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Adipose tissue is one of the major sites for fatty acid synthesis and lipid storage. We generated adipose (fat)-specific ACC1 knockout (FACC1KO) mice using the aP2-Cre/loxP system. FACC1KO mice showed prenatal growth retardation; after weaning, however, their weight gain was comparable to that of wild-type (WT) mice on a normal diet. Under lipogenic conditions of fasting/re-feeding a fat-free diet, lipid accumulation in adipose tissues of FACC1KO mice was significantly decreased; this is consistent with a 50-66% reduction in the ACC activity in these tissues compared with that of WT mice. Surprisingly, FACC1KO mice manifested skeletal growth retardation phenotype accompanied by decreased chondrocyte proliferation in the growth plate and lower trabecular bone density. In addition, there was about a 30% decrease in serum insulin-like growth factor I (IGF1), and while the serum leptin level was decreased by about 50%, it did not counteract the osteopenic effects of IGF1 on the bone. Fatty acid analyses of mutant bone lipids revealed relatively higher levels of C18:2n-6 and C18:3n-3 and lower levels of their elongation C20 homologs than that of WT cohorts, leading to lower levels of C20 homologs and bone development. Moreover, aP2-Cre-mediated ACC1 inactivation in bone tissue led to a decreased number of osteoblasts but not of osteoclasts. The downregulation of ACC1 on osteoblastogenesis may be the cause for the osteopenia phenotype of FACC1KO bone homeostasis.

Urocortin-1 (UCN) a corticotropin releasing-factor (CRF) related peptide, has been found to be expressed in many different tissues like the central nervous system, the cardiovascular system, adipose tissue, and skeletal muscle. The effects of UCN are mediated via stimulation of CRF-receptors 1 and 2 (CRFR1 and 2, CRFR's) with a high affinity for CRFR2. It has been shown that the CRF-related peptides and CRFR's are involved in the regulation of stress-related endocrine, autonomic and behavioural responses. Using immunocytochemistry, immunohistochemistry and RT-PCR, we now can show the differentiation dependent expression of UCN mRNA and peptide in human mesenchymal progenitor cells (MSCs) directed to the osteoblastic phenotype for the first time. UCN expression was down regulated by TGF-beta and BMP-2 in the early proliferation phase of osteoblast development, whereas dexamethasone (dex) minimally induced UCN gene expression during matrix maturation after 24 h stimulation. Stimulation of MSCs for 28 days with ascorbate/beta-glycerophosphate (asc/bGp) induced UCN gene expression at day 14. This effect was prevented when using 1,25-vitamin D3 or dex in addition. There was no obvious correlation to osteocalcin (OCN) gene expression in these experiments. In MSCs from patients with metabolic bone disease (n = 9) UCN gene expression was significantly higher compared to MSCs from normal controls (n = 6). Human MSCs did not express any of the CRFR's during differentiation to osteoblasts. Our results indicate that UCN is produced during the development of MSCs to osteoblasts and differentially regulated during culture as well as by differentiation factors. The expression is maximal between proliferation and matrix maturation phase. However, UCN does not seem to act on the osteoblast itself as shown by the missing CRFR's. Our results suggest new perspectives on the role of urocortin in human skeletal tissue in health and disease.

Hereditary forms of renal phosphate wasting have been studied thoroughly in the past years. X-linked Hypophosphatemic rickets (XLH), autosomal dominant hypophosphatemic rickets/osteomalacia (ADHR) and autosomal recessive hypophosphatemic rickets (ARHR) are known genetic disorders in which a disturbance of phosphatonins is a causative factor in the pathogenesis. We describe a comparable but yet undescribed disorder in a family in which a 53 year old man presented with a spontaneous fracture after suffering for years with severe fatigue and musculoskeletal pains. A low serum phosphate was discovered. The two subsequent generations of this family developed the same symptoms but at an earlier age. Almost all family members have been investigated and the majority appears to have low bone density and/or renal phosphate wasting and/or low serum phosphate. Remarkably no rickets was found. No elevation of FGF23 or mutations in the gene encoding FGF23 were found. We believe this is a new familial disorder of bone metabolism and phosphate homeostasis in which a disturbance of bone modulators may play a central role., (2009 European Federation of Internal Medicine.)

Aim: Secondary osteoporosis with subsequent fractures are well-known symptoms in ankylosing spondylitis. As a possible genetic component of osteoporosis, an association between bone mineral density (BMD) and polymorphisms of the vitamin D receptor (VDR) gene has been reported., Method: In the present study, we investigated relationships between these polymorphisms, BMD, biochemical markers of bone metabolism and markers of disease activity in ankylosing spondylitis., Results: AS patients showed osteoporosis more frequently than healthy controls. A mapping of activity indices and BMD showed an association of lumbar and peripheral BMD as well as of the current and median C-reactive protein over the last 3 years with the used FokI-genotypes of the VDR in male patients with AS. Female AS patients showed no significant association., Conclusion: The pathophysiological mechanisms of this association are unclear, especially with respect to the inflammatory activity. An early assessment of the risk for osteopenia/osteoporosis in AS patients using molecular biological tests could make possible timely preventive measures or therapy.

Cbl proteins are multifunctional adaptor molecules that modulate cellular activity by targeting the ubiquitylating system, endocytic complexes, and other effectors to a wide variety of regulatory proteins, especially activated receptor and nonreceptor tyrosine kinases. Cbl and Cbl-b perform unique functions in various cells, in addition to redundant functions that are required for embryonic development. We previously showed that eliminating Cbl impaired osteoclast motility, which modestly delayed embryonic bone development. We now report that Cbl-b(-/-) mice are osteopenic, because of increased bone resorption with little compensating increase in bone formation. In vitro bone-resorbing activity and differentiation of osteoclast-like cells (OCLs) were increased, as were some RANKL-induced signaling events (activation of NF-kappaB and the mitogen-activated protein kinases extracellular signal-regulated kinase [ERK] and p38), suggesting that specific RANKL-activated mechanisms contribute to the increased rate of differentiation and bone-resorbing activity. Re-expressing Cbl-b in Cbl-b(-/-) OCLs normalized the increased bone-resorbing activity and overexpressing Cbl-b in wildtype OCLs inhibited bone resorption. Cbl was without effect in either wildtype or Cbl-b(-/-) OCLs. Functional tyrosine kinase binding (TKB) and RING finger domains were required for the rescue by Cbl-b. Thus, both Cbl and Cbl-b perform regulatory functions in osteoclasts that are unique to one or the other protein (i.e., functions that cannot be compensated by the other homolog). One of Cbl-b's unique functions in osteoclasts is to downregulate bone resorption.

CCAAT/enhancer binding proteins (C/EBPs) are expressed by osteoblasts and adipocytes during differentiation. C/EBP beta is critical for adipogenesis; however, its role in osteoblastogenesis is unclear, and its function in the postnatal skeleton is not known. To study C/EBP beta in osteoblasts in vivo, we created transgenic mice expressing full length C/EBP beta under the control of a 3.8 kb fragment of the human osteocalcin promoter. Two transgenic lines were established in a friend leukemia virus strain B genetic background, and compared with wild type littermate controls. Both C/EBP beta transgenic lines exhibited osteopenia, with a 30% decrease in bone volume, due to a decrease in trabecular number. The number of osteoblasts and osteoclasts per bone perimeter was not changed. Bone marrow stromal cells from C/EBP beta transgenics showed reduced mineralization, and reduced alkaline phosphatase mRNA levels. Calvarial osteoblasts from C/EBP beta transgenics displayed reduced alkaline phosphatase activity. To determine the consequences of the Cebpb deletion in vivo, the phenotype of Cebpb null mice was compared with that of wild type controls of identical genetic composition. Cebpb null mice exhibited reduced weight, body fat, and bone mineral density, and decreased bone volume, due to a decrease in trabecular number. The number of osteoblasts and osteoclasts per bone perimeter was not changed. C/EBP beta downregulation by RNA interference in calvarial osteoblasts had no effect on osteoblast differentiation/function. The phenotype of the Cebpb inactivation may be secondary to systemic indirect effects, and to direct effects of C/EBP beta in osteoblasts. In conclusion, C/EBP beta plays a role in mesenchymal cell differentiation and its misexpression in vivo causes osteopenia.

