Serotonin Is Involved in Autoimmune Arthritis through Th17 Immunity and Bone Resorption.

Rheumatoid arthritis is a chronic disease that results in a disabling and painful condition as it progresses to destruction of the articular cartilage and ankylosis of the joints. Although the cause of the disease is still unknown, evidence argues that autoimmunity plays an important part. There are increasing but contradictory views regarding serotonin being associated with activation of immunoinflammatory pathways and the onset of autoimmune reactions. We studied serotonin's involvement during collagen-induced arthritis in wild-type and Tph1(-/-) mice, which have markedly reduced peripheral serotonin levels. In wild-type mice, induction of arthritis triggered a robust increase in serotonin content in the paws combined with less inflammation. In Tph1(-/-) mice with arthritis, a marked increase in the clinical and pathologic arthritis scores was noticed. Specifically, in Tph1(-/-) mice with arthritis, a significant increase in osteoclast differentiation and bone resorption was observed with an increase in IL-17 levels in the paws and in Th17 lymphocytes in the draining lymph nodes, whereas T-regulatory cells were dampened. Ex vivo serotonin and agonists of the 5-HT2A and 5-HT2B receptors restored IL-17 secretion from splenocytes and Th17 cell differentiation in Tph1(-/-) mice. These findings indicate that serotonin plays a fundamental role in arthritis through the regulation of the Th17/T-regulatory cell balance and osteoclastogenesis.

Ankle-brachial index and bone turnover in patients on dialysis.

An association between atherosclerosis and osteoporosis has been reported in several studies. This association could result from local intraosseous atherosclerosis and ischemia, which is shown by limb osteoporosis in patients with peripheral artery disease (PAD), but also could result from bidirectional communication between the skeleton and blood vessels. Systemic bone disorders and PAD are frequent in ESRD. Here, we investigated the possible interaction of these disorders. For 65 prevalent nondiabetic patients on hemodialysis, we measured ankle-brachial pressure index (ABix) and evaluated mineral and bone disorders with bone histomorphometry. In prevalent patients on hemodialysis, PAD (ABix<0.9 or >1.4/incompressible) was associated with low bone turnover and pronounced osteoblast resistance to parathyroid hormone (PTH), which is indicated by decreased double-labeled surface and osteoblast surface (P<0.001). Higher osteoblast resistance to PTH in patients with PAD was characterized by weaker correlation coefficients (slopes) between serum PTH and double-labeled surface (P=0.02) or osteoblast surface (P=0.03). The correlations between osteoclast number or eroded surface and serum mineral parameters, including PTH, did not differ for subjects with normal ABix and PAD. Common vascular risk factors (dyslipidemia, smoking, and sex) were similar for normal, low, and incompressible ABix. Patients with PAD were older and had high C-reactive protein levels and longer hemodialysis vintage. These results indicate that, in prevalent nondiabetic patients with ESRD, PAD associates with low bone turnover and pronounced osteoblast resistance to PTH.

Decreased osteoclastogenesis in serotonin-deficient mice.

Peripheral serotonin, synthesized by tryptophan hydroxylase-1 (TPH(1)), has been shown to play a key role in several physiological functions. Recently, controversy has emerged about whether peripheral serotonin has any effect on bone density and remodeling.We therefore decided to investigate in detail bone remodeling in growing and mature TPH(1) knockout mice (TPH(1)(-/-)). Bone resorption in TPH(1)(-/-) mice, as assessed by biochemical markers and bone histomorphometry, was markedly decreased at both ages. Using bone marrow transplantation, we present evidence that the decrease in bone resorption in TPH(1)(-/-) mice is cell-autonomous. Cultures from TPH(1)(-/-) in the presence of macrophage colony-stimulating factor and receptor activator for NF-KB ligand (RANKL) displayed fewer osteoclasts, and the decreased differentiation could be rescued by adding serotonin. Our data also provide evidence that in the presence of RANKL, osteoclast precursors express TPH(1) and synthesize serotonin. Furthermore, pharmacological inhibition of serotonin receptor 1B with SB224289, and of receptor 2A with ketanserin, also reduced the number of osteoclasts. Our findings reveal that serotonin has an important local action in bone, as it can amplify the effect of RANKL on osteoclastogenesis.

