In an effort to identify genetic polymorphisms in potential candidate genes for osteoporosis, 10 variants were identified in the OSCAR gene using direct DNA sequencing, and 560 postmenopausal women were genotyped at five SNP loci, using the TaqMan method. The rare allele (G allele) of OSCAR-2322A>G (SNP in the 5′ flanking region) showed significant association with lower BMD at various bone sites in postmenopausal women (n = 560).
Introduction: BMD is the major factor for determining bone strength and osteoporotic fracture risk and is determined by both environmental and multiple genetic factors. The osteoclast-associated receptor (OSCAR) plays a critical role in osteoclast differentiation and thus is an important candidate gene for the modulation of BMD.
Materials and Methods: Through direct sequencing in 24 Korean individuals, 10 sequence variants were identified: 2 in the 5′ flanking region, 7 in the exons (including 6 nonsynonymous single-nucleotide polymorphisms [SNPs]), and 1 in an intron. Five of these polymorphisms were selected for larger-scale genotyping in postmenopausal women (n = 560). Areal BMD (g/cm2) of the anterior-posterior lumbar spine and the nondominant proximal femur was measured using DXA (Lunar Expert XL and Hologic QDR 4500-A). Lateral thoracolumbar radiographs were obtained in all subjects.
Results: Using multiple regression analysis and controlling for age, years since menopause, height, weight, and evaluation machine as covariates, the rare allele (G allele) of OSCAR-2322A>G showed significant association with lower BMD at various bone sites in postmenopausal women.
Conclusion: These findings suggest that the promoter variant in OSCAR gene (OSCAR-2322A>G) might be one of genetic determinants of BMD in postmenopausal women.
OSTEOPOROSIS IS A skeletal disorder characterized by compromised bone strength predisposing a person to an increased risk of fracture. BMD is frequently used as a proxy measure and accounts for ∼70% of bone strength.(1) Although multiple environmental risk factors are involved in the pathogenesis, genetic factors are also implicated and account for about 50–85% of the variance in BMD based on twin and family studies.(2–4) Therefore, it would be important to identify genes that act as regulators of BMD, which could then be used to identify subjects at risk for osteoporosis and to target preventive treatment.
Osteoclasts, the only cells that are capable of resorbing bone, are essential for bone homeostasis. They are derived from hematopoietic cells of the monocyte-macrophage lineage that give rise to macrophages and dendritic cells.(5–8) The macrophage-colony stimulating factor and RANKL produced by osteoblasts/stromal cells are essential factors for supporting osteoclast differentiation.(9, 10) Recently, the osteoclast-associated receptor (OSCAR), a novel member of the leukocyte receptor complex, has been identified as a regulator of osteoclastogenesis. The human OSCAR (MIM 606862) gene has been mapped to the leukocyte receptor complex (LRC) 21 on chromosome 19q13.4.(11–13) It is widely expressed in cells of myeloid lineage, continually expressed during differentiation of CD14+ monocytes into dendritic cells, and maintained after maturation.(14) The formation of osteoclasts from bone marrow precursor cells is substantially inhibited by the addition of a soluble form of OSCAR in co-culture with osteoblasts in the presence of bone-resorbing factors,(12, 13) suggesting a critical role of OSCAR in osteoclast differentiation. Therefore, the OSCAR gene can be considered as a potential candidate for the modulation of BMD and the risk of osteoporosis.
Despite the important role of OSCAR in bone metabolism, its genetic effects on the determination of bone mass have not been studied thus far. In this study, we performed extensive screening of the gene by direct sequencing to detect polymorphisms, and we analyzed associations with BMD and the risk of radiological vertebral fracture in Korean postmenopausal women.
