International Cartilage Repair Society

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1 OsteoArthritis and Cartilage (2007) 15, 972e978 ª 2007 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved. doi: /j.joca Estrogen receptor alpha genotype is associated with a reduced prevalence of radiographic hip osteoarthritis in elderly Caucasian women 1 K. Lian M.D.y, L. Lui M.S.z, J. M. Zmuda Ph.D.x, M. C. Nevitt Ph.D.yz, M. C. Hochberg M.D.k{, J. M. Lee M.S.#, J. Li Ph.D.# and N. E. Lane M.D.yy* y Department of Epidemiology, University of California at San Francisco, San Francisco, CA 94143, USA z Department of Biostatistics, University of California at San Francisco, San Francisco, CA 94143, USA x Department of Epidemiology, University of Pittsburgh, Pittsburgh, PA, USA k Department of Medicine, University of Maryland, Baltimore, MD, USA { Department of Epidemiology and Preventive Medicine, University of Maryland, Baltimore, MD, USA # Department of Human Genetics, Roche Molecular Systems, Alameda, CA, USA yy Department of Medicine, Center for Aging, University of California at Davis, Sacramento, CA 94817, USA Summary International Cartilage Repair Society Purpose: This study evaluated the association between polymorphisms in the estrogen receptor (ER) alpha gene (ESR1) and prevalent and incident radiographic hip osteoarthritis (RHOA) in a large, well-defined prospective cohort of elderly Caucasian women. Methods: Prevalent and incident RHOA was evaluated from all available pelvis X-rays obtained from the Study of Osteoporotic Fractures at baseline and after a mean of 8.3 years. Evaluable DNA samples were available from 4746 of these subjects. RHOA cases were defined by published methods. The ESR1 polymorphisms at intron I (Pvu II for a T/C substitution and Xba I for an A/G substitution) were genotyped in the context of a multiplex polymerase chain reaction (PCR) amplification followed by allele-specific single nucleotide polymorphism (SNP) detection with immobilized oligonucleotide probes in linear arrays. Multiple logistic regression was performed to estimate odds ratios (ORs) and 95% confidence intervals (95% CI) associated with the T/C and A/G polymorphisms. Results: RHOA was present in 12.1% of subjects, of whom 325 had joint space narrowing (JSN) score 3 and 130 had an osteophyte score 2 and JSN score 2. There was a significant reduction in the odds of prevalent RHOA for individuals with the C/C compared to T/T genotype at the Pvu II site with an OR of 0.71 (95% CI: 0.55e0.92) (P ¼ 0.01). Adjustments for age, weight, height, hip Bone mineral density (BMD) and estrogen use did not alter the relationship between the C/C genotype and reduced risk of RHOA, with an OR of 0.71 (95% CI: 0.54e0.94) (P ¼ 0.01). The risk of incident RHOA was reduced for the Pvu II C/C compared to the T/T genotype (P ¼ 0.11). Also, the reduced risk of incident RHOA in C/C subjects varied by estrogen use. There was no association between the Xba I G/G or G/A genotypes and RHOA with OR of 0.82 (95% CI: 0.61e1.10) (P ¼ 0.19) compared to women with A/A genotype. Conclusions: We conclude that the C/C genotype of the ER alpha Pvu II polymorphism was associated with a modestly reduced risk of prevalent and incident RHOA in elderly Caucasian women. Additional work is required to understand how the intron I ESR1 polymorphism may alter joint degeneration. ª 2007 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved. Key words: Osteoarthritis, Estrogen receptors, Polymorphisms. Introduction Osteoarthritis (OA) of the hip is a common disease. It increases with age among the elderly population in the United States and accounts for a significant portion of health care burden 1. 1 This work was supported by grants from the NIH 1R01AG05407, 1R01AR40431, the Doris Duke Clinical Research Fellowship (CRF) Program for Medical Students # , the Rosalind Russell Arthritis Research Foundation, and funding from Roche Molecular Systems. *Address correspondence and reprint requests to: Dr Nancy Lane, Department of Medicine, University of California at Davis, nd Avenue, Suite 2600, Sacramento, CA 95817, USA. Tel: ; Fax: ; nancy.lane@ ucdmc.ucdavis.edu Received 2 October 2006; revision accepted 19 February Multiple risk factors including age, obesity, tissue injury, and gender, among others, have been well established for hip OA 1. With further investigation, genetics appears to be playing an increasingly larger role in the etiopathogenesis of hip OA 2,3. Estrogen receptors (ERs) are known to be associated with disease of bone and articular cartilage. The ER has two isoforms, alpha and beta. The ER alpha gene (ESR1) is an important mediator of signal transduction, and this protein is expressed in cells of the musculoskeletal system including bone cells and chondrocytes 4. Epidemiology studies have shown that women treated with hormone replacement therapy have a reduced risk of hip OA and hand OA 5e10 but other studies have found short-term estrogen replacement may increase the risk of hand and hip OA 11e14. The investigators speculated that long-term estrogen use was protective of articular cartilage loss or prevented increased bone remodeling around the joint 5e

2 Osteoarthritis and Cartilage Vol. 15, No. 8 To date, the associations of the ESR1 polymorphisms and knee and hip OA have varied. While some investigators have reported an increased risk of OA of the knee and generalized OA associated with ESR1 polymorphisms 10,15e17, others have not 18,19. The mechanism for this increased risk of OA and the ESR1 polymorphism is not known; however, it could be through its effects of bone or articular cartilage metabolism 18. However, reports of ESR1 polymorphisms and bone metabolism are conflicting with one study reporting increased odds of bone loss and a lower hip bone mineral density (BMD) over time in individuals with an intronic ESR1 minor allele polymorphism (Pvu II) compared to a control group 20, while another study failed to find an association 21. The purpose of this study was to further evaluate the association between the two widely studied polymorphisms in intron I of the ESR1 (Pvu II and Xba I) with prevalent and incident radiographic hip OA (RHOA) in a large population of elderly Caucasian women from the Study of Osteoporotic Fractures (SOF). Patients and methods STUDY POPULATION All subjects were participants in the SOF, a multi-center cohort study initiated in 1986 to determine risk factors for osteoporotic fractures in elderly women 6,22. Participants were all aged 65 and older at baseline and were recruited from population-based listings at four clinical centers in the United States. Exclusion criteria for the parent study, SOF, included bilateral hip replacement and an inability to walk unassisted; African-American women were excluded because of their low risk of hip fracture. The study was approved by the Institutional Review Board at each of the institutions involved, and all subjects provided written informed consent at enrollment and each clinical examination 22,23. Buffy coat specimens were collected from a total of 6975 participants at either visit 2 (between 1989 and 1990) or visit 6 (between 1997 and 1998), and genotyping was performed in all women who provided adequate consent for genetic studies (Fig. 1). Bilateral supine AeP pelvic radiographs were obtained at baseline and at visit 5 (mean of 8.3 years of follow-up) 24. The present analysis utilized the visit 5 radiographs to evaluate prevalent RHOA and both the baseline and follow-up radiographs to evaluate incident RHOA. ER ALPHA GENOTYPING 973 The ESR1 polymorphisms were genotyped in the context of a multiplex polymerase chain reaction (PCR) amplification followed by allele-specific single nucleotide polymorphism (SNP) detection with immobilized oligonucleotide probes in linear arrays, as previously described 19,20. Primers were modified at the 5 0 phosphate by conjugation with biotin. Purified human genomic DNA (10e50 ng) was amplified in a reaction volume of 50 ml with AmpliTaq 9704 subjects satisfied SOF entry criteria and enrolled 6975 subjects with available and sufficient DNA DNA consent and buffy coat collected Subjects with both Visit 1 and Visit 5 pelvis radiographs* RHOA case criteria (worst hip): MJS 1.5mm Summary grade 3 radiographic features or Definite osteophytes and JSN both grade subjects with available ER alpha genotype and Visit 1 and Visit 5 pelvis x-ray readings 569 subjects satisfied RHOA case criteria 4175 controls 325 RHOA cases had JSN grade RHOA cases with any osteophyte grade 2, JSN 2 Fig. 1. Flow sheet of study subjects. *Study subjects were excluded if they had Paget s disease, bilateral hip replacements, or rheumatoid arthritis. If a patient has bilateral RHOA, the most diseased hip was used in this analysis.

3 974 K. Lian et al.: Hip OA, ER alpha, and genetics Gold DNA polymerase using a GeneAmp PCR System 9600 thermal cycler (PE Biosystems, Foster City, CA) with the following cycling profile: an initial hold at 94 C for 7 min, then 33 two-step amplification cycles of 15 s at 95 C for denaturation and 60 s at 60 C for annealing/extension, and a final 5-min product extension step at 68 C. Chromogenic detection of allelic variants following stringent hybridization of the biotinylated PCR products to the immobilized sequence-specific probes was performed on a Profiblot II T24 (Tecan, Research Triangle Park, NC). RMS inhouse software was used to scan the linear arrays on an Epson Perfection 1670 scanner (Epson, Long Beach, CA) and to assign genotypes. Three thousand one hundred and eighty-two samples were genotyped for ER alpha Pvu II twice and 188 for Xba I twice. The agreement between the duplicate genotyping was greater than 95% 23,24. RADIOGRAPHIC ASSESSMENTS Prevalent RHOA Details of radiographic scoring methods for hip OA have been previously described 9,18. Subjects were considered to have prevalent RHOA at visit 5 if they satisfied at least one of the following three criteria: joint space narrowing (JSN) grade 3 (range 0e4) which was equivalent to a minimal joint space measurement of 1.5 mm or less at either the medial or lateral joint space, a summary grade 3 (3 of five individual radiographic features), or both definite (grade 2) osteophytes (range 0e3) and JSN (range 0e4) in the same hip. For the present study, subjects with a diagnosis of rheumatoid arthritis, Paget s disease of bone, or bilateral hip replacements were excluded from this analysis 18,24,25. In this study, 32 subjects had unilateral THRs and for these subjects we used the contralateral hip (non-thr hip) to define RHOA from the visit 5 radiograph 23,24. For each subject, the worst RHOA hip was used in this analysis. In addition, we further characterized the radiographic phenotype of the RHOA subjects into those with JSN score 3 (n ¼ 325) and those RHOA cases (n ¼ 130) that had a femoral osteophyte grade 2 with JSN 2 to try to further assess RHOA by phenotype (Fig. 1) 23. Reproducibility was good with kappa scores for inter-rater reliability of 0.66 for definite JSN (2), 0.71 for definite osteophytes, and 0.65 for summary grades 2. The intraclass correlation coefficient was 0.85 for the continuous measurement of minimal joint space 6,23,24. Incident RHOA A hip without baseline RHOA was eligible to develop RHOA at the follow-up visit. A hip was defined as having developed RHOA (incident disease) if any of the three findings were present at the 8.3-year follow-up visit in a hip free of all of these findings at baseline, (1) summary grade 2, (2) lateral JSN grade 2 or medial JSN grade 3, or (3) definite osteophytes 2 in any location and definite JSN 2 in any location 26,27. COVARIATE ASSESSMENT All study participants completed a self-administered questionnaire at the baseline and follow-up visits that included age, northern European ancestry, self-reported health status, age at menopause, current medication use including current estrogen replacement therapy (ERT), multivitamins, other supplements, and current smoking history. Data presented for study subject characteristics are from visit 5. Height was measured using a wall-mounted Harpenden statiometer (Holtain, Dyfed, UK) and weight was measured with a balance beam scale; methods have been previously described 6,26,27. Protocols for BMD measurements of the hip were performed using dual-energy absorptiometry (DXA) (QDR 1000, Hologic, Waltham, MA) and have been previously described as well 9. STATISTICAL ANALYSIS Differences in subject characteristics and BMD variables between RHOA cases and participants without RHOA were assessed by Student s t test for continuous variables and Chi-square tests for dichotomous variables. HardyeWeinberg equilibrium was assessed in both groups by Chisquare tests, and allele frequencies were calculated using the gene counting method. Logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CI) for each genotype (for Pvu II TT, TC and CC; for Xba I AA, AG, GG) and the risk of any RHOA, RHOA defined by JSN 3 or RHOA defined by femoral osteophytosis of 2, and incident RHOA. All analyses for the association of ESR1 Pvu II (C/C and C/T) and Xba I (G/G and G/A) genotypes and RHOA definitions were performed both with and without adjustments for known and potentially confounding variables including age, height, weight, estrogen use, and femoral neck BMD. Statistical analysis was performed using the statistical software program SAS version 8.2 (SAS Institute, Inc., Cary, NC). Results CHARACTERISTICS OF STUDY SUBJECTS AND GENOTYPE FREQUENCIES Among the 4703 study subjects, 569 satisfied the criteria for RHOA, of which 325 subjects had JSN grade 3, and 130 had moderate to severe osteophytes (Fig. 1). When compared with the 4134 control subjects who did not satisfy the RHOA criteria, the RHOA cases were older, poorer overall health status, and had higher femoral neck BMD (Table I). All other subject characteristics and BMD variables were not significantly different between the two groups. The frequency of the Pvu II C allele in the cohort was 21.1%, and the frequency of the Xba I G allele in the cohort was 11.8% and both were similar to frequencies observed in similar populations 15e17. Genotype frequencies did not deviate from HardyeWeinberg equilibrium. ORs FOR PREVALENT RHOA There was nearly a 30% reduction in the odds of RHOA between the C/C genotype when compared to the T/T genotype at the Pvu II site (P < 0.01) (Table II). Adjustment for age, weight, height, femoral neck BMD, and estrogen use did not alter this association between the C/C genotype and reduced RHOA (P < 0.01). A similar reduction was observed with the RHOA phenotype characterized by moderate to severe JSN with reduced odds of nearly 35% (P < 0.01) for the C/C genotype compared to the T/T, and this remained after adjustment for covariates. There was also a dose response with each copy of the C allele and Pvu II, although this association was not significant. No association was observed for the Pvu II genotype and RHOA characterized by moderate to severe osteophytosis. There was an inverse association observed between the Xba I G/G or G/A genotypes and RHOA when compared to

4 Osteoarthritis and Cartilage Vol. 15, No. 8 Table I Subject characteristics with RHOA and controls at visit 5 Characteristic RHOA cases Controls P value Number e Age (years) <0.01 Height (cm) Weight (kg) BMI (kg/m 2 ) Age at menopause (years) Current estrogen use (%) Current vitamin D use (%) Smoking (%) Health status, good/excellent (%) <0.01 Areal BMD by DXA (g/cm 2 ) Total hip Femoral neck <0.01 Genotype frequencies Pvu II TT (%) 188 (33.2) 1162 (28.3) 0.03 TC (%) 277 (48.9) 2067 (50.3) CC (%) 102 (18.0) 884 (21.5) Xba I AA (%) 257 (45.2) 1700 (41.2) 0.20 AG (%) 250 (43.9) 1932 (46.9) GG (%) 62 (10.9) 491 (11.9) Continuous values are noted in mean SD, dichotomous values noted as percentage. P values are from Chi-square test for dichotomous variables and from Student s t test for continuous variables. the A/A genotype (Table III), although it did not reach statistical significance. INCIDENT RHOA There were 2451 subjects who had no RHOA at the baseline visit and 305 who developed incident RHOA at the follow-up visit. Subjects with the Pvu II C/C genotype had a reduced risk of incident RHOA, of nearly 25% compared to those with the T/T genotype but this difference was not significant (P ¼ 0.11). However, subjects with the Pvu II C/C genotype who were on estrogen during the follow-up period (n ¼ 452) had over a 30% reduction in risk of incident RHOA compared to subjects with T/T genotype (n ¼ 584) (P < 0.05) (Table IV). The interaction of prior estrogen use and Pvu II genotype age-adjusted was P ¼ 0.73 and for Xba I P ¼ Discussion 975 The present study addressed the association between polymorphisms of ESR1 and the risk of RHOA. We found that ESR1 Pvu II SNP (C/C) was associated with reduced prevalence of RHOA and incidence of RHOA by nearly 30%. The reduced odds of prevalent RHOA was most significant in the subgroup of RHOA subjects with moderate to severe JSN. Since radiographic evidence of JSN is a surrogate marker for loss of articular cartilage, this polymorphism may protect against cartilage loss. Interestingly, there was no association with the Xba I SNP and RHOA in this population. Lastly, we report that subjects with the Pvu II C/C genotype on estrogen treatment have a modest but significant reduction in the development of incident RHOA compared to subjects without this genotype. This may be the first study to find a reduced risk of RHOA with the ESR1 Pvu II polymorphism. Ushiyama et al. reported