Differentially expressed genes in autosomal dominant osteopetrosis type II osteoclasts reveal known and novel pathways for osteoclast biology.

Autosomal dominant osteopetrosis type II (ADO II) is a rare, heritable bone disorder characterized by a high bone mass and insufficient osteoclast activity. Mutations in the CLCN7 gene have been reported to cause ADO II. To gain novel insights into the pathways dysregulated in ADOII osteoclasts, we identified changes in gene expression in osteoclasts from patients with a heterozygous mutation of CLCN7. To do this, we carried out a transcriptomic study comparing gene expression in the osteoclasts of patients with ADO II and healthy donors. Our data show that, according to our selection criteria, 182 genes were differentially expressed in osteoclasts from patients and controls. From the 18 displaying the highest change in microarray, we confirmed differential expression for seven by qPCR. Although two of them have previously been found to be expressed in osteoclasts (ITGB5 and SERPINE2), the other five (CES1 (carboxyl esterase 1), UCHL1 (ubiquitin carboxy-terminal esterase L1, also known as ubiquitin thiolesterase), WARS (tryptophanyl-tRNA synthetase), GBP4 (guanylate-binding protein 4), and PRF1) are not yet known to have a role in this cell type. At the protein level, we confirmed elevated expression of ITGB5 and reduced expression of WARS, PRF1, and SERPINE2. Transfection of ClC-7 harboring the G215R mutation into osteoclasts resulted in an increased ITGB5 and reduced PRF1 expression of borderline significance. Finally, we observed that the ADO II patients presented a normal or increased serum level of bone formation markers, demonstrating a coupling between dysfunctional osteoclasts and osteoblasts. Sphingosine kinase 1 mRNA was expressed at the same level in ADO II and control osteoclasts. In conclusion, these data suggest that in addition to an acidification dysfunction caused by the CLCN7 mutation, a change in ITGB5, PRF1, WARS, and SERPINE2 expression could be part of the osteoclastic phenotype of ADO II.

An activating Fgfr3 mutation affects trabecular bone formation via a paracrine mechanism during growth.

The fibroblast growth factor receptor 3 (FGFR3) plays a critical role in the regulation of endochondral ossification. Fgfr3 gain-of-function mutations cause achondroplasia, the most common form of dwarfism, and a spectrum of chondrodysplasias. Despite a significant number of studies on the role of FGFR3 in cartilage, to date, none has investigated the influence of Fgfr3-mediated effects of the growth plate on bone formation. We studied three mouse models, each expressing Fgfr3 mutation either ubiquitously (CMV-Fgfr3(Y367C/+)), in chondrocytes (Col II-Fgfr3(Y367C/+)) or in mature osteoblasts (Col I-Fgfr3(Y367C/+)). Interestingly, we demonstrated that dwarfism with a significant defect in bone formation during growth was only observed in mouse models expressing mutant Fgfr3 in the cartilage. We observed a dramatic reduction in cartilage matrix mineralization and a strong defect of primary spongiosa. Anomalies of primary spongiosa were associated with an increase in osteoclast recruitment and a defect of osteoblasts at the mineralization front. A significant decrease in bone volume, trabecular thickness and number was also observed in the trabecular bone. Interestingly, no anomalies in proliferation and differentiation of primary osteoblasts from CMV-Fgfr3(Y367C/+) mice were observed. Based on these data, we excluded a potential function of Fgfr3 directly on osteoblasts at 3 weeks of age and we obtained evidence that the disorganization of the growth plate is responsible for the anomalies of the trabecular bone during bone formation. Herein, we propose that impaired FGFR3 signaling pathways may affect trabecular bone formation via a paracrine mechanism during growth. These results redefine our understanding of endochondral ossification in FGFR3-related chondrodysplasias.

