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Osteoprotegerin Lys3Asn Polymorphism and the Risk of Fracture in Older Women

Department of Epidemiology (S.P.M., J.I.O., J.A.C., J.M.Z.), University of Pittsburgh, Pittsburgh, Pennsylvania 15261; San Francisco Coordinating Center (L.Y.L., S.R.C.), California Pacific Medical Center Research Institute, San Francisco, California 94115; Center for Chronic Disease Outcomes Research (K.E.E., B.C.T.), Veterans Affairs Medical Center, and University of Minnesota, Minneapolis, Minnesota 55455; Kaiser Permanente Center for Health Research Northwest/Hawaii (T.A.H.), Portland, Oregon 97227-1098; Department of Medicine and Epidemiology and Preventive Medicine (M.C.H.), University of Maryland School of Medicine, Baltimore, Maryland 21201; Department of Human Genetics (J.L., S.C., J.M.L.), Roche Molecular Systems, Alameda, California 94501; and Genetics and Genomics (G.P.), Roche Palo Alto, Palo Alto, California 94304

Address all correspondence and requests for reprints to: Joseph M. Zmuda, Ph.D., Department of Epidemiology, Graduate School of Public Health, University of Pittsburgh, 130 DeSoto Street, Pittsburgh, Pennsylvania 15261. E-mail: zmudaj{at}edc.pitt.edu.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Context: Osteoprotegerin (OPG) is a soluble decoy receptor for receptor activator nuclear factor -β that blocks osteoclastic bone resorption.

Objective: We investigated the association between a Lys3Asn polymorphism in the OPG gene and bone mineral density (BMD), and the risk of fracture in 6695 women aged 65 yr and older participating in the Study of Osteoporotic Fractures.

Design: BMD was measured using either single-photon absorptiometry (Osteon Osteoanalyzer; Dove Medical Group, Los Angeles, CA) or dual-energy x-ray absorptiometry (Hologic QDR 1000; Hologic, Inc., Bedford, MA). Incident fractures were confirmed by physician adjudication of radiology reports. Genotyping was performed using an immobilized probe-based assay.

Results: Women who were homozygous for the minor G (Lys) allele had significantly lower BMD at the intertrochanter, distal radius, lumbar spine, and calcaneus than those with the C (Asn) allele. There were 701 incident hip fractures during 13.6-yr follow-up (91,249 person-years), including 362 femoral neck and 333 intertrochanteric hip fractures. Women with the C/C (Asn-Asn) genotype had a 51% higher risk of femoral neck fracture (95% confidence interval, 1.13–2.02) and 26% higher risk of hip fracture (95% confidence interval, 1.02–1.54) than those with the G/G (Lys-Lys) genotype. These associations were independent of BMD. Intertrochanteric fractures were not associated with the Lys3Asn polymorphism.

Conclusion: These results require confirmation but suggest a role for the OPG Lys3Asn polymorphism in the genetic susceptibility to hip fractures among older white women.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The number of osteoporotic hip fractures is expected to increase considerably during the next 25 yr, primarily as a result of increases in the older population (1, 2). Hip fractures are the most serious consequence of osteoporosis, and a major cause of disability and death among elderly women. Osteoporotic hip fractures represent a substantial economic burden with recent annual health care costs estimated at $8.6 billion in the United States alone (3).

Osteoporosis and hip fractures appear to be under strong genetic control (4, 5, 6). Maternal history of fracture confers a 2-fold increased risk of hip fracture in elderly women that is independent of other risk factors, including bone mineral density (BMD) (5). In recent years much progress has been made in identifying genes and alleles that may contribute to variation in BMD, but the genetic etiology of fracture, particularly hip fractures, remains poorly defined (7).

Osteoprotegerin (OPG) (TNFRSF11B) has a key role in bone remodeling. Receptor activated nuclear factor -β ligand (RANKL) stimulates osteoclastogenesis by binding receptor activator nuclear factor -β (RANK) (8). OPG is a decoy receptor for RANK and inhibits bone resorption. In the animal model, mice that do not express OPG develop severe osteoporosis, whereas those overexpressing OPG develop osteopetrosis (9, 10, 11).

