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Lon is an ATP-dependent protease belonging to the "ATPase associated with diverse cellular activities" (AAA+) protein family. In humans, Lon is translated as a precursor and imported into the mitochondria matrix through deletion of the first 114 amino acid residues. In mice, embryonic knockout of lon is lethal. In humans, some dysfunctional lon mutations are tolerated but they cause a developmental disorder known as the CODAS syndrome. To gain a better understanding on the enzymology of human mitochondrial Lon, this study compares the structure-function relationship of the WT versus one of the CODAS mutants R721G to identify the mechanistic features in Lon catalysis that are affected. To this end, steady-state kinetics were used to quantify the difference in ATPase and ATP-dependent peptidase activities between WT and R721G. The K m values for the intrinsic as well as protein-stimulated ATPase were increased whereas the k cat value for ATP-dependent peptidase activity was decreased in the R721G mutant. The mutant protease also displayed substrate inhibition kinetics. In vitro studies revealed that R721G did not degrade the endogenous mitochondrial Lon substrate pyruvate dehydrogenase kinase isoform 4 (PDK4) effectively like WT hLon. Furthermore, the pyruvate dehydrogenase complex (PDH) protected PDK4 from hLon degradation. Using hydrogen deuterium exchange/mass spectrometry and negative stain electron microscopy, structural perturbations associated with the R721G mutation were identified. To validate the in vitro findings under a physiologically relevant condition, the intrinsic stability as well as proteolytic activity of WT versus R721G mutant towards PDK 4 were compared in cell lysates prepared from immortalized B lymphocytes expressing the respective protease. The lifetime of PDK4 is longer in the mutant cells, but the lifetime of Lon protein is longer in the WT cells, which corroborate the in vitro structure-functional relationship findings., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Familial mutations of the protein kinase A (PKA) R1α regulatory subunit lead to a generalized predisposition for a wide range of tumors, from pituitary adenomas to pancreatic and liver cancers, commonly referred to as Carney complex (CNC). CNC mutations are known to cause overactivation of PKA, but the molecular mechanisms underlying such kinase overactivity are not fully understood in the context of the canonical cAMP-dependent activation of PKA. Here, we show that oligomerization-induced sequestration of R1α from the catalytic subunit of PKA (C) is a viable mechanism of PKA activation that can explain the CNC phenotype. Our investigations focus on comparative analyses at the level of structure, unfolding, aggregation, and kinase inhibition profiles of wild-type (wt) PKA R1α, the A211D and G287W CNC mutants, as well as the cognate acrodysostosis type 1 (ACRDYS1) mutations A211T and G287E. The latter exhibit a phenotype opposite to CNC with suboptimal PKA activation compared with wt. Overall, our results show that CNC mutations not only perturb the classical cAMP-dependent allosteric activation pathway of PKA, but also amplify significantly more than the cognate ACRDYS1 mutations nonclassical and previously unappreciated activation pathways, such as oligomerization-induced losses of the PKA R1α inhibitory function., Competing Interests: The authors declare no competing interest.

