Mutations in LIFR rewire the JAK/STAT signaling pathway: A study unveiling mechanistic details of Stüve-Wiedemann syndrome.

Stüve-Wiedemann syndrome (SWS), a rare autosomal recessive disorder, characterized by diminutive size, curvature of the elongated bones, bent fingers, episodes of heightened body temperature, respiratory distress or periods of breath-holding, and challenges with feeding, especially causes fatality in infants. SWS is an outcome of potential missense mutations in the leukemia inhibitory factor receptor gene reflected as numerous amino acid mutations at protein level. Employing in silico tools and techniques like mutational screening with Pred_MutHTP, I-Mutant2.0, PANTHER.db, PolyPhen, to classify mutations as deleterious/destabilizing, in conjunction with experimental data analysis, P136A and S279P emerged as 'effect'-causing mutations. Pre-existing knowledge suggests, SWS progression is effectuated conformationally altered and dysfunctional LIFR, unable to bind to LIF and further form the LIF/LIFR/gp130 signalling complex. To gain functional insights into the effect of the said mutations on the wild type protein, an all-atom, explicit, solvent molecular dynamics simulation was performed following docking approaches. Consequently, referring to the RMSD, RMSF, protein dynamic network analysis, energy landscape plots and domain motion analysis, it was revealed that unbound LIFR_WT was more prone to LIF binding as usual whereas the mutants exhibited considerable domain closure to inhibit LIF binding. We conducted binding affinity analysis via MM/GBSA and dissociation constant estimation after LIFR-LIF docking and found the WT_complex to be more stable and compact as a whole when compared to the flexible mutant complexes thus being associated with SWS. Our study offers a route for understanding molecular level implications upon LIFR mutations which opens an avenue for therapeutic interventions.

Osteochondroma formation is independent of heparanase expression as revealed in a mouse model of hereditary multiple exostoses.

Hereditary multiple exostoses (HME) is a rare, pediatric disorder characterized by osteochondromas that form along growth plates and provoke significant musculoskeletal problems. HME is caused by mutations in heparan sulfate (HS)-synthesizing enzymes EXT1 or EXT2. Seemingly paradoxically, osteochondromas were found to contain excessive extracellular heparanase (Hpse) that could further reduce HS levels and exacerbate pathogenesis. To test Hpse roles, we asked whether its ablation would protect against osteochondroma formation in a conditional HME model consisting of mice bearing floxed Ext1 alleles in Agr-CreER background (Ext1f/f ;Agr-CreER mice). Mice were crossed with a new global Hpse-null (Hpse-/- ) mice to produce compound Hpse-/- ;Ext1f/f ;Agr-CreER mice. Tamoxifen injection of standard juvenile Ext1f/f ;Agr-CreER mice elicited stochastic Ext1 ablation in growth plate and perichondrium, followed by osteochondroma formation, as revealed by microcomputed tomography and histochemistry. When we examined companion conditional Ext1-deficient mice lacking Hpse also, we detected no major decreases in osteochondroma number, skeletal distribution, and overall structure by the analytical criteria above. The Ext1 mutants used here closely mimic human HME pathogenesis, but have not been previously tested for responsiveness to treatments. To exclude some innate therapeutic resistance in this stochastic model, tamoxifen-injected Ext1f/f ;Agr-CreER mice were administered daily doses of the retinoid Palovarotene, previously shown to prevent ectopic cartilage and bone formation in other mouse disease models. This treatment did inhibit osteochondroma formation compared with vehicle-treated mice. Our data indicate that heparanase is not a major factor in osteochondroma initiation and accumulation in mice. Possible roles of heparanase upregulation in disease severity in patients are discussed.

A Novel Intronic Splicing Mutation in the EXT2 Gene of a Chinese Family with Multiple Osteochondroma.

Background: Multiple osteochondroma (MO), an autosomal dominant genetic disease, is caused by heterozygous mutations in the EXT1 and EXT2 genes. Approximately 80% of pathogenic mutations are nonsense/missense mutations, small indels, and splicing mutations. Splicing mutations, particularly at the 3' and 5' splice sites, disrupt normal mRNA processing and cause exon skipping or aberrant splicing, ultimately resulting in protein truncation and loss of function. Methods: Polymerase chain reaction (PCR) and Sanger sequencing were applied to detect subtle mutations in a Chinese family with MO, the pathogenicity of a splicing variant was predicted by bioinformatics and further verified using a minigene splicing assay. Results: A novel and heterozygous splicing mutation, c.626 + 2_626 + 5delTAGG, was identified in the EXT2 gene of the proband and the father by PCR and Sanger sequencing, whereas the unaffected mother and brother had wild-type alleles at the same site. Bioinformatics predicted that the 5' splicing site of exon 3 in the EXT2 gene was destroyed due to this mutation. A hybrid minigene splicing assay (HMSA) indicated that the mutation disturbed the normal splicing of the EXT2 gene mRNA and led to a deletion of 79 bp at the 5' end of exon 3, which resulted in aberrant splicing of exon 3 and introduced an earlier stop codon in the EXT2 gene. Conclusion: A novel splicing mutation was identified that produced the MO phenotype through aberrant splicing in a Chinese family. This observation, expands our knowledge of the spectrum of molecular pathogenic mechanisms leading to aberrant mRNA splicing.

Identification of Novel Mutations in the EXT1 and EXT2 Genes of Chinese Patients with Hereditary Multiple Osteochondromas.

Aim: To detect mutations in the EXT1 and EXT2 genes in four Chinese families with hereditary multiple osteochondromas (HMO). HMO is an autosomal dominant disorder characterized by the overgrowth of multiple cartilage-capped bones in the metaphysis of long bones and flat bones. Methods: Polymerase chain reaction-based amplification followed by DNA sequencing of the complete coding sequences of EXT1 and EXT2 was performed for four Chinese families with HMO. Results: The mutant allele was found in six patients: three mutations were found in EXT1 and two in EXT2. A novel frameshift mutation, which generates a premature stop codon at codon 586 and causes partial loss of the glycosyltransferase domain, was detected in exon 9 of EXT1 (F579Yfs*8). We hypothesize that F579Yfs*8 is a pathogenic mutation. Two novel missense mutations (G339S and V545D) were found in EXT1. The variant c.1634T>A (V545D) is apparently benign. In addition we found a novel deletion mutation in EXT2, c.856_864 del TTCCTCCTG, which results in the deletion of 286Phe, 287Leu, and 288Leu, that is likely pathogenic. Finally, we identified a likely benign variant in exon 13 of EXT2. c.2035-41T>C (rs3740878). Conclusions: We found three novel, potentially pathogenic mutations in EXT1 and EXT2, including a novel frameshift mutation. More importantly, our study results have expanded the spectrum of EXT mutations conducive to the genetic diagnosis and counseling of patients with HMO.

Absence of GP130 cytokine receptor signaling causes extended Stüve-Wiedemann syndrome.

The gene IL6ST encodes GP130, the common signal transducer of the IL-6 cytokine family consisting of 10 cytokines. Previous studies have identified cytokine-selective IL6ST defects that preserve LIF signaling. We describe three unrelated families with at least five affected individuals who presented with lethal Stüve-Wiedemann-like syndrome characterized by skeletal dysplasia and neonatal lung dysfunction with additional features such as congenital thrombocytopenia, eczematoid dermatitis, renal abnormalities, and defective acute-phase response. We identified essential loss-of-function variants in IL6ST (a homozygous nonsense variant and a homozygous intronic splice variant with exon skipping). Functional tests showed absent cellular responses to GP130-dependent cytokines including IL-6, IL-11, IL-27, oncostatin M (OSM), and leukemia inhibitory factor (LIF). Genetic reconstitution of GP130 by lentiviral transduction in patient-derived cells reversed the signaling defect. This study identifies a new genetic syndrome caused by the complete lack of signaling of a whole family of GP130-dependent cytokines in humans and highlights the importance of the LIF signaling pathway in pre- and perinatal development.

Bone development and remodeling in metabolic disorders.

There are many metabolic disorders that present with bone phenotypes. In some cases, the pathological bone symptoms are the main features of the disease whereas in others they are a secondary characteristic. In general, the generation of the bone problems in these disorders is not well understood and the therapeutic options for them are scarce. Bone development occurs in the early stages of embryonic development where the bone formation, or osteogenesis, takes place. This osteogenesis can be produced through the direct transformation of the pre-existing mesenchymal cells into bone tissue (intramembranous ossification) or by the replacement of the cartilage by bone (endochondral ossification). In contrast, bone remodeling takes place during the bone's growth, after the bone development, and continues throughout the whole life. The remodeling involves the removal of mineralized bone by osteoclasts followed by the formation of bone matrix by the osteoblasts, which subsequently becomes mineralized. In some metabolic diseases, bone pathological features are associated with bone development problems but in others they are associated with bone remodeling. Here, we describe three examples of impaired bone development or remodeling in metabolic diseases, including work by others and the results from our research. In particular, we will focus on hereditary multiple exostosis (or osteochondromatosis), Gaucher disease, and the susceptibility to atypical femoral fracture in patients treated with bisphosphonates for several years.

A novel splice mutation induces exon skipping of the EXT1 gene in patients with hereditary multiple exostoses.

The molecular mechanism of hereditary multiple exostoses (HME) remains ambiguous and a limited number of studies have investigated the pathogenic mechanism of mutations in patients with HME. In the present study, a novel heterozygous splice mutation (c.1284+2del) in exostosin glycosyltransferase 1 (EXT1) gene was identified in a three­generation family with HME. Bioinformatics and TA clone­sequencing indicated that the splice site mutation would result in exon 4 skipping. Reverse transcription­quantitative polymerase chain reaction (RT­qPCR) revealed that the expression levels of wild­type EXT1/EXT2 mRNA in patients with HME were significantly decreased, compared with normal control participants (P<0.05). Abnormal EXT1 transcript lacking exon 4 (EXT1­DEL) and full­length EXT1 mRNA (EXT1­FL) were overexpressed in 293­T cells and Cos­7 cells using lentivirus infection. RT­qPCR demonstrated that the expression level of EXT1­DEL was significantly increased, compared with EXT1­FL (17.032 vs. 6.309, respectively; P<0.05). The protein encoded by EXT1­DEL was detected by western blot analysis, and the level was increased, compared with EXT1­FL protein expression. Immunofluorescence indicated that the protein encoded by EXT1­DEL was located in the cytoplasm of Cos­7 cells, which was consistent with the localization of the EXT1­FL protein. In conclusion, the present study identified a novel splice mutation that causes exon 4 skipping during mRNA splicing and causes reduced expression of EXT1/EXT2. The mutation in EXT1­DEL generated a unique peptide that is located in the cytoplasm in vitro, and it expands the mutation spectrum and provides molecular genetic evidence for a novel pathogenic mechanism of HME.

Heparan sulfate antagonism alters bone morphogenetic protein signaling and receptor dynamics, suggesting a mechanism in hereditary multiple exostoses.

Hereditary multiple exostoses (HME) is a pediatric disorder caused by heparan sulfate (HS) deficiency and is characterized by growth plate-associated osteochondromas. Previously, we found that osteochondroma formation in mouse models is preceded by ectopic bone morphogenetic protein (BMP) signaling in the perichondrium, but the mechanistic relationships between BMP signaling and HS deficiency remain unclear. Therefore, we used an HS antagonist (surfen) to investigate the effects of this HS interference on BMP signaling, ligand availability, cell-surface BMP receptor (BMPR) dynamics, and BMPR interactions in Ad-293 and C3H/10T1/2 cells. As observed previously, the HS interference rapidly increased phosphorylated SMAD family member 1/5/8 levels. FACS analysis and immunoblots revealed that the cells possessed appreciable levels of endogenous cell-surface BMP2/4 that were unaffected by the HS antagonist, suggesting that BMP2/4 proteins remained surface-bound but became engaged in BMPR interactions and SMAD signaling. Indeed, surface mobility of SNAP-tagged BMPRII, measured by fluorescence recovery after photobleaching (FRAP), was modulated during the drug treatment. This suggested that the receptors had transitioned to lipid rafts acting as signaling centers, confirmed for BMPRII via ultracentrifugation to separate membrane subdomains. In situ proximity ligation assays disclosed that the HS interference rapidly stimulates BMPRI-BMPRII interactions, measured by oligonucleotide-driven amplification signals. Our in vitro studies reveal that cell-associated HS controls BMP ligand availability and BMPR dynamics, interactions, and signaling, and largely restrains these processes. We propose that HS deficiency in HME may lead to extensive local BMP signaling and altered BMPR dynamics, triggering excessive cellular responses and osteochondroma formation.

Signaling systems affecting the severity of multiple osteochondromas.

Multiple osteochondromas (MO) syndrome is a dominant autosomal bone disorder characterized by the formation of cartilage-capped bony outgrowths that develop at the juxtaposition of the growth plate of endochondral bones. MO has been linked to mutations in either EXT1 or EXT2, two glycosyltransferases required for the synthesis of heparan sulfate (HS). The establishment of mouse mutants demonstrated that a clonal, homozygous loss of Ext1 in a wild type background leads to the development of osteochondromas. Here we investigate mechanisms that might contribute to the variation in the severity of the disease observed in human patients. Our results show that residual amounts of HS are sufficient to prevent the development of osteochondromas strongly supporting that loss of heterozygosity is required for osteochondroma formation. Furthermore, we demonstrate that different signaling pathways affect size and frequency of the osteochondromas thereby modulating the severity of the disease. Reduced Fgfr3 signaling, which regulates proliferation and differentiation of chondrocytes, increases osteochondroma number, while activated Fgfr3 signaling reduces osteochondroma size. Both, activation and reduction of Wnt/ß-catenin signaling decrease osteochondroma size and frequency by interfering with the chondrogenic fate of the mutant cells. Reduced Ihh signaling does not change the development of the osteochondromas, while elevated Ihh signaling increases the cellularity and inhibits chondrocyte differentiation in a subset of osteochondromas and might thus predispose osteochondromas to the transformation into chondrosarcomas.

Advances in the pathogenesis and possible treatments for multiple hereditary exostoses from the 2016 international MHE conference.

Multiple hereditary exostoses (MHE) is an autosomal dominant disorder that affects about 1 in 50,000 children worldwide. MHE, also known as hereditary multiple exostoses (HME) or multiple osteochondromas (MO), is characterized by cartilage-capped outgrowths called osteochondromas that develop adjacent to the growth plates of skeletal elements in young patients. These benign tumors can affect growth plate function, leading to skeletal growth retardation, or deformations, and can encroach on nerves, tendons, muscles, and other surrounding tissues and cause motion impairment, chronic pain, and early onset osteoarthritis. In about 2-5% of patients, the osteochondromas can become malignant and life threatening. Current treatments consist of surgical removal of the most symptomatic tumors and correction of the major skeletal defects, but physical difficulties and chronic pain usually continue and patients may undergo multiple surgeries throughout life. Thus, there is an urgent need to find new treatments to prevent or reverse osteochondroma formation. The 2016 International MHE Research Conference was convened to provide a forum for the presentation of the most up-to-date and advanced clinical and basic science data and insights in MHE and related fields; to stimulate the forging of new perspectives, collaborations, and venues of research; and to publicize key scientific findings within the biomedical research community and share insights and relevant information with MHE patients and their families. This report provides a description, review, and assessment of all the exciting and promising studies presented at the Conference and delineates a general roadmap for future MHE research targets and goals.

Palovarotene Inhibits Osteochondroma Formation in a Mouse Model of Multiple Hereditary Exostoses.

Multiple hereditary exostoses (MHE), also known as multiple osteochondromas (MO), is an autosomal dominant disorder characterized by the development of multiple cartilage-capped bone tumors (osteochondromas). The large majority of patients with MHE carry loss-of-function mutations in the EXT1 or EXT2 gene, which encodes a glycosyltransferase essential for heparan sulfate (HS) biosynthesis. Increasing evidence suggests that enhanced bone morphogenetic protein (BMP) signaling resulting from loss of HS expression plays a role in osteochondroma formation in MHE. Palovarotene (PVO) is a retinoic acid receptor γ selective agonist, which is being investigated as a potential drug for fibrodysplasia ossificans progressiva (FOP), another genetic bone disorder with features that overlap with those of MHE. Here we show that PVO inhibits osteochondroma formation in the Fsp1Cre ;Ext1flox/flox model of MHE. Four-week daily treatment with PVO starting at postnatal day (P) 14 reduced the number of osteochondromas that develop in these mice by up to 91% in a dose-dependent manner. An inhibition of long bone growth observed in animals treated from P14 was almost entirely abrogated by delaying the initiation of treatment to P21. We also found that PVO attenuates BMP signaling in Fsp1Cre ;Ext1flox/flox mice and that aberrant chondrogenic fate determination of Ext1-deficient perichondrial progenitor cells in these mice is restored by PVO. Together, the present data support further preclinical and clinical investigations of PVO as a potential therapeutic agent for MHE. © 2017 American Society for Bone and Mineral Research.

The pathogenic roles of heparan sulfate deficiency in hereditary multiple exostoses.

Heparan sulfate (HS) is an essential component of cell surface and matrix proteoglycans (HS-PGs) that include syndecans and perlecan. Because of their unique structural features, the HS chains are able to specifically interact with signaling proteins -including bone morphogenetic proteins (BMPs)- via their HS-binding domain, regulating protein availability, distribution and action on target cells. Hereditary Multiple Exostoses (HME) is a rare pediatric disorder linked to germline heterozygous loss-of-function mutations in EXT1 or EXT2 that encode Golgi-resident glycosyltransferases responsible for HS synthesis, resulting in a systemic HS deficiency. HME is characterized by cartilaginous/bony tumors -called osteochondromas or exostoses- that form within perichondrium in long bones, ribs and other elements. This review examines most recent studies in HME, framing them in the context of classic studies. New findings show that the spectrum of EXT mutations is larger than previously realized and the clinical complications of HME extend beyond the skeleton. Osteochondroma development requires a somatic "second hit" that would complement the germline EXT mutation to further decrease HS production and/levels at perichondrial sites of osteochondroma induction. Cellular studies have shown that the steep decreases in local HS levels: derange the normal homeostatic signaling pathways keeping perichondrium mesenchymal; cause excessive BMP signaling; and provoke ectopic chondrogenesis and osteochondroma formation. Data from HME mouse models have revealed that systemic treatment with a BMP signaling antagonist markedly reduces osteochondroma formation. In sum, recent studies have provided major new insights into the molecular and cellular pathogenesis of HME and the roles played by HS deficiency. These new insights have led to the first ever proof-of-principle demonstration that osteochondroma formation is a druggable process, paving the way toward the creation of a clinically-relevant treatment.

Transcriptome Analysis Revealed Impaired cAMP Responsiveness in PHF21A-Deficient Human Cells.

Potocki-Shaffer Syndrome is a rare neurodevelopmental syndrome associated with microdeletion of a region of Chromosome 11p11.2. Genetic evidence has implicated haploinsufficiency of PHF21A, a gene that encodes a histone-binding protein, as the likely cause of intellectual disability and craniofacial abnormalities in Potocki-Shaffer Syndrome. However, the molecular consequences of reduced PHF21A expression remain elusive. In this study, we analyzed by RNA-Sequencing (RNA-Seq) two patient-derived cell lines with heterozygous loss of PHF21A compared to unaffected individuals and identified 1,885 genes that were commonly misregulated. The patient cells displayed down-regulation of key pathways relevant to learning and memory, including Cyclic Adenosine Monophosphate (cAMP)-signaling pathway genes. We found that PHF21A is required for full induction of a luciferase reporter carrying cAMP-responsive elements (CRE) following stimulation by the cAMP analog, forskolin. Finally, PHF21A-deficient patient-derived cells exhibited a delayed induction of immediate early genes following forskolin stimulation. These results suggest that an impaired response to cAMP signaling might be involved in the pathology of PHF21A deficiency. This article is part of a Special Issue entitled: [SI: Molecules & Cognition].

Interactions of signaling proteins, growth factors and other proteins with heparan sulfate: mechanisms and mysteries.

Heparan sulfate (HS) is a component of cell surface and matrix-associated proteoglycans (HSPGs) that, collectively, play crucial roles in many physiologic processes including cell differentiation, organ morphogenesis and cancer. A key function of HS is to bind and interact with signaling proteins, growth factors, plasma proteins, immune-modulators and other factors. In doing so, the HS chains and HSPGs are able to regulate protein distribution, bio-availability and action on target cells and can also serve as cell surface co-receptors, facilitating ligand-receptor interactions. These proteins contain an HS/heparin-binding domain (HBD) that mediates their association and contacts with HS. HBDs are highly diverse in sequence and predicted structure, contain clusters of basic amino acids (Lys and Arg) and possess an overall net positive charge, most often within a consensus Cardin-Weintraub (CW) motif. Interestingly, other domains and residues are now known to influence protein-HS interactions, as well as interactions with other glycosaminoglycans, such as chondroitin sulfate. In this review, we provide a description and analysis of HBDs in proteins including amphiregulin, fibroblast growth factor family members, heparanase, sclerostin and hedgehog protein family members. We discuss HBD structural and functional features and important roles carried out by other protein domains, and also provide novel conformational insights into the diversity of CW motifs present in Sonic, Indian and Desert hedgehogs. Finally, we review progress in understanding the pathogenesis of a rare pediatric skeletal disorder, Hereditary Multiple Exostoses (HME), characterized by HS deficiency and cartilage tumor formation. Advances in understanding protein-HS interactions will have broad implications for basic biology and translational medicine as well as for the development of HS-based therapeutics.

Heparanase stimulates chondrogenesis and is up-regulated in human ectopic cartilage: a mechanism possibly involved in hereditary multiple exostoses.

Hereditary multiple exostoses is a pediatric skeletal disorder characterized by benign cartilaginous tumors called exostoses that form next to growing skeletal elements. Hereditary multiple exostoses patients carry heterozygous mutations in the heparan sulfate (HS)-synthesizing enzymes EXT1 or EXT2, but studies suggest that EXT haploinsufficiency and ensuing partial HS deficiency are insufficient for exostosis formation. Searching for additional pathways, we analyzed presence and distribution of heparanase in human exostoses. Heparanase was readily detectable in most chondrocytes, particularly in cell clusters. In control growth plates from unaffected persons, however, heparanase was detectable only in hypertrophic zone. Treatment of mouse embryo limb mesenchymal micromass cultures with exogenous heparanase greatly stimulated chondrogenesis and bone morphogenetic protein signaling as revealed by Smad1/5/8 phosphorylation. It also stimulated cell migration and proliferation. Interfering with HS function both with the chemical antagonist Surfen or treatment with bacterial heparitinase up-regulated endogenous heparanase gene expression, suggesting a counterintuitive feedback mechanism that would result in further HS reduction and increased signaling. Thus, we tested a potent heparanase inhibitor (SST0001), which strongly inhibited chondrogenesis. Our data clearly indicate that heparanase is able to stimulate chondrogenesis, bone morphogenetic protein signaling, cell migration, and cell proliferation in chondrogenic cells. These properties may allow heparanase to play a role in exostosis genesis and pathogenesis, thus making it a conceivable therapeutic target in hereditary multiple exostoses.

Effects of leukemia inhibitory receptor gene mutations on human hypothalamo-pituitary-adrenal function.

BACKGROUND: Stuve-Wiedemann syndrome (STWS) (MIM #601559) is a rare autosomal recessive disorder caused by mutations in the leukemia inhibitory factor receptor (LIFR) gene. STWS has a diverse range of clinical features involving hematopoietic, skeletal, neuronal and immune systems. STWS manifests a high mortality due to increased risk of sudden death. Heterodimerization of the LIFR mediates leukemia inhibitory factor (LIF) signalling through the intracellular Janus kinase (JAK)/STAT3 signalling cascade. The LIF/LIFR system is highly expressed in and regulates the hypothalamo-pituitary-adrenal (HPA) axis. OBJECTIVES: HPA function was investigated in three STWS patients to characterise consequences of impaired LIF/LIFR signalling on adrenal function. DESIGN: Six genetically proven STWS patients from four unrelated Turkish families were included in the study. Sudden death occurred in three before 2 years of age. Basal adrenal function tests were performed by measurement of early morning serum cortisol and plasma ACTH concentrations on at least two different occasions. Low dose synacthen stimulation test and glucagon stimulation tests were performed to explore adrenal function in three patients who survived. RESULTS: All patients carried the same LIFR (p.Arg692X) mutation. Our oldest patient had attenuated morning serum cortisol and plasma ACTH levels at repeated measurements. Two of three patients had attenuated cortisol response (<18 µg/dl) to glucagon, one of whom also had borderline cortisol response to low dose (1 µg) ACTH stimulation consistent with central adrenal insufficiency. CONCLUSIONS: STWS patients may develop central adrenal insufficiency due to impaired LIF/LIFR signalling. LIF/LIFR system plays a role in human HPA axis regulation.

Carriers of loss-of-function mutations in EXT display impaired pancreatic beta-cell reserve due to smaller pancreas volume.

Exotosin (EXT) proteins are involved in the chain elongation step of heparan sulfate (HS) biosynthesis, which is intricately involved in organ development. Loss of function mutations (LOF) in EXT1 and EXT2 result in hereditary exostoses (HME). Interestingly, HS plays a role in pancreas development and beta-cell function, and genetic variations in EXT2 are associated with an increased risk for type 2 diabetes mellitus. We hypothesized that loss of function of EXT1 or EXT2 in subjects with hereditary multiple exostoses (HME) affects pancreatic insulin secretion capacity and development. We performed an oral glucose tolerance test (OGTT) followed by hyperglycemic clamps to investigate first-phase glucose-stimulated insulin secretion (GSIS) in HME patients and age and gender matched non-affected relatives. Pancreas volume was assessed with magnetic resonance imaging (MRI). OGTT did not reveal significant differences in glucose disposal, but there was a markedly lower GSIS in HME subjects during hyperglycemic clamp (iAUC HME: 0.72 [0.46-1.16] vs. controls 1.53 [0.69-3.36] nmol·l-1·min-1, p<0.05). Maximal insulin response following arginine challenge was also significantly attenuated (iAUC HME: 7.14 [4.22-10.5] vs. controls 10.2 [7.91-12.70] nmol·l-1·min-1 p<0.05), indicative of an impaired beta-cell reserve. MRI revealed a significantly smaller pancreatic volume in HME subjects (HME: 72.0±15.8 vs. controls 96.5±26.0 cm3 p = 0.04). In conclusion, loss of function of EXT proteins may affect beta-cell mass and insulin secretion capacity in humans, and render subjects at a higher risk of developing type 2 diabetes when exposed to environmental risk factors.

Immunohistochemical Localization of Bone Morphogenetic Proteins (BMPs) and their Receptors in Solitary and Multiple Human Osteochondromas.

The expression of bone morphogenetic proteins (BMPs) and their cognate receptors (BMPRs) in osteochondromas has not been investigated. We determined the immunohistochemical localization and distribution of BMP-2/4, -6 and -7; BMP receptors BMPR-1A, BMPR-1B and BMPR-2; signal transducing proteins phosphorylated Smad1/5/8; and BMP antagonist noggin in the cartilaginous cap of solitary (SO) and multiple (MO) human osteochondromas and compared these with bovine growth plate and articular cartilage. The distribution and localization patterns for BMP-6, BMP-7, BMPR-1A and BMPR-2 were similar between the cartilaginous cap and the growth plate. BMP-2/4 and BMPR-1B were present throughout the growth plate. However, BMP-2/4 and phosphorylated Smad1/5/8 were mainly detected in proliferating chondrocytes of the cartilaginous cap. Also, BMPR-1B was found in hypertrophic chondrocytes of SO and proliferating chondrocytes of MO. Noggin was observed in resting chondrocytes and, to a lesser extent, in clustered proliferating chondrocytes in SO. On the other hand, noggin in MO was observed in proliferating chondrocytes. Since BMPs can stimulate proliferation and hypertrophic differentiation of chondrocytes, these findings suggest that there is an imbalance of BMP-2/4 and noggin interactions that may lead to abnormal regulation of chondrocyte proliferation and differentiation in the cartilaginous cap of human osteochondromas.

Multiple hereditary exostoses (MHE): elucidating the pathogenesis of a rare skeletal disorder through interdisciplinary research.

Abstract An interdisciplinary and international group of clinicians and scientists gathered in Philadelphia, PA, to attend the fourth International Research Conference on Multiple Hereditary Exostoses (MHE), a rare and severe skeletal disorder. MHE is largely caused by autosomal dominant mutations in EXT1 or EXT2, genes encoding Golgi-associated glycosyltransferases responsible for heparan sulfate (HS) synthesis. HS chains are key constituents of cell surface- and extracellular matrix-associated proteoglycans, which are known regulators of skeletal development. MHE affected individuals are HS-deficient, can display skeletal growth retardation and deformities, and consistently develop benign, cartilage-capped bony outgrowths (termed exostoses or osteochondromas) near the growth plates of many skeletal elements. Nearly 2% of patients will have their exostoses progress to malignancy, becoming peripheral chondrosarcomas. Current treatments are limited to the surgical removal of symptomatic exostoses. No definitive treatments have been established to inhibit further formation and growth of exostoses, prevent transition to malignancy, or address other medical problems experienced by MHE patients, including chronic pain. Thus, the goals of the Conference were to assess our current understanding of MHE pathogenesis, identify key gaps in information, envision future therapeutic strategies and discuss ways to test and implement them. This report provides an assessment of the exciting and promising findings in MHE and related fields presented at the Conference and a discussion of the future MHE research directions. The Conference underlined the critical usefulness of gathering experts in several research fields to forge new alliances and identify cross-fertilization areas to benefit both basic and translational biomedical research on the skeleton.