Vitamin D receptor gene polymorphisms and osteoarthritis of the hand, hip, and knee: acase–control study in Japan

J. Huang, T. Ushiyama, K. Inoue, T. Kawasaki and S. Hukuda

Department of Orthopaedic Surgery, Shiga University of Medical Science, Seta, Otsu,520-2192, Japan

Correspondence to: T. Ushiyama.


    Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Objective. To investigate the association between vitamin D receptor (VDR) gene polymorphisms andJapanese female patients with osteoarthritis (OA) of the hand, hip, and knee.

Methods.BsmI,ApaI, andTaqI restriction fragment length polymorphisms (RFLPs) of the VDR gene were analysed in 270Japanese female patients with radiographic OA of the hand, hip, tibiofemoral (TF) joint, andpatellofemoral (PF) joint, as well as in female controls.

Results. There was no significant association between the VDR gene RFLPs and OA of the hand, hip, TFjoint, PF joint, or polyarticular involvement. The previously detected preventive genotype of the VDRgene was uncommon in our test population.

Conclusion. The relative importance of VDR gene polymorphism in the development of OA may vary betweenethnic groups.

KEY WORDS: Osteoarthritis, Hand, Hip, Knee, Vitamin D receptor gene, Polymorphism


    Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Osteoarthritis (OA) is a common disease that is most prevalent in aged people [1]. It is considered to be a group of clinically heterogeneous disorders sharing common pathologicalfeatures of articular cartilage loss and subchondral bone reaction. Although the aetiology of OA isunknown, abundant epidemiological evidence suggests the importance of genetic factors in somesubgroups of OA, especially in hand and knee OA [26]. Until now, some candidate genes have been proposed in OA of the hand, hip, knee, spine, andpolyarticular involvement [711].

Vitamin D receptor (VDR) gene polymorphism is known for its association with osteoporosis [12], and the inverse relationship between osteoporosis and OA suggests that VDR genepolymorphism might be associated with both diseases [13,14]. Recent studies have revealed the association of VDR gene polymorphism with some OA sites.Uitterlindenet al. [15] demonstrated that VDR gene polymorphism is related to radiographic OA of the knee, especiallyin osteophyte formation, in subjects in The Netherlands. A study by Keenet al. [16] also showed that this gene influenced the risk of early knee OA within the British population andthat it had no relationship with nodal arthritis. Joneset al. [17] reported that allelic variations of the VDR gene and lifestyle factors were both associated with thepresence and severity of spinal degenerative disease. Aerssenset al. [18] and Tamaiet al. [19] reported the lack of association between OA of the hip and VDR gene polymorphisms in Belgianwomen and Japanese women, respectively.

Since there are inter-racial differences in the frequency of distribution of VDR gene polymorphisms [12,20,21] and because previous reports suggest that its association with OA depends on the joints, theassociation between the VDR gene and OA of various joint sites should be examined in differentraces. In this study, we investigated the frequency distributions of the VDR geneBsmI,ApaI, andTaqI restriction fragment length polymorphisms (RFLPs) in Japanese female patients with OA of thehand, hip, tibiofemoral (TF) joint, and patellofemoral (PF) joint, and compared them with those ofJapanese female controls.


    Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Definition of radiographic OA
OA was defined radiographically using the Kellgren–Lawrence scale [22] as follows. Regarding hand OA, subjects with symmetrical polyarticular involvement of theinterphalangeal joints of the hands, a characteristic feature of generalized OA, were chosen ashaving hand OA. Thus, hand OA was radiographically defined as having grade 2 or more changesaccording to the Kellgren–Lawrence scale in at least three interphalangeal joints of eachhand in a radiograph of the hands (posteroanterior view), as previously described by others [23]. Hip OA, TF joint OA, and PF joint OA were defined as having grade 2 or more changes accordingto the Kellgren–Lawrence scale in at least one side of the respective joint in radiographs ofthe pelvis (non-weight-bearing anteroposterior view) and knees (weight-bearing anteroposterior viewand non-weight-bearing lateral view), respectively. Polyarticular OA was defined as having hand, hip,and TF OA.

Subjects with OA of each joint were further divided into two groups, mild and severe. Amongpatients with hand OA, those who showed grade 3 or more changes according to theKellgren–Lawrence scale in one or more IP joints of each hand were defined as having severehand OA. For the hip, TF, and PF joints, severe OA of the respective joint was defined as havinggrade 3 or more changes according to the Kellgren–Lawrence scale in at least one side ofthe respective joint. The remainder with OA were defined as having mild OA of the respective joint.