A deficit in bone formation is a major factor in diabetes-related osteopenia. We examined here whether diabetes-associated changes in osteoblast phenotype might in part result from a decrease in PTH-related protein (PTHrP). We used a bone marrow ablation model in diabetic mice by multiple streptozotocin injections. PTHrP (1-36) (100 microg/kg, every other day) or vehicle was administered to mice for 13 d starting 1 wk before marrow ablation. Diabetic mice showed bone loss in both the intact femur and the regenerating tibia on d 6 after ablation; in the latter, this was related to decreased bone-forming cells, osteoid surface, and blood vessels, and increased marrow adiposity. Moreover, a decrease in matrix mineralization occurred in ex vivo bone marrow cultures from the unablated tibia from diabetic mice. These skeletal alterations were associated with decreased gene expression (by real-time PCR) of Runx2, osterix, osteocalcin, PTHrP, the PTH type 1 receptor, vascular endothelial growth factor and its receptors, and osteoprotegerin to receptor activator of nuclear factor-kappaB ligand mRNA ratio, and increased peroxisome proliferator-activated receptor-gamma2 mRNA levels. Similar changes were induced by hyperosmotic (high glucose or mannitol) medium in osteoblastic MC3T3-E1 cells, which were mimicked by adding a neutralizing anti-PTHrP antibody or PTH type 1 receptor antagonists to these cells in normal glucose medium. PTHrP (1-36) administration reversed these changes in both intact and regenerating bones from diabetic mice in vivo, and in MC3T3-E1 cells exposed to high glucose. These findings strongly suggest that PTHrP has an important role in the altered osteoblastic function related to diabetes.

Summary: In 87 patients with hereditary hemochromatosis, osteoporosis was detected in 25%, and osteopenia in 41%. Bone mineral density was independently associated with BMI, ALP levels, hypogonadism/menopause, and the amount of iron removed to reach depletion, but not with cirrhosis. Osteoporosis is influenced by iron overload in hemochromatosis., Introduction: To analyze prevalence, clinical characteristics and genetic background associated with osteoporosis in a retrospective study in Italian patients with hereditary hemochromatosis (HHC)., Methods: In 87 consecutive patients with HHC, bone mineral density was systematically evaluated by dual energy x-ray absorptiometry of the lumbar spine (n = 87) and femoral neck (n = 66)., Results: Osteoporosis was detected in 22 (25.3%), and osteopenia in 36 (41.4%) patients. Mean Z scores were -0.92 +/- 1.42 at lumbar spine and -0.35 +/- 1.41 at femoral neck. Lumbar spine T-score was independently associated with total ALP (p = 0.002), hypogonadism/menopause (p = 0.026), and iron overload (p = 0.033 for ferritin and p = 0.017 for iron removed). We observed a borderline significance for BMI (p = 0.069) and smoking status (p = 0.086). Lumbar spine osteoporosis was independently associated with lower BMI (OR 0.73, 95% CI 0.54-0.94), total ALP (OR 1.17, 95% CI 1-1.39 per 10 unit increase) and the amount of iron removed (OR 1.53, 95% CI 1-2.5 per 5 g increase). HFE genotypes did not differ between patients with and without osteoporosis., Conclusions: Osteoporosis is observed in a quarter of unselected patients with HHC, independently of the genetic background, and is associated with ALP, hypogonadism, body weight, and severity of iron overload.

Mutations in GBA1 gene that encodes lysosomal glucocerebrosidase result in Type 1 Gaucher Disease (GD), the commonest lysosomal storage disorder; the most prevalent disease mutation is N370S. We investigated the heterogeneity and natural course of N370S GD in 403 patients. Demographic, clinical, and genetic characteristics of GD at presentation were examined in a cross-sectional study. In addition, the relative risk (RR) of cancer in patients compared with age-, sex-, and ethnic-group adjusted national rates of cancer was determined. Of the 403 patients, 54% of patients were homozygous (N370S/N370S) and 46% were compound heterozygous for the N370S mutation (N370S/other). The majority of N370S/N370S patients displayed a phenotype characterized by late onset, predominantly skeletal disease, whereas the majority of N370S/other patients displayed early onset, predominantly visceral/hematologic disease, P < 0.0001. There was a striking increase in lifetime risk of multiple myeloma in the entire cohort (RR 25, 95% CI 9.17-54.40), mostly confined to N370S homozygous patients. The risk of other hematologic malignancies (RR 3.45, 95% CI 1.49-6.79), and overall cancer risk (RR 1.80, 95% CI 1.32-2.40) was increased. Homozygous N370S GD leads to adult-onset progressive skeletal disease with relative sparing of the viscera, a strikingly high risk of multiple myeloma, and an increased risk of other cancers. High incidence of gammopathy suggests an important role of the adaptive immune system in the development of GD. Adult patients with GD should be monitored for skeletal disease and cancers including multiple myeloma., (Copyright 2009 Wiley-Liss, Inc.)

Although induced mutations in traditional laboratory animals have been valuable as models for human diseases, they have some important limitations. Here, we propose a complementary approach to discover genes and mechanisms that might contribute to human disorders: the analysis of evolutionary mutant models in which adaptive phenotypes mimic maladaptive human diseases. If the type and mode of action of mutations favored by natural selection in wild populations are similar to those that contribute to human diseases, then studies in evolutionary mutant models have the potential to identify novel genetic factors and gene-by-environment interactions that affect human health and underlie human disease.

Thalassemias represent the most common single-gene disorder causing a major public health problem in India. Thalassemia and hemoglobinopathies probably developed over 7000 years ago as a defense against malaria. In simple terms, thalassemia is caused by a mutation in either the â-globin chain or the á-globin chain which combine equally in red cells to form hemoglobin. These mutations lead to varying degree of anemia resulting into thalassemia minor, intermedia or major. Present write up relates to advances in the management of â-thalassemia major.

Bone loss occurs as early as the third decade and its cumulative effect throughout adulthood may impact risk for osteoporosis in later life, however, the genes and environmental factors influencing early bone loss are largely unknown. We investigated the role of genes in the change in bone mineral density (BMD) in participants in the San Antonio Family Osteoporosis Study. BMD change in 327 Mexican Americans (ages 25-45 years) from 32 extended pedigrees was calculated from DXA measurements at baseline and follow-up (3.5 to 8.9 years later). Family-based likelihood methods were used to estimate heritability (h(2)) and perform autosome-wide linkage analysis for BMD change of the proximal femur and forearm and to estimate heritability for BMD change of lumbar spine. BMD change was significantly heritable for total hip, ultradistal radius, and 33% radius (h(2) = 0.34, 0.34, and 0.27, respectively; p < 0.03 for all), modestly heritable for femoral neck (h(2) = 0.22; p = 0.06) and not heritable for spine BMD. Covariates associated with BMD change included age, sex, baseline BMD, menopause, body mass index, and interim BMI change, and accounted for 6% to 24% of phenotype variation. A significant quantitative trait locus (LOD = 3.6) for femoral neck BMD change was observed on chromosome 1q23. In conclusion, we observed that change in BMD in young adults is heritable and performed one of the first linkage studies for BMD change. Linkage to chromosome 1q23 suggests that this region may harbor one or more genes involved in regulating early BMD change of the femoral neck.