MATERIALS AND METHODS
The study population comprised 560 postmenopausal women of Korean ethnicity who visited Asan Medical Center (AMC, Seoul, Korea). Menopause was defined as the absence of menstruation for at least 6 months and was confirmed by measurement of serum follicle-stimulating hormone (FSH). Women who were prematurely menopausal (<40 years of age) were excluded. Women who had taken drugs that might affect bone metabolism for >6 months or within the previous 12 months were also excluded. Subjects were excluded if they had suffered from any disease that might affect bone metabolism. Women who had had a stroke or dementia were also excluded because of concerns related to their limited physical activity. Women were also excluded if they had osteophyte formation above the fourth grade of the Nathan classification(15) and/or severe facet joint osteoarthritis in the lumbar spine using conventional spine radiographs. The study was approved by the AMC Ethics Review Committee, and written informed consent was obtained from all subjects. For a comparison of allele frequencies of OSCAR single-nucleotide polymorphisms (SNPs) identified in the Korean population with other major ethnic groups, we also genotyped 50 white and 50 black DNA samples obtained from the Human Genetic Cell Repository (http://locus.umdnj.edu/nigms/).
Areal BMD (g/cm2) of the anterior-posterior lumbar spine (L2-L4) and proximal femur (femoral neck, femoral shaft, total femur, trochanter, and Ward's triangle) was measured using DXA (Expert XL with software version 1.90; Lunar, Madison, WI, USA) in 431 women. In 129 women, BMD was measured using the QDR 4500-A with software version 4.84 (Hologic, Waltham, MA, USA). The BMD values were measured only at the lumbar spine and femoral neck before January 2001 in our institution, and the Hologic machine did not measure BMD at the femoral shaft. Therefore, BMD values at the femoral shaft and at other proximal femur sites (total femur, trochanter, and Ward's triangle) were not available in 229 of the 431 subjects and in 100 of the 129 subjects. Based on the upper-extremity dominance, the BMD at the proximal femur was measured at the nondominant sites. Short-term in vivo measurement precision for the Lunar and Hologic machines, expressed as CV, was 0.82% and 0.85% for the lumbar spine, respectively, and 1.12% and 1.20% for the femoral neck, respectively. These values were obtained by scanning 17 volunteers who were not part of the study. These volunteers underwent five scans on the same day, getting off and back on the table between examinations. The T score was used to diagnose osteopenia and osteoporosis at each site according to the World Health Organization definition (−2.5 < T score < −1.0 SD, and T score ≤ −2.5 SD, respectively), and the subjects having a T score more than −1.0 were classified as normal controls. Lateral thoracolumbar (T4-L4) radiographs were obtained for all subjects. A vertebral fracture was defined quantitatively as a loss of 15% or more in the anterior, posterior, or middle height of one or more vertebral sites in subjects without previous history of major trauma such as traffic accidents.
Sequencing analysis of the OSCAR gene
Genomic DNA was extracted from peripheral blood leukocytes using a commercial kit (Wizard Genomic DNA purification kit; Promega, Madison, WI, USA). We sequenced exons and their boundaries in the OSCAR gene, including the promoter region (∼1.5 kb), to discover genetic variants in 24 Korean DNA samples using a DNA analyzer (ABI PRISM 3700; Applied Biosystems, Foster City, CA, USA). Eight primer sets of the OSCAR gene for the amplification and sequencing analysis were designed based on GenBank sequences (reference genome sequence for OSCAR: NT_011109.15 released in February 2004). Sequence variants were verified by chromatograms.
Genotyping with fluorescence polarization detection
For genotyping of polymorphic sites, amplifying primers and probes were designed for TaqMan.(16) Primer Express (Applied Biosystems) was used to design both the PCR primers and the MGB TaqMan probes. One allelic probe was labeled with the FAM dye and the other with the fluorescent VIC dye. PCRs were run in the TaqMan Universal Master mix without UNG (Applied Biosystems), with PCR primer concentrations of 900 nM and TaqMan MGB-probe concentrations of 200 nM. Reactions were performed in a 384-well format in a total reaction volume of 5 μl using 20 ng of genomic DNA. The plates were placed in a thermal cycler (PE 9700; Applied Biosystems) and heated at 50°C for 2 minutes and 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 s and 60°C for 1 minute. The TaqMan assay plates were transferred to a Prism 7900HT instrument (Applied Biosystems) where the fluorescence intensity in each well of the plate was read. Fluorescence data files from each plate were analyzed using automated software (SDS 2.1; Applied Biosystems).