Camurati-Engelmann disease with obesity in a newly identified family carrying a missense p.Arg156Cys mutation in the TGFB1 gene.

We report on a family affected by Camurati-Engelmann disease, characterized by radiological signs limited to the tibia, and associated with overweight or obesity, which is not a known feature of this disorder. The affected patients were heterozygous for a c.466C > T mutation (which predicts p.Arg156Cys) in the latency associated protein (LAP)-coding domain of the TGFB1 gene. This mutation had previously been reported once in another family with a similar, atypical phenotype, which suggests a possible phenotype/genotype relationship.

Does hormone replacement therapy prevent lateral rotatory spondylolisthesis in postmenopausal women?

PURPOSE: Degenerative scoliosis usually begins at menopause and lateral rotatory olisthesis (LRO) might be a triggering factor in the onset of degenerative scoliosis in postmenopausal women. We set out to evaluate the influence of hormone replacement therapy (HRT) on degenerative scoliosis and on LRO. METHODS: A cross-sectional study was conducted in 146 postmenopausal women: 75 women had received HRT for more than 1 year (HRT > 1) and 71 women had never received HRT or less than 1 year (HRT < 1). Scoliotic curve, LRO, sacral slope, lordosis, kyphosis were measured. The excess risk of LRO associated with age, BMI, isometric strength of brachial biceps, bone mineral density, lean mass and HRT was evaluated using a multiple logistic regression model. RESULTS: No difference was found in sacral slope, lumbar lordosis or thoracic kyphosis between both groups or in the presence of scoliosis. The prevalence of LRO was significantly lower in HRT >1 than HRT <1 (8 vs. 30%) while the risk was dependent on age, HRT and their interaction. LRO increased with age only in HRT <1 (11% when aged ≤66 years vs. 39% when aged >66 years, p = 0.013), whereas the prevalence of LRO remained stable in HRT >1. CONCLUSIONS: LRO was significantly lower in women who received HRT. The excess risk of LRO was dependent on both age and HRT status. These findings suggest that HRT might prevent the onset of LRO, and therefore might contribute to the prevention of low back pain.

Assessment of dynamic humeral centering in shoulder pain with impingement syndrome: a randomised clinical trial.

OBJECTIVES: Treatment for degenerative rotator cuff disease of the shoulder includes physiotherapy. Dynamic humeral centering (DHC) aims at preventing subacromial impingement, which contributes to the disease. The goal of this study was to assess the effectiveness of DHC. METHOD: 69 patients with shoulder pain and impingement syndrome were prospectively included in a single-centre randomised trial with a 12-month follow-up. Patients and assessor were blinded to the study hypothesis and treatment, respectively. DHC and non-specific mobilisation as control were performed for 6 weeks, in 15 supervised individual outpatient sessions, and patients performed daily home exercises. The planned primary outcome was the Constant score including subscores for pain, activity, mobility and strength at 3 months. Secondary outcomes were the Constant score and subscores at 12 months, and medication use for pain at 3 and 12 months. RESULTS: The DHC group did not differ from the control group in the total Constant score at 3 months. However, the DHC group showed a higher Constant subscore for pain (12.2 (SD 2.8) vs 9.9 (2.9), least square means difference 2.1, 95% CI 0.7 to 3.5, p=0.004). At 3 months, the DHC group also showed a higher rate of no medication use (96.7% vs 71%, proportional difference 25.7, 95% CI 3.7 to 51.9, p=0.012). There was no other intergroup difference. CONCLUSIONS: There was no difference in the total Constant score between DHC and controls. However, pain was improved at 3 months after DHC. The differences found in subscores for pain should be explored in future studies. Trial registration clinicaltrials.gov Identifier: NCT 01022775.