Objective: Recently the significance of genetic traits, influencing hormonal and environmental factors, in susceptibility to osteopenia and osteoporosis development has been indicated. Much attention to the polymorphic variants of vitamin D receptor (VDR) gene was paid. The restriction polymorphisms in VDR gene could be involved in the modulation of vitamin D action and modulate the level of bone mineral density (BMD) and the risk to develop osteopenia and osteoporosis., Materials and Methods: Total 321 postmenopausal women (mean age 63.26 +/- 8.90 years), including women with osteoporosis (163 patients) and osteopenia (95) have been compared to 63 women with normal t-score value. For detection of VDR polymorphisms PCR/RFLP (polymerase chain reaction/restriction fragment length polymorphism) assay have been used., Results: The frequency of BsmI, ApaI, and TaqI polymorphic variants of VDR gene detected in investigated groups was not statistically different. The slight, not significant tendency to prevalence of a allele (ApaI polymorphism) in the controls comparing to women with osteoporosis and osteopenia have been noted. Higher prevalence of homozygous TT genotype (TaqI polymorphism) the in the both groups with lower BMD value (47.9 : 49.5 vs. 34.9% in the controls) and higher prevalence of T allele in these both groups (65.9 : 68.4 vs. 57.9) was been also observed., Conclusions: The presence of T allele of TaqI polymorphism could predict the higher risk to develop osteoporosis in postmenopausal woman; consequently t allele could have protective effect. The presence of A allele (ApaI polymprphism) seems to be weakly connected with osteoporosis susceptibility.

Survey radiographs of all the growing dogs aged up to 6 months, which were presented to the IVRI polyclinics during the 10 year period were screened to study the determinants of bone mass, density and growth. On the basis of clinical history and radiographic evaluation of long bones, the cases were categorizes as normal or osteopenic. The relative cortical density (RCD), cortical index (CI), diameter of bone at the distal metaphysis (DDFM) and the width of the growth plate (WFGP) were determined by taking the femur as a model bone in German shepherd, Doberman and Spitz breeds of dogs at different age groups. The results showed that RCD was the least in 0-2 month old normal growing dogs in all the breeds. As the age advanced up to 6 months the RCD increased 20-25% and at 6 months, Spitz and Doberman showed significant increase (P<0.05) in the RCD. In osteopenic bones, RCD remained less (25-50%) than that of normal animals at all age groups, and at 2-6 months of age, RCD in osteopenic bones was significantly lesser than in normal animals in GSD and Spitz breeds. The CI was also the least at 0-2 months of age in normal dogs. The CI increased about 50% at 4-6 months of age in GSD and Spitz. Whereas in Dob., there was no appreciable change in the CI at different age groups, and at 2-6 months it was significantly (P<0.05) lesser than that of Spitz. In osteopenic bones, the CI was 25-75% lesser than that of normal animals at different age groups, and at 4-6 months there was significant difference (P<0.05) between the normal and osteopenic bones in GSD and Spitz. The DDFM was the least in 0-2 month old normal growing dogs, and as the age advanced, it increased 10-20% up to 6 months. However, no significant difference in the DDFM was seen between breeds and also between the normal and osteopenic bones at different age groups. In normal animals, the WFGP was the highest in the early age, subsequently it reduced 50-75% and at the 4-6 months there was a significant decrease (P<0.05) in all breeds of dogs. And at the 4-6 months, there was significant (P<0.05) difference in the WFGP between breeds, it was the least in Spitz and maximum in Dob., suggesting faster growth plate closure in Spitz than in GSD and Dob. breeds. In osteopenic bones, WFGP was generally more than in normal animals, and at 4-6 months (about 3-5 times more) there was significant difference (p<0.05) between the normal and osteopenic bones in all breeds, indicating that physeal closure may be delayed in osteopenic bones. The results indicate that among different breeds Doberman breed has the least bone mass and may be more prone to osteopenia; whereas Spitz has the strongest bone.

Mice with interleukin (IL)-7 transgene under the control of E(alpha) promoter over-express IL-7 in MHC class II-positive cells and develop specific immune phenotype, marked by an increase in CD45R(+) cells in both the bone marrow and peripheral blood. We show that IL-7 transgenic mice have a bone phenotype characterized by an age-related loss of trabecular bone in both axial and long bones. Osteopenia was the result of increased number of active osteoclasts on the surface of trabecular bone. Furthermore, IL-7 transgenic mice showed increased osteoclastic but unchanged osteoblastic potential of the bone marrow in vitro. IL-7 over-expression also created osteoclastogenic microenvironment within the bone marrow which promoted the commitment of precursors towards the osteoclast lineage. These findings are important for immunological disturbances where IL-7 is involved and where alterations in the immune system are accompanied by changes in bone metabolism, such as multiple myeloma, rheumatoid arthritis and postmenopausal osteoporosis.

Osteoclast inhibitory lectin (OCIL or clrb) is a member of the natural killer cell C-type lectins that have a described role mostly in autoimmune cell function. OCIL was originally identified as an osteoblast-derived inhibitor of osteoclast formation in vitro. To determine the physiological function(s) of OCIL, we generated ocil(-/-) mice. These mice appeared healthy and were fertile, with no apparent immune function defect, and phenotypic abnormalities were limited to bone. Histomorphometric analysis revealed a significantly lower tibial trabecular bone volume and trabecular number in the 10- and 16-week-old male ocil(-/-) mice compared with wild type mice. Furthermore, ocil(-/-) mice showed reduced bone formation rate in the 10-week-old females and 16-week-old males while Static markers of bone formation showed no significant changes in male or female ocil(-/-) mice. Examination of bone resorption markers in the long bones of ocil(-/-) mice indicated a transient increase in osteoclast number per unit bone perimeter. Enhanced osteoclast formation was also observed when either bone marrow or splenic cultures were generated in vitro from ocil(-/-) mice relative to wild type control cultures. Loss of ocil therefore resulted in osteopenia in adult mice primarily as a result of increased osteoclast formation and/or decreased bone formation. The enhanced osteoclastic activity led to elevated serum calcium levels, which resulted in the suppression of circulating parathyroid hormone in 10-week-old ocil(-/-) mice compared with wild type control mice. Collectively, our data suggest that OCIL is a physiological negative regulator of bone.

Connective tissue growth factor (CTGF), a member of the CCN family of proteins, is expressed in skeletal cells, and the ctgf null mutation leads to neonatal lethality due to defects in skeletal development. To define the function of CTGF in the postnatal skeleton, we created transgenic mice overexpressing CTGF under the control of the human osteocalcin promoter. CTGF transgenic female and male mice exhibited a significant decrease in bone mineral density, compared with wild-type littermate controls. Bone histomorphometry revealed that CTGF overexpression caused decreased trabecular bone volume due to impaired osteoblastic activity because mineral apposition and bone formation rates were decreased. Osteoblast and osteoclast number and bone resorption were not altered. Calvarial osteoblasts and stromal cells from CTGF transgenics displayed decreased alkaline phosphatase and osteocalcin mRNA levels and reduced bone morphogenetic protein (BMP) signaling mothers against decapentaplegic, Wnt/beta-catenin, and IGF-I/Akt signaling. In conclusion, CTGF overexpression in vivo causes osteopenia, secondary to decreased bone formation, possibly by antagonizing BMP, Wnt, and IGF-I signaling and activity.

