An intermediate form of juvenile Paget's disease caused by a truncating TNFRSF11B mutation

Juvenile Paget's disease (JPD) is a rare condition with an autosomal recessive mode of inheritance. Typically presenting in infancy or early childhood, the disorder is characterized by a generalized widening of the long bones and thickening of the skull combined with sustained elevation of serum alkaline phosphatase levels. The extremely rapid bone turnover results in osteopenia, fractures, and progressive skeletal deformity. In 2002, mutations in TNFRSF11B, the gene encoding osteoprotegerin, were described as underlying JPD. We evaluated a patient with JPD at the clinical, biochemical, radiological, and molecular level. Mutation analysis of TNFRSF11B revealed a homozygous insertion/deletion in exon 5, predicted to result in truncation of the protein at amino acid 325. The residual activity of the mutated protein product was investigated by Western blotting and ELISA upon transient overexpression. Absence of the C-terminal domain abolished homodimerization and was shown to lead to a decreased capacity of the mutant protein to bind its ligand RANKL. We conclude that truncation of the C-terminal part of osteoprotegerin negatively affects functional activity. As a consequence, osteoclast formation and function are up-regulated, causing the increased bone turnover seen in this patient.     

Juvenile Paget's disease in an Iranian kindred with vitamin D deficiency and novel homozygous TNFRSF11B mutation

Juvenile Paget's disease (JPD) is a rare heritable osteopathy characterized biochemically by markedly increased serum alkaline phosphatase (ALP) activity emanating from generalized acceleration of skeletal turnover. Affected infants and children typically suffer bone pain and fractures and deformities, become deaf, and have macrocranium. Some who survive to young adult life develop blindness from retinopathy engendered by vascular microcalcification. Most cases of JPD are caused by osteoprotegerin (OPG) deficiency due to homozygous loss‐of‐function mutations within the TNFRSF11B gene that encodes OPG. We report a 3‐year‐old Iranian girl with JPD and craniosynostosis who had vitamin D deficiency in infancy. She presented with fractures during the first year‐of‐life followed by bone deformities, delayed development, failure‐to‐thrive, and pneumonias. At 1 year‐of‐age, biochemical studies of serum revealed marked hyperphosphatasemia together with low‐normal calcium and low inorganic phosphate and 25‐hydroxyvitamin D levels. Several family members in previous generations of this consanguineous kindred may also have had JPD and vitamin D deficiency. Mutation analysis showed homozygosity for a unique missense change (c.130T>C, p.Cys44Arg) in TNFRSF11B that would compromise the cysteine‐rich domain of OPG that binds receptor activator of NF‐κB ligand (RANKL). Both parents were heterozygous for this mutation. The patient's serum OPG level was extremely low and RANKL level markedly elevated. She responded well to rapid oral vitamin D repletion followed by pamidronate treatment given intravenously. Our patient is the first Iranian reported with JPD. Her novel mutation in TNFRSF11B plus vitamin D deficiency in infancy was associated with severe JPD uniquely complicated by craniosynostosis. Pamidronate treatment with vitamin D sufficiency can be effective therapy for the skeletal disease caused by the OPG deficiency form of JPD.     

Deletion of aspartate 182 in OPG causes juvenile Paget's disease by impairing both protein secretion and binding to RANKL.

Mutations in the OPG gene cause idiopathic hyperphosphatasia. We characterized the effects of one such mutation and found that the mutant OPG is poorly secreted and has reduced biological activity compared with the wildtype protein. Therefore, correct structure and cellular processing of OPG is essential for normal bone remodeling. INTRODUCTION: Inactivating mutations in osteoprotegerin (OPG) cause juvenile Paget's disease (JPD). We recently reported a family with JPD in which affected members were homozygous for an in-frame mutation resulting in the deletion of aspartate 182 in OPG. Here we report the structural and functional characterization of the OPGdeltaD182 mutant protein. MATERIALS AND METHODS: Inhibition of osteoclastogenesis by the recombinant OPG proteins was studied in a murine bone marrow culture. Binding of wildtype and mutant OPG to RANKL was measured in two experimental systems: glutathione-S-transferase (GST) pull-down assay and surface plasmon resonance. Site-directed mutagenesis was used to study the glycosylation of OPGdeltaD182 in two potential glycosylation sites adjacent to the deleted aspartate residue at position 182. ELISA and Western blots were used to determine OPG concentrations in cell lysates and conditioned media from transiently transfected cells. RESULTS: OPGdeltaD182 inhibited the generation of osteoclasts less effectively than the wildtype protein and had a reduced ability to bind to RANKL. The apparent higher molecular weight of OPGdeltaD182 compared with the wildtype is a result of hyperglycosylation of asparagine residues at positions 178 and 183. Glycosylation at N183 has the potential to disrupt OPG structure by interfering with disulphide bond formation and correct protein folding. Transient transfection experiments in SaOS2 cells suggest that OPGdeltaD182 is retained within the cell, a typical response to unstable or incorrect protein folding. CONCLUSIONS: Taken together, these data suggest that the deletion of aspartate 182 impairs both the secretion and activity of OPG, which in turn provides an explanation for the increased osteoclastogenesis and high bone turnover observed in JPD patients with this mutation.     