Effects of risedronate in Runx2 overexpressing mice, an animal model for evaluation of treatment effects on bone quality and fractures.

Young mice overexpressing Runx2 specifically in cells of the osteoblastic lineage failed to gain bone mass and exhibited a dramatic increase in bone resorption, leading to severe osteopenia and spontaneous vertebral fractures. The objective of the current study was to determine whether treatment with a bisphosphonate (risedronate, Ris), which reduces fractures in postmenopausal as well as in juvenile osteoporosis, was able to improve bone quality and reduce vertebral fractures in mice overexpressing Runx2. Four-week-old female Runx2 mice received Ris at 2 and 10 µg/kg subcutaneously twice a week for 12 weeks. Runx2 and wild-type mice received vehicle (Veh) as control. We measured the number of new fractures by X-ray and bone mineral density (BMD) by DEXA. We evaluated bone quality by histomorphometry, micro-CT, and Fourier transform infrared imaging (FTIRI). Ris at 20 µg/kg weekly significantly reduced the average number of new vertebral fractures compared to controls. This was accompanied by significantly increased BMD, increased trabecular bone volume, and reduced bone remodeling (seen in indices of bone resorption and formation) in the vertebrae and femoral metaphysis compared to Runx2 Veh. At the femur, Ris also increased cortical thickness. Changes in collagen cross-linking seen on FTIRI confirmed that Runx2 mice have accelerated bone turnover and showed that Ris affects the collagen cross-link ratio at both forming and resorbing sites. In conclusion, young mice overexpressing Runx2 have high bone turnover-induced osteopenia and spontaneous fractures. Ris at 20 µg/kg weekly induced an increase in bone mass, changes in bone microarchitecture, and decreased vertebral fractures.

Compromised Volumetric Bone Density and Microarchitecture in Men With Congenital Hypogonadotropic Hypogonadism.

CONTEXT: Men with congenital hypogonadotropic hypogonadism (CHH) and Kallmann syndrome (KS) have both low circulating testosterone and estradiol levels. Whether bone structure is affected remains unknown. OBJECTIVE: To characterize bone geometry, volumetric density and microarchitecture in CHH/KS. METHODS: This cross-sectional study, conducted at a single French tertiary academic medical center, included 51 genotyped CHH/KS patients and 40 healthy volunteers. Among CHH/KS men, 98% had received testosterone and/or combined gonadotropins. High-resolution peripheral quantitative computed tomography (HR-pQCT), dual-energy x-ray absorptiometry (DXA), and measurement of serum bone markers were used to determine volumetric bone mineral density (vBMD) and cortical and trabecular microarchitecture. RESULTS: CHH and controls did not differ for age, body mass index, and levels of vitamin D and PTH. Despite long-term hormonal treatment (10.8 ± 6.8 years), DXA showed lower areal bone mineral density (aBMD) in CHH/KS at lumbar spine, total hip, femoral neck, and distal radius. Consistent with persistently higher serum bone markers, HR-pQCT revealed lower cortical and trabecular vBMD as well as cortical thickness at the tibia and the radius. CHH/KS men had altered trabecular microarchitecture with a predominant decrease of trabecular thickness. Moreover, CHH/KS men exhibited lower cortical bone area, whereas total and trabecular areas were higher only at the tibia. Earlier treatment onset (before age 19 years) conferred a significant advantage for trabecular bone volume/tissue volume and trabecular vBMD at the tibia. CONCLUSION: Both vBMD and bone microarchitecture remain impaired in CHH/KS men despite long-term hormonal treatment. Treatment initiation during adolescence is associated with enhanced trabecular outcomes, highlighting the importance of early diagnosis.

Bone loss induced by Runx2 over-expression in mice is blunted by osteoblastic over-expression of TIMP-1.