Notch receptors are determinants of cell fate decisions. To define the role of Notch in the adult skeleton, we created transgenic mice overexpressing the Notch intracellular domain (NICD) under the control of the type I collagen promoter. First-generation transgenics were small and osteopenic. Bone histomorphometry revealed that NICD caused a decrease in bone volume, secondary to a reduction in trabecular number; osteoblast and osteoclast number were decreased. Low fertility of founder mice and lethality of young pups did not allow the complete establishment of transgenic lines. To characterize the effect of Notch overexpression in vitro, NICD was induced in osteoblasts and stromal cells from Rosa(notch) mice, in which a STOP cassette flanked by lox(P) sites is upstream of NICD, by transduction with an adenoviral vector expressing Cre recombinase (Cre) under the control of the cytomegalovirus (CMV) promoter (Ad-CMV-Cre). NICD impaired osteoblastogenesis and inhibited Wnt/beta-catenin signaling. To determine the effects of notch1 deletion in vivo, mice in which notch1 was flanked by lox(P) sequences (notch1(loxP/loxP)) were mated with mice expressing Cre recombinase under the control of the osteocalcin promoter. Conditional null notch1 mice had no obvious skeletal phenotype, possibly because of rescue by notch2; however, 1-month-old females exhibited a modest increase in osteoclast surface and eroded surface. Osteoblasts from notch1(loxP/loxP) mice, transduced with Ad-CMV-Cre and transfected with Notch2 small interfering RNA, displayed increased alkaline phosphatase activity. In conclusion, Notch signaling in osteoblasts causes osteopenia and impairs osteo-blastogenesis by inhibiting the Wnt/beta-catenin pathway.

Unlabelled: This study evaluated the association of a polymorphism in the CD40 gene with BMD and risk of osteopenia or osteoporosis in a population of 602 postmenopausal women. Results showed that women with the TT genotype had lower BMD at femoral neck and spine sites and increased risk of osteopenia or osteoporosis., Introduction: Recent findings have demonstrated that the CD40/CD40L system, which is of main importance for the immune system, can also be implied in the regulation of bone metabolism. The main objective of the present work has been to clarify whether single nucleotide polymorphisms (SNPs) affecting genes of CD40/CD40L system could be linked with abnormalities in the level of bone mineral density (BMD) in menopausal women., Methods: We performed an association study of BMD values with a SNP located at position -1 of the Kozak consensus sequence of CD40 gene (rs1883832; C>T) in a population of 602 postmenopausal women., Results: Women with the TT genotype (8.6% of women) displayed a reduction in femoral neck BMD (FN BMD) and lumbar spine BMD (LS BMD) of 6.2% and of 6.3%, respectively, as compared to women with CC + CT genotype. Logistic regression analysis adjusted for age, weight, and height showed that women with the TT genotype had increased risk for FN (odds ratio: 2.34; 95% CI: 1.12-4.89) and LS (odds ratio: 2.49; 95% CI: 1.19-5.24) osteopenia or osteoporosis., Conclusions: Women with the TT genotype in rs1883832 SNP affecting to Kozak consensus sequence of CD40 gene had lower BMD at FN and at LS sites and increased risk of osteopenia or osteoporosis.

ICER is a member of the CREM family of basic leucine zipper transcription factors that acts as a dominant negative regulator of gene transcription. Four different isoforms of ICER (I, Igamma, II and IIgamma) are transcribed from the P2 promoter of the Crem gene. We previously found that each of the ICER isoforms is induced by parathyroid hormone in osteoblasts. The goal of the present study was to assess the function of ICER in bone by overexpressing ICER in osteoblasts of transgenic mice. ICER I and ICER II cDNAs, each containing an N-terminal FLAG epitope tag, were cloned downstream of a fragment containing 3.6 kb of the rat Col1a1 promoter and most of the rat Col1a1 first intron to produce pOBCol3.6-ICER I and pOBCol3.6-ICER II transgenes, respectively. Multiple lines of mice were generated bearing the ICER I and ICER II transgenes. At 8 weeks of age, ICER I and ICER II transgenic mice had lower body weights and decreased bone mineral density of femurs and vertebrae. Further studies were done with ICER I transgenic mice, which had greatly reduced trabecular bone volume and a markedly decreased bone formation rate in femurs. Osteoblast differentiation and osteocalcin expression were reduced in ex vivo bone marrow cultures from ICER I transgenic mice. ICER I antagonized the activity of ATF4 at its consensus DNA binding site in the osteocalcin promoter in vitro. Thus, transgenic mice with osteoblast-targeted overexpression of ICER exhibited osteopenia caused primarily by reduced bone formation. We speculate that ICER regulates the activity and/or expression of ATF/CREB factors required for normal bone formation.

Bone-related complications in Gaucher disease are considered to be poorly responsive to specific enzyme replacement therapy. Polymorphisms of candidate genes associated with low bone density were investigated to see whether they are correlated with bone mineral density (BMD) and bone involvement in Gaucher disease. Genotyping for polymorphisms in candidate genes (interleukins 1alpha and 1beta, interleukin-1 receptor antagonist; cytochrome P450; collagen 1A1; low-density Lipoprotein Receptor; bone morphogenic protein 4; vitamin D receptor; and estrogen receptor 2beta) were performed using standard methodologies. BMD was measured by dual energy X-ray absorptiometry (DXA). One hundred and ninety-four patients and 100 controls were genotyped for the above polymorphisms. Thirteen haplotypes were obtained, with several correlations with BMD in patients; also, a haplotype (T889-T3954-C511-240VNTR of IL1) was significantly correlated with T-scores and Z-score for femur neck and lumbar spine (p = 0.01) in patients. Haplotypes of bone-specific candidate genes associated with BMD may predict severity of these features in Gaucher disease.

Five members from 3 generations, including a 35-year-old woman and her 2 sons, both mentally impaired to a different degree, were studied in a tertiary care hospital. Anamnestic, clinical, neurological, and radiological evaluations were used to describe phenotypes. A and B postaxial polydactyly, transmitted likely as autosomal dominant, was associated with an extensive variability of phenotypic features: (1) cutaneous syndactyly, (2) nail-teeth dysplasia, (3) osteopenia, and (4) mental delay. The likelihood that the constellation of observations we report here is caused by mutation of a single gene that subsequently affects multiple physiological activities, although fascinating, remains to be proven. Instead, we hypothesize that it likely develops as a contiguous gene syndrome.

The vitamin D endocrine system is important for skeletal homeostasis. 1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] impacts bone indirectly by promoting intestinal absorption of calcium and phosphate and directly by acting on osteoblasts and osteoclasts. Despite the direct actions of 1,25(OH)(2)D(3) in bone, relatively little is known of the mechanisms or target genes that are regulated by 1,25(OH)(2)D(3) in skeletal cells. Here, we identify semaphorin 3B (SEMA3B) as a 1,25(OH)(2)D(3)-stimulated gene in osteoblastic cells. Northern analysis revealed strong induction of SEMA3B mRNA by 1,25(OH)(2)D(3) in MG-63, ST-2, MC3T3, and primary osteoblastic cells. Moreover, differentiation of these osteogenic cells enhanced SEMA3B gene expression. Biological effects of SEMA3B in the skeletal system have not been reported. Here, we show that osteoblast-derived SEMA3B alters global skeletal homeostasis in intact animals and osteoblast function in cell culture. Osteoblast-targeted expression of SEMA3B in mice resulted in reduced bone mineral density and aberrant trabecular structure compared with nontransgenic littermates. Histomorphometry studies indicated that this was likely due to increased osteoclast numbers and activity. Indeed, primary osteoblasts obtained from SEMA3B transgenic mice stimulated osteoclastogenesis to a greater extent than nontransgenic osteoblasts. This study establishes that SEMA3B is a 1,25(OH)(2)D(3)-induced gene in osteoblasts and that osteoblast-derived SEMA3B impacts skeletal biology in vitro and in vivo. Collectively, these studies support a putative role for SEMA3B as an osteoblast protein that regulates bone mass and skeletal homeostasis.

