[Preimplantation genetic diagnosis of infantile malignant osteopetrosis in a Chinese family].

OBJECTIVE: To explore the application of preimplantation genetic diagnosis (PGD) for infantile malignant osteopetrosis (IMO). METHODS: For a family affected with IMO, PGD was provided using combined parental mutation detection and haplotype constructions with microsatellite markers spanning the TCIRG1 gene. Prenatal diagnosis was performed on the chorionic villus and amniocentesis samples by direct sequencing. RESULTS: Prenatal diagnosis showed that the fetus by the third pregnancy has carried the parental mutations [c.242delC (p.Pro81Argfs*85) and c.1114C>T (p.Gln372*)], and the pregnancy was terminated. PGD was subsequently performed through mutations detection and haplotype analyses following whole genome amplification (WGA) of each of 13 cells. The results showed that 6 of the 13 embryos were unaffected, 3 were carriers and 4 were affected. Well developed unaffected/carrier embryos were selected and transferred into the uterus. A single pregnancy was confirmed. Subsequently pre- and post-natal diagnoses have confirmed development of a healthy child. CONCLUSION: The study demonstrated the advantage of PGD over prenatal diagnosis when natural pregnancies have repeatedly produced IMO children/fetuses.

Osteopetrosis in obese female rats is site-specifically inhibited by physical training.

NEW FINDINGS: What is the central question of this study? Clinical studies suggest that obesity 'protects' against osteoporosis. However, these studies used only bone densitometry and assessed only one bone site, which is insufficient to enable conclusions to be drawn about the response of the whole skeleton. Furthermore, the effects of exercise on bone responses in obesity have not been explored previously. What is the main finding and what is its importance? We show that obesity causes osteopetrosis. Therefore, the classical perspective of 'protective effects of obesity' needs to be reviewed, and exercise is an important tool to avoid these alterations and to maintain the homeostasis of bone. A sedentary lifestyle and obesity induce systemic inflammatory responses. Although the effects of physical inactivity on osseous tissue have been well established, the effects of obesity on bone tissue remain controversial. Furthermore, the effects of physical training on bone tissue responses in the presence of diet-induced obesity are unknown. Our aim was to investigate the effects of obesity and physical training at multiple bone sites in rats. Female Wistar rats were divided into the following four groups: (i) control diet, non-trained (C-NT); (ii) high-refined carbohydrate-containing diet, non-trained (HC-NT); (iii) control diet, trained (C-T); and (iv) high-refined carbohydrate-containing diet, trained (HC-T). At 5 months of age, the rats were submitted to daily exercise for 30 min day(-1). After 13 weeks, blood samples, adipose and skeletal tissues were harvested. Two-way ANOVA was applied to detect differences (significance accepted when P ≤ 0.05). The HC-NT group exhibited increased body mass, adiposity, serum leptin, serum insulin, insulin resistance index and concentrations of tumour necrosis factor-α and interleukin-6. Obese rats (HC-NT) exhibited thickening of nasal bones, trabecular bones in the lumbar vertebrae and long bones in a site-dependent manner. The HC-T group exhibited similar adiposity and inflammatory results. Morphological analysis of the lumbar vertebrae in rats fed the HC diet revealed characteristics of osteopetrosis that were inhibited by exercise. In conclusion, the HC diet induced obesity and inflammatory/hormonal alterations and increased the trabecular bone in a site-dependent manner. However, obesity caused osteopetrosis in the lumbar vertebrae, which could be inhibited by physical training. Although exercise inhibited the development of bone alterations, physical training did not inhibit the HC diet-induced obesity responses.

Genetic predispositions and the short- and long-term effects of hormonal therapy on bone mineral density in girls with functional hypothalamic amenorrhoea.

INTRODUCTION: The aim of this study was to verify if genetic factors influence the short- and long-term therapeutic responses to oestroprogestagen (OP) therapy, implemented in girls with functional hypothalamic amenorrhoea (FHA) in order to improve their bone mineral density (BMD). MATERIAL AND METHODS: The study included 78 FHA girls who underwent a four-year sequential OP therapy with 17-beta oestradiol and didrogesterone. Changes in the lumbar spine BMD were determined at the end of the therapy and six years after its discontinuation, and analysed in regards to PvuII and XbaI polymorphisms of oestrogen receptor-alpha gene, BsmI polymorphism of vitamin D3 receptor gene, and Sp1 polymorphism of the type-1 collagen gene. RESULTS: After four years of OP therapy, a significant increase in BMD was documented in the studied group. Follow-up densitometry performed six years after completing the therapy revealed a significant decrease in BMD level; nonetheless, the values of this parameter were still significantly higher compared to pretreatment level. Neither the particular polymorphisms nor their combinations influenced the relative change in BMD at the end of the therapy and after a six-year follow-up. CONCLUSIONS: Variability of genes involved in oestrogen, vitamin D3 and collagen metabolism does not influence the short- and long-term results of OP therapy in girls with FHA.