A G-to-C polymorphism at codon 3 in exon 1 of the OPG gene causes an amino acid substitution from lysine to asparagine (Lys3Asn; rs2073618). Several studies have reported associations between the Asn (C) allele and increased lumbar spine BMD, lower risk of osteopenia or osteoporosis, and reduced risk of fracture (12, 13, 14). However, the number of fractures in these studies was small, and the inconsistent findings require further validation (15). In the current study, we analyzed the relationship between Lys3Asn genotype, BMD, and the risk of fractures during 13.6-yr follow-up in 6658 community dwelling, older Caucasian women.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Subjects

All women were participants in the Study of Osteoporotic Fractures (SOF), a prospective study of 9704 community dwelling white women who were at least 65 yr of age at study entry. Women were recruited for the SOF between 1986 and 1988 from population-based listings, such as voter registration lists, at four clinical centers in the United States: Baltimore, Maryland; Minneapolis, Minnesota; Monongahela Valley, Pennsylvania; and Portland, Oregon (16). Women were excluded from enrolling in the SOF if they reported bilateral hip replacements or were unable to walk unassisted. African-American women were excluded because of their low risk of hip fracture. The institutional review boards at each institution approved the study. All women provided written informed consent at entry into the study and at each clinical examination.

In 1989–1990, buffy coat specimens were collected from participants who returned for a second examination. In addition, whole blood samples were collected during 1997–1998 in women who had not previously provided buffy coat samples. A total of 6704 participants provided samples for DNA extraction. Genotyping was completed in 6658.

BMD

Peripheral BMD (distal and proximal radius and calcaneus) was measured at baseline using single-photon absorptiometry using the Osteon Osteoanalyzer (Dove Medical Group, Los Angeles, CA). Proximal femur and lumbar spine BMD were measured by dual-energy x-ray absorptiometry (QDR 1000; Hologic, Inc., Bedford, MA) at a second clinic visit in 1989–1990. Details of these methods and quality control procedures have been reported elsewhere (17, 18).

Fracture ascertainment

Details of the method for identifying fractures have been published (19). Briefly, participants were contacted every 4 months by postcard or telephone to ask whether they had sustained a fracture. More than 95% of these contracts were completed. All fractures were validated by a radiographic report. Fractures that occurred because of major trauma such as motor vehicle accidents were excluded. In the current study, all nonvertebral fractures were analyzed as a group, and hip, femoral neck, intertrochanter, and distal forearm fractures were analyzed separately.

Incident spine fractures were assessed by comparing baseline spine radiographs to repeat radiographs taken an average of 3.7 yr later. A fracture was defined as a reduction in any vertebral height measure (anterior, medial, or posterior) of any vertebral body (T4–L4) of at least 20%, and at least 4 mm between baseline and follow-up. More complete details of these measures have been published (20, 21).

Other measures

Body weight was measured (after removal of shoes and heavy outer clothing) using a balance beam scale. Height was measured without shoes using a Harpenden stadiometer (Holtain Ltd., Dyved, UK). Height and weight were used to calculate body mass index (BMI) (kg/m²). Maternal history of fractures, ancestry, self-reported health status, smoking status, and physical activity were assessed by a questionnaire that was reviewed with the participant by a trained interviewer. Women were also asked about whether they were currently taking oral estrogen or calcium supplements.

Genotyping

The Lys3Asn polymorphism was selected for genotyping because it is the only common, nonsynonymous single nucleotide polymorphism in the OPG gene and had previously been associated with several osteoporosis outcomes. Women were genotyped for the Lys3Asn polymorphism in exon 1 of OPG at Roche Molecular Systems (Alameda, CA) in the context of a multiplex PCR amplification, followed by allele-specific single nucleotide polymorphism detection with immobilized oligonucleotide probes in linear arrays, similar to methods described previously (22). Primers were modified at the 5' phosphate by conjugation with biotin. Ten to 50 ng purified human genomic DNA was amplified in a reaction volume of 50 µl with AmpliTaq Gold DNA polymerase using a GeneAmp PCR System 9600 thermal cycler (PE Biosystems, Foster City, CA) and the following cycling profile: an initial hold at 94 C for 7 min; 33 cycles of 95 C for 15 sec and 60 C for 60 sec; and a final extension step of 68 C for 5 min. Chromogenic detection of allelic variants after 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). In-house software was used to scan the linear arrays on an Epson Perfection 1670 scanner (Epson, Long Beach, CA) and to assign genotypes. Of samples, 1% was genotyped twice, and all results were concordant.