Excessive skin wrinkling and cutis laxa are seen in many genetic conditions and overlapping features can make a clinical diagnosis difficult. Here we report on 22 Omani patients from 11 consanguineous families with the diagnosis of wrinkly skin syndrome (WSS, OMIM 278250) or geroderma osteodysplasticum hereditaria (GO, OMIM 231070). The WSS phenotype evolves during early childhood and includes a generalized and excessive skin wrinkling, dental problems, herniae, foot deformities, hip dislocations, growth retardation, and a large anterior fontanelle. The facial gestalt is characterized by a broad nasal bridge, hypertelorism, and downslanting palpebral fissures. We were unable to differentiate between WSS and cutis laxa with growth and developmental delay (CLGDD, OMIM 219200) suggesting that both can be considered as one entity. Distinct hallmarks of GO were skin wrinkling limited to the dorsum of hands and feet and to the abdomen, normal fontanelles, maxillary hypoplasia, bowed long bones, and osteopenia with frequent fractures. In contrast to the attenuation of the skin phenotype with age in WSS, adult patients with GO appeared prematurely aged. A serum sialotransferrin type 2 pattern was found in all four WSS patients tested. Apolipoprotein CIII (a marker for O-glycosylation) was normal suggesting that WSS is frequently associated with a N-protein glycosylation defect, probably at the level of processing (CDG-II). All four investigated GO patients showed normal sialotransferrin patterns. The known loci for cutis laxa and WSS on 2q31, 5q23-q31, 7q11, 11q13, and 14q32 were excluded. We suggest that WSS and GO are distinct entities with overlapping features., (Copyright 2008 Wiley-Liss, Inc.)

The biological actions of androgens are mediated by transcriptional control of target genes through binding to the androgen receptor (AR). The AR belongs to the nuclear receptor superfamily and acts as a ligand-inducible transcriptional factor. Androgens/AR system is thought to be central to male physiology and behaviors to the development of clinical pathology. The physiological importance of AR function currently recognized in female reproduction also has been verified in mouse genetic model. Here we review functions of AR in male and female physiology as revealed by mice lacking AR.

The mechanisms that regulate bone mass are important in a variety of complex diseases such as osteopenia and osteoporosis. Regulation of bone mass is a polygenic trait and is also influenced by various environmental and lifestyle factors, making analysis of the genetic basis difficult. As an effort toward identifying novel genes involved in regulation of bone mass, N-ethyl-N-nitrosourea (ENU) mutagenesis in mice has been utilized. Here we describe a mouse mutant termed Yoda that was identified in an ENU mutagenesis screen for dominantly acting mutations. Mice heterozygous for the Yoda mutation exhibit craniofacial abnormalities: shortened snouts, wider skulls, and deformed nasal bones, underlined by altered morphology of frontonasal sutures and failure of interfrontal suture to close. A major feature of the mutant is reduced bone mineral density. Homozygosity for the mutation results in embryonic lethality. Positional cloning of the locus identified a missense mutation in a highly conserved region of the ankyrin repeat domain 11 gene (Ankrd11). This gene has not been previously associated with bone metabolism and, thus, identifies a novel genetic regulator of bone homeostasis.

Estrogen (E) is critical for the maintenance of bone mass in both female and male mice and steroid receptor coactivator (SRC)-1 has been shown to be important for mediating E effects on bone, at least in female mice. In the present study, we defined the skeletal phenotype of male SRC-1 knock out (KO) mice and compared it with their female littermates. Further, to determine the role of SRC-1 in mediating effects of E on bone in male mice, we examined the skeletal effects of gonadectomy (gnx) with or without E replacement in male mice and placed these findings in the context of our previous studies in female SRC-1 KO mice. Analysis of a large group of male (WT, n=67; SRC-1 KO, n=56) and female (WT, n=66; SRC-1 KO, n=70) mice showed a significant decrease in trabecular volumetric bone mineral density (vBMD) in SRC-1 KO mice compared to their WT littermates in both genders (male SRC-1 KO, 275+/-3 vs. WT, 295+/-3 mg/cm(3), P<0.001; female SRC-1 KO, 210+/-2 vs. WT, 221+/-2 mg/cm(3), P<0.001). Following gnx and E replacement (10 microg/kg/day), we previously demonstrated that SRC-1 KO female mice have a defect in E action in trabecular, but not in cortical bone. In contrast, we now demonstrate that the same dose of E administered to gnx'd male SRC-1 KO mice was sufficient to prevent trabecular bone loss in these mice. For example, in WT female mice, gnx followed by E replacement maintained spine BMD (1.2+/-3.4% vs. baseline) as compared to gnx without E replacement (-12.7+/-2.6%, P<0.001 vs. sham); this effect of E was absent in SRC-1 KO female mice. By contrast, the identical dose of E was equally effective in maintaining spine BMD in E-treated gnx'd male WT (-5.2+/-5.1% vs. baseline) and male SRC-1 KO (-5.4+/-5.3%) mice, respectively, as compared to gnx'd mice without E treatment (WT, -17.6+/-2.5%, P=0.02; SRC-1 KO, -28.6+/-2.6%, P<0.001 vs. sham). E treatment was effective in suppressing cancellous bone turnover in both gnx'd WT and SRC-1 KO male mice as determined by significant reductions in osteoblast and osteoclast numbers; however, in female mice, E treatment only suppressed bone turnover in WT but not in SRC-1 KO mice. Collectively, these findings demonstrate that loss of SRC-1 results in trabecular osteopenia in male and female mice, but in contrast to female mice, this is not due to any detectable resistance to E action in trabecular bone in male SRC-1 KO mice.

The monoamine serotonin (5-HT), a well-known neurotransmitter, is also important in peripheral tissues. Several studies have suggested that 5-HT is involved in bone metabolism. Starting from our original observation of increased 5-HT(2B) receptor (5-HT(2B)R) expression during in vitro osteoblast differentiation, we investigated a putative bone phenotype in vivo in 5-HT(2B)R knockout mice. Of interest, 5-HT(2B)R mutant female mice displayed reduced bone density that was significant from age 4 months and had intensified by 12 and 18 months. This histomorphometrically confirmed osteopenia seems to be due to reduced bone formation because 1) the alkaline phosphatase-positive colony-forming unit capacity of bone marrow precursors was markedly reduced in the 5-HT(2B)R mutant mice from 4 to 12 months of age, 2) ex vivo primary osteoblasts from mutant mice exhibited reduced proliferation and delayed differentiation, and 3) calcium incorporation was markedly reduced in osteoblasts after 5-HT(2B)R depletion (produced genetically or by pharmacological inactivation). These findings support the hypothesis that the 5-HT(2B)R receptor facilitates osteoblast recruitment and proliferation and that its absence leads to osteopenia that worsens with age. We show here, for the first time, that the 5-HT(2B)R receptor is a physiological mediator of 5-HT in bone formation and, potentially, in the onset of osteoporosis in aging women.