Idiopathic hyperphosphatasia and TNFRSF11B mutations: relationships between phenotype and genotype.

Homozygous mutations in TNFRSF11B, the gene encoding osteoprotegerin, were found in affected members from six of nine families with idiopathic hyperphosphatasia. The severity of the phenotype was related to the predicted effects of the mutations on osteoprotegerin function. INTRODUCTION: Idiopathic hyperphosphatasia (IH) is a rare high bone turnover congenital bone disease in which affected children are normal at birth but develop progressive long bone deformities, fractures, vertebral collapse, skull enlargement, and deafness. There is, however, considerable phenotypic variation from presentation in infancy with severe progressive deformity through to presentation in late childhood with minimal deformity. Two recent reports have linked idiopathic hyperphosphatasia with deletion of, or mutation in, the TNFRSF11B gene that encodes osteoprotegerin (OPG), an important paracrine modulator of RANKL-mediated bone resorption. MATERIALS AND METHODS: We studied subjects with a clinical diagnosis of IH and unaffected family members from nine unrelated families. Clinical, biochemical, and radiographic data were collected, and genomic DNA examined for mutations in TNFRSF11B. The relationship between the mutations, their predicted effects on OPG function, and the phenotype were then examined. RESULTS: Of the nine families studied, affected subjects from six were homozygous for novel mutations in TNFRSF11B. Their parents were heterozygous, consistent with autosomal recessive inheritance. Four of the six mutations occurred in the cysteine-rich ligand-binding domain and are predicted to disrupt binding of OPG to RANKL. Missense mutations in the cysteine residues, predicted to cause major disruption to the ligand-binding region, were associated with a severe phenotype (deformity developing before 18 months age and severe disability), as was a large deletion mutation. Non-cysteine missense mutations in the ligand-binding domain were associated with an intermediate phenotype (deformity recognized around the age of 5 years and an increased rate of long bone fracture). An insertion/deletion mutation at the C-terminal end of the protein was associated with the mildest phenotype. CONCLUSION: Mutations in TNFRSF11B account for the majority of, but not all, cases of IH, and there are distinct genotype-phenotype relationships.     

Loss of chaotic trabecular structure in OPG-deficient juvenile Paget's disease patients indicates a chaogenic role for OPG in nonlinear pattern formation of trabecular bone.

The RANK-RANKL-OPG system of osteoclast regulation may play a key role in determining chaotic structure in trabecular bone. Iliac trabecular bone from juvenile Paget's disease patients deficient in functional OPG shows parallel, anisotropic structure instead of normal chaotic structure. Evidence from experimental systems suggests that RANK-RANKL-OPG controls key nonlinear "chaogenic" parameters, such as friction, forcing frequency, feedback, and boundary forcing. The RANK-RANKL-osteoprotegerin (OPG) system of osteoclast regulation may play a key role in determining chaotic structure in trabecular bone. Iliac trabecular bone from juvenile Paget's disease (JPD) patients deficient in functional OPG shows parallel, anisotropic structure instead of normal chaotic structure. Evidence from experimental systems suggests that RANK-RANKL-OPG controls key nonlinear "chaogenic" parameters, such as friction, forcing frequency, feedback, and boundary forcing. The Belousov-Zhabotinsky reaction-diffusion system, the catalytic oxidation of CO on platinum surfaces, and thermal diffusion in liquid helium allow visualization of nonlinear emergent patterns such as labyrinthine structures, turbulence, and cellular structures, all of which bear some resemblance to trabecular bone. In JPD, the gene for OPG (TNFRSF11B) is subject to an inactivating mutation, leading to increased resorption and accelerated remodeling. Histomorphometric images of iliac crest trabecular bone from teenagers suffering from JPD show a highly unusual array of parallel, regular trabecular plates, instead of the typical chaotic, fractal patterns of normal trabecular bone. Loss of OPG function is associated with a change from chaotic to regular structure, suggesting that the RANK-RANKL-OPG system is controlling key nonlinear "chaogenic" parameters. Looking at trabecular bone from the perspective of nonlinear pattern formation may help understand other phenomena, such as the marked dependence of trabecular bone's architectural and mechanical quality on remodeling rate independent of the trabecular bone mass.     