Pathogenicity of tibial dyschondroplasia (TD) in broiler chickens is not detected yet. Janus Kinase/Signal Transducer and Activator of Transcription (JAK-STAT) signaling pathway-related genes were investigated in thiram induced TD chickens. Real-time qPCR and immunohistochemical (IHC) technique were used to observe the expression changes of STAT3 and SOSC3 gene on days 1, 2, 4, 6 after feeding 100 mg·kg -1 thiram. Morphological, pathological, and histological results of this study suggested that chondrocyte cells were observed more damaged on day 6 than day 1, 2, and 4. Therefore, Lameness and damaged chondrocytes gradually increased from day 1 to 6. The mRNA expression level of STAT3 was observed insignificant (P > 0.05) in thiram induced TD chickens' group of day 1. However, on days 2, 4, and 6, the expression was significant (P < 0.05). SOCS3 increased in thiram group on days 1, 2 and 6, decreased on day 4 (P < 0.05). The p-STAT3 and SOCS3 protein's protein localization was evaluated in the control and thiram-induced TD broiler chickens through IHC, suggesting that SOSC3 protein was observed significantly higher on days 1, 2, and 6 and down-regulated on day 4. p-STAT3 protein on thiram induced group was observed significantly upregulated on days 4 and 6. In conclusion, the differential expression of STAT3 and SOCS3 showed that the JAK-STAT signaling pathway might play an important role in regulating an abnormal proliferation, differentiation, or apoptosis of chondrocytes in TD at an early stage., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Matrix metalloproteinase 2 (MMP2) is a well‑characterized protein that is indispensable for extracellular matrix remodeling and other pathological processes, such as tumor progression and skeletal dysplasia. Excessive activation of MMP2 promotes osteolytic metastasis and bone destruction in late‑stage cancers, while its loss‑of‑function mutations result in the decreased bone mineralization and generalized osteolysis occurring progressively in skeletal developmental disorders, particularly in multicentric osteolysis, nodulosis and arthropathy (MONA). Either upregulation or downregulation of MMP2 activity can result in the same osteolytic effects. Thus, different functions of MMP2 have been recently identified that could explain this observation. While MMP2 can degrade bone matrix, facilitate osteoclastogenesis and amplify various signaling pathways that enhance osteolysis in bone metastasis, its role in maintaining the number of bone cells, supporting osteocytic canalicular network formation and suppressing leptin‑mediated inhibition of bone formation has been implicated in osteolytic disorders caused by MMP2 deficiency. Furthermore, the proangiogenic activity of MMP2 is one of the potential mechanisms that are associated with both pathological situations. In the present article, the latest research on MMP2 in bone homeostasis is reviewed and the mechanisms underlying the role of this protein in skeletal metastasis and developmental osteolysis are discussed.

Biallelic mutations in ACP5, encoding tartrate-resistant acid phosphatase (TRACP), have recently been identified to cause the inherited immuno-osseous disorder, spondyloenchondrodysplasia (SPENCD). This study was undertaken to characterize the eight reported missense mutations in ACP5 associated with SPENCD on TRACP expression. ACP5 mutant genes were synthesized, transfected into human embryonic kidney (HEK-293) cells and stably expressing cell lines were established. TRACP expression was assessed by cytochemical and immuno-cytochemical staining with a panel of monoclonal antibodies. Analysis of wild (WT) type and eight mutant stable cell lines indicated that all mutants lacked stainable enzyme activity. All ACP5 mutant constructs were translated into intact proteins by HEK-293 cells. The mutant TRACP proteins displayed variable immune reactivity patterns, and all drastically reduced enzymatic activity, revealing that there is no gross inhibition of TRACP biosynthesis by the mutations. But they likely interfere with folding thereby impairing enzyme function. TRACP exists as two isoforms. TRACP 5a is a less active monomeric enzyme (35kD), with the intact loop peptide and TRACP 5b is proteolytically cleaved highly active enzyme encompassing two subunits (23 kD and 16 kD) held together by disulfide bonds. None of the mutant proteins were proteolytically processed into isoform 5b intracellularly, and only three mutants were secreted in significant amounts into the culture medium as intact isoform 5a-like proteins. Analysis of antibody reactivity patterns revealed that T89I and M264K mutant proteins retained some native conformation, whereas all others were in "denatured" or "unfolded" forms. Western blot analysis with intracellular and secreted TRACP proteins also revealed similar observations indicating that mutant T89I is amply secreted as inactive protein. All mutant proteins were attacked by Endo-H sensitive glycans and none could be activated by proteolytic cleavage in vitro. In conclusion, determining the structure-function relationship of the SPENCD mutations in TRACP will expand our understanding of basic mechanisms underlying immune responsiveness and its involvement in dysregulated bone metabolism., Competing Interests: The authors have declared that no competing interests exist.