OA patients
Among consecutive female patients visiting our out-patient clinic between April 1996 and February1999, 270 women (mean age 63.8 yr, range 29–87 yr) who were diagnosed as havingradiographic OA in either the hand, hip, or TF joint were included in this study as cases. Fourradiographs were taken for each case: the hands, the pelvis, and the knees as described above.One hundred and nine of them returned for a post-operative follow-up of orthopaedic surgery, ofwhich 71 underwent hip surgery, 34 knee surgery, two spine surgery, and two both hip and kneesurgery. The remaining non-operated patients visited our clinic for consultation regarding anoperation, or with hip, knee, and hand complaints. The majority of women who had radiographichand OA voiced minor complaints of hand symptoms. All radiographs were assessed by a singleblind observer. If the patient had undergone surgery for the hip or knee, pre-operative radiographs ofthese joints were reviewed.

According to the definition of radiographic OA, 270 cases were classified into one or more of thefollowing categories (Table1Go): hand OA, 134 cases (mean age 66.6 yr, range 45–87 yr) with 38 mild cases and 96 severecases; hip OA, 146 cases (mean age 62.8 yr, range 29–84 yr) with 21 mild cases and 125severe cases; TF joint OA, 180 cases (mean age 67.6 yr, range 42–87 yr) with 58 mildcases and 122 severe cases; PF joint OA, 167 cases (mean age 67.6 yr, range 42–87 yr)with 108 mild cases and 59 severe cases; and polyarticular OA, 46 cases (mean age 68.8 yr, range55–84 yr).


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TABLE 1.  Case characteristics
 
The height and body weight were measured for all OA subjects, but clinical evidence of nodes wasnot systematically recorded.

Control subjects
Women aged 60 yr or more, who were routine visitors to three municipal daycare centres for theelderly, located in our catchment population, and who responded to our invitation for a health checkexamination, were chosen as control subjects. They were not hospitalized, had independent dailylives, and were able to walk without support. Medical history-taking, measurements of body weightand height, and a physical examination were performed. Rheumatic complaints were systematicallyrecorded by joint site on a chart. The presence and severity of bony swelling in the interphalangealjoints were clinically assessed by a single observer, according to the previous report by Hartet al. [24]. The grades of clinical nodes were defined as follows: grade 0, no bony swelling; grade 1, possiblebony swelling; grade 2, definite bony swelling, not severe; grade 3, severe bony swelling but nodeformity; grade 4, severe bony swelling with deformity [24]. Having clinical nodes was defined as having at least one joint of grade 2 or more. Radiographswere not taken of the control subjects.

Women with a history of rheumatoid arthritis or other inflammatory joint disease were excluded fromthis study. A total of 161 women (mean age 72.2 yr, range 60–90 yr) were included in thestudy and placed into at least one of the following control groups. Of these, 127 women withoutclinical nodes of the hands were chosen as the controls for hand OA, 158 without present hip painwere chosen as the controls for hip OA, and 105 without present knee pain were chosen as thecontrols for both TF and PF OA. Eighty-eight women in whom clinical nodes of the hands wereabsent, and who had no present pain in either the hip or knee, were chosen as the controls forpolyarticular OA. The characteristics of the control subjects are shown inTable2Go.


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TABLE 2.  Control characteristics
 
Analysis of VDR gene polymorphisms
Peripheral blood was collected from the 270 cases and 161 control subjects. Analysis of VDR geneRFLPs was performed according to the method of the previous report, with a slight modification [21]. Briefly, 100 ng of genomic DNA, extracted from peripheral blood, was amplified by polymerasechain reaction (PCR) using specific primers in the same PCR conditions [21]. The amplified PCR product was digested with restriction endonucleasesBsmI,ApaI, andTaqI (Nippongene, Tokyo, Japan) and electrophoresed on a 1.0% agarose gel. The RFLPs wererepresented by Bb (BsmI), Aa (ApaI), and Tt (TaqI) with upper- and lower-case letters signifying the absence or presence of restriction sites,respectively.

Statistical analysis
An odds ratio (OR) and 95% confidence interval (CI) were calculated with respect to thepresence of the reference VDR genotype as compared with other genotypes. A multivariate analysisusing logistic regression was performed to control for body mass index. The software used for thisanalysis was SPSS (SPSS Inc., Chicago, IL, USA).


    Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The frequencies ofBsmI,ApaI, andTaqI RFLPs of the VDR gene in the control subjects were similar to those previously described forJapanese women [20] and essentially obeyed the Hardy–Weinberg law.

There was no significant difference in VDR genotype frequency in eachBsmI,ApaI, andTaqI site between the control and each OA group: OA of the hand, the hip, the TF joint, and the PF joint(Table3Go). Furthermore, no significant difference could be found when the patients with polyarticular OA werecompared with the controls (Table4Go). The distribution of the VDR genotypes by a combination ofBsmI,ApaI, andTaqI sites also revealed no significant differences between the controls and OA of various sites (Tables5 and6GoGo). Mild and severe OA cases were compared with the controls separately, again yielding nosignificant differences (data not shown).