Statistical analysis

Allele frequencies were estimated by the gene counting method, and departures from Hardy-Weinberg equilibrium were tested using a ² test. We compared characteristics of women by genotype using ANOVA or covariance for continuous measures and ² tests for categorical variables. To determine the relationship between genotype and subsequent fracture risk, we used Cox proportional hazard models to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for the associations between OPG genotype and fracture with the G/G (Lys/Lys) genotype of the Lys3Asn polymorphism as the referent group. We report the models unadjusted and adjusted for distal radius BMD. Distal radial BMD is presented in this analysis because it was measured at baseline before any recorded fractures occurred; however, models were also run adjusting for other BMD sites. Because the characteristics of women did not differ by genotype, we did not include further covariates in the model. Population attributable risk (PAR) for fracture was calculated as PAR = p(RR – 1)/1 + p(RR – 1), where p is the prevalence of the risk factor, and RR is the relative risk. For this analysis we have used the HR as an estimate of the relative risk. The risk factors included in the PAR analysis were the lowest quartile of distal radius BMD, maternal history of fracture, lowest quartile of body weight, current corticosteroid use, and current smoking. The HRs for these risk factors were estimated using Cox proportional hazard models and were adjusted for age. All analyses were preformed using SAS version 8.2 (SAS Institute Inc., Cary, NC).

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The Lys3Asn polymorphism showed genotypical frequencies of G/G = 0.20, G/C = 0.50, and C/C = 0.30, and an allele frequency of 0.45 for the minor G (Lys) allele. Observed and expected genotype counts were consistent with Hardy-Weinberg equilibrium. Age, anthropometric measures, including weight, height, and BMI, smoking status, walking for exercise, mother’s history of fracture, use of estrogen ever, calcium supplements and steroids, and overall health status were not significantly different by OPG genotype (Table 1).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
In a prospective analysis of nearly 7000 older postmenopausal women, we found that the Asn3Asn genotype for the Lys3Asn polymorphism in the OPG gene was significantly associated with both increased BMD and increased risk of hip fracture. The fracture associations were independent of BMD and had a PAR for femoral neck fractures that was comparable to or higher than several established risk factors for fracture. The present findings require confirmation but support a role for OPG in the etiology of osteoporosis in older Caucasian women.

Few previous genetic association studies have had adequate statistical power to analyze hip fractures in general, and those that did examine hip fracture considered these fractures as a homogeneous outcome. However, hip fractures include intertrochanteric and femoral neck fractures that may have different etiologies. For example, the composition of bone in the intertrochanter region contains more trabecular bone than the femoral neck region (23). Epidemiological risk factors for femoral neck and intertrochanter fractures are also known to differ (24). Maternal history of hip fracture, in particular, nearly doubled the risk of femoral neck fractures but was not related to intertrochanteric fractures (25). Consistent with these prior results, women with the OPG Lys3Asn polymorphism had an increased risk of femoral neck but not intertrochanteric fractures in the present analyses. This is the first study, to our knowledge, of a specific genetic risk factor for femoral neck fractures. However, our results are consistent with studies in mice that indicate that there are skeletal site-specific genetic loci for bone mass and strength (26, 27, 28). Even the distribution of bone mineral into the cortical and trabecular compartments within a skeletal region appears to be under genetic regulation, with unique genetic factors contributing to trabecular and cortical bone mass (26). Identifying the loci for specific types of fracture may have practical implications by allowing individuals at increased risk for fracture to be identified and treated with interventions directed at the specific molecular defects contributing to site-specific skeletal fragility.