RANKL/RANK/OPG system and bone status in females with anorexia nervosa

Minimal data exist concerning the relationship between osteokines of the RANKL/RANK/OPG system, especially RANKL, and bone status in females with anorexia nervosa (AN). For this reason we investigated the relationship between bone metabolism (as assessed based on serum levels of OC and CTx), and OPG and sRANKL concentrations in females with AN. Ninety-one female patients with AN and 29 healthy female subjects aged 13 to 18 years of age participated in the study. Serum OC, CTx, OPG and sRANKL were measured by ELISA. The female patients with AN demonstrated an essential suppression of OC and CTx, increased OPG and sRANKL levels, and a reduced OPG/sRANKL ratio. OC, CTx and the OPG/sRANKL ratio correlated positively with body mass and BMI in these patients, whereas in the case of OPG and sRANKL the relationship was negative. A significant positive correlation was observed between OPG and sRANKL and also between bone markers and the OPG/sRANKL ratio, and negative between CTx and sRANKL. In female patients with AN, the OPG/RANKL ratio was a significant and independent predictor of osteocalcin, a bone formation marker — OC (R² = 0.065, p = 0.012) whereas the OPG/sRANKL ratio and BMI were significant and independent predictors of a bone resorption marker — CTx (R² = 0.095; p = 0.012). In conclusion, the body mass, BMI values, and bone markers suppression observed in female patients with AN might be associated with an increase in OPG and sRANKL levels and a significant decrease of the OPG/sRANKL ratio. Although higher OPG levels may compensate for excessive bone resorption in female patients with AN, the lower OPG/sRANKL ratio seems to indicate that some inadequacies exist regarding this compensation effect, which might contribute to low bone density in these patients. The OPG/sRANKL ratio might prove a more relevant marker to predict bone metabolism in female patients with AN than sRANKL and/or OPG alone.     

Susceptibility to Paget's disease of bone is influenced by a common polymorphic variant of osteoprotegerin.

To clarify the role of the TNFRSF11B gene encoding osteoprotegerin (OPG), in Paget's disease of bone (PDB) we studied TNFRSF11B polymorphisms in an association study of 690 UK subjects and in a worldwide familial study of 66 kindreds. We found that the G1181 allele of TNFRSF11B, encoding lysine at codon 3 of the OPG protein, predisposes to both sporadic and familial PDB. INTRODUCTION: Paget's disease of bone (PDB) is a common disorder characterized by focal abnormalities of bone turnover. Genetic factors are important in the pathogenesis of PDB, and studies have shown that inactivating mutations of the TNFRSF11B gene, encoding osteoprotegerin (OPG), cause the rare syndrome of juvenile Paget's disease. In this study, we sought to determine whether polymorphisms of the TNFRSF11B gene contribute to the pathogenesis of classical PDB. MATERIALS AND METHODS: We screened for polymorphisms of the TNFRSF11B gene by DNA sequencing of the proximal promoter, coding exons, and intron-exon boundaries in 20 PDB patients and 10 controls. Informative single nucleotide polymorphisms (SNPs), including a G1181C SNP, which predicts a lysine-asparagine substitution at codon 3 of the OPG signal peptide and haplotypes, were related to the presence of PDB in 312 cases compared with 378 controls and to transmission of PDB in 140 affected offspring from 66 kindreds with familial PDB. RESULTS AND CONCLUSIONS: The G1181 allele was significantly over-represented in PDB patients (chi(2) = 5.7, df = 1, p = 0.017, adjusted alpha = 0.024), equivalent to an odds ratio for PDB of 1.55 (95% CI: 1.11-2.16). The distribution of TNFRSF11B haplotypes significantly differed in sporadic PDB cases and controls (chi(2) = 30.2, df = 9, p < 0.001) because of over-representation of haplotypes containing the G1181 allele in cases. The family study showed that the most common haplotype containing the G1181 allele was transmitted more frequently than expected to 140 individuals with familial PDB (chi(2) = 7.35, df = 1, p < 0.01), and the transmission disequilibrium was even more pronounced in a subgroup of 78 familial PDB patients who did not carry mutations of the SQSTM1 gene (chi(2) = 8.44, df = 1, p < 0.005). We conclude that the G1181 allele of TNFRSF11B, encoding lysine at codon 3 of the OPG protein, predisposes to the development of sporadic PDB and familial PDB that is not caused by SQSTM1 mutations.     

Serum osteoprotegerin and receptor activator of nuclear factor-κB ligand (RANKL) concentrations in allogeneic stem cell transplant-recipients: a role in bone loss?