The Runx2 gene is essential for osteoblast differentiation and function. In vivo over-expression of Runx2 in osteoblasts increases bone resorption, and blocks terminal osteoblast differentiation. Several lines of evidence suggest that osteoblastic matrix metalloproteinases (MMPs) could contribute to the increased bone resorption observed in mice over-expressing Runx2 (Runx2 mice). The goal of our study was to use a transgenic approach to find out whether the inhibition of osteoblastic MMPs can reduce the bone loss induced by the over-expression of Runx2. We analyzed the effect of the in vivo over-expression of the TIMP-1 in osteoblasts on the severe osteopenic phenotype in Runx2 mice. Females with the different genotypes (WT, Runx2, TIMP-1 and TIMP-1/Runx2) were analyzed for bone density, architecture, osteoblastic and osteoclastic activity and gene expression using qPCR. TIMP-1 over-expression reduces the bone loss in adult Runx2 mice. The prevention of the bone loss in TIMP-1/Runx2 mice was due to a combination of reduced bone resorption and sustained bone formation. We present evidence that the ability of osteoblastic cells to induce osteoclastic differentiation is lower in TIMP-1/Runx2 mice than in Runx2 mice, probably due to a reduction in the expression of RANK-L and of the Runx2 transgene. Osteoblast primary cells from TIMP-1/Runx2 mice, but not from Runx2 mice, were able to differentiate into fully mature osteoblasts producing high osteocalcin levels. In conclusion, our findings suggest that osteoblastic MMPs can affect osteoblast differentiation. Our work also indicates that osteoblastic MMPs are partly responsible for the bone loss observed in Runx2 transgenic mice.

Histamine promotes osteoclastogenesis through the differential expression of histamine receptors on osteoclasts and osteoblasts.

In addition to the numerous roles of histamine in both the immune and nervous systems, previous studies have suggested that this bioamine might also be involved in bone metabolism. Following our observations of impaired bone resorption in ovariectomized rats after histamine receptor antagonist treatment, we focused in this study on osteoclasts and osteoclast precursors. We looked for a direct action of histamine on these cells using both in vivo and in vitro approaches. In vivo, we triggered a remodeling sequence in rat mandibular bone and treated the animals with either histamine or histamine receptor antagonists. Histamine was shown to increase the number of osteoclasts and osteoclast precursors whereas antagonists of histamine receptor-1 and -2 decreased both osteoclast recruitment and resorption. In vitro, spleen cells from histamine-deficient mice were treated with receptor activator for nuclear factor kappa B ligand and macrophage colony stimulating factor, giving rise to both reduced numbers of osteoclasts and decreased resorption on dentin slices. Histamine enhanced resorption in these cultures in a dose-dependent manner. In addition, we identified osteoclast precursors as a source of histamine. In contrast, histamine increased the receptor activator for nuclear factor kappa B ligand/osteoprotegerin ratio in primary osteoblasts that did not secrete histamine. We observed a differential expression of histamine receptor-1 and -2 mRNAs in both primary osteoclasts and osteoblasts, confirming their functional roles with selective antagonists. Thus, histamine acts directly on osteoclasts, osteoclast precursors, and osteoblasts, promoting osteoclastogenesis through autocrine/paracrine mechanisms.

The effect of treatment based on a diuretic (indapamide) +/- ACE inhibitor (perindopril) on fractures in the Hypertension in the Very Elderly Trial (HYVET).

BACKGROUND: fractures may have serious implications in an elderly individual, and fracture prevention may include a careful choice of medications. DESIGN: the Hypertension in the Very Elderly Trial (HYVET) was a double-blind placebo-controlled trial of a thiazide-like diuretic (indapamide 1.5 mg SR) with the optional addition of the angiotensin-converting enzyme (ACE) inhibitor (perindopril 2-4 mg). Fracture was a secondary end point of the trial. SETTING: HYVET recruited participants from Eastern and Western Europe, China, Australasia, and Tunisia. SUBJECTS: all participants were > or =80 years of age and hypertensive. METHODS: participants were randomised to receive a thiazide-like diuretic (indapamide 1.5 mg SR) +/- ACE inhibitor (perindopril 2-4 mg) or matching placebos. Incident fractures were validated and analysed based on time to first fracture. RESULTS: there were 3,845 participants in HYVET and a total 102 reported fractures (42 in the active and 60 in the placebo group). When taking only validated first fractures, 90 were included in the analyses (38 in the active and 52 in the placebo group). Cox proportional hazard regression, adjusted for key baseline risk factors, resulted in a point estimate of 0.58 (95% CI 0.33-1.00, P = 0.0498). CONCLUSIONS: despite the lowering of blood pressure, treatment with a thiazide-like diuretic and an ACE inhibitor does not increase and may decrease fracture rate.