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TABLE 3.  Frequency of vitamin D receptor (VDR) genotype distribution in female patients with handosteoarthritis (OA), hip OA, tibiofemoral (TF) OA and patellofemoral (PF) OA
 

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TABLE 4.  Frequency of vitamin D receptor (VDR) genotype distribution in patients with polyarticular osteoarthritis (OA)
 

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TABLE 5.  Combination ofBsmI,ApaI, andTaqI restriction fragment length polymorphisms (RFLPs) of the vitamin D receptor (VDR) gene in female patients with hand osteoarthritis (OA) hip OA, tibiofemoral (TF) OA and patellofemoral (PF) OA
 

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TABLE 6.  Combination ofBsmI,ApaI, andTaqI restriction fragment length polymorphisms (RFLPs) of the vitamin D receptor (VDR) gene in female patients with polyarticular osteoarthritis (OA)
 
VDR genotypes `bb' and `bbaaTT', which were reported as the preventive genotypes for osteoporosisin Japanese pre-menopausal women [20], did not reveal any association for any site of OA.

In a separate analysis, both VDR genotypes and body mass index were included as dependentvariables in a logistic regression model to control for body mass index. However, the results wereessentially similar to those without adjusting for obesity (data not shown).


    Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
In this study, we failed to find any significant relationship between VDR gene polymorphism and OAin Japanese women. In the study in The Netherlands, which included both men and women, the`baT' haplotype was over-represented and the `bAT' haplotype under-represented in OA of the TFjoint [15], while in the British study, the risk of TF joint OA was associated with the presence of the `T' allelein women [16]. Neither VDR genotype was related to TF OA in this study. Furthermore, we also could not find any significant relationship between VDR gene polymorphism and OA of the hand, hip, PF joint, orpolyarticular involvement. Our results for hip OA were in accordance with previous studies in Belgium and Japan [18,19].

In previous TF OA studies, cases were sampled from the general population, whereas our studywas of hospital attenders, which resulted in a greater percentage of symptomatic and severe casesbeing included, causing the inevitable selection bias to occur. Spectoret al. noted that the prevalence of symptomatic knee OA was only 2.3 per 100 compared with that ofradiographically defined knee OA which was 17 per 100 [25]. From those results, the majority of radiographically detected cases from general populationsamples may be asymptomatic. Interestingly, increasing osteophytosis was not associated with aworsening of symptoms or function in the study by Ledinghamet al. [26]. In previous studies, the association between the VDR gene and TF OA appeared to be related toosteophyte presence rather than cartilage loss [15,16]. Taken together, it is possible to speculate that VDR gene polymorphism may not be related toprogression or severe cases of TF OA.

In the present study, radiographs were not taken for the control subjects, possibly weakening itsstatistical power, and we did not control for possible confounders, such as occupation and sportsactivity. We believe, nevertheless, that these limitations did not influence the nature of our studyresults. To detect a three-fold increased risk of OA, with the chance of not detecting the relative riskas significant being 0.2 (type II error rate), the required sample sizes [27] in the Japanese population are estimated to be from 54 (for genotype `Aa') to 124 (for genotype`TT'), except for genotypes `BB' and `tt' which were extremely rare in our population. Inconsideration of the required sample sizes, it would appear that the study was adequately poweredto detect an effect attributable to theApaI locus but not theBsmI norTaqI loci. Given that the `T' allele is responsible for developing OA with a prevalence of knee OA, atleast TF joint OA should have been much higher in our population than in Europe, because thepreviously detected preventive genotype `tt' was hardly detectable in our population. However, thepopulation-based radiographic study of knee OA in Japan did not show a significant difference fromthose in Europe and North America. For example, the prevalence of knee OA, defined as havinggrade 2 or more changes by the Kellgren–Lawrence scale, among women aged between 55and 64 yr was 7.3% in US National Health Surveys [28], 18.6% in the Zoetermer Community Survey in The Netherlands [29], and 9.8% in the Japanese study [30]. It is therefore probable that VDR gene polymorphism is in linkage disequilibrium with a nearbysusceptibility locus, and the strength of the relationship between the VDR gene and OA may varybetween ethnic groups.


    Acknowledgments
 
We wish to thank Dr J. Nishioka, Dr M. Nabae, Dr T. Mori, Dr T. Takase, Mrs K. Taniguchi, and MrsK. Nishikawa for collecting the blood samples. This study was supported in part by Grants-in-Aid forScientific Research (10671355) from the Ministry of Education, Science, and Culture of Japan.


    References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Submitted 15 June 1998; revised version accepted 9 August 1999.