Purpose: Osteoporosis is a long-term complication of allogeneic stem cell transplantation (SCT). Receptor activator of nuclear factor-κB ligand (RANKL) increases osteoclast activity, while osteoprotegerin (OPG) neutralizes RANKL. A deficiency of OPG or an excess of RANKL may contribute to post-SCT bone loss. Methods: Serum OPG and soluble RANKL (sRANKL) concentrations were determined in 30 patients who received calcium, vitamin D and sex steroids – with or without pamidronate – prior to SCT and 1, 3, 6, and 12 months post-SCT and compared to those in healthy controls. Results: Despite all treatments patients lost bone at the hip. At baseline, serum OPG was similar in patients and controls; in the two patient groups it increased by 26–27% at 6 months post-SCT (p=0.002–0.028) and over the control level (p=0.002). Serum sRANKL concentrations were also similar in patients and controls at baseline. In those patients receiving pamidronate sRANKL concentrations decreased by 42% (p=0.0007) at 3 months post-SCT. The findings on the effect of SCT on OPG and sRANKL serum levels were ascertained in 28 additional patients who did not receive pamidronate, at a median of 122 days after SCT. In this latter group, OPG but not sRANKL concentrations were clearly elevated (p<0.001) in comparison to healthy controls. In conclusion, the present study fails to support the view that an excess of sRANKL or a deficiency of OPG would have a substantial impact on bone loss in SCT-recipients. Conclusion: Serum sRANKL concentrations may be modulated by bisphosphonates.     

Variations along the 24-hour cycle of circulating osteoprotegerin and soluble RANKL: a rhythmometric analysis

Summary The variability of serum osteoprotegerin (OPG) and soluble RANKL (sRANKL) along the 24-h cycle was assessed in 20 healthy women. No rhythmic variations of serum OPG, sRANKL or sRANKL/OPG ratio were detected as a group phenomenon. Timing of sampling is unlikely to influence the results of measurements of circulating OPG and sRANKL. Introduction Physiological bone turnover shows diurnal variations. The aim of the study was to assess variability of OPG and sRANKL serum levels along the 24-h cycle. Methods Blood was collected from 20 healthy women (median age 31 years, range 25–65 years) at 4-h intervals between 08:00 and 24:00 and at 2-h intervals between 24:00 and 08:00. Serum albumin, cortisol, osteocalcin (OC), C-terminal telopeptide of type I collagen (CTX), OPG and total sRANKL were measured. Temporal variations were assessed by the COSINOR model. Results Circadian rhythms of cortisol and albumin documented a normal synchronization within the circadian structure. Serum OC and CTX showed rhythmic variations, peaking at night-time. Rhythmic variations of serum OPG, sRANKL and sRANKL/OPG ratio were not detected as a group phenomenon. On an individual basis, rhythmic changes were detected in ten patients for OPG and eight patients for sRANKL, with very small amplitudes and heterogeneous acrophases. Conclusions The absence of consistent rhythmic variations of circulating OPG and sRANKL levels may reflect the absence of rhythmic variations of their expression in the bone microenvironment. Were this the case, the nocturnal rise of bone resorption should be accounted for by different, not RANKL/OPG-mediated factors. Since circulating OPG and sRANKL may derive from sources other than bone, rhythmicity could be masked by non-rhythmic or non-synchronized rhythmic expression in these sources. Timing of sampling is unlikely to influence the results of measurements of circulating OPG and sRANKL. Andrea Dovio and Daniele Generali contributed equally to the study.     

OPG and sRANKL Serum Concentrations in Osteopenic,Postmenopausal Women After 2‐Year Genistein Administration

Introduction : RANKL and its decoy receptor osteoprotegerin (OPG) constitute a complex physiological mediator system involved in the regulation of bone resorption and may be responsible for the homeostatic mechanism of normal bone remodeling. Genistein, an isoflavone representing 1–5% of total phytoestrogen content in soybean products, may positively regulate cellular bone metabolism, but its mechanism of action on bone is not yet fully understood. Materials and Methods : We studied the serum levels of both soluble RANKL (sRANKL) and OPG and the sRANKL/OPG ratio in 389 postmenopausal women (age, 49–67 yr) with a femoral neck BMD <0.795 g/cm² and no significant comorbid conditions after 24‐mo therapy with genistein, (n = 198; 54 mg/d) or placebo (n = 191). Both intervention and placebo contained calcium and vitamin D₃. All patients received dietary instruction in an isocaloric fat‐reduced diet. Results : In comparison with placebo, sRANKL level was lower (p < 0.001 versus placebo) and OPG higher in genistein recipients (p < 0.001 versus placebo) at 1 and 2 yr, respectively. Moreover, at the end of 24 mo, genistein produced a significant reduction in the sRANKL/OPG ratio compared with placebo (genistein = ?0.021, 95% CI, ?0.020 to ?0.022; placebo = +0.004, 95% CI, 0.003–0.005; difference = ?0.020, 95% CI, ?0.015 to ?0.025, p < 0.001). Conclusions : Our findings suggest that genistein plus calcium and vitamin D₃ as part of a healthy diet is able to positively modulate bone turnover in a cohort of osteopenic, postmenopausal women and improve sRANKL‐OPG balance after 24 mo of treatment.     

A nonsynonymous TNFRSF11A variation increases NFκB activity and the severity of Paget's disease

Mutations in the SQSTM1 gene were identified as a common cause of Paget's disease of bone (PDB) but experimental evidence demonstrated that SQSTM1 mutation is not sufficient to induce PDB in vivo. Here, we identified two nonsynonymous single nucleotide polymorphisms (SNPs) (C421T, H141Y and T575C, V192A) in the TNFRSF11A gene, associated with PDB and with the severity of phenotype in a large population of 654 unrelated patients that were previously screened for SQSTM1 gene mutations. The largest effect was found for the T575C variant, yielding an odds ratio of 1.29 (p = 0.003), with the C allele as the risk allele. Moreover, an even more significant p-value (p = 0.0002) was observed in the subgroup of patients with SQSTM1 mutation, with an odds ratio of 1.71. Interestingly, patients with the C allele also showed an increased prevalence of polyostotic disease (68%, 53%, and 51% in patients with CC, CT, and TT genotypes, respectively; p = 0.01), as well as an increased number of affected skeletal sites (2.9, 2.5, and 2.0 in patients with CC, CT, and TT genotypes, respectively, p = 0.008). These differences increased when analyses were restricted to cases with SQSTM1 mutation. In human cell lines, cotrasfection with mutated SQSTM1 and TNFRSF11A(A192) produced a level of activation of NFκB signaling greater than cotrasfection with wild-type SQSTM1 and TNFRSF11A(V192), confirming genetics and clinical evidences. These results provide the first evidence that genetic variation within the OPG/RANK/RANKL system influences the severity of PBD in synergistic action with SQSTM1 gene mutations.     

Mutation Screening of the TNFRSF11A Gene Encoding Receptor Activator of NFkB (RANK) in Familial and Sporadic Paget's Disease of Bone and Osteosarcoma

Paget's disease of bone (PDB) is a common disorder characterized by focal areas of increased and disorganized osteoclastic bone resorption, leading to bone pain, deformity, pathological fracture, and an increased risk of osteosarcoma. Genetic factors play an important role in the pathogenesis of Paget's disease. In some families, the disease has been found to be linked to a susceptibility locus on chromosome 18q21-22, which also contains the gene responsible for familial expansile osteolysis (FEO)--a rare bone dysplasia with many similarities to Paget's disease. Insertion mutations of the TNFRSF11A gene encoding Receptor Activator of NF kappa B (RANK) have recently been found to be responsible for FEO and rare cases of early onset familial Paget's disease. Loss of heterozygosity (LOH) affecting the PDB/FEO critical region has also been described in osteosarcomas suggesting that TNFRSF11A might also be involved in the development of osteosarcoma. In order to investigate the possible role of TNFRSF11A in the pathogenesis of Paget's disease and osteosarcoma, we conducted mutation screening of the TNFRSF11A gene in patients with familial and sporadic Paget's disease as well as DNA extracted from Pagetic bone lesions, an osteosarcoma arising in Pagetic bone and six osteosarcoma cell lines. No specific abnormalities of the TNFRSF11A gene were identified in a Pagetic osteosarcoma, the osteosarcoma cell lines, DNA extracted from Pagetic bone lesions, or DNA extracted from peripheral blood in patients with familial or sporadic Paget's disease including several individuals with early onset Paget's disease. These data indicate that TNFRSF11A mutations contribute neither to the vast majority of cases of sporadic or familial PDB, nor to the development of osteosarcoma.     