χ2 tests were used to determine whether individual variants were in equilibrium at each locus in the population (Hardy-Weinberg equilibrium). We examined Lewontin's D′ (|D′|) and the linkage disequilibrium (LD) coefficient r2 between all pairs of biallelic loci.(17, 18) Haplotypes (ht) of each individual were inferred using the algorithm developed by Stephens et al.(19) (PHASE), which uses a Bayesian approach incorporating a priori expectations of haplotypic structure based on population genetics and coalescent theory. Phase probabilities of all polymorphic sites for haplotypes were calculated for each individual using this software. Individuals with phase probabilities <97% were excluded in the analysis. The genetic effects of inferred haplotypes were analyzed in the same way as the polymorphisms. Multiple regression analyses were performed for BMD controlling for age (continuous variable), years since menopause (YSM; continuous variable), weight, height, and type of evaluation machine as covariates. The genotype distributions of OSCAR polymorphisms and haplotypes between patient groups including osteopenic and osteoporotic subjects and normal subjects were analyzed with logistic regression models controlling for age, YSM, weight, height, and type of evaluation machine as covariates. In addition, the genotype and haplotype distributions between patients with and without vertebral fractures were also analyzed with logistic regression model controlling for age, YSM, weight, and height. The effective number of independent marker loci in OSCAR was calculated to correct for multiple testing. The effective number in OSCAR was calculated using the software SNPSpD, which is based on the spectral decomposition (SpD) of matrices of pairwise LD between SNPs.(20) The resulting number of independent marker loci was applied to correct for multiple testing. Putative transcription factor sites were examined using the software-TFSEARCH: Searching Transcription Factor Binding Sites (version 1.3; putative score > 0.9).(21)
The mean age of the participants was 59.4 ± 7.2 years (range, 46–83 years), and the mean YSM was 10.4 ± 8.2 years (range, 1–35 years; Table 1). BMD was measured using two machines, Lunar Expert XL and Hologic QDR 4500-A, in 431 (77.0%) and 129 (23.0%) subjects, respectively. As expected, age (p = 0.01 and p = 0.005), weight (p < 0.0001 and p < 0.0001), height (p = 0.01 and p = 0.09), and YSM (p = 0.02 and p < 0.0001) were significantly correlated with BMD at the lumbar spine and femoral neck (Table 1). The BMD values measured by the Hologic machine (0.764 ± 0.116 and 0.606 ± 0.098 mg/cm2 in the two groups of subjects, respectively) were lower than those measured by the Lunar machine (0.870 ± 0.182 and 0.723 ± 0.128 mg/cm2, respectively; each p < 0.0001). The types of machine and the software versions clearly affect the BMD values,(22) and ethnicity may also contribute to BMD because of genetic differences in skeletal structure,(23) which requires a cross-calibration of the two machines. Because cross-calibration data were not available in Korean women thus far, we analyzed associations of the genetic variations with BMD values using statistical adjustments with the type of machine as a covariate.
Table Table 1.. Clinical Profiles and Multiple Regression Analyses on BMD (g/cm2) in Korean Postmenopausal Women (n = 560)
Through direct sequencing of all exons and their boundaries in the OSCAR gene, including the −1500 bp of the 5′ flanking region, 10 SNPs were identified: 2 in the 5′ flanking region, 7 in the exons (including 6 nonsynonymous SNPs), and 1 in an intron (Fig. 1A). The frequencies in the Korean population are shown in Fig. 1A. Pairwise comparisons among SNPs revealed one set of absolute LD (|D′| = 1 and r2 = 1; −2322A>G:−111C>G, e.g., the A allele of −2322A>G always travels with the C allele of −111C>G and the G allele of −2322A>G always travels with the G allele of −111C>G) and two sets of complete LD (|D′| = 1 and r2 ≠ 1 (e.g., only three haplotypes are constructed among four possible haplotypes by two SNPs; Figs. 1A and 1C). Genotype distributions of all loci were in Hardy-Weinberg equilibrium (p > 0.05; Table 2). Five SNPs were selected for larger-scale genotyping based on LDs, frequencies, and haplotype tagging status. Haplotypes in the OSCAR gene were constructed using the PHASE software(19) (Fig. 1B).
Table Table 2.. SNPs and Allele Frequencies of the OSCAR Gene in Korean (n = 560), African-American (n = 50), and White (n = 50) Subjects
The associations with BMD of the lumbar spine and femoral neck were performed using multiple regression analysis, controlling for age, YSM, height, weight, and evaluation machines as covariates. The obtained p values were corrected for multiple testing by the effective number of independent marker loci (4.74) in OSCAR (4.74) (pcor). One SNP in the 5′ flanking region, OSCAR-2322A>G, was associated with BMD at both the lumbar spine and femoral neck (pcor = 0.033 and pcor = 0.023, respectively; Table 3). The genetic effects of OSCAR −2322 A>G on BMD at the lumbar spine were gene-dose dependent; the highest BMD was found in homozygotes for the common allele (0.86 ± 0.19 mg/cm2), intermediate BMD was found in heterozygotes (0.84 ± 0.17 mg/cm2), and the lowest BMD was found in homozygotes for the rare allele (0.82 ± 0.17 mg/cm2). However, at the femoral neck, the OSCAR −2322 A>G showed dominant effect on BMD values; a higher BMD was found in individuals bearing the GG genotype than in others (AA and AG genotypes; Table 3). The genetic effects of OSCAR −2322 A>G on BMD were also detected at other femoral sites (femoral shaft and Ward's triangle; Table 4). In further haplotype analysis, OSCAR-ht1 was significantly associated with BMD at both bone sites (pcor = 0.047 at the lumbar spine; Table 3). However, these genetic effects likely come from OSCAR-2322A>G because OSCAR-ht1 is mostly (>90%) tagged by OSCAR-2322A>G (Fig. 1B).
Table Table 3.. Regression Analysis of BMD at Lumbar Spine (L2-L4) and Femoral Neck With OSCAR Polymorphisms in Korean Postmenopausal Women
Table Table 4.. Regression Analysis of BMD of Femoral Shaft, Lumbar Spine (L2-L4), Total Femur, Femoral Neck, Trochanter, and Ward's Triangle With OSCAR−2322A>G Polymorphisms in Korean Postmenopausal Women, Adjusted for Age, YSM, Weight, Height, and Evaluation Machine
The genotype distributions were analyzed among normal subjects and the patient group (osteopenic or osteoporotic subjects). The rare allele frequency of OSCAR-2322 A>G was found to be higher in the patient group (Table 1; p = 0.03; not significant after correction for multiple testing). In addition, the genetic effects of OSCAR polymorphisms on the risk of radiological vertebral fracture were also analyzed. The fractures were detected in 98 (16.9%) subjects in this study. Although it could not retain significance after correction for multiple testing, the rare allele frequency of OSCAR-2322 A>G was higher in subjects with vertebral fracture (frequency = 0.515) than in those without vertebral fracture (frequency = 0.441). This increased risk of vertebral fracture might be explained by the decreased lumbar spine BMD in individuals with OSCAR-2322G (the G allele of OSCAR-2322A>G). Although significant differences in frequencies of polymorphisms were observed among three ethnic groups (p = 0.04 <0.0001; Table 2), the frequencies of OSCAR-2322A>G, which was the only SNP associated with BMD and risk of fractures, were not as different among the ethnic groups (p = 0.04) as other SNPs.