Objective: Chronic treatment with antiepileptic medication is associated with reduced bone mineral density (BMD), which may underlie the two- to sixfold increase in fracture rates observed in patients with epilepsy. The objective was to determine the timing of the BMD deficit in ambulatory children with epilepsy., Methods: A cross-sectional evaluation was conducted in 82 ambulatory children aged 6 to 18 years (12.4 +/- 3.3 years) with epilepsy for <1 year (n = 18), 1 to 5 years (n = 37), and 6 or more years (n = 27). Controls were 32 healthy children aged 12.8 +/- 2.6 years. Age- and sex-corrected total body BMD Z-score was measured., Results: Total BMD Z-score was lower in children with epilepsy (0.10 +/- 0.96; CI = -0.08, 0.34) compared to controls (0.57 +/- 0.74; CI = 0.3, 0.84; p = 0.03). Increasing duration of epilepsy was associated with a progressive reduction in BMD compared to controls (Spearman r = -0.197; p = 0.03). Compared to controls, those with epilepsy for 1 to 5 years had a mean BMD Z-score of 0.13 +/- 0.78 (CI = -0.13, 0.39; p = 0.04) and in those treated for 6 or more years BMD was 0.06 +/- 1.11 (CI = -0.38, 0.5; p = 0.04). For those with epilepsy for <1 year BMD was 0.23 +/- 1.1 (CI = -0.31, 0.77; p = 0.21)., Conclusions: Children treated for epilepsy sustain significant bone mineral density (BMD) deficit compared to controls during the initial 1 to 5 years of treatment which progressively worsens thereafter. This progressive BMD deficit may be a contributing factor to the increased fracture risk observed in patients with epilepsy and may accelerate aging-related osteoporosis.

Background: GPRC6A is a widely expressed orphan G-protein coupled receptor that senses extracellular amino acids, osteocalcin and divalent cations in vitro. The physiological functions of GPRC6A are unknown., Methods/principal Findings: In this study, we created and characterized the phenotype of GPRC6A(-/-) mice. We observed complex metabolic abnormalities in GPRC6A(-/-) mice involving multiple organ systems that express GPRC6A, including bone, kidney, testes, and liver. GPRC6A(-/-) mice exhibited hepatic steatosis, hyperglycemia, glucose intolerance, and insulin resistance. In addition, we observed high expression of GPRC6A in Leydig cells in the testis. Ablation of GPRC6A resulted in feminization of male GPRC6A(-/-) mice in association with decreased lean body mass, increased fat mass, increased circulating levels of estradiol, and reduced levels of testosterone. GPRC6A was also highly expressed in kidney proximal and distal tubules, and GPRC6A(-/-) mice exhibited increments in urine Ca/Cr and PO(4)/Cr ratios as well as low molecular weight proteinuria. Finally, GPRC6A(-/-) mice exhibited a decrease in bone mineral density (BMD) in association with impaired mineralization of bone., Conclusions/significance: GPRC6A(-/-) mice have a metabolic syndrome characterized by defective osteoblast-mediated bone mineralization, abnormal renal handling of calcium and phosphorus, fatty liver, glucose intolerance and disordered steroidogenesis. These findings suggest the overall function of GPRC6A may be to coordinate the anabolic responses of multiple tissues through the sensing of extracellular amino acids, osteocalcin and divalent cations.

Sparc null mutants have been generated independently via targeted mutations in exons 4 and 6. Previous studies have identified low-turnover osteopenia in the 129Sv/C57BL/6 exon 4 knockout. Since both Sparc null mutations result in complete absence of Sparc protein, similar phenotypic outcomes are likely. However, genetic background (strain) and/or linkage disequilibrium effects can influence phenotype. Different inactivating mutations should be tested in various mouse strains; similar phenotypic outcomes can then confidently be assigned to the mutated gene. We have evaluated the bone phenotype in the 129Sv/EvSparc(tm1cam) exon 6 knockout at 4 and 9 mo, using physical measurement, mechanical strength tests, and DXA scanning. We have also quantified bone marrow adiposity and circulating leptin levels to assess adipose tissue metabolism. 129Sv/EvSparc(tm1cam) null mice show decreased bone mineral density and bone mineral content and increased mechanical fragility of bone, in line with previous studies. Differences were also noted. Increased body weight and levels of bone marrow adiposity but decreased circulating leptin concentrations were identified at 4, but not 9 mo, and 129Sv/EvSparc(tm1cam) null mice also had shorter femurs. Molecular phenotyping was carried out using mouse HGMP NIA microarrays with cortical femur samples at various ages, using semiquantitative RT-PCR validation. We identified 429 genes highly expressed in normal bone. Six genes (Sparc, Zfp162, Bysl, E2F4, two ESTs) are differentially regulated in 129Sv/EvSparc(tm1cam) cortical femur vs. 129Sv/Ev controls. We confirm low-turnover osteopenia as a feature of the Sparc null phenotype, identifying the usefulness of this mouse as a model for human osteoporosis.

We explored the role of G alpha(q)-mediated signaling on skeletal homeostasis by selectively expressing a constitutively active G alpha(q) (mutation of Q209L) in osteoblasts. Continuous signaling via G alpha(q) in mouse osteoblastic MC3T3-E1 cells impaired differentiation. Mice that expressed the constitutively active G alpha(q) transgene in cells of the osteoblast lineage exhibited severe osteopenia in cortical and trabecular bones. Osteoblast number, bone volume, and trabecular thickness were reduced in transgenic mice, but the osteoclasts were unaffected. Osteoblasts from transgenic mice showed impaired differentiation and matrix formation. In the presence of a protein kinase C inhibitor GF109203X, this impairment was not seen, indicating mediation by the protein kinase C pathway. We propose that continuous activation of the G alpha(q) signal in osteoblasts plays a crucial, previously unrecognized role in bone formation.

Osteogenic disorder Shionogi (ODS) rats are genetically defective in ascorbic acid biosynthesis. They exhibit a gait abnormality due to dysfunctional bone formation and display various dental abnormalities. Conditions of the oral cavity and tooth quality both influence the development of dental caries. This study was designed to determine the characteristics of dental caries in ODS/ ShiJclod/od rats. Caries were scored and compared among ODS/ShiJclod/od, ODS/ShiJcl+/+, and Jcl:Wistar retired breeders. Among male rats, the caries scores of the ODS/ShiJclod/od and ODS/ShiJcl+/+ groups were similar to each other but greater than those in Jcl:Wistar rats, whereas among female rats, caries scores in ODS/ShiJclod/od animals were equivalent to or somewhat greater than those in ODS/ShiJcl+/+ rats, whose scores were markedly greater than those of Jcl:Wistar rats. The results suggest that ODS/ShiJcl rats were more susceptible to dental caries than were Jcl:Wistar rats. Under the conditions of the study, caries scores between ODS/ ShiJclod/od and ODS/ShiJcl+/+ rats differed only among parous females.

Background & Aims: Peptide YY (PYY) and pancreatic polypeptide (PPY) are members of the neuropeptide Y peptide family. The neuropeptide Y receptor signaling pathway has been implicated in a number of physiologic processes, including the regulation of energy balance and bone mass. To investigate the contribution of endogenous PYY and PPY to these processes, we generated both Pyy- and Ppy-deficient mice., Methods: Pyy(-/-) and Ppy(-/-) mice and their respective wild-type littermates were studied from 8 weeks to 9 months of age. Food intake, metabolic parameters, and locomotor activity were monitored using indirect calorimetry. Body composition and bone parameters were analyzed using dual energy x-ray absorptiometry, histomorphometry, and vertebral compression testing., Results: Studies in these mice showed an osteopenic phenotype specific to the Pyy-deficient line, which included a reduction in trabecular bone mass and a functional deficit in bone strength. Furthermore, female Pyy(-/-) mice showed a greater sensitivity to ovariectomy-induced bone loss compared with wild-type littermates. No food intake or metabolic phenotype was apparent in male or female Pyy(-/-) mice on standard chow. However, female Pyy(-/-) mice on a high-fat diet showed a greater propensity to gain body weight and adiposity. No metabolic or osteopenic phenotype was observed in Ppy-deficient mice., Conclusions: These results indicate that endogenous PYY plays a critical role in regulating bone mass. In comparison, its role in regulating body weight is minor and is confined to situations of high-fat feeding.