OPG and sRANKL serum levels and incident hip fracture in postmenopausal Caucasian women in the Women's Health Initiative Observational Study

PurposeThe osteoprotogerin/receptor activator of NF-kappa β/receptor activator of NF-kappa β ligand (OPG/RANK/RANKL) pathway plays a critical role in bone remodeling. This study investigated associations between serum levels of OPG, soluble RANKL (sRANKL), and the ratio of OPG/sRANKL to risk of incident hip fracture.MethodsA nested case–control study was conducted among postmenopausal, Caucasian women aged 50–79 at baseline (1993–1998), followed for hip fracture through March 2005 in the Women's Health Initiative Observational Study. 400 incident hip fracture cases were selected and individually matched to 400 controls with no prior fracture or incident hip fracture. Matching factors were baseline age, enrollment date and hormone therapy (HT) exposure. Baseline serum OPG and sRANKL levels were measured using high sensitivity ELISA. Odds ratios were computed for quartiles of each biomarker adjusting for matching factors and hip fracture risk factors.ResultsSerum OPG was significantly associated with older age, low physical activity and poorer physical function in control women. sRANKL was inversely associated with total calcium intake in control women, but not associated with age or other fracture risk factors. The odds ratio for hip fracture comparing the highest to lowest quartiles of OPG was 2.28 (95% confidence interval (CI), 1.45–3.61) after adjusting for the matching variables (p-value for linear trend < 0.001), and 1.87 (95% CI, 1.15–3.04; p for linear trend = 0.02) after adjusting for self-rated health status, physical activity and physical functioning. No significant associations between sRANKL or the ratio of OPG/sRANKL and hip fracture risk were observed.ConclusionSerum OPG levels were independently associated with a nearly twofold increased risk of hip fracture in postmenopausal women.     

Plasma osteoprotegerin is associated with mortality in hemodialysis patients

Expression of bone proteins resulting from transdifferentiation of vascular smooth muscle cells into osteoblasts suggests that vascular calcifications are a bioactive process. Regulating molecules such as osteoprotegerin (OPG) and receptor activator of NF-kappaB ligand (RANKL) could play a key role in bone-vascular calcification imbalance. This study investigated the contribution of these proteins as well as mineral metabolism disorders in hemodialysis (HD) patient outcome. A total of 185 HD patients were followed up prospectively for 2 yr. In addition to clinical characteristics, mineral metabolism markers as well as OPG and soluble RANKL (sRANKL) were measured at baseline. After 2 yr, survival rates were described with Kaplan-Meier and compared with Cox regression analyses; 50 patients died (27 from cardiovascular diseases). Calcium, phosphate, and calcium x phosphate product were not associated with mortality. Both hyperparathyroidism (parathyroid hormone > or =300 pg/ml) and hypoparathyroidism (parathyroid hormone <150 pg/ml) were poorly associated with all-cause and cardiovascular mortality. By contrast, elevated OPG levels predicted all-cause (relative risk [RR] 2.67; 95% confidence interval [CI] 1.32 to 5.41; P = 0.006) and cardiovascular mortality (RR 3.15; 95% CI 1.14 to 8.69; P = 0.03). Low levels of sRANKL were associated with a protective effect for all-cause mortality (RR 0.45; 95% CI 0.21 to 0.94; P = 0.03). The association of OPG with all-cause mortality was stronger in patients with C-reactive protein > or =12.52 mg/L. In this condition, both highest (RR 5.68; 95% CI 1.48 to 22.73; P = 0.01) and lowest tertiles (RR 5.37; 95% CI 147 to 1968; P = 0.01) significantly predicted poor outcome. These results show that regulating-bone molecules, especially OPG, are strong predictors of mortality in HD patients, suggesting that OPG is a vascular risk factor, in particular in patients who have high C-reactive protein levels. OPG determination therefore should be added to the biologic follow-up of these patients.     

Plasma and drainage fluid levels of soluble receptor activator of nuclear factor-kB (sRANK), soluble receptor activator of nuclear factor-kB ligand (sRANKL) and osteoprotegerin (OPG) during proximal humerus fracture healing

Fracture healing is an ordered process that restores the structural integrity of the bone. Soluble receptor activator of nuclear factor-kB (sRANK), its ligand (sRANKL) and osteoprotegerin (OPG) are involved in bone remodelling, thus they may play a role in fracture repair. OPG, soluble RANK and RANKL levels were measured in plasma and in drainage fluid, collected from pre-surgery phase to healing in ten patients of both genders (age range 26–65 years) with proximal humerus fracture needing osteosynthesis. All patients showed fracture healing. No significant modifications in the concentrations of sRANKL and OPG were observed, while sRANK showed a significant increase in drainage fluid 24 hours post-surgery compared with intra-surgery time. OPG levels were higher in plasma and drainage fluid than sRANK and sRANKL at each time point. Since there are no published data about sRANK involvement in fracture healing, our study represents the first preliminary indication about a local increase of this marker concentration immediately after surgery.     