Increased bone resorption and osteopenia in Dlx5 heterozygous mice.

Distal-less (Dlx) homeobox transcription factors play a central role in the control of osteogenesis. In particular, Dlx5 regulates osteoblasts/osteoclasts coupling during perinatal bone formation. We analyze here the effect of Dlx5 allelic reduction in the control of bone remodeling. We first show that Dlx5 expression persists during postnatal bone development. We then compare the skeletal phenotype of 10- and 20-week-old Dlx5(+/-) mice to that of wild-type (WT) littermates. Dlx5(+/-) male mice exhibit lower bone mineral density (BMD) at both ages while only 20-week-old females are affected. microCT analyses reveal a reduction in cortical thickness of femoral midshafts in Dlx5(+/-) mice. Histomorphometry on distal femora shows no changes in trabecular structure and confirms a reduction in Dlx5(+/-) cortical thickness. The cortical decrease of 10-week-old mice does not derive from a reduction in periosteal bone apposition, but results from increased bone resorption with a significantly higher number of endosteal osteoclasts per bone surface and a larger marrow diameter. Urinary level of deoxypyridinoline is also higher in heterozygous mice confirming an increase in bone resorption activity. Our findings might be relevant for understanding complex, multifactorial diseases such as osteoporosis in which quantitative deregulation of gene expression leads to disruption of bone homeostasis.

Dlx5, a positive regulator of osteoblastogenesis, is essential for osteoblast-osteoclast coupling.

The homeodomain protein Dlx5 is an activator of Runx2 (a key regulator of osteogenesis) and is thought to be an important regulator of bone formation. At present, however, the perinatal lethality of Dlx5-null mice has hampered the elucidation of its function in osteogenesis. Here we provide the first analysis of the effects of Dlx5 inactivation on bone development. Femurs of Dlx5-null mouse embryos at the end of gestation exhibit a reduction in both total and trabecular bone volume associated with increased trabecular separation and reduced trabecular number. These parameters are often associated with pathological conditions characterized by reduced osteoblast activity and increased bone resorption. Dlx5(-/-) osteoblasts in culture display reduced proliferation and differentiation rate and reduction of Runx2, Osx, Osteocalcin and Bone Sialoprotein expression. In addition to impaired osteoblast function, Dlx5(-/-) femurs exhibit significant increases in osteoclast number. As Dlx5 is not expressed by osteoclasts, we suggest that its osteoblastic expression might control osteoblast/osteoclast coupling. Cultured Dlx5(-/-) osteoblasts displayed a higher RANKL/OPG ratio. Furthermore, Dlx5(-/-) osteoblasts induced a higher number of TRAP-positive multinucleated cells in normal spleen cultures with a globally increased resorption activity. These findings suggest that Dlx5 is a central regulator of bone turnover as it activates bone formation directly and bone resorption indirectly.

Sociodemographic and lifestyle risk factors for incident dementia and cognitive decline in the HYVET.