Mice lacking GPR103A expression display osteopenia. Analysis of mouse quantitative trait loci literature associated with bone mineral density suggested GPR103A ligand P518/Qrfp (chromosome 2qB) as a candidate osteoporosis gene. Promoter and coding regions of mouse P518/Qrfp were sequenced from genomic DNA obtained from the osteoporosis-prone strain SAMP6 and control strains SAMR1, A/J, AKR/J, BALB/c, C3H/HeJ, C57BL/6J, and DBA/2J. Four single-nucleotide polymorphisms (SNPs) were identified in only SAMP6 genomic DNA, g.-1773 T-->C, g.110 A-->G (N37S), g.188 G-->A (R63K), and g.135 T-->C (H45H). The promoter SNP generated a novel neuron-restrictive silencing factor binding site, a repressor that decreases gene expression in nonneuronal tissues. TaqMan analysis demonstrated fivefold lower P518/Qrfp liver expression in SAMP6 versus SAMR1 or C57BL/6J control strains. Tissue distribution of human, mouse, and rat P518/Qrfp and its receptors showed expression in bone and spinal cord. A direct role for P518/Qrfp function in maintaining bone mineral density is suggested.

Unlabelled: Metabolic bone disease is an important complication among infants very-low-birth-weight (< 1500 g). In adults, osteoporosis has been shown to be associated with polymorphisms of vitamin D receptor, estrogen receptor, and collagen Ialpha1 receptor genes., Aim: The primary goal of the study was to investigate the possible association between metabolic bone disease and the allelic polymorphisms of these three genes., Method: 104 infants very-low-birth-weight were enrolled to the study. Bone formation (serum alkaline phosphatase, osteocalcin) and bone resorption (urinary excretion of calcium and pyridinium crosslink) markers were determined and x-rays of the chest and wrist (together with the distal portions of associated long bones) were obtained., Results: Thirty infants (28,8%) were diagnosed with metabolic bone disease based on high activity of bone formation, bone resorption markers, and positive radiologic signs. Statistically significant correlation between thymine-adenine repeat [(TA) n ] allelic variant of estrogen receptor gene and bone disease was observed. Infants with metabolic bone disease more often carried low number of repeats [(TA) n < 19] [odds ratio (OR): 5.82, 95% confidence interval (CI): 2.26-14.98]. Significantly higher number of repeats [(TA)n > 18] was found more frequently in the control group (OR: 0.20, 95% CI: 0.05-0.82). Furthermore significant interaction between vitamin D receptor and collagen Ialpha1 receptor genotypes ( p = 0.023) was observed. In a forward stepwise logistic regression model, bone disorder of preterms correlated with male gender ( p = 0.001), duration of hospitalization ( p = 0.007), homozygous allelic variants of high number of (TA) n repeats ( p = 0.025) and interaction between vitamin D receptor (Tt) and estrogen receptor (homozygous allelic variants of low number of repeats) genotype ( p = 0.037)., Conclusion: The results suggest that the development of metabolic bone disease in infants very-low-birth-weight may be associated with genetic polymorphisms.

Although the etiology of scoliosis is multifactorial, genetic factors play an important role. Recent linkage studies on familial idiopathic scoliosis revealed multiple putative predisposition loci. A genetic association study is complementary to linkage studies in defining the genetic basis of complex traits of diseases like idiopathic scoliosis. The onset and progression of adolescent idiopathic scoliosis is a manifestation of aberrant growth in the spine. Meanwhile, a high proportion of patients with adolescent idiopathic scoliosis have additional phenotypes, which suggest systemic growth dys-regulation during puberty. The growth hormone-insulin-like growth factor axis plays the principle role in skeletal growth regulation. Because growth hormone receptor alleles were associated with body stature and response to growth hormone treatment, we hypothesized the growth hormone receptor is a candidate predisposing and disease modifier gene for adolescent idiopathic scoliosis. Five hundred ten girls with adolescent idiopathic scoliosis and 363 normal subjects were recruited. Curve severity, arm span, and bone mineral densities were recorded. Five polymorphisms were studied by polymerase chain reaction-restriction fragment length polymorphism. All the genotype and allele frequencies were comparable between groups. The Cobb angles among patients of different genotypes were similar. The growth hormone receptor did not appear to be a predisposing gene or disease modifier gene of adolescent idiopathic scoliosis.

Introduction: Osteoporotic fracture (OF) is a public health problem. It is a common practice in the genetics of osteoporosis that bone mineral density (BMD) was studied as a major surrogate phenotype in gene search for risk of OF (ROF) because of their high phenotypic correlation. However, some studies indicate that the genetic correlation between BMD and ROF is very low. This implies that most genes found important for BMD may not be relevant to ROF. Ideally, employing OF per se as a direct study phenotype can directly find the relevant genes underlying ROF., Evidence: Here, we summarized some evidence supporting ROF under moderate genetic control, and the current progress of molecular genetic studies employing OF as the direct study phenotype, then give our consideration on the future prospects in the genetics of ROF.

Nephroblastoma overexpressed (Nov), a member of the Cyr 61, connective tissue growth factor, Nov (CCN) family of proteins, is expressed by osteoblasts, but its function in cells of the osteoblastic lineage is not known. We investigated the effects of Nov overexpression by transducing murine ST-2 stromal and MC3T3 osteoblastic cells with a retroviral vector where Nov is under the control of the cytomegalovirus promoter. We also examined the skeletal phenotype of transgenic mice expressing Nov under the control of the human osteocalcin promoter. Overexpression of Nov in ST-2 cells inhibited the appearance of mineralized nodules and decreased alkaline phosphatase activity and osteocalcin mRNA levels. Nov overexpression inhibited the effect of bone morphogenetic protein (BMP)-2 on the phosphorylation of Smad 1/5/8; on the transactivation of 12xSBE-Oc-pGL3, a BMP/Smad signaling reporter construct, and of Wnt 3 on cytoplasmic beta-catenin levels; and on the transactivation of the Wnt/beta-catenin signaling reporter construct 16xTCF-Luc. Nov overexpression did not activate Notch or transforming growth factor beta signaling. Glutathione S-transferase pulldown assays demonstrated direct Nov-BMP interactions. Nov transgenic mice exhibited osteopenia. In conclusion, Nov binds BMP-2 and antagonizes BMP-2 and Wnt activity, and its overexpression inhibits osteoblastogenesis and causes osteopenia.

IL-1alpha transgenic (Tg) mice exhibit chronic inflammatory arthritis and subsequent osteopenia, with IL-1-induced GM-CSF playing an important role in the pathogenesis. This study analyzed the mechanisms underlying osteopenia in Tg mice, and the therapeutic effects of the cyclooxygenase-2 inhibitor celecoxib on such osteopenia. Inhibited osteoclast formation was observed in RANKL-treated bone marrow cell (BMC) cultures from Tg mice and coculture of Tg-derived BMCs and wild-type-derived primary osteoblasts (POBs). FACS analysis indicated that this inhibition was attributable to a decreased number of osteoclast precursors within Tg-derived BMCs. Moreover, in coculture of Tg-derived POBs and either Tg- or wild-type-derived BMCs, osteoclast formation was markedly inhibited because Tg-derived POBs produced abundant GM-CSF, known as a potent inhibitor of osteoclast differentiation. Histomorphometric analysis of Tg mice revealed that both bone formation and resorption were decreased, with bone formation decreased more prominently. Interestingly, administration of celecoxib resulted in further deterioration of osteopenia where bone formation was markedly suppressed, whereas bone resorption remained unchanged. These results were explained by our in vitro observation that celecoxib dose-dependently and dramatically decreased osteogenesis by Tg mouse-derived POBs in culture, whereas mRNA expressions of GM-CSF and M-CSF remained unchanged. Consequently, blockade of PGE(2) may exert positive effects on excessively enhanced bone resorption observed in inflammatory bone disease, whereas negative effects may occur mainly through reduced bone formation, when bone resorption is constitutively down-regulated as seen in Tg mice.