Polymorphism of the vitamin D3 receptor gene and bone mineral density in girls with functional hypothalamic amenorrhea subjected to oestroprogestagen treatment.

BACKGROUND: We investigated whether the vitamin D3 receptor gene (VDR) polymorphism can modulate therapeutic response of functional hypothalamic amenorrhea (FHA) patients to the oestroprogestagen (EP) treatment. MATERIAL AND METHODS: The study included 84 FHA girls and 50 controls. FHA patients underwent a four-year sequential EP therapy with 17-ß oestradiol (2 mg from the 2(nd) to 25(th) day of the menstrual cycle) and didrogesterone (10 mg from the 16(th) to the 25(th) day). Their hormonal parameters were monitored along with bone turnover marker levels and bone mineral density (BMD). Additionally, the VDR gene BsmI polymorphism was determined. RESULTS: Hormonal therapy was reflected by a substantial improvement of BMD. However, the values of BMD observed after four years of treatment in FHA patients were still significantly lower than baseline bone mineral density determined in the control group (1.007 ± 0.100 vs. 1.141 ± 0.093 g/cm(2), respectively; p < 0.001). No significant effects of the VDR genotype were observed on the dynamics of BMD during consecutive years of hormonal treatment and mean bone mineral density determined after completing the therapy (1.006 ± 0.101 vs. 1.013 ± 0.114 vs. 1.006 ± 0.094 g/cm(2) for BB, bb and Bb genotypes, respectively; p = 0.973). CONCLUSIONS: This study did not confirm that VDR polymorphism can modulate therapeutic outcome of FHA girls subjected to the hormonal treatment. Nonetheless, this study confirmed the effectiveness of EP therapy in the simultaneous treatment of menstrual disorders and the normalisation of bone mineral density in FHA patients.

Dissociation of bone resorption and bone formation in adult mice with a non-functional V-ATPase in osteoclasts leads to increased bone strength.

Osteopetrosis caused by defective acid secretion by the osteoclast, is characterized by defective bone resorption, increased osteoclast numbers, while bone formation is normal or increased. In contrast the bones are of poor quality, despite this uncoupling of formation from resorption.To shed light on the effect of uncoupling in adult mice with respect to bone strength, we transplanted irradiated three-month old normal mice with hematopoietic stem cells from control or oc/oc mice, which have defective acid secretion, and followed them for 12 to 28 weeks.Engraftment levels were assessed by flow cytometry of peripheral blood. Serum samples were collected every six weeks for measurement of bone turnover markers. At termination bones were collected for µCT and mechanical testing. An engraftment level of 98% was obtained. From week 6 until termination bone resorption was significantly reduced, while the osteoclast number was increased when comparing oc/oc to controls. Bone formation was elevated at week 6, normalized at week 12, and reduced onwards. µCT and mechanical analyses of femurs and vertebrae showed increased bone volume and bone strength of cortical and trabecular bone.In conclusion, these data show that attenuation of acid secretion in adult mice leads to uncoupling and improves bone strength.

Effect of the anti-diabetic drug metformin on bone mass in ovariectomized rats.

Recent studies have reported bone loss in the patients with diabetes and a direct osteogenic effect of metformin on osteoblast-like cells in culture. In this study, we investigated the action of metformin on bone mass in ovariectomized (OVX) rats. Three months after either a sham surgery or bilateral ovariectomy, thirty-two female Sprague-Dawley rats were randomly assigned into four groups: (1) Sham group; (2) OVX group; (3) OVX+metformin (50mg/kg/day) group; and (4) OVX+metformin (100mg/kg/day) group. After 2 months of oral administration with or without metformin, tibiae were harvested for dual energy X-ray absorptiometry, micro-computed tomography (micro-CT) and histology analysis, while the bone marrow cells from tibiae were collected for measurement of the mRNAs expressions for three osteoblast genes and estrogen receptors alpha by the use of real-time RT-PCR. We found that the impaired bone density and quality induced by bilateral ovariectomy were significantly improved by the treatment of metformin (both 50 and 100mg/kg/day), and this action could be partly mediated by regulating bone marrow cells development through induction of mechanisms regulating osteoblast markers core binding factor a1 and LDL receptor-related protein 5. These findings provide new evidence that the anti-diabetic drug metformin has a direct inhibition effect on bone loss in OVX rats, in addition to its well-documented osteogenic potency in vitro.

Lymphocytes and the Dap12 adaptor are key regulators of osteoclast activation associated with gonadal failure.