Non-ossifying fibromas are seen in different disorders recognizable by specific features. Indeed, osteoglophonic dysplasia (OD) is characterized by radiolucent bone lesions associated with severe short stature, dysmorphism and failure of dental eruption. This syndrome is caused by heterozygous activating mutations in the immunoglobulin-like D3 domain of the FGFR1 gene, encoding a tyrosine kinase. Here, we report three patients from the same family presenting with radiolucent bone lesions and teeth retentions. Exome sequencing allowed identification of a novel mutation c.917C > T, p. Pro306Leu in exon 7 of the FGFR1 gene. Our patients present with normal stature and no severe dysmorphism. This report describes a mild form of OD and expands the phenotype related to FGFR1 mutations. These findings emphasize the need to consider FGFR1 variants in the case of multiple non-ossifying bone lesions associated with dental eruption anomalies., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

SMARCAL1 is an ATP-driven DNA annealing helicase that is similar in structure to the chromatin regulators in the subfamily A group of the SWI/SNF-related matrix-associated actin-dependent chromatin regulators. SMARCAL1 catalyzes the formation of dsDNA by annealing the single-stranded binding protein RPA coated ssDNA with its complementary strand both in vitro and in vivo . In humans, different mutations of Smarcal1 gene are found to be closely related to different symptoms shown in individuals with Schimke immuno-osseous dysplasia (SIOD). This paper reviews the recent research progress of SMARCAL1 functions in remodeling DNA replication forks at damaged DNA sites, working in classical non-homologous end joining (NHEJ) repair of DNA double-stranded breaks, and in maintaining chromosomal telomere integrity. The relationships between the mutations of Smarcal1 gene in different SIOD symptoms, and the possible involvements of SMARCAL1 in neuromuscular degenerative diseases associated with trinucleotide repeats expansions are also updated and discussed to better understand the roles and mechanisms of the annealing helicase in genome stability maintenance.

Tibial dyschondroplasia (TD) negatively affects broilers all over the world, in which the accretion of the growth plate (GP) develops into tibial proximal metaphysis. Plastrum testudinis extract (PTE) is renowned as a powerful antioxidant, anti-inflammatory, and bone healing agent. The current study was conducted to evaluate the efficacy of PTE for the treatment of thiram-induced TD chickens. Broilers (day old; n = 300) were raised for 3 days with normal feed. On the 4th day, three groups ( n = 100 each) were sorted, namely, the control (normal diet), TD, and PTE groups (normal diet+ thiram 50 mg/kg). On the 7th day, thiram was stopped in the TD and PTE group, and the PTE group received a normal diet and PTE (30 mg/kg/day). Plastrum testudinis extract significantly restored ( p < 0.05) the liver antioxidant enzymes, inflammatory cytokines, serum biochemicals, GP width, and tibia weight as compared to the TD group. The PTE administration significantly increased ( p < 0.05) growth performance, vascularization, AKT (serine/threonine-protein kinase), and PI3K expressions and the number of hepatocytes and chondrocytes with intact nuclei were enhanced. In conclusion, PTE has the potential to heal TD lesions and act as an antioxidant and anti-inflammatory drug in chickens exposed to thiram via the upregulation of AKT and PI3K expressions.

Genetic causes of skeletal disorders are manifold and affect, among others, enzymes of bone and connective tissue synthesis pathways. We present a twelve-year-old boy with a mild skeletal dysplasia, hypermobility of joints and axial malalignment of lower limbs and feet. Exome sequencing revealed a biallelic loss of function mutation in CSGALNACT1, which encodes chondroitin sulfate N-acetylgalactosaminyltransferase 1 and plays a major role in the chondroitin sulfate chain biosynthesis and therefore in the synthesis of glycosaminoglycans. Recently, the first case of a pediatric patient with a mild skeletal dysplasia due to a compound heterozygous large intragenic deletion and a damaging missense variant in CSGALNACT1 was reported. We here identify a second case and the first juvenile patient with a homozygous frameshift variant in CSGALNACT1 which corroborates its role in mild and non-progressive skeletal dysplasia with joint laxity., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In humans, the pathogenesis of diabetes is characterised by two major pancreatic β cell defects: a reduction in β cell mass and the failure of β cells to produce enough insulin. Over the past two decades, multiple studies involving cell cultures, animal models and human subjects have established the importance of the protein kinase RNA-like endoplasmic reticulum kinase (PERK) in the adaptive functional capacