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Skeletal deformities are typical AD-HIES manifestations, which are mainly caused by heterozygous and loss-of-function mutations in Signal transducer and activator of transcription 3 (STAT3). However, the mechanism is still unclear and the treatment strategy is limited. Herein, we reported that the mice with Stat3 deletion in osteoblasts, but not in osteoclasts, induced AD-HIES-like skeletal defects, including craniofacial malformation, osteoporosis, and spontaneous bone fracture. Mechanistic analyses revealed that STAT3 in cooperation with Msh homeobox 1(MSX1) drove osteoblast differentiation by promoting Distal-less homeobox 5(Dlx5) transcription. Furthermore, pharmacological activation of STAT3 partially rescued skeletal deformities in heterozygous knockout mice, while inhibition of STAT3 aggravated bone loss. Taken together, these data show that STAT3 is critical for modulating skeletal development and maintaining bone homeostasis through STAT3-indcued osteogenesis and suggest it may be a potential target for treatments., (© 2021. The Author(s).)

Skeletal ciliopathies (e.g., Jeune syndrome, short rib polydactyly syndrome, and Sensenbrenner syndrome) are frequently associated with nephronophthisis-like cystic kidney disease and other organ manifestations. Despite recent progress in genetic mapping of causative loci, a common molecular mechanism of cartilage defects and cystic kidneys has remained elusive. Targeting two ciliary chondrodysplasia loci ( ift80 and ift172 ) by CRISPR/Cas9 mutagenesis, we established models for skeletal ciliopathies in Xenopus tropicalis Froglets exhibited severe limb deformities, polydactyly, and cystic kidneys, closely matching the phenotype of affected patients. A data mining-based in silico screen found ttc30a to be related to known skeletal ciliopathy genes. CRISPR/Cas9 targeting replicated limb malformations and renal cysts identical to the models of established disease genes. Loss of Ttc30a impaired embryonic renal excretion and ciliogenesis because of altered posttranslational tubulin acetylation, glycylation, and defective axoneme compartmentalization. Ttc30a/b transcripts are enriched in chondrocytes and osteocytes of single-cell RNA-sequenced embryonic mouse limbs. We identify TTC30A/B as an essential node in the network of ciliary chondrodysplasia and nephronophthisis-like disease proteins and suggest that tubulin modifications and cilia segmentation contribute to skeletal and renal ciliopathy manifestations of ciliopathies in a cell type-specific manner. These findings have implications for potential therapeutic strategies., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Transportin-2 (TNPO2) mediates multiple pathways including non-classical nucleocytoplasmic shuttling of >60 cargoes, such as developmental and neuronal proteins. We identified 15 individuals carrying de novo coding variants in TNPO2 who presented with global developmental delay (GDD), dysmorphic features, ophthalmologic abnormalities, and neurological features. To assess the nature of these variants, functional studies were performed in Drosophila. We found that fly dTnpo (orthologous to TNPO2) is expressed in a subset of neurons. dTnpo is critical for neuronal maintenance and function as downregulating dTnpo in mature neurons using RNAi disrupts neuronal activity and survival. Altering the activity and expression of dTnpo using mutant alleles or RNAi causes developmental defects, including eye and wing deformities and lethality. These effects are dosage dependent as more severe phenotypes are associated with stronger dTnpo loss. Interestingly, similar phenotypes are observed with dTnpo upregulation and ectopic expression of TNPO2, showing that loss and gain of Transportin activity causes developmental defects. Further, proband-associated variants can cause more or less severe developmental abnormalities compared to wild-type TNPO2 when ectopically expressed. The impact of the variants tested seems to correlate with their position within the protein. Specifically, those that fall within the RAN binding domain cause more severe toxicity and those in the acidic loop are less toxic. Variants within the cargo binding domain show tissue-dependent effects. In summary, dTnpo is an essential gene in flies during development and in neurons. Further, proband-associated de novo variants within TNPO2 disrupt the function of the encoded protein. Hence, TNPO2 variants are causative for neurodevelopmental abnormalities., Competing Interests: Declaration of interests The Department of Molecular and Human Genetics at Baylor College of Medicine receives revenue from clinical genetic testing completed at Baylor Genetics Laboratories. Y.S. and A.B. are employees of GeneDx, Inc., (Copyright © 2021 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Wingless-type MMTV integration site family, member 16 ( wnt16 ), is a wnt ligand that participates in the regulation of vertebrate skeletal development. Studies have shown that wnt16 can regulate bone metabolism, but its molecular mechanism remains largely undefined. We obtained the wnt16 -/- zebrafish model using the CRISPR-Cas9-mediated gene knockout screen with 11 bp deletion in wnt16 , which led to the premature termination of amino acid translation and significantly reduced wnt16 expression, thus obtaining the wnt16 -/- zebrafish model. The expression of wnt16 in bone-related parts was detected via in situ hybridization. The head, spine, and tail exhibited significant deformities, and the bone mineral density and trabecular bone decreased in wnt16 -/- using light microscopy and micro-CT analysis. RNA sequencing was performed to explore the differentially expressed genes (DEGs). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis found that the down-regulated DEGs are mainly concentrated in mTOR, FoxO, and VEGF pathways. Protein-protein interaction (PPI) network analysis was performed with the detected DEGs. Eight down-regulated DEGs including akt1, bnip4, ptena, vegfaa, twsg1b, prkab1a, prkab1b, and pla2g4f.2 were validated by qRT-PCR and the results were consistent with the RNA-seq data. Overall, our work provides key insights into the influence of wnt16 gene on skeletal development.

Homozygous mutation of the Csf1r locus (Csf1rko) in mice, rats and humans leads to multiple postnatal developmental abnormalities. To enable analysis of the mechanisms underlying the phenotypic impacts of Csf1r mutation, we bred a rat Csf1rko allele to the inbred dark agouti (DA) genetic background and to a Csf1r-mApple reporter transgene. The Csf1rko led to almost complete loss of embryonic macrophages and ablation of most adult tissue macrophage populations. We extended previous analysis of the Csf1rko phenotype to early postnatal development to reveal impacts on musculoskeletal development and proliferation and morphogenesis in multiple organs. Expression profiling of 3-week old wild-type (WT) and Csf1rko livers identified 2760 differentially expressed genes associated with the loss of macrophages, severe hypoplasia, delayed hepatocyte maturation, disrupted lipid metabolism and the IGF1/IGF binding protein system. Older Csf1rko rats developed severe hepatic steatosis. Consistent with the developmental delay in the liver Csf1rko rats had greatly-reduced circulating IGF1. Transfer of WT bone marrow (BM) cells at weaning without conditioning repopulated resident macrophages in all organs, including microglia in the brain, and reversed the mutant phenotypes enabling long term survival and fertility. WT BM transfer restored osteoclasts, eliminated osteopetrosis, restored bone marrow cellularity and architecture and reversed granulocytosis and B cell deficiency. Csf1rko rats had an elevated circulating CSF1 concentration which was rapidly reduced to WT levels following BM transfer. However, CD43hi non-classical monocytes, absent in the Csf1rko, were not rescued and bone marrow progenitors remained unresponsive to CSF1. The results demonstrate that the Csf1rko phenotype is autonomous to BM-derived cells and indicate that BM contains a progenitor of tissue macrophages distinct from hematopoietic stem cells. The model provides a unique system in which to define the pathways of development of resident tissue macrophages and their local and systemic roles in growth and organ maturation., Competing Interests: The authors have declared that no competing interests exist.

Distal hereditary motor neuropathies (HMNs) and axonal Charcot-Marie-Tooth neuropathy (CMT2) are clinically and genetically heterogeneous diseases characterized primarily by motor neuron degeneration and distal weakness. The genetic cause for about half of the individuals affected by HMN/CMT2 remains unknown. Here, we report the identification of pathogenic variants in GBF1 (Golgi brefeldin A-resistant guanine nucleotide exchange factor 1) in four unrelated families with individuals affected by sporadic or dominant HMN/CMT2. Genomic sequencing analyses in seven affected individuals uncovered four distinct heterozygous GBF1 variants, two of which occurred de novo. Other known HMN/CMT2-implicated genes were excluded. Affected individuals show HMN/CMT2 with slowly progressive distal muscle weakness and musculoskeletal deformities. Electrophysiological studies confirmed axonal damage with chronic neurogenic changes. Three individuals had additional distal sensory loss. GBF1 encodes a guanine-nucleotide exchange factor that facilitates the activation of members of the ARF (ADP-ribosylation factor) family of small GTPases. GBF1 is mainly involved in the formation of coatomer protein complex (COPI) vesicles, maintenance and function of the Golgi apparatus, and mitochondria migration and positioning. We demonstrate that GBF1 is present in mouse spinal cord and muscle tissues and is particularly abundant in neuropathologically relevant sites, such as the motor neuron and the growth cone. Consistent with the described role of GBF1 in Golgi function and maintenance, we observed marked increase in Golgi fragmentation in primary fibroblasts derived from all affected individuals in this study. Our results not only reinforce the existing link between Golgi fragmentation and neurodegeneration but also demonstrate that pathogenic variants in GBF1 are associated with HMN/CMT2., (Copyright © 2020 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

PIK3CA-related overgrowth spectrum is caused by mosaicism mutations in the PIK3CA gene. These mutations, which are also observed in various types of cancer, lead to a constitutive activation of the PI3K/AKT/mTOR pathway, increasing cell proliferation. Heat shock transcription factor 1 (HSF1) is the major stress-responsive transcription factor. Recent findings indicate that AKT phosphorylates and activates HSF1 independently of heat-shock in breast cancer cells. Here, we aimed to investigate the role of HSF1 in PIK3CA-related overgrowth spectrum. We observed a higher rate of proliferation and increased phosphorylation of AKT and p70S6K in mutant fibroblasts than in control cells. We also found elevated phosphorylation and activation of HSF1, which is directly correlated to AKT activation. Specific AKT inhibitors inhibit HSF1 phosphorylation as well as HSF1-dependent gene transcription. Finally, we demonstrated that targeting HSF1 with specific inhibitors reduced the proliferation of mutant cells. As there is currently no curative treatment for PIK3CA-related overgrowth spectrum, our results identify HSF1 as a new potential therapeutic target., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

In humans, mutations in the PIEZO2 gene, which encodes for a mechanosensitive ion channel, were found to result in skeletal abnormalities including scoliosis and hip dysplasia. Here, we show in mice that loss of Piezo2 expression in the proprioceptive system recapitulates several human skeletal abnormalities. While loss of Piezo2 in chondrogenic or osteogenic lineages does not lead to human-like skeletal abnormalities, its loss in proprioceptive neurons leads to spine malalignment and hip dysplasia. To validate the non-autonomous role of proprioception in hip joint morphogenesis, we studied this process in mice mutant for proprioceptive system regulators Runx3 or Egr3. Loss of Runx3 in the peripheral nervous system, but not in skeletal lineages, leads to similar joint abnormalities, as does Egr3 loss of function. These findings expand the range of known regulatory roles of the proprioception system on the skeleton and provide a central component of the underlying molecular mechanism, namely Piezo2.

Skeletal dysplasias are a diverse group of heritable diseases affecting bone and cartilage growth. Throughout the years, the molecular defect underlying many of the diseases has been identified. These identifications led to novel insights in the mechanisms regulating bone and cartilage growth and homeostasis. One of the pathways that is clearly important during skeletal development and bone homeostasis is the Wingless and int-1 (WNT) signaling pathway. So far, three different WNT signaling pathways have been described, which are all activated by binding of the WNT ligands to the Frizzled (FZD) receptors. In this review, we discuss the skeletal disorders that are included in the latest nosology of skeletal disorders and that are caused by genetic defects involving the WNT signaling pathway. The number of skeletal disorders caused by defects in WNT signaling genes and the clinical phenotype associated with these disorders illustrate the importance of the WNT signaling pathway during skeletal development as well as later on in life to maintain bone mass. The knowledge gained through the identification of the genes underlying these monogenic conditions is used for the identification of novel therapeutic targets. For example, the genes underlying disorders with altered bone mass are all involved in the canonical WNT signaling pathway. Consequently, targeting this pathway is one of the major strategies to increase bone mass in patients with osteoporosis. In addition to increasing the insights in the pathways regulating skeletal development and bone homeostasis, knowledge of rare skeletal dysplasias can also be used to predict possible adverse effects of these novel drug targets. Therefore, this review gives an overview of the skeletal and extra-skeletal phenotype of the different skeletal disorders linked to the WNT signaling pathway., (Copyright © 2020 Huybrechts, Mortier, Boudin and Van Hul.)

Excess selenium entering the aquatic environment from anthropogenic activities has been associated with developmental abnormalities in fish including skeletal deformities of the head and spine. However, mechanisms of this developmental toxicity have not been well-characterized. In this study, Japanese medaka (Oryzias latipes) embryos were exposed to seleno-l-methionine (Se-Met) in a range of concentrations. Gene expression was evaluated for sex-determining region Y (SRY)-related box (Sox9a and Sox9b), runt-related transcription factor 2 (Runx2), and melatonin receptor (Mtr). Alterations in the length of Meckel's cartilage, tail curvature, and decreased calcification were observed in skeletal stains at 10- and 22-days post-fertilization (dpf). Embryonic exposure of Osterix-mCherry transgenic medaka resulted in fewer teeth. Sox9a and Sox9b were up-regulated, while Runx2 and Mtr were down-regulated by Se-Met prior to hatch. Whole mount in situ hybridization (WISH) localized gene expression to areas observed to be affected in vivo. In addition, Se-Met exposures of a Mtr morpholino (Mtr-MO) as well as Luzindole exposed embryos developed similar skeletal malformations, supporting involvement of Mtr. These findings demonstrate that Se-Met modulates expression of key genes involved in chondrogenic differentiation and bone formation during development., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Purpose: To determine the relationship between the bone formation-related functions of GPR126 and the structural asymmetry of spine in adolescent idiopathic scoliosis (AIS)., Methods: Vertebral body samples were obtained from 51 AIS patients during spinal surgery between October 2014 and November 2017, and the expression pattern of GPR126 in the convex/concave sides of AIS spine was identified by RT-qPCR. Next, we explored the bone formation-related functions of GPR126 by knocking down and overexpressing GPR126 in human mesenchymal stem cells (hMSC) and further performing osteogenic differentiation. We also applied overexpression of N-terminal fragments derived from GPR126 (GPR126-NTFs) and osteogenic differentiation experiments to determine the functional part of GPR126 in skeletal development., Results: We provided evidence that GPR126 showed a marked convex/concave asymmetric expression in the spine of AIS. Further RNA detection found that exon6-included transcripts of GPR126 (GPR126-exon6 in ) were significantly higher expressed in the convex side of AIS patients. Knocking down of GPR126 accelerated ossification of hMSCs during osteogenic differentiation, and overexpression of GPR126-exon6 in delayed this process. Overexpression of GPR126-NTFs revealed that NTF is a functional fragment and exon6-included NTF (NTF-exon6 in ) delayed ossification of hMSCs., Conclusion: Our findings indicated that GPR126-NTFs play a role in skeletal development, and the inclusion/exclusion of exon6 may regulate the bone formation-related functions of GPR126. The convex/concave asymmetric expression of GPR126-exon6 in may be an important factor in abnormal bone formation of AIS. These slides can be retrieved under Electronic Supplementary Material.

Background: The mutant chondrodysplasia (cho) is a cartilage-targeting disorder in C57BL mice that results in dwarfing and other malformations stemming from this collagenopathy. Clarke Fraser made the discovery of the mutation accidentally in the early 1960s during the thalidomide tragedy., Methods: For this review we identified key research on cho as since its discovery. Relevant data were compiled to make a comprehensive review that details discoveries associated with the cho mutation, that describes the associated phenotypes and molecular mechanisms, and that provides a discussion surrounding its current clinical relevance., Results: Mechanistically, cho acts by hindering chondrogenesis and endochondral bone formation. The phenotype results from a 1-nt deletion in the gene encoding the alpha 1 chain of type XI collagen. For more than half a century, researchers have studied the pathogenesis of the cho mutation in relation to a variety of mouse models of human birth defects and disease. These studies have resulted in several discoveries linking cho with such human disorders as dwarfism, tracheal stenosis, cleft palate, pulmonary hypoplasia, and osteoarthritis (OA)., Conclusion: The study of cho has led to numerous advances in understanding human birth defects, congenital disorders, and adult human disease. The most recent studies have suggested a role for the TGF-Beta, HtrA1, Ddr2, and Mmp-13 pathway in the degradation of articular cartilage and the development of OA in cho/+ mice. We have shown that the anti-hypertension drug Losartan is a TGF-Beta blocker that could be used to treat OA in Stickler syndrome, and thereby rescue the WT phenotype., (© 2019 Wiley Periodicals, Inc.)

Individuals with acromegaloid physical appearance or tall stature may be referred to endocrinologists to exclude growth hormone (GH) excess. While some of these subjects could be healthy individuals with normal variants of growth or physical traits, others will have acromegaly or pituitary gigantism, which are, in general, straightforward diagnoses upon assessment of the GH/IGF-1 axis. However, some patients with physical features resembling acromegaly - usually affecting the face and extremities -, or gigantism - accelerated growth/tall stature - will have no abnormalities in the GH axis. This scenario is termed pseudoacromegaly, and its correct diagnosis can be challenging due to the rarity and variability of these conditions, as well as due to significant overlap in their characteristics. In this review we aim to provide a comprehensive overview of pseudoacromegaly conditions, highlighting their similarities and differences with acromegaly and pituitary gigantism, to aid physicians with the diagnosis of patients with pseudoacromegaly., (Copyright © 2018 Elsevier Inc. All rights reserved.)

MiR-214 belongs to a family of microRNA (small, highly conserved noncoding RNA molecules) precursors that play a pivotal role in biological functions, such as cellular function, tissue development, tissue homeostasis, and pathogenesis of diseases. Recently, miR-214 emerged as a critical regulator of musculoskeletal metabolism. Specifically, miR-214 can mediate skeletal muscle myogenesis and vascular smooth muscle cell proliferation, migration, and differentiation. MiR-214 also modulates osteoblast function by targeting specific molecular pathways and the expression of various osteoblast-related genes; promotes osteoclast activity by targeting phosphatase and tensin homolog (Pten); and mediates osteoclast-osteoblast intercellular crosstalk via an exosomal miRNA paracrine mechanism. Importantly, dysregulation in miR-214 expression is associated with pathological bone conditions such as osteoporosis, osteosarcoma, multiple myeloma, and osteolytic bone metastasis of breast cancer. This review discusses the cellular targets of miR-214 in bone, the molecular mechanisms governing the activities of miR-214 in the musculoskeletal system, and the putative role of miR-214 in skeletal diseases. Understanding the biology of miR-214 could potentially lead to the development of miR-214 as a possible biomarker and a therapeutic target for musculoskeletal diseases., (© 2018 Wiley Periodicals, Inc.)

The most impressive phenotypic appearance of sirenomelia is the presence of a 180°-rotated, axially positioned, single lower limb. Associated gastrointestinal and genitourinary anomalies are almost always present. This rare anomaly is still the subject of ongoing controversies concerning its nosology, pathogenesis, and possible genetic etiology. Sirenomelia can be part of a syndromic continuum, overlapping with other complex conditions including caudal dysgenesis and VATER/VACTERL/VACTERL-H associations, which could all be part of a heterogeneous spectrum, and originate from an early defect in blastogenesis. It is imaginable that different "primary field defects," whether or not genetically based, induce a spectrum of caudal malformations. In the current study, we review the contemporary hypotheses and conceptual approaches regarding the etiology and pathogenesis of sirenomelia, especially in the context of concomitant conditions. To expand on the latter, we included the external and internal dysmorphology of one third trimester sirenomelic fetus from our anatomical museum collection, in which multiple concomitant but discordant anomalies were observed compared with classic sirenomelia, and was diagnosed as VACTERL-H association with sirenomelia. Birth Defects Research 109:791-804, 2017. © 2017 The Authors. Birth Defects Research Published by Wiley Periodicals, Inc., (© 2017 The Authors. Birth Defects Research Published by Wiley Periodicals, Inc.)

Bruck syndrome (BS) is a disorder characterized by joint flexion contractures and skeletal dysplasia that shows strong clinical overlap with the brittle bone disease osteogenesis imperfecta (OI). BS is caused by biallelic mutations in either the FKBP10 or the PLOD2 gene. PLOD2 encodes the lysyl hydroxylase 2 (LH2) enzyme, which is responsible for the hydroxylation of lysine residues in fibrillar collagen telopeptides. This hydroxylation directs crosslinking of collagen fibrils in the extracellular matrix, which is necessary to provide stability and tensile integrity to the collagen fibrils. To further elucidate the function of LH2 in vertebrate skeletal development, we created a zebrafish model harboring a homozygous plod2 nonsense mutation resulting in reduced telopeptide hydroxylation and crosslinking of bone type I collagen. Adult plod2 mutants present with a shortened body axis and severe skeletal abnormalities with evidence of bone fragility and fractures. The vertebral column of plod2 mutants is short and scoliotic with compressed vertebrae that show excessive bone formation at the vertebral end plates, and increased tissue mineral density in the vertebral centra. The muscle fibers of mutant zebrafish have a reduced diameter near the horizontal myoseptum. The endomysium, a layer of connective tissue ensheathing the individual muscle fibers, is enlarged. Transmission electron microscopy of mutant vertebral bone shows type I collagen fibrils that are less organized with loss of the typical plywood-like structure. In conclusion, plod2 mutant zebrafish show molecular and tissue abnormalities in the musculoskeletal system that are concordant with clinical findings in BS patients. Therefore, the plod2 zebrafish mutant is a promising model for the elucidation of the underlying pathogenetic mechanisms leading to BS and the development of novel therapeutic avenues in this syndrome. © 2016 American Society for Bone and Mineral Research., (© 2016 American Society for Bone and Mineral Research.)

Thrombospondins (TSPs) are widely known as a family of five calcium-binding matricellular proteins. While these proteins belong to the same family, they are encoded by different genes, regulate different cellular functions and are localized to specific regions of the body. TSP-5 or Cartilage Oligomeric Matrix Protein (COMP) is the only TSP that has been associated with skeletal disorders in humans, including pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED). The pentameric structure of COMP, the evidence that it interacts with multiple cellular proteins, and the recent reports of COMP acting as a 'lattice' to present growth factors to cells, inspired this review of COMP and its interacting partners. In our review, we have compiled the interactions of COMP with other proteins in the cartilage extracellular matrix and summarized their importance in maintaining the structural integrity of cartilage as well as in regulating cellular functions., (Copyright © 2014 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.)

Background: Congenital anomalies or birth defects are among the leading causes of infant mortality and morbidity around the world. The impact of congenital anomalies is particularly severe in middle- and low-income countries where health care resources are limited. The prevalence of congenital anomalies varies in different parts of the world, which could reflect different aetiological factors in different geographical regions., Methods: Between October 2012 and January 2013, a cross-sectional study was conducted involving young infants below 2 months of age, admitted at a university teaching hospital in Tanzania. Face-to-face interviews with parents/caretakers of young infants were carried out to collect socio-demographic and clinical information. Physical examinations were performed on all young infants. Echocardiography, X-ray, cranial as well as abdominal ultrasonographies were performed when indicated., Results: Analysis of the data showed that among 445 young infants enrolled in the study, the prevalence of congenital anomalies was 29%, with the Central Nervous System (CNS) as the most commonly affected organ system. Maternal factors that were significantly associated with congenital anomalies included the lack of peri-conceptional use of folic acid (OR = 3.1; 95% CI = 1.4-6.7; p = 0.005), a maternal age of above 35 years (OR = 2.2; 95% CI = 1.1-4.3; p = 0.024) and an inadequate attendance to antenatal clinic (OR = 2.1; 95% CI = 1.4-3.3; p < 0.001). Infant factors that were significantly associated with congenital anomalies were female sex, a birth weight of 2.5 kg or more, singleton pregnancy and a birth order above 4., Conclusions: Due to the high prevalence of congenital anomalies observed in this particular context, the hospital should mobilize additional resources for an optimal and timely management of the patients with congenital anomalies. In this study, the proportion of women taking folic acid supplements during early pregnancy was very low. Efforts should be made to ensure that more women use folic acid during the peri-conceptional period, as the use of folic acid supplement has been linked by several authors to a reduced occurrence of some congenital anomalies.

All TGF-beta family members have a prodomain that is important for secretion. Lack of secretion of a TGF-beta family member GDF5 is known to underlie some skeletal abnormalities, such as brachydactyly type C that is characterized by a huge and unexplained phenotypic variability. To search for potential phenotypic modifiers regulating secretion of GDF5, we compared cells overexpressing wild type (Wt) GDF5 and GDF5 with a novel mutation in the prodomain identified in a large Pakistani family with Brachydactyly type C and mild Grebe type chondrodyslplasia (c527T>C; p.Leu176Pro). Initial in vitro expression studies revealed that the p.Leu176Pro mutant (Mut) GDF5 was not secreted outside the cells. We subsequently showed that GDF5 was capable of forming a complex with latent transforming growth factor binding proteins, LTBP1 and LTBP2. Furthermore, secretion of LTBP1 and LTBP2 was severely impaired in cells expressing the Mut-GDF5 compared to Wt-GDF5. Finally, we demonstrated that secretion of Wt-GDF5 was inhibited by the Mut-GDF5, but only when LTBP (LTBP1 or LTBP2) was co-expressed. Based on these findings, we suggest a novel model, where the dosage of secretory co-factors or stabilizing proteins like LTBP1 and LTBP2 in the microenvironment may affect the extent of GDF5 secretion and thereby function as modifiers in phenotypes caused by GDF5 mutations.

To study pathological changes of fibromuscular system and the role of TGF-β1/Smad pathway in the urethra of a parturition-induced stress urinary incontinence (SUI) rat model. Twenty-eight 8-week-old Sprague-Dawley female rats at gestational day 16 were used and randomized into two groups: sham group and SUI group. After delivery, rats in the SUI group underwent postpartum vaginal balloon dilation and bilateral ovariectomy. 1 month after ovariectomy, urodynamics was assessed. Histological examination (Masson's trichrome stain, picrosirius red stain, Hart's elastin stain, Gordon & Sweet's stain, and immunohistochemical stain) and Western blot were performed on urethral tissues. Both leak point pressure and maximal bladder capacity were significantly decreased in the balloon-injured ovariectomized rats, compared with the sham rats. Muscle was significantly decreased in the urethra of SUI rats compare with sham rats. Collagen I/III and reticular fibers from SUI group were also significantly lower than sham group. Meanwhile, elastic fibers and reticular fibers showed fragmentation and disorganization indicating impairment in the fibromuscular system in SUI rats. TGF-β1, MMP-9, and phosphorylated Smad2 (p-Smad2) were expressed significantly higher in SUI than in sham rats. Simulated birth trauma and menopause induced an upregulation of the TGF-β1/Smad pathway and impairment of the fibromuscular system in the urethra.

Runx2 is a transcription factor controlling skeletal development, and is also expressed in extraskeletal tissues where its function is not well understood. Existing Runx2 mutant and transgenic mouse models do not allow the necessary control of Runx2 expression to understand its functions in different tissues. We generated conditional, doxycyline-inducible, triple transgenic mice (CMV-Cre;ROSA26-neo(flox/+)-rtTA;Tet-O-Runx2) to investigate the effects of wide spread overexpression of Runx2. Osteoblasts isolated from CMV-Cre;ROSA26-neo(flox/+)-rtTA; Tet-O-Runx2 mice demonstrated a dose-dependent effect of doxycycline to stimulate Runx2 transgene expression. Doxycycline administration to CMV-Cre;ROSA26-neo(flox/+)-rtTA;Tet-O-Runx2 mice induced Runx2 transgene expression in all tissues tested, with the highest levels observed in kidney, ovary, and bone. Runx2 overexpression resulted in deceased body size and reduced viability. With regard to bone, Runx2 overexpressing mice paradoxically displayed profound osteopenia and diminished osteogenesis. Induced expression of Runx2 in extraskeletal tissues resulted in ectopic calcification and induction of the osteogenic program in a limited number of tissues, including lung and muscle. In addition, the triple transgenic mice showed evidence of a myeloproliferative disorder and an apparent inhibition of lymphocyte development. Thus, overexpression of Runx2 both within and outside of the skeleton can have diverse biological effects. Use of tissue specific Cre mice will allow this model to be used to conditionally and inducibly overexpress Runx2 in different tissues and provide a means to study the post-natal tissue- and cell context-dependent functions of Runx2., (Copyright © 2010 Wiley-Liss, Inc.)

High-dose bolus exposure of rats to ethylene glycol (EG) causes developmental toxicity mediated by a metabolite, glycolic acid (GA), whose levels increase disproportionately when its metabolism is saturated. However, low-level exposures that do not saturate GA metabolism have a low potential for developmental effects. Toward the goal of developing EG risk assessments based on internal dose metrics, this study examined the differences between fast (bolus) and slow (continuous infusion) dose-rate exposures to EG on developmental outcome and pharmacokinetics. Time-mated female CD rats received sc bolus injections of 0, 1000, or 2000 mg/kg/day of EG on gestation day (GD) 6-15 once daily, whereas three corresponding groups were given the same daily doses as an infusion administered continuously from GD 6-15 via an sc implantable pump. In the sc bolus groups, increases in 11 fetal malformations (major defects) and 12 variations (minor alterations) were seen at the 2000 mg/kg/day dose level, whereas increases in 2 malformations and 2 variations occurred at 1000 mg/kg/day. In contrast, equivalent daily doses of EG given slowly via infusion did not cause any developmental effects. A pharmacokinetics time course was then conducted to compare GD 11-12 kinetics from oral bolus (gavage) exposure versus sc infusion of EG. Although dose rate had a modest impact (8- to 11-fold difference) on peak EG levels, peak levels of GA in maternal blood, kidney, embryo, and exocoelomic fluid were 59, 100, 49, and 56 times higher, respectively, following gavage versus the same dose given by infusion. These data illustrate how high-dose bolus exposure to EG causes a dramatic shift to nonlinear GA kinetics, an event which is highly unlikely to occur following exposures to humans associated with consumer and worker uses.

Down syndrome has a prevalence of one in 500 to one in 1,000 live births and is the most common cause of mental retardation. Most patients are treated in childhood and adolescence for mental or growth retardation. Studies that evaluate bone mass in Down syndrome are limited, and many are small case series in pediatric and adult populations who live either in the community or in residential institutions. Several environmental and hormonal factors contribute to low bone mineral density in such patients. Muscle hypotonia, low amounts of physical activity, poor calcium and vitamin D intakes, hypogonadism, growth retardation and thyroid dysfunction contribute to substantial impairments in skeletal maturation and bone-mass accrual that predispose these patients to fragility fractures. Here, we review indications and limitations of bone-mass measurements in children, summarize the endocrine and skeletal abnormalities in patients presenting with Down syndrome, and review studies that investigate therapeutic strategies for such patients.

Here, we present evidence that Lrp6, a coreceptor for Wnt ligands, is required for the normal formation of somites and bones. By positional cloning, we demonstrate that a novel spontaneous mutation ringelschwanz (rs) in the mouse is caused by a point mutation in Lrp6, leading to an amino acid substitution of tryptophan for the evolutionarily conserved residue arginine at codon 886 (R886W). We show that rs is a hypomorphic Lrp6 allele by a genetic complementation test with Lrp6-null mice, and that the mutated protein cannot efficiently transduce signals through the Wnt/beta-catenin pathway. Homozygous rs mice, many of which are remarkably viable, exhibit a combination of multiple Wnt-deficient phenotypes, including dysmorphologies of the axial skeleton, digits and the neural tube. The establishment of the anteroposterior somite compartments, the epithelialization of nascent somites, and the formation of segment borders are disturbed in rs mutants, leading to a characteristic form of vertebral malformations, similar to dysmorphologies in individuals suffering from spondylocostal dysostosis. Marker expression study suggests that Lrp6 is required for the crosstalk between the Wnt and notch-delta signaling pathways during somitogenesis. Furthermore, the Lrp6 dysfunction in rs leads to delayed ossification at birth and to a low bone mass phenotype in adults. Together, we propose that Lrp6 is one of the key genetic components for the pathogenesis of vertebral segmentation defects and of osteoporosis in humans.

Identifying the molecular basis for genotype-phenotype correlations in human diseases has direct implications for understanding the disease process and hence for the identification of potential therapeutic targets. To this end, we performed microarray expression analysis on benign (pheochromocytomas) and malignant (medullary thyroid carcinomas, MTCs) tumors from patients with multiple endocrine neoplasia (MEN) type 2A or 2B, related syndromes that result from distinctive mutations in the RET receptor tyrosine kinase. Comparisons of MEN 2B and MEN 2A MTCs revealed that genes involved in the process of epithelial to mesenchymal transition, many associated with the tumor growth factor beta pathway, were up-regulated in MEN 2B MTCs. This MEN 2B MTC profile may explain the early onset of malignancy in MEN 2B compared with MEN 2A patients. Furthermore, chondromodulin-1, a known regulator of cartilage and bone growth, was expressed at high levels specifically in MEN 2B MTCs. Chondromodulin-1 mRNA and protein expression was localized to the malignant C cells, and its high expression was directly associated with the presence of skeletal abnormalities in MEN 2B patients. These findings provide molecular evidence that associate the previously unexplained skeletal abnormalities and early malignancy in MEN 2B compared with MEN 2A syndrome.