European knee OA stratum appeared not significant (P-value = 0.063) and had a significant lower effect size (P = 0.029) when compared with the Asian studies. In addition, we could not detect a significant combined effect for hip OA. These data are in line with the recent findings of Valdes et al. (13). The different results within the European studies may in part be caused by the OA selection criteria varying from symptomatic, radiographic OA to total knee replacement (TKR) and/or differences in the control selection varying from random individuals to individuals without OA symptoms, underscoring the need of additional standardization of the OA phenotypes.

Global meta-analysis including the original Asian samples showed significant association for the common G-allele of DVWA SNP rs7639618 with knee OA, which persisted when we tested sensitivity by removing the initial Japanese discovery cohort (set B). In contrast to the other analyses, the global analysis (including the Japanese discovery cohort) showed a moderate heterogeneity as expressed by the I2 of 40%. This indicates that the effect observed in the Japanese discovery cohort (set B) may be an overestimation due to selection strategies applied in GWA analyses towards the lowest P-values. Similar to the Japanese and Chinese cohorts, we could not find more or other significant effects with the other DVWA SNPs rs11718863 or rs9864422 (results not shown).

As has been reported previously in OA replication studies (10), we observed a large allele frequency difference between Asians and Europeans for the functional relevant rs7639618, with the G-allele of the SNP having a frequency of ∼50% in Asians and 85% in Europeans. This much higher frequency of the G-allele in Europeans does reduce the power of the European cohorts to detect significant association. Alternatively, if the rs7639618 SNP in combination with rs11718863 are not the ‘true functional’ relevant SNPs, the observed difference between Asian and European populations may be due to an underlying difference in the LD. However, the LD block surrounding the SNPs in Asian and Caucasian populations, as given by HapMap (14), indicates a similar structure and length.

Altogether our results demonstrate a significant global effect on knee OA for the DVWA SNP rs7639618 which is not independently replicated in a large number of European knee OA cases and controls. It should be noted, however, that the observed effect size in Europeans knee OA cases is significantly lower and the frequency of the risk allele is significantly higher when compared with the Asians which, in combination with possible differences in yet unknown environmental factors, may have again hampered our efforts to robustly replicate in one ethnic group, an OA-associated allele identified in a different ethnic group. To embark on these differences, future meta-analyses may benefit from the inclusion of such environmental factors or OA cases of additional ethnicities.

MATERIALS AND METHODS

DNA samples

The four European collections have been described in detail elsewhere (15–19). In short, the UK (15) and Spanish cases include patients with severe primary OA who were due to undergo, or had undergone, TKR or total hip replacement, whereas controls were 55 years or older and free of OA-related complaints. The samples from Greece consisted of TKR patients and controls older than 45 years and free of clinical OA. The Genetics OsteoArthritis and Progression (GARP) study from Leiden, the Netherlands, consisted of sibling pairs concordant for clinical and radiographically (Kellgren/Lawrence) confirmed OA at two or more joint sites among hand, spine (cervical or lumbar), knee or hip (18), random controls were partners of the offspring of the Leiden longevity study (20). To comply with the OA phenotypes used in the original study, GARP patients with symptomatic in addition to radiographic knee or hip OA were selected. The European individuals were of white ethnicity and DNA samples were obtained after the donors had provided their informed consent and with the approval of the respective Ethics Committees.

Genotyping

New genotype data on the European samples of the DVWA SNPs rs7639618, rs1171863 and rs9864422 were generated at three centres: Santiago for the Spanish collection, Leiden for the GARP and UK collections, and the RIKEN institute of Tokyo for the Greek collection. In Santiago, a fluorescent 5’ exonuclease assay designed as a TaqMan Genotyping Assay was used (Applied Biosystems, Foster City, CA, USA). In Leiden, mass spectrometry (the homogeneous MassARRAY system; Sequenom, San Diego, CA, USA) was used following standard protocols. In Tokyo, sequencing of PCR products using the ABI3700 DNA analyser (Applied Biosystems) was used according to the manufacturer’s instructions. For the Asian population, we used data as described by Miyamoto et al. (12).

At Santiago, no duplicates were genotyped, whereas the genotyping success rate of subjects was 94.7% and the subsequent SNP genotyping success rate was 99.88%. For the samples that were genotyped by the Sequenom system in Leiden, ∼8% of the samples were genotyped in duplicate and compared. Error rates were 1.55% for the UK and 0.57% for the Leiden collection. The subjects showed an average success rate of 99.3% and a subsequent SNP success rate of 99.97%. At the RIKEN institute, all genotyping data were checked manually and ∼5% of the samples (both cases and controls) were checked by direct sequencing of genomic DNAs, error rate was 0%.

Statistical analysis

To test for replication and a global effect of the findings of Miyamoto et al. (12), we examined the effects of allele frequency differences of the DVWA SNP rs7639618 by calculating summary ORs and 95% CIs for knee OA in each group, each ethnic group as well as for the entire set. As there is a general tendency for discovery samples to have a high OR due to a ‘winners curse’, we tested sensitivity of the association by excluding the Japanese discovery cohort (set B). Next, we explored possible association for hip OA in the newly generated data of the European samples. Due to the relatively low frequency of the rare allele in Europeans (∼15%), we were not able to robustly test the mode of inheritance. To adjust for the family relationship among sibling pairs of the GARP study, standard errors (SEs) were estimated from the variance between the sibling pairs (robust SEs) and used in all the comparisons between the GARP subjects and controls including the meta-analyses (21). We performed robust SE analyses using Stata SE8 software (Stata Corporation, USA). Meta-analyses were performed in R (http://www.r-project.org/). Summary ORs were estimated using the random-effects model of DerSimonian and Laird (22). The heterogeneity was quantified using the I2 statistic for inconsistency (23) and its statistical significance was tested with the χ2 distributed Cochran Q statistic (24). I2 describes the proportion of variation that is unlikely to be due to chance and is considered large for values over 50% (23).Two-tailed P-values are reported for all analyses. For the distribution of genotypes, HWE was tested by using the HWE program of LINKUTIL. The difference in the combined effect size of, respectively, Asian and European samples was determined by the difference in the β and its statistical significance was tested with the χ2 distribution of this difference divided by the SE of this difference.

FUNDING

This study was supported by Research into Ageing, the Arthritis Research Campaign, the Leiden University Medical Centre, the Dutch Arthritis Association and Pfizer Inc., Groton, CT, USA support the GARP study. The genotypic work was supported by the Netherlands Organization of Scientific Research (MW 904-61-095, 911-03-016, 917 66344 and 911-03-012), Leiden University Medical Centre and the Centre of Medical System Biology in the framework of the Netherlands Genomics Initiative. The contribution of the Santiago’s group to this project was financed by the Foundation Mutua Madrilena Madrid, Spain. The UK study was supported by Research into Ageing and by the Arthritis Research Campaign.

ACKNOWLEDGEMENT

The authors thank Jeanine Houwing Duistermaat (LUMC, Department of Medical Statistics, Leiden, The Netherlands) for statistical support.

Conflict of Interest statement. None declared.

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