an increased risk of generalized OA with the Pvu II and Xba I minor genotypes present with 65 OA patients and 318 controls 16, and Loughlin et al. 19 found no increased risk of idiopathic OA of the hip and knee with the same ESR1 SNPs with 371 OA patients and 369 controls. Bergink et al. reported an increased risk of knee OA with Pvu II and Xba I polymorphisms with an OR of 2.2 for homozygotes (Pvu II C/C) and especially for the subgroup of knee OA subjects with osteophytes as the primary radiographic feature 17. There have been reports that the polymorphisms of the ESR1 are associated with differences in BMD 20,21. Indeed, Bergink et al. found a relationship between osteophytes and increased risk of knee OA with ESR1 Pvu II and Xba I polymorphisms 17. They further hypothesized that variations in the ESR1 may be associated with changes in juxtaarticular bone or its response to external forces, rather than articular cartilage and this might lead to more severe OA later in life 17. Interestingly, our group has reported that hip BMD is associated with RHOA in this cohort; however, adjustment for femoral neck BMD did not alter the influence of the Pvu II C/C genotype to reduce the risk of RHOA in our study. Table II Association between ER alpha (Pvu II) and RHOA status Cases/Total (%) OR (95% CI)* OR (95% CI)y RHOA 567/4680 (12.1) e e TT 188/1350 (13.9) 1.0 (ref) 1.0 (ref) TC 277/2344 (11.8) 0.84 (0.69, 1.03) 0.87 (0.71, 1.08) CC 102/986 (10.3) 0.71 (0.55, 0.92) 0.71 (0.54, 0.94) Osteophytosis 130/4243 (3.1) e e TT 40/1202 (3.3) 1.0 (ref) 1.0 (ref) TC 65/2132 (3.1) 0.92 (0.62, 1.38) 0.93 (0.62, 1.41) CC 25/909 (2.8) 0.82 (0.49, 1.36) 0.85 (0.50, 1.42) JSN 324/4437 (7.3) e e TT 111/1273 (8.7) 1.0 (ref) 1.0 (ref) TC 158/2225 (7.1) 0.82 (0.63, 1.05) 0.86 (0.65, 1.13) CC 55/939 (5.9) 0.65 (0.46, 0.91) 0.67 (0.46, 0.96) *Age-adjusted. yadjusted for age, weight, height, ERT use, femoral neck BMD.

5 976 K. Lian et al.: Hip OA, ER alpha, and genetics Table III Association between ER alpha (Xba I) and RHOA status Cases/Total (%) OR (95% CI)* OR (95% CI)y RHOA 569/4692 (12.1) e e AA 257/1957 (13.1) 1.0 (ref) 1.0 (ref) AG 250/2182 (11.5) 0.87 (0.72, 1.05) 0.88 (0.73, 1.08) GG 62/553 (11.2) 0.82 (0.61, 1.10) 0.77 (0.56, 1.05) Osteophytosis 130/4253 (3.1) e e AA 52/1752 (3.0) 1.0 (ref) 1.0 (ref) AG 65/1997 (3.3) 1.11 (0.77, 1.61) 1.16 (0.79, 1.69) GG 13/504 (2.6) 0.86 (0.46, 1.59) 0.85 (0.46, 1.60) JSN 325/4448 (7.3) e e AA 153/1853 (8.3) 1.0 (ref) 1.0 (ref) AG 137/2069 (6.6) 0.80 (0.63, 1.02) 0.79 (0.61, 1.03) GG 35/526 (6.7) 0.78 (0.53, 1.14) 0.72 (0.47, 1.09) *Age-adjusted. yadjusted for age, weight, height, ERT use, femoral neck BMD. Our observation of a reduced risk of RHOA with ESR1 Pvu II polymorphism was associated with the protection of loss of joint space, a radiographic surrogate for articular cartilage. As this ER is found on articular chondrocytes it may be that this polymorphism may be associated with enhanced transcriptional activity or this polymorphism may be in linkage disequilibrium with a functional polymorphism at another location within the ESR1 gene region 9,10. Alternatively, subjects with the C allele of ESR1 may develop more articular cartilage than those without it. However, we did not find a significant difference in articular minimal joint space width, a surrogate measure for the amount of articular cartilage in subjects with the C allele of Pvu II compared to the T/T genotype in our study population. Since epidemiologic studies report hormone replacement therapy reduce the incidence of hip OA 13, we evaluated if the development of incident RHOA in subjects with the ESR1 Pvu II polymorphism (C/C vs T/T or C/T) was influenced by ERT. The risk of incident RHOA was reduced about 25% in the subjects with the Pvu II C/C vs T/T genotype overall, but ERT reduced the risk to greater than 30% in C/C vs T/T subjects. Other investigators have found that women on ERT with the Pvu II C allele had more protection against fracture risk than the T/T group 28. Also, mesenchymal-stem cell derived osteoblasts (MSCOs) obtained from individuals with the C allele of ESR1 had a higher basal differentiation capacity of these MSCOs than individuals without the C allele, however those with the C allele had higher response to sex steroid treatment. These results could assist in explaining the contradictory effects of ERT on osteoblasts in vitro and in