Expression of bone resorption genes in osteoarthritis and in osteoporosis

Cathepsin K and MMP-9 are considered to be the most abundant proteases in osteoclasts. TRAP is a marker for osteoclasts, and there is increasing evidence of its proteolytic role in bone resorption. RANKL is a recently discovered regulator of osteoclast maturation and activity and induces expression of many genes. This study compared cathepsin K, MMP-9, TRAP, RANKL, OPG, and osteocalcin gene expression in the proximal femur of patients with osteoarthritis with that of patients with femoral neck fracture. Fifty-six patients undergoing arthroplasty because of osteoarthritis or femoral neck fracture were included in the study. Total mRNA was extracted from the bone samples obtained from the intertrochanteric region of the proximal femur. Real-time RT-PCR was used to quantify CTSK (cathepsin K), MMP-9 (matrix metalloproteinase 9), ACP5 (TRAP), TNFSF11 (RANKL), TNFRSF11B (OPG), and BGLAP (osteocalcin) mRNAs. The levels of mRNAs coding for MMP-9 and osteocalcin indicated higher expression in the osteoarthritic group (P = 0.011, P = 0.001, respectively), whereas RANKL expression and the ratio RANKL/OPG were both significantly lower in the osteoarthritic group than in the fracture group. Expression of cathepsin K, MMP-9, and TRAP relative to RANKL was significantly higher in the osteoarthritic group. Ratios of all three proteolytic enzymes relative to formation marker osteocalcin were higher in the fracture group. Gene expression of cathepsin K, MMP-9, TRAP, RANKL, OPG, and osteocalcin and the association between their mRNA levels pointed to higher bone resorption and bone formation in osteoarthritis, differences in balance between them, and differences in regulation of bone resorption in osteoarthritic and osteoporotic bone.     

Serum OPG and RANKL levels before and after intravenous bisphosphonate treatment in Paget's disease of bone

Paget's disease of bone (PDB) is a focal disorder of bone remodeling characterized by increased osteoclast-mediated bone resorption. Even though increasing evidence indicates enhanced nuclear factor-kB (NF-kB) signaling as a common mechanism involved in PDB and other related disorders, few studies investigated circulating osteoprotegerin (OPG) and receptor of activator of NF-kB-ligand (RANKL) levels in PDB patients. In this study we explored the relationships between OPG or RANKL levels and bone turnover markers in a group of patients with PDB, before and after intravenous bisphosphonate treatment (pamidronate 60 mg). Both OPG and RANKL were markedly elevated in PDB patients with respect to control groups (healthy or osteoporotic postmenopausal women and elderly men) and were positively associated with bone turnover markers. Higher levels of these cytokines were observed in polyostotic than monostotic PDB cases. The ratio between RANKL and OPG was more than 3-fold higher in PDB patients than in controls. Interestingly, in the group of patients treated with pamidronate, we found an increase in OPG levels that become statistically significant after 3 and 6 months from treatment. A trend toward a decrease in RANKL levels after treatment was also observed. The RANKL/OPG ratio was significantly reduced after 3 and 6 months of therapy. In contrast, in patients classified as non-responders, OPG and RANKL levels after pamidronate infusion did not significantly differ with respect to pre-treatment values. Thus, the positive effect of amino bisphosphonates in the treatment of PDB may be due to either direct or indirect suppression of RANKL-induced bone resorption through decreased RANKL and increased OPG production.     

血液透析患者血管钙化、骨密度与骨保护素及其配体的相互关系

目的 研究维持性血液透析（MHD）患者血管钙化、骨密度与血清骨保护素（OPG）及其配体sRANKL的相互关系。 方法 采用酶联免疫吸附法测定血清OPG、sRANKL水平；X线平片检测腹主动脉、股动脉及桡动脉部位血管钙化，计算血管钙化积分；双能X线骨密度仪测定腰椎及股骨骨密度。对各参数的相互关系进行统计分析。 结果 （1）39例MHD患者中25例（64.1%）在不同部位有不同程度的血管钙化，其中轻度钙化16例（41.0%），中重度钙化9例（23.1%）。中重度钙化者血清OPG水平、OPG/sRANKL比值显著高于轻度钙化者[（342.50±171.53） ng/L比（206.21±137.88） ng/L，t = -2.253，P = 0.025；454.65±455.63比135.31±136.81，t = 59，P = 0.035]，而sRANKL水平差异无统计学意义[（0.10±0.08） pmol/L比(0.12±0.08) pmol/L，t = 0.534，P > 0.05]。多元线性回归分析显示 OPG/sRANKL是血管钙化评分的独立影响因素。（2）与骨量正常者比较，骨量异常者血清OPG水平升高[（249.05±137.66） ng/L比（226.67±170.12） ng/L]，sRANKL水平降低[（0.11±0.08） pmol/L比（0.12±0.02） pmol/L]，OPG/sRANKL比值升高（202.31±219.24比148.08±210.10），但差异均无统计学意义。多元线性回归分析显示OPG/sRANKL是腰椎T值的独立影响因素。（3）多元线性回归分析显示血管钙化评分是腰椎和股骨T值的独立影响因素。 结论 MHD患者血管钙化程度是腰椎及股骨骨密度的独立影响因素， OPG/sRANKL可能在血管钙化和骨密度的关系中起了纽带作用。     