INTRODUCTION: previous studies have suggested that smoking, living alone and having a high body mass index may increase risk of developing dementia whereas a normal body mass index, having received education and moderate alcohol consumption may decrease risk. Dementia risk also increases with age and is thought to be higher in hypertensives. METHOD: we used data collected in the Hypertension in the Very Elderly Trial (HYVET), and cognitive function was assessed using the Mini-Mental State Examination (MMSE) at baseline and annually. Participants with a fall in MMSE to <24 or with a fall of 3 points in any 1 year were investigated further. The association of baseline sociodemographic, medical and lifestyle factors with incident dementia or decline in MMSE scores was assessed by regression models. RESULTS: incident dementia occurred in 263 of 3,336 participants over a mean follow-up of 2 years. In multivariate analyses, being underweight, BMI < 18.5 (HR 1.90, 95% CI 1.06-3.39) or obese, BMI >30 (HR 1.84, 95% CI 1.24-2.72), increased risk of incident dementia as did piracetam use (HR 2.72, 95% CI 1.60-4.63). Receiving formal education was associated with a reduced risk (HR 0.59, 95% CI 0.45-0.78). There was no association with smoking, alcohol and gender. Similar results were found when examining mean annual change in the MMSE score. DISCUSSION: our results for BMI and education agree with those from other studies. The increased risk associated with piracetam may reflect awareness of memory problems before any diagnosis of dementia has been made. Trial participants may be healthier than the general population and further studies in the general population are required.

Association of bone activity, calcium load, aortic stiffness, and calcifications in ESRD.

An inverse relationship between arterial calcifications and bone activity has been documented in patients with ESRD. Calcium overload is associated with arterial calcification, which is associated with arterial stiffening. Whether bone activity interacts with calcium load, aortic stiffness, or arterial calcification is unknown. This study assessed the impact of bone activity on the relationships between the dosage of calcium-containing phosphate binders and aortic stiffness (measured by pulse wave velocity) or abdominal aorta calcification score. Aortic stiffness and calcification were both positively associated with calcium load and negatively associated with bone activity. A significant interaction was found between dosage of calcium-containing phosphate binders and bone activity such that calcium load had a significantly greater influence on aortic calcifications and stiffening in the presence of adynamic bone disease. Independent of any other factor, including dosage of calcium-containing phosphate binders, adynamic bone was associated with greater aortic stiffening, suggesting cross-talk between the bone and arterial walls.

Role of the captured retroviral envelope syncytin-B gene in the fusion of osteoclast and giant cell precursors and in bone resorption, analyzed ex vivo and in vivo in syncytin-B knockout mice.

Syncytin-A and -B are envelope genes of retroviral origin that have been captured in evolution for a role in placentation. They trigger cell-cell fusion and were shown to be essential for the formation of the syncytiotrophoblast layer during mouse placenta formation. Syncytin-A and -B expression has been described in other tissues and their highly fusogenic properties suggested that they might be involved in the fusion of other cell types. Here, taking advantage of mice knocked out for syncytin-B, SynB-/- mice, we investigated the potential role of syncytin-B in the fusion of cells from the monocyte/macrophage lineage into multinucleated osteoclasts (OCs) -in bone- or multinucleated giant cells -in soft tissues. In ex vivo experiments, a significant reduction in fusion index and in the number of multinucleated OCs and giant cells was observed as soon as Day3 in SynB-/- as compared to wild-type cell cultures. Interestingly, the number of nuclei per multinucleated OC or giant cell remained unchanged. These results, together with the demonstration that syncytin-B expression is maximal in the first 2 days of OC differentiation, argue for syncytin-B playing a role in the fusion of OC and giant cell mononucleated precursors, at initial stages. Finally, ex vivo, the observed reduction in multinucleated OC number had no impact on the expression of OC differentiation markers, and a dentin resorption assay did not evidence any difference in the osteoclastic resorption activity, suggesting that syncytin-B is not required for OC activity. In vivo, syncytin-B was found to be expressed in the periosteum of embryos at embryonic day 16.5, where TRAP-positive cells were observed. Yet, in adults, no significant reduction in OC number or alteration in bone phenotype was observed in SynB-/- mice. In addition, SynB-/- mice did not show any change in the number of foreign body giant cells (FBGCs) that formed in response to implantation of foreign material, as compared to wild-type mice. Altogether the results suggest that in addition to its essential role in placenta formation, syncytin-B plays a role in OCs and macrophage fusion; yet it is not essential in vivo for OC and FBGC formation, or maintenance of bone homeostasis, at least under the conditions tested.