vivo and also help to explain the different responses to ERT 29. In addition, Herrington et al. found women with the Pvu II polymorphism of ESR1 had a greater increase in high density lipoprotein (HDL) and greater reduction in E-selectin, a soluble adhesion molecule involved in inflammation, on ERT 30,31. These investigators went on to determine whether this intronic SNP had some functional significance and found the C allele produced a favorable and functionally significant binding site for the myb transcription factor. To date there are no data that this genotype is not the functional polymorphism responsible for the observed differential responses. Nonetheless, it could be that this polymorphism is in linkage disequilibrium with another polymorphism that is the functional variant 32. In addition, if OA is a chronic low grade inflammatory disease 33, then the modest reduction in systemic inflammation, e.g., reduced E- selectin levels, in subjects on ERT and with the Pvu II C allele might be the pathway in which these subjects either develop less or have slower RHOA disease progression 30. Additional studies are needed to elucidate these findings. This study offers several strengths, including its large community-based cohort of elderly Caucasian women and a validated radiographic scoring system that allowed for the construction of phenotype-specific definitions to fully characterize the extent of RHOA. However, there are also several potential limitations. Our radiographic definitions of RHOA may not be comparable to other grading schemes. Also, despite the large number of subjects with DNA and a hip radiograph available for scoring OA, we only had 569 subjects with RHOA and the numbers of participants were smaller when we subdivided by either genotype, individual radiographic features or disease progression. However, with our study sample size we had over 90% power Table IV Association between ER alpha (Pvu II) and incident RHOA on and off ERT ERT No ERT Cases/Total (%) OR (95% CI)* OR (95% CI)y Cases/Total (%) OR (95% CI)* OR (95%CI)y RHOA 254/2136 (11.9) e e 305/2451 (12.4) e e TT 81/584 (13.9) 1.0 (ref) 1.0 (ref) 104/737 (14.1) 1.0 (ref) 1.0 (ref) TC 128/1100 (11.6) 0.82 (0.61, 1.10) 0.81 (0.59, 1.12) 144/1199 (12.0) 0.85 (0.65, 1.12) 0.91 (0.68, 1.21) CC 45/452 (10.0) 0.68 (0.46, 1.01) 0.66 (0.44, 0.99)þ 57/515 (11.1) 0.75 (0.53, 1.06) 0.78 (0.54, 1.13) þp < *Age-adjusted. yadjusted for age, weight, height, femoral neck BMD.

6 Osteoarthritis and Cartilage Vol. 15, No. 8 confirm or refute our stated hypothesis for prevalent disease and greater than 80% for incident disease. This study population was limited to Caucasian women aged 65 years and older and we only evaluated one anatomic site for OA. Our findings may not be generalized to other populations or to other anatomic sites for OA such as the knee and the hand. Also, population stratification is an often cited limitation of candidate gene studies because population stratification can contribute to false-positive associations and an inability to reproduce findings 34. Women in the present study were recruited from several clinical centers in the United States and any resultant population stratification could have in principle produced false-positive results. However, adjusting for study center and northern European ancestry had no effect on our findings. In summary, we found that the Pvu II C/C genotype of the ESR1 was associated with a modestly reduced risk of prevalent and incident RHOA. Our findings differ from those reported for this polymorphism in knee OA and generalized OA in other populations. Additional work is now required to determine why this polymorphism prevents RHOA development. References 1. Felson DT, Lawrence RC, Dieppe PA, Hirsch R, Helmick CG, Jordan JM, et al. Osteoarthritis: new insights. Ann Intern Med 2000;133:635e Spector TD, MacGregor AJ. Risk factors for osteoarthritis: genetics. Osteoarthritis Cartilage 2004;12(Suppl A):S39e Loughlin J. Genetic epidemiology of primary osteoarthritis. Curr Opin Rheumatol 2001;13:111e6. 4. Kusec V, Virdi AS, Prince R, Triffitt JT. Localization of estrogen receptor-alpha in human and rabbit skeletal tissues. J Clin Endocrinol Metab Jul 1998;83(7):2421e8. 5. Cauley JA, Kwoh CK, Egeland G, Nevitt MC, Cooperstein L, Rohay J. Serum sex hormones and severity of OA of the hand. 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