Recessive inactivating mutations in human matrix metalloproteinase 2 (MMP2, gelatinase A) are associated with syndromes that include abnormal facial appearance, short stature, and severe bone loss. Mmp2(-/-) mice have only mild aspects of these abnormalities, suggesting that MMP2 function is redundant during skeletal development in the mouse. Here, we report that Mmp2(-/-) mice with additional mutations that render type I collagen resistant to collagenase-mediated cleavage to TC(A) and TC(B) fragments (Col1a1(r/r) mice) have severe developmental defects resembling those observed in MMP2-null humans. Composite Mmp2(-/-);Col1a1(r/r) mice were born in expected Mendelian ratios but were half the size of wild-type, Mmp2(-/-), and Col1a1(r/r) mice and failed to thrive. Furthermore, composite Mmp2(-/-);Col1a1(r/r) animals had very abnormal craniofacial features with shorter snouts, bulging skulls, incompletely developed calvarial bones and unclosed cranial sutures. In addition, trabecular bone mass was reduced concomitant with increased numbers of bone-resorbing osteoclasts and osteopenia. In vitro, MMP2 had a unique ability among the collagenolytic MMPs to degrade mutant collagen, offering a possible explanation for the genetic interaction between Mmp2 and Col1a1(r). Thus, because mutations in the type I collagen gene alter the phenotype of mice with null mutations in Mmp2, we conclude that type I collagen is an important modifier gene for Mmp2. Developmental Dynamics 236:1683-1693, 2007. (c) 2007 Wiley-Liss, Inc., (Copyright 2007 Wiley-Liss, Inc.)

Context: Estrogens regulate bone mass by binding to the estrogen receptor (ER)-alpha as well as ER-beta. The specific ERalpha cofactor retinoblastoma-interacting zinc finger protein (RIZ)-1 enhances ERalpha function in the presence of estrogen., Objective: The objective of the study was to determine whether a RIZ P704 insertion (+)/deletion (-) (indel) polymorphism modulates the impact of estradiol on bone mineral density (BMD) and study the association between the polymorphism and BMD in elderly subjects., Design: This was a population-based, prospective, and cross-sectional study, the Swedish MrOS Study, and the Malmö OPRA Study, respectively., Setting: The study was conducted at three academic medical centers: Sahlgrenska Academy in Gothenburg, Malmö University Hospital, and Uppsala University Hospital., Participants: In total, 4058 men and women, aged 69-81 yr, were randomly selected from population registries., Main Outcome Measures: BMD (grams per square centimeter) was measured at femoral neck, trochanter, lumbar spine, and total body., Results: The RIZ P704(+/+) genotype was associated with low BMD in both women (femoral neck, P < 0.001; trochanter, P < 0.01; lumbar spine, P < 0.05; total body, P < 0.01) and men (lumbar spine, P < 0.05). However, the association between the polymorphism and BMD was dependent on estradiol status. The positive correlation between serum estradiol and BMD was significantly modulated by the genotype with a stronger correlation in the P704(+/+) group than the P704(-/-) group (r = 0.19 vs. r = 0.08, P < 0.05)., Conclusions: These large-scale studies of elderly men and women indicate that the ERalpha cofactor RIZ gene has a prominent effect on BMD, and the P704 genotype modulates the impact of estradiol on BMD. Further studies are required to determine whether this polymorphism modulates the estrogenic response to estradiol treatment.

Mutations in the COL1A1 and COL1A2 genes, encoding the proalpha1 and 2 chains of type I collagen, cause osteogenesis imperfecta (OI) or Ehlers-Danlos syndrome (EDS) arthrochalasis type. Although the majority of missense mutations in the collagen type I triple helix affect glycine residues in the Gly-Xaa-Yaa repeat, few nonglycine substitutions have been reported. Two arginine-to-cysteine substitutions in the alpha1(I)-collagen chain are associated with classic EDS [R134C (p.R312C)] or autosomal dominant Caffey disease with mild EDS features [R836C (p.R1014C)]. Here we show alpha1(I) R-to-C substitutions in three unrelated patients who developed iliac or femoral dissection in early adulthood. In addition, manifestations of classic EDS in Patient 1 [c.1053C>T; R134C (p.R312C); X-position] or osteopenia in Patients 2 [c.1839C>T; R396C (p.R574C); Y-position] and 3 [c.3396C>T; R915C (p.R1093C); Y-position] are seen. Dermal fibroblasts from the patients produced disulfide-bonded alpha1(I)-dimers in approximately 20% of type I collagen, which were efficiently secreted into the medium in case of the R396C and R915C substitution. Theoretical stability calculations of the collagen type I heterotrimer and thermal denaturation curves of monomeric mutant alpha1(I)-collagen chains showed minor destabilization of the collagen helix. However, dimers were shown to be highly unstable. The R134C and R396C caused delayed procollagen processing by N-proteinase. Ultrastructural findings showed collagen fibrils with variable diameter and irregular interfibrillar spaces, suggesting disturbed collagen fibrillogenesis. Our findings demonstrate that R-to-C substitutions in the alpha1(I) chain may result in a phenotype with propensity to arterial rupture in early adulthood. This broadens the phenotypic range of nonglycine substitutions in collagen type I and has important implications for genetic counseling and follow-up of patients carrying this type of mutation., (Copyright 2007 Wiley-Liss, Inc.)

Androgen receptor (AR) gene has been extensively studied in diverse clinical conditions. In addition to the point mutations, trinucleotide repeat (CAG and GGN) length polymorphisms have been an additional subject of interest and controversy among geneticists. The polymorphic variations in triplet repeats have been associated with a number of disorders, but at the same time contradictory findings have also been reported. Further, studies on the same disorder in different populations have generated different results. Therefore, combined analysis or review of the published studies has been of much value to extract information on the significance of variations in the gene in various clinical conditions. AR genetics has been reviewed extensively but until now review articles have focused on individual clinical categories such as androgen insensitivity, male infertility, prostate cancer, and so on. We have made the first effort to review most the aspects of AR genetics. The impact of androgens in various disorders and polymorphic variations in the AR gene is the main focus of this review. Additionally, the correlations observed in various studies have been discussed in the light of in vitro evidences available for the effect of AR gene variations on the action of androgens.

A recessive form of severe osteogenesis imperfecta that is not caused by mutations in type I collagen has long been suspected. Mutations in human CRTAP (cartilage-associated protein) causing recessive bone disease have been reported. CRTAP forms a complex with cyclophilin B and prolyl 3-hydroxylase 1, which is encoded by LEPRE1 and hydroxylates one residue in type I collagen, alpha1(I)Pro986. We present the first five cases of a new recessive bone disorder resulting from null LEPRE1 alleles; its phenotype overlaps with lethal/severe osteogenesis imperfecta but has distinctive features. Furthermore, a mutant allele from West Africa, also found in African Americans, occurs in four of five cases. All proband LEPRE1 mutations led to premature termination codons and minimal mRNA and protein. Proband collagen had minimal 3-hydroxylation of alpha1(I)Pro986 but excess lysyl hydroxylation and glycosylation along the collagen helix. Proband collagen secretion was moderately delayed, but total collagen secretion was increased. Prolyl 3-hydroxylase 1 is therefore crucial for bone development and collagen helix formation.