Bone resorption by osteoclasts is necessary to maintain bone homeostasis. Osteoclast differentiation from hematopoietic progenitors and their activation depend on M-CSF and RANKL, but also requires co-stimulatory signals acting through receptors associated with DAP12 and FcRgamma adaptors. Dap12 mutant mice (KDelta75) are osteopetrotic due to inactive osteoclasts but, surprisingly, these mice are more sensitive than WT mice to bone loss following an ovariectomy. Because estrogen withdrawal is known to disturb bone mass, at least in part, through lymphocyte interaction, we looked at the role of mature lymphocytes on osteoclastogenesis and bone mass in the absence of functional DAP12. Lymphocytes were found to stimulate an early osteoclast differentiation response from Dap12-deficient progenitors in vitro. In vivo, Rag1-/- mice lacking mature lymphocytes did not exhibit any bone phenotype, but lost their bone mass after ovariectomy like KDelta75 mice. KDelta75;Rag1-/- double mutant female mice exhibited a more severe osteopetrosis than Dap12-deficient animals but lost their bone mass after ovariectomy, like single mutants. These results suggest that both DAP12 and mature lymphocytes act synergistically to maintain bone mass under physiological conditions, while playing similar but not synergistic co-stimulatory roles in protecting bone loss after gonadal failure. Thus, our data support a role for lymphocytes during osteoclast differentiation and suggest that they may function as accessory cells when regular osteoclast function is compromised.

Assessment of osteoclast number and function: application in the development of new and improved treatment modalities for bone diseases.

Numerous experimental and clinical observations suggest that overall changes in bone resorption during menopause or treatment with hormone replacement therapy (HRT) are combined effects of changes in osteoclast number and function. Moreover, due to a coupling between osteoclastic bone resorption and osteoblastic bone formation, pronounced alteration of osteoclast number will eventually lead to alteration of osteoblastic bone formation. Fragments of type I collagen, such as the C- and N-terminal telopeptides of collagen type I (CTX and NTX, respectively), are generated during bone resorption and hence can be used as surrogate markers of osteoclast function. Circulating levels of different enzymes in the serum, such as TRAP 5b and cathepsin K are proportional to the number of osteoclasts, and hence can be used as surrogate markers of osteoclast number. Since antiresorptive effects can be obtained in different ways, we felt it was timely to discuss the different scenarios, highlight differences specific to different pharmacological interventions with different mechanisms of action, and discuss how these bone markers can assist us in a deeper analysis of the pharmacodynamics and safety profile of existing and upcoming drug candidates.

Delayed hematopoietic development in osteopetrotic (op/op) mice.

Changes in structure, cellularity, hematopoietic progenitor cell and macrophage content, and osteoclast activity were investigated in the hematopoietic organs of the colony-stimulating factor 1(CSF-1)-less osteopetrotic (op/op) mouse. The data indicated that op/op mice undergo an age-related hematopoietic recovery and resolution of osteopetrosis, suggesting that the hematopoietic system has the capacity to use alternative mechanisms to compensate for the absence of an important multifunctional growth factor, CSF-1. In young animals, op/op femurs were heavily infiltrated with bone, and marrow cellularity was significantly reduced. After 6 wk of age, there was an increase in the marrow space available for hematopoiesis. The femoral cavity of op/op mice progressively enlarged, and by 22 wk of age its appearance and marrow cellularity was comparable to that of controls. The percentage of op/op mononuclear phagocytes, defined by F4/80 antigen expression, progressively increased to normal levels by 35 wk of age. There was no difference in the incidence of both primitive and mononuclear phagocyte-committed, CSF-1-responsive progenitor cells in op/op marrow, but their femoral content was significantly reduced in young mice. During the period of reduced hematopoiesis in the marrow of young op/op mice, splenic hematopoietic activity was elevated. This mutant mouse represents a system for the study of the CSF-1-independent regulatory mechanisms involved in hematopoietic regulation.

Pathogenesis of virus-induced osteopetrosis in the chicken.

Infection of chicken embryos with a nondefective avian leukosis virus MAV-2(0) induced bone hyperplasia and profound suppression of bursal and thymic lymphoid cell development within 2- to 3 weeks after hatching. A time-course study of infectivity during embryonal life showed a gradual loss of susceptibility between the 10th day of incubation and hatching. This finding suggests that an embryonal stem cell either before or early after seeding into the bone and primary lymphoid organs is a virus-susceptible target cell. Histologic observations of the bursa indicated only mild damage during the 1st week and severely impaired proliferation of follicular lymphoid cells during subsequent weeks of development. Thus, viral infection conferred a cytostatic rather than a cytolytic effect upon the lymphoid target cell. Infection of stem cells, which are progenitors of lymphoid cells as well as of bone osteoclasts, is compatible with the manifestations of the disease. A model of cellular disorders of the bone and lymphoid tissues is proposed. Adoptive transfer of lymphoid cells from adult MAV-2(0)-immunized chickens into histocompatible virus-infected juvenile recipients abrogated the manifestation of osteopetrosis. Passive injection of antiserum containing virus-neutralizing antibodies was also effective. Anti-gp85 viral surface glycoprotein-binding antibodies in sera from adoptively protected chickens receiving immune cells were exclusively of cell donor IgG allotype.