Changes in the serum sex steroids, IL-7 and RANKL-OPG system after bone marrow transplantation: influences on bone and mineral metabolism

This study prospectively investigated the changes of the serum levels of the sex steroids, IL-7, soluble receptor activator of nuclear factor κB ligand (sRANKL) and osteoprotegerin (OPG) in bone marrow transplantation (BMT) recipients. This study also examined whether the changes of these cytokine levels and sex steroids actually influence bone turnover and post-BMT bone loss by correlation analysis. Data were analyzed from 39 patients (33.6 ± 6.4 years, 19 men and 20 women) who had DXA performed before BMT and at 1 year after BMT. The bone turnover markers, sex steroids and the cytokine levels were measured before BMT and serially after BMT.

The mean bone loss in the lumbar spine and the total proximal femur was 5.9% (P < 0.01) and 11.3% (P < 0.01), respectively. During the immediate post-BMT period, bone formation decreased, whereas the bone resorption increased. For the female recipients, the estradiol levels declined at 1 week after BMT, and they did not recover to the basal levels. For the male recipients, the testosterone levels decreased at 1 week and then it increased to its baseline level. The IL-7 levels reached their maximum at 1 week and then declined to baseline level by 3 months. The serum sRANKL, OPG levels and the sRANKL/OPG ratio showed their peak at post-BMT 3 weeks.

The mean daily dose of steroid was associated with suppressed bone formation, enhanced bone resorption and increased sRANKL levels. The IL-7 levels were also noted to be either positively correlated with the levels of ICTP or they were negatively correlated with the levels of osteocalcin at 1 and 3 weeks after BMT. Bone loss at the lumbar spine and the proximal femur was influenced by the decreased sex steroids and increased IL-7 levels. During the observation period, the IL-7 levels showed positive correlations with the sRANKL levels and the sRANKL/OPG ratio. For the female patients, the serum IL-7 levels were negatively associated with the estradiol levels at 1 and 3 weeks after BMT.

All these findings suggest that IL-7 plays an important role for post-BMT bone loss, and this possibly happens via the RANKL pathway. These data also suggest that the up-regulation of IL-7 during the early post-BMT period may result from a deficiency of estrogen.     

Relationship between coronary and abdominal calcification score, serum osteoprotegerin (OPG), and serum tartrate-resistant acid phosphatase (TRACP) -5b in pre-dialysis CKD patients

Osteoprotegerin (OPG) inhibits interaction of the receptor-activator of nuclear factor-kappaB (RANK) ligand (RANKL) with its receptor RANK, which is expressed on osteoclasts. OPG appeared to accelerate vascular calcification in vitro by the inhibition of vascular osteoclast-like cells. On the contrary, early-onset arterial calcification was observed in OPG-deficient mice. We measured the coronary artery calcification score (CACS) and abdominal aortic calcification score (AAoCS) by multi-detector computed tomography in 30 pre-dialysis CKD patients (eGFR 20 mL/min on average). Biomarkers were measured, including serum OPG, soluble RANKL (sRANKL) and tartrate-resistant acid phosphatase (TRACP) -5b (the biomarker of osteoclasts independent of renal function). The median values of CACS and AAoCS were 54.4 and 1,088 Agatston units (AU), respectively. Serum OPG was increased and serum sRANKL was decreased. In a multivariate logistic regression analysis using CACS > or = 100 AU as the outcome variable, CACS was found to be positively correlated with serum corrected Ca x iP product and serum OPG, though it was not correlated with serum TRACP-5b. ROC curve analysis showed that the serum OPG cutoff value predicting CACS > or = 100 AU was 5.2 pmol/L (624 pg/mL). In a stepwise regression analysis, log (AAoCS + 1) was positively correlated with serum OPG alone, but it was not correlated with age, eGFR, serum albumin and bone alkaline phosphatase (BAP). No correlation was found between serum OPG and serum TRACP-5b. In conclusion, vascular calcification in pre-dialysis CKD patients was correlated with an increase in OPG, but was independent of serum TRACP-5b. The decrease in serum sRANKL may have been caused by the increase in OPG production.