Sclerosing bone dysplasias with hallmarks of dysosteosclerosis in four patients carrying mutations in SLC29A3 and TCIRG1.

The osteopetroses and related sclerosing bone dysplasias can have a broad range of manifestations. Especially in the milder forms, sandwich vertebrae are an easily recognizable and reliable radiological hallmark. We report on four patients from three families presenting with sandwich vertebrae and platyspondyly. The long bone phenotypes were discordant with one patient showing modeling defects and patchy osteosclerosis, while the second displayed only metaphyseal sclerotic bands, and the third and fourth had extreme metaphyseal flaring with uniform osteosclerosis. Two of the four patients had experienced pathological fractures, two had developmental delay, but none showed cranial nerve damage, hepatosplenomegaly, or bone marrow failure. According to these clinical features the diagnoses ranged between intermediate autosomal recessive osteopetrosis and dysosteosclerosis. After exclusion of mutations in CLCN7 we performed gene panel and exome sequencing. Two novel mutations in SLC29A3 were found in the first two patients. In the third family a TCIRG1 C-terminal frameshift mutation in combination with a mutation at position +4 in intron 2 were detected. Our study adds two cases to the small group of individuals with SLC29A3 mutations diagnosed with dysosteosclerosis, and expands the phenotypic variability. The finding that intermediate autosomal recessive osteopetrosis due to TCIRG1 splice site mutations can also present with platyspondyly further increases the molecular heterogeneity of dysosteosclerosis-like sclerosing bone dysplasias.

Genome-wide linkage screen of bone mineral density (BMD) in European pedigrees ascertained through a male relative with low BMD values: evidence for quantitative trait loci on 17q21-23, 11q12-13, 13q12-14, and 22q11.

CONTEXT: Bone mass is under strong genetic control, with heritability estimates greater than 50% and is likely determined by complex interactions between genetic and environmental factors. OBJECTIVE: The objective of the study was to localize genes contributing to bone mineral density (BMD) variation. DESIGN: An autosomal genome-wide scan for BMD at the lumbar spine and femoral neck was conducted with variance components linkage methods. PARTICIPANTS: A total of 103 pedigrees (Network in Europe on Male Osteoporosis Family Study) ascertained through a male relative with low (Z-score < or = -2) BMD values at either lumbar spine or femoral neck. MAIN OUTCOME MEASURES: Nonparametric multipoint logarithm of the odds ratio scores for lumbar spine and femoral neck BMD values adjusted for age, gender, and body mass index. RESULTS: We identified a total of eight chromosomal regions with logarithm of the odds ratio score of 1.5 or greater (P < or = 5 x 10(-3)): on 1q42-43, 11q12-13, 12q23-24, 17q21-23, 21q22, and 22q11 for lumbar spine and on 5q31-33 and 13q12-14 for femoral neck BMD. CONCLUSIONS: Four of our detected quantitative trait loci (QTL) reached the genome-wide criteria for significant (17q,21-23, P < or = 2 x 10(-5)) or suggestive (11q12-13, 22q11, and 13q12-14, P < or = 7 x 10(-4)) linkage. Apart from 22q11, which is a novel QTL, all other loci provide consistent replication for previously reported QTLs for BMD and other bone-related traits. Finally, several of our specific-linkage areas encompass prominent candidate genes: type 1 collagen (COL1A1) and the sclerosteosis/van Buchem disease (SOST) genes on 17q21-23; the low-density lipoprotein receptor-related protein 5 (LRP5) gene on 11q12-13; and the rank ligand gene on 13q12-14. Further analysis of these positive regions by fine linkage disequilibrium mapping is thus warranted.