Several previous studies have examined the association between the OPG Lys3Asn polymorphism and osteoporosis-related phenotypes. Most of the published studies have found associations between the CC (Asn) genotype and increased BMD, especially at the lumbar spine (12, 13, 14, 29, 30, 31). For example, Langdahl et al. (12) found that the CC genotype (Asn) was less common among osteoporosis patients than in normal controls and was also associated with significantly higher BMD at the lumbar spine in all participants combined. In another study of 136 postmenopausal Slovenian women, subjects with the CC genotype also had significantly higher lumbar spine BMD (13). In a study of 205 postmenopausal Chinese women, BMD at the lumbar spine of the Asn-Asn(C/C) genotype was significantly higher (9.5–12.6%) than Lys-Asn (G/C) and Lys-Lys (G/G) genotypes (P = 0.012), with evidence for an allele dose effect (P = 0.008). The Lys-Lys (G/G) genotype was associated with a 2.7-times higher risk of osteopenia/osteoporosis than the Asn-Asn (C/C) genotype (14). However, not all of these studies have reported consistent findings, and several studies found no association between the Lys3Asn polymorphism and osteoporosis phenotypes (15, 32, 33, 34). In contrast, Irish postmenopausal women with one or more C (Asn) alleles had 14.8% lower BMD (P = 0.05) at the lumbar spine and 14.4% lower BMD (P = 0.04) at the femoral neck (15). More recently, Ueland et al. (34) found no significant association between the Lys3Asn polymorphism and total hip BMD, femoral neck BMD, or fracture risk in a study of 1101 elderly Australian women. We confirm and extend the findings of most of these cross-sectional studies by showing that the Asn3Asn (C/C) genotype is associated with higher BMD at several skeletal sites, and by demonstrating that this polymorphism is also significantly associated with increased femoral neck fractures over 13.6 yr in nearly 7000 older Caucasian American women.

Our study found that the Asn3Asn (C/C) genotype was associated with higher BMD at the calcaneus, distal radius, intertrochanter, and lumbar spine while also being associated with an increased risk of hip and, particularly, femoral neck hip fractures. It is important to note that many of the skeletal sites showing an increase in BMD did not show a subsequent increase in fracture risk (e.g. lumbar spine and intertrochanter), whereas the largest increase in fracture risk was seen at the femoral neck, where there was no significant association with BMD.

Bone health is not only determined by BMD but also by the structural and material properties of bone, including trabecular microarchitecture, cortical thickness, porosity, mineral and collagen composition, and microdamage (35). Dual-energy x-ray absorptiometry provides a two-dimensional assessment of the skeleton and may not adequately reflect these other aspects of bone health. Thus, it is possible that the Lys3Asn OPG polymorphism may influence fracture propensity through skeletal characteristics other than BMD. In addition, OPG has specifically been implicated in vascular calcification and might interfere with arterial blood flow, thus contributing to altered bone metabolism (36). This is particularly relevant in the femoral neck region where the femoral artery is quite susceptible to vascular disease.

OPG influences bone metabolism by blocking RANKL from binding to RANK on osteoclast precursor cells, thus inhibiting differentiation and subsequent bone resorption (37, 38, 39). With aging, a reduction in OPG levels would allow RANKL to bind more freely with RANK and promote osteoclast maturation, which may mediate some of the osteoclastic resorption at the endosteal bone surface, and contribute to cortical thinning and cortical instability (40, 41). However, studies attempting to relate serum concentrations of OPG to BMD or fracture risk in humans have yielded conflicting findings (42, 43, 44). Part of the difficulty in evaluating OPG as a potential biomarker of osteoporotic risk may be that serum concentrations of OPG do not provide a reliable indicator of OPG levels or activity within the bone microenvironment. On the other hand, genetically determined differences in OPG expression or function may be a more reliable indicator of long-term OPG activity. This possibility is supported by the association between the OPG Lys3Asn polymorphism and the risk of fracture over an average of 13.6-yr follow-up in the present study. In addition, the Lys3Asn missense polymorphism occurs in the signal peptide region of the gene as well as in a potential exonic splicing enhancer site. Changes in these sequences are predicted to have a functional effect on the protein (http://fastsnp.ibms.sinica.edu.tw) (45).

The potential genetic influence on fracture susceptibility has been estimated in past twin and cohort studies (4). In one study a positive maternal history of hip fracture doubled a woman’s risk of suffering a hip fracture (5). In analyses of a large cohort of Swedish twin pairs, genetic variation explained nearly 50% of the variation in hip fracture risk; however, the genetic contribution to fracture diminished with increasing age at first fracture (6). As with most complex diseases, the majority of the specific genetic variants that underlie the familial component to osteoporotic fracture risk have not been identified, and the allelic architecture of osteoporotic fracture is still a matter of debate. One hypothesis proposes that the genetic architecture for common conditions, such as osteoporotic fractures, is conferred by genetic variants (alleles) that occur at high frequency (common-disease/common-variant hypothesis) (46, 47, 48). These alleles are each thought to increase disease susceptibility by conferring a small to modest increase in risk. However, because these susceptibility alleles are common, the population-attributable risk (the percentage of disease that can be attributed to them) is high. Others hypothesize that the genetic architecture of common diseases may be explained by the effects of a large number of loci, each with multiple disease-predisposing alleles of low frequency (49, 50). There is currently very limited empirical evidence to prove or disprove either model for the genetic architecture of most common diseases and particularly for osteoporotic fractures. Our findings for the OPG Lys3Asn polymorphism are consistent with the common disease, common variant model in which high frequency alleles may contribute a modest relative risk but an appreciable proportion of disease burden in the population. Moreover, the relatively high PAR of femoral neck fracture with the Lys3Asn polymorphism raises the possibility that a limited number of disease susceptibility genes with common variants might explain a major proportion of fracture risks in the population. However, our findings do not exclude the possible contribution of rare variants, and it is likely that both common and rare variants contribute to osteoporotic fractures. Comprehensive studies of other candidate genes for bone metabolism are clearly needed to determine the relative contributions of both common and rare variants.

Our study has several strengths, including its large, prospective design with excellent follow-up rates and nearly complete adjudication of fractures over an extended period of time. There are also several potential limitations. Participants in the present analysis were community dwelling North American Caucasian women aged 65 yr and older, and our findings may not be generalizable to younger women, women of other races, men, or institutionalized subjects. However, Caucasian women experience the greatest risk of osteoporosis in the population, and the majority of this risk comes after age 65, when hip fracture rates begin an exponential increase. Nonetheless, additional studies of the OPG Lys3Asn polymorphism and the OPG locus in general will be needed to replicate and extend our findings.

In summary, our findings suggest that older white women with the OPG Asn3Asn genotype have both increased BMD and an increased risk of hip fractures. The hip fractures associated with this polymorphism occurred primarily in the femoral neck region of the hip and were not explained by differences in BMD. Our findings require confirmation but suggest that the nonsynonymous coding polymorphism, or a closely linked allelic variant, may be a novel genetic marker for femoral neck fracture risk in older Caucasian women. The identification of genetic markers of femoral neck fractures may be especially useful as life-long indicators of risk and may ultimately help to identify optimal therapies in patients at increased risk.

	Footnotes

 
This study was supported by Public Health Service Grants AG05407, AR35582, AG05394, AR35584, AR35583, R01 AG005407, R01 AG027576–22, 2 R01 AG005394–22A1, and 2 R01 AG027574–22A1. J.I.O. and S.P.M. were supported by National Institute on Aging Grant T32 AG00181–13.

Disclosure Summary: J.A.C. receives funding from Merck Co., Eli Lily Co., Pfizer Pharmaceuticals, and Novartis Pharmaceuticals. K.E.E. is a federal employee of the Veterans Affairs Medical Center in Minneapolis, MN, and has received research support from California Pacific Medical Center, which receives funding from Roche Molecular Systems. M.C.H. acts as a consultant for Amgen, Inc. J.L., S.C., and J.M.L. are all employees of Roche Molecular Systems, which provided genotyping reagents and services for this study at no cost under a research collaboration. G.P. is an employee of Roche Palo Alto. S.R.C. is an employee of the California Pacific Medical Center and receives research support from Roche Molecular Systems. All other authors have no conflicts of interest.

First Published Online March 4, 2008

¹ S.P.M. and J.I.O. authors contributed equally to this manuscript.

Abbreviations: BMD, Bone mineral density; BMI, body mass index; CI, confidence interval; HR, hazard ratio; OPG, osteoprotegerin; PAR, population attributable risk; RANK, receptor activator nuclear factor -β; RANKL, receptor activator nuclear factor -β ligand; SOF, Study of Osteoporotic Fractures.

Received May 7, 2007.

Accepted February 26, 2008.

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