In summary, examination of the genetic effects of polymorphisms in OSCAR has revealed that OSCAR-2322G is significantly associated with decreased BMD in postmenopausal women.
In this study, we identified 10 polymorphisms of OSCAR in a Korean population, and noted that one SNP (OSCAR-2322A>G) and one haplotype (OSCAR-ht1) were significantly associated with BMD values after controlling for confounding variables. To our knowledge, this is the first clinical report suggesting the role of OSCAR in bone metabolism, and our observations suggest that OSCAR may one of genetic determinants of BMD postmenopausal women.
OSCAR was first found to be specifically expressed in osteoclast precursors and mature osteoclasts. In co-culture with osteoblasts, osteoclast formation was inhibited by OSCAR-Fc (Fragment, crystallizable) in a murine model, indicating that OSCAR could be an important regulator of osteoclastogenesis.(12) Subsequently, the human OSCAR gene was found to be expressed widely in cells of myeloid lineage, including all stages of differentiation and maturation of dendritic cells as well as osteoclasts.(24) The dendritic cells could stimulate a T-cell clone specific for an epitope of mouse immunoglobulin G after uptake and processing of the human OSCAR-specific antibody, showing the capacity of this receptor to mediate antigen presentation.(14) This suggests that the human OSCAR gene may play a crucial role in the regulation of the immune system in addition to its role in osteoclastogenesis.
Bone mass is determined by the differences between bone resorption by osteoclasts and formation by osteoblasts. An imbalance in the remodeling process, in which resorption exceeds formation, results in the accelerated bone loss.(8, 25, 26) Research has shown that the immune system plays an important role in bone remodeling.(25, 26) For example, proinflammatory cytokines, which are stimulated by immune responses, regulate bone metabolism, even in individuals without immunological diseases.(27) Furthermore, the activation of RANKL, an essential factor for osteoclast differentiation, is also regulated by the immune system.(28) Therefore, it is suggested that OSCAR regulates osteoclastogenesis both directly and indirectly through the immune system.
In this study, we scrutinized the presence of polymorphisms in the promoter region (∼1.5 kb), as well as exons and their boundaries, and noted that the OSCAR-2322A>G polymorphism in the promoter was significantly associated with BMD at all regions tested. This suggests that the OSCAR promoter polymorphism could modulate BMD by influencing transcription and/or expression levels of OSCAR. Recently, So et al.(13) reported that the binding of microphthalmia transcription factor (MITF) to its cognate sites in the mouse OSCAR promoter is critical for OSCAR expression. Similarly, the promoter region of the human OSCAR gene also contains several putative transcription factor binding sites (Fig. 1A; TFSEARCH: Searching Transcription Factor Binding Sites, version 1.3; putative score > 0.9).(20) Interestingly, the sequences flanking the OSCAR-2322A>G site contains a putative (91%) binding site for transcription factor CREB, which binds to the cAMP response element (CRE) and activates gene transcription in response to a wide variety of extracellular signals.(29)
It would be needed that correction for multiple testing be applied to the p values obtained from analyses of multiple genetic markers. However, when a background LD exists between SNPs but they are assumed to be completely independent, strict correction, such as Bonferroni's correction, would markedly overcorrect for the inflated false-positive rate, resulting in a reduction in power. In our study, several p values could retain significances after correction for multiple testing using SNPSpD.(19) In addition, the fact that consistent positive signals at the same site (OSCAR-2322 A>G) with related BMD in various bone sites were detected might support the reliability of the obtained significances.
In summary, in an effort to examine the possible involvement of genetic polymorphisms of OSCAR in osteoporosis, 10 polymorphisms in OSCAR were newly identified, and five common sites were genotyped in Korean postmenopausal women (n = 560). Using statistical analyses, genetic association of OSCAR-2322A>G polymorphisms with BMD was elucidated. These results offer an important piece in understanding the genetic background of bone metabolism.
This study was supported by a grant from the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (Project 01-PJ3-PG6–01GN11–0002).