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Osteoprotegerin Gene Polymorphisms in Men with Coronary Artery Disease

Departments of Internal Medicine and Cardiology (M.So., M.Sc., A.M.S., M.H., B.M., J.R.S.), and Gastroenterology, Endocrinology, and Metabolism (L.C.H.), Philipps University, D-35033 Marburg, Germany

Address all correspondence and requests for reprints to: Dr. Michael Schoppet, Department of Internal Medicine and Cardiology, Philipps University, Baldingerstrasse, D-35033 Marburg, Germany. E-mail: schoppet{at}mailer.uni-marburg.de.

	Abstract

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Osteoprotegerin (OPG) antagonizes receptor activator of nuclear factor-B ligand (RANKL), the principal regulator of osteoclasts. Of note, OPG-deficient mice display osteoporosis and arterial calcification. Recently, OPG gene polymorphisms have been associated with osteoporosis and early predictors of cardiovascular disease. In this study, we examined OPG gene polymorphisms in 468 men who had absence of coronary artery disease (CAD) or single-, double-, or triple-vessel disease on coronary angiography. Denaturing gradient gel electrophoresis followed by DNA sequencing revealed nucleotide substitutions 149 TC, 163 AG, 209 GA, 245 TG, 950 TC (all promoter), 1181 GC (exon 1), and 6890 AC (intron 4), respectively. Although single polymorphisms were not associated with CAD, linkage of polymorphisms 950 and 1181 revealed that haplotypes were overrepresented in men with CAD (² = 17.05; P = 0.03) with an increased risk of CAD in carriers of genotypes 950 TC/1181 GC and 950 CC/1181 CC (odds ratio, 1.67; 95% confidence interval, 1.02–2.72; P = 0.04). Furthermore, serum OPG levels were correlated with the presence of a C allele at position 950 (P = 0.02). In summary, linkage of genetic variations of the OPG gene at positions 950 and 1181 may confer an increased risk of CAD in Caucasian men.

	Introduction

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OSTEOPROTEGERIN (OPG) IS a secreted basic glycoprotein that belongs to the TNF receptor family. OPG acts by competing with receptor activator of nuclear factor-B (RANK), a surface receptor expressed on osteoclasts and dendritic cells, for binding to RANK ligand (RANKL). RANKL, a member of the TNF family, is a critical cytokine for the differentiation, activation, and survival of osteoclasts and acts as regulator of osteoblast-osteoclast cross-talks and bone homeostasis (1). OPG is expressed by a variety of organs and tissues, such as the heart, lung, kidney, bone, and vessel wall (2), whereas RANKL is mainly expressed by osteoblastic lineage cells and activated T cells (1).

OPG is found as a 60-kDa monomeric and a disulfide-linked homodimeric form of 120 kDa composed of 401 amino acid residues, as deduced from cDNA nucleotide sequencing with a signal peptide of 21 amino acids (3). The mouse and human OPG genes have been cloned and characterized, and the human OPG gene located on chromosome 8 represents a single copy gene with 5 exons spanning 29 kb of the human genome (4, 5).

OPG-deficient mice exhibit osteoporosis and vascular calcification of the medial layer of the aorta and renal arteries (6), a phenotype that can be rescued by an OPG transgene delivered from midgestation through adulthood (7). Furthermore, the induction of vascular calcification by warfarin or toxic doses of vitamin D can be prevented by parenteral administration of an OPG-Fc fusion protein in an animal model (8), and OPG can be detected by immunohistochemistry in atherosclerotic tissue specimens (9). In addition, elevated OPG serum levels are positively correlated with the presence and severity of CAD and with overall cardiovascular mortality (10, 11, 12). Because OPG gene polymorphisms have been found to be associated with osteoporosis and vascular impairment in recent studies (13), we tested the hypothesis of whether OPG polymorphisms are associated with CAD in men.

	Subjects and Methods

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Patients

The patients examined in this study represent a subset of a study cohort of 522 men, in whom increased serum OPG levels were associated with the presence and severity of CAD (11). The study included a population of 468 Caucasian men undergoing diagnostic coronary angiography for suspected CAD (for patient characteristics, see Table 1). The study was approved by the local institutional review board, and informed consent was obtained from all patients participating in this study. Patients with malignant diseases, osteoporosis, or chronic nephropathy (creatinine levels, >2.0 mg/dl) and those receiving systemic glucocorticoids, immunosuppressants, or other drugs known to affect bone metabolism were excluded from the study. CAD was defined as narrowing of more than 50% of at least one major coronary artery, and coronary angiographies were interpreted by at least two experienced cardiologists.

Blood samples were collected before coronary angiography of fasting patients and were subsequently stored at –20 C until analysis. Serum OPG concentrations were determined by an ELISA system purchased from Immundiagnostik (Bensheim, Germany), following exactly the instructions of the manufacturer (11).

Arterial hypertension was defined as systolic blood pressure measurements repeatedly greater than 140 mm Hg, diastolic blood pressure greater than 90 mm Hg, or current treatment with antihypertensive drugs. Patients were considered diabetic if fasting glucose was repeatedly above 120 mg/dl or if patients received oral antidiabetic drugs or insulin injections. Hypercholesterolemia was defined as low density lipoprotein (LDL) cholesterol level greater than 100 mg/dl in men with CAD and greater than 130 mg/dl in men without CAD.

Preparation of genomic DNA and PCR amplification

Genomic DNA was isolated from whole blood by standard procedures. Oligonucleotide primers for the amplification of the OPG gene were designed based on the OPG sequence available in GenBank (accession no. AB008821). PCR amplifications were carried out in a total volume of 50 µl with a reaction mixture containing 1.5 ng genomic DNA, 10 mM Tris-HCl (pH 8.3), 2 mM MgCl₂, 50 mM KCl, 200 µM of each deoxy-NTP, 10 pmol of each primer, and 1 U platinum Taq DNA polymerase (Invitrogen Life Technologies, Karlsruhe, Germany). The PCR amplification conditions included an initial denaturation at 94 C for 3 min, followed by 30 cycles with denaturation at 94 C for 30 sec, annealing at 62 C for 30 sec, and extension at 72 C for 2 min, followed by a final extension step at 72 C for 10 min. Afterward, a short denaturation/renaturation program was performed that consisted of 5 min at 95 C, 5 min at 65 C, 5 min at 37 C, and final cooling to 4 C.

Denaturing gradient gel electrophoresis (DGGE)

DGGE analysis for OPG gene mutations was performed in a modified Protean II electrophoresis chamber (Bio-Rad Laboratories, Munich, Germany). PCR samples (5 µl) were run through 1-mm-thick 7.5% acrylamide gels (acrylamide/bisacrylamide, 37.5:1) containing a linear gradient of 38–75% denaturant (100% = 7 M urea, 40% formamide, and 10% glycerol). Electrophoresis was performed at 120 V for 5 h at a constant temperature of 60 C. After electrophoresis, the gels were stained with ethidium bromide and visualized under UV light.

DNA sequencing

For DNA sense and antisense sequencing, PCR products were purified from agarose gels using Qiaex II gel extraction kit (Qiagen, Hilden, Germany). Forward and reverse sequencings were performed with the BigDye terminator cycle sequencing ready reaction kit (Applied Biosystems, Warrington, UK) on an ABI PRISM 377 DNA sequencer (Applied Biosystems) in all identified subjects with abnormal DGGE mobility patterns.

PCR-restriction fragment length polymorphism analysis

Genotyping of the 950 TC OPG promoter polymorphism was performed by PCR amplification of the OPG promoter sequence with oligonucleotide primers 5'-TGCGTCCGGATCTTGGCTGGATCGG-3' (sense) and 5'-GGGCGCGGCGGGCGCGCCCAGGGACTTACCACGAGCGCGCAGCACAGCAA-3' (antisense), followed by restriction endonuclease digestion with 3 U HincII (New England Biolabs, Frankfurt, Germany) for 16 h at 37 C and resolution by electrophoresis on a 2.5% agarose gel. The 570-bp PCR product is cleaved into 288- and 282-bp fragments only in the presence of a C nucleotide at position 950.

Genotyping of the 1181 GC OPG exon 1 polymorphism was performed using a mismatched oligonucleotide approach. A 570-bp fragment was amplified with primers 5'-TGCGTCCGGATCTTGGCTGGATCGG-3' (sense) and 5'-GGGCGCGGCGGGCGCGCCCAGGGACTTACCACGAGCGCGCAGCACAGCTA-3' (antisense), containing a T instead of an A nucleotide two bases before the 3' end that corresponds to the third base of codon 3 that encodes lysine in exon 1 of the OPG gene, thereby introducing an artificial XspI restriction site in the presence of the mutant allele. After PCR amplification, products were digested with 3 U XspI (Cambrex Bio Science, Apen, Germany) for 16 h and subsequently analyzed on a on a 2.5% agarose gel. In the presence of a C nucleotide at position 1181, the 570-bp PCR product was cleaved into 522- and 48-bp fragments, respectively (Fig. 1).

View larger version (50K): [in this window] [in a new window]   FIG. 1. A, Graphical view of the OPG gene structure. The region containing the OPG promoter and exon 1 is shown. Potential binding sites for transcription factors AP2 and SP1 are indicated as boxes. The location of the 950 TC promoter and 1181 GC exon 1 polymorphism regions are marked with asterisks. B, Genotyping of the 950 TC OPG promoter and 1181 GC OPG exon 1 polymorphisms. The 570-bp PCR-based mismatched oligonucleotide approach amplified products were either digested with HincII (950 TC) or XspI (1181 GC) and resolved on a 2.5% agarose gel. Lane 1, Wild-type carrier (TT) of the 950 TC OPG promoter polymorphism; lane 2, heterozygous carrier (TC) of the 950 TC OPG promoter polymorphism; lane 3, homozygous carrier (CC) of the 950 TC OPG promoter polymorphism; lane 4, wild-type carrier (GG) of the 1181 GC OPG exon 1 polymorphism; lane 5, heterozygous carrier (GC) of the 1181 GC OPG exon 1 polymorphism; lane 6, homozygous carrier (CC) of the 1181 GC OPG exon 1 polymorphism. M, Molecular weight marker (Promega Corp., Mannheim, Germany) used as a size standard.

Statistical analysis was performed using the software SPSS for Windows (version 11.0.1, SPSS, Inc., Chicago, IL). Summary statistics for continuous variables were recorded as means and SDs. Categorical data were summarized as frequencies and percentages. Comparisons of genotype frequencies were performed by Pearsons ² test. Multiple group comparisons of serum OPG levels were performed using the Kruskal-Wallis test; differences between two groups were compared using the Mann-Whitney U test. Binary logistic regression analysis was performed with presence of CAD as the dependent variable and the presence of one of the haplotypes (950 TC/1181 GC or 950 CC/1181 CC), hypertension, body mass index, age, or LDL cholesterol as independent variables. Results were recorded as the odds ratio (OR) with the 95% confidence interval (CI) for each independent variable. P < 0.05 was considered statistically significant.

	Results

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Based on combined DGGE, DNA sequencing, and PCR-restriction fragment length polymorphism analysis, a total of seven different OPG gene polymorphisms were identified in our cohort of 468 male patients with or without CAD. OPG gene polymorphisms were located at positions 149, 163, 209, 245, and 950 in the promoter region; at position 1181 in exon 1; and at position 6890 in intron 4 (only one patient with genotype 6890 CC). Of note, polymorphisms 149, 209, and 245 were found to be in complete linkage, as assessed by DGGE and DNA sequencing. OPG alleles were distributed equally between patients with CAD and patients without CAD (polymorphisms, 149, 209, and 245: ² = 0.26, P = 0.6; 163: ² = 0.31, P = 0.86; 950: ² = 2.38, P = 0.3; and 1181: ² = 2.79, P = 0.25). The frequencies of OPG genotypes in the study cohort are summarized in Table 2.

We further assessed the possible association of serum OPG levels with promoter polymorphisms and polymorphism 1181 located in exon 1, respectively. None of the polymorphisms at position 149, 209, 245 (all P = 0.655), 163 (P = 0.237), or 1181 (P = 0.47) was associated with circulating OPG concentrations. However, the presence of a C allele in the promoter region at position 950 was associated with higher OPG serum levels (950 TT, 5.5 ± 2.0 pmol/liter; 950 TC, 6.0 ± 2.4 pmol/liter; 950 CC, 6.2 ± 2.1 pmol/liter; P = 0.02).

	Discussion

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Recently, we and others found serum OPG levels to be positively correlated with the presence and severity of CAD (10, 11). Therefore, we studied potential OPG gene polymorphisms in a subgroup of our study cohort (all of them underwent coronary angiography), in whom serum OPG levels, established CAD risk factors, and coronary findings have been previously determined (11). Linkage analysis of polymorphisms 950 and 1181 revealed that genotypes 950 TC/1181 GC and 950 CC/1181 CC are overrepresented in men with CAD and associated with an increased risk of CAD in multivariate regression analysis. Furthermore, subjects with a C allele in the promoter region at position 950 (TC and CC) exhibited significantly higher circulating OPG serum levels.

The association between bone metabolism and vascular disease, which are most prevalent in postmenopausal women and elderly people, has been suggested for several years and is supported by a number of studies (14, 15, 16). Lipid alterations and hormonal changes, including altered lipid oxidation, and the effects of glucocorticoids, PTH, and estrogen deficiency have been implicated to increase vascular calcification and decrease bone mass (17, 18). Because OPG knockout mice concurrently exhibit osteoporosis and arterial calcification of the great arteries (6), OPG has become another candidate gene to link both diseases (19).

A number of polymorphisms of the OPG gene in the promoter region, in exons 1–5, and in introns 1–4, have been described in prior investigations (20, 21, 22, 23, 24), including polymorphisms 149 TC, 163 AG, 209 GA, 245 TG, 950 TC (all promoter), 1181 GC (exon 1), and 6890 AC (intron 4), which have been confirmed in our study with similar frequencies as those reported previously (21, 22, 24). We failed to detect the promoter polymorphism at position 889, which has been reported by Arko et al. (21) in 1.9% of their cohort and which has not been found in the study by Langdahl et al. (22). Of interest, we also confirmed complete linkage of polymorphisms at position 149, 209, and 245, as reported previously (22).

Although a recent study did not find a significant association of OPG polymorphisms and osteoporosis in a Japanese population (20), polymorphisms 209 GA and 245 TG in the promoter region of OPG appear to be significantly negatively correlated with bone mineral density at the lumbar spine, but not at the femoral neck, in a cohort of postmenopausal Slovenian women with osteoporosis (21). The researchers conclude that the 209 GA/245 TG haplotype may represent a risk factor for genetic susceptibility to osteoporosis in postmenopausal women. Another genetic study demonstrated that polymorphisms 163 AG and 245 TG were more common in Danish patients with vertebral fractures (22). However, neither study found an unambiguous association of polymorphism 950 TC with bone mineral density, osteoporotic fractures, or markers of bone turnover, in accordance with studies of Irish or Swedish populations (23, 24). In contrast, the homozygous polymorphism 950 CC was found to be concurrently associated with increased intima-media thickness in the common carotid artery and a reduced maximal postischemic forearm blood flow, both of which are early structural and functional indicators of atherosclerosis in a Swedish cohort (25). Of interest, the 950 polymorphism is located 129 bp upstream from the TATA box, 13 bp downstream from an activating protein 2-binding site, and 32 bp upstream from a specificity protein 1-binding site. However, promoter prediction program analysis did not indicate that a nucleotide substitution at position 950 directly affects any known transcription factor recognition motif.

The 1181 GC polymorphism is located in the upstream region of exon 1, which encodes the signal peptide of the OPG protein and results in a lysine to asparagine substitution. However, protein prediction program analysis did not reveal altered secondary structure or altered signal peptide cleavage of the OPG protein. The polymorphism was associated with a preserved bone mass at the lumbar spine in 1181 CC homozygosity, whereas 1181 GG homozygosity and 1181 GC heterozygosity were more common in patients with osteoporosis (22). The association of polymorphisms at position 1181 with vascular disease has not been reported to date.

In our study population of Caucasian men, only one patient had the homozygous base pair substitution at position 6890 (intron 4), whereas a number of subjects revealed nucleotide changes at positions 149, 163, 209, 245, 950 (all within the promoter), and 1181 (exon 1). Although none of these polymorphisms was associated with the presence of CAD separately, linkage of the 950 and 1181 polymorphisms exhibited a significantly different distribution in patients with CAD compared with patients without CAD. Genotypes 950 TC/1181 GC and 950 CC/1181 CC were overrepresented in men with CAD and were associated with an increased risk of CAD, suggesting that these genotypes may contribute to an increased susceptibility of atherosclerosis. Sequence variations in the OPG promoter in concert with polymorphisms in the exon encoding for the signal peptide of this secretory protein may act synergistically to modulate transcription, intracellular trafficking, or secretion of the OPG protein. Thus, we studied the association of polymorphisms with serum OPG concentrations and found serum levels to be significantly higher in genotypes 950 TC and 950 CC compared with the wild-type genotype.

Our study has several limitations. Circulating OPG levels may inadequately reflect OPG protein synthesis in or sequestration to the vessel wall. Furthermore, sequence variations may influence not only serum levels but also protein activity or ligand and receptor binding characteristics, which we did not specifically address. Finally, only men were studied, and bone metabolism was not simultaneously evaluated.

In summary, our data indicate that genetic variations in the OPG gene, a calcification-inhibiting protein, may be associated with increased susceptibility to CAD in Caucasian men.

	Acknowledgments

 
We thank Ms. U. Freund, S. Motzny, and U. Otte for excellent technical assistance.

	Footnotes

 
This work was supported by a Heisenberg fellowship from the Deutsche Forschungsgemeinschaft (to L.C.H.; Ho 1875/3-1 and Ho 1875/4-1).

M.So. and M.Sc. contributed equally to this work.

Abbreviations: CAD, Coronary artery disease; CI, confidence interval; DGGE, denaturing gradient gel electrophoresis; LDL, low-density lipoprotein; OPG, osteoprotegerin; OR, odds ratio; RANK, receptor activator of nuclear factor-B; RANKL, RANK ligand.

Received November 30, 2003.

Accepted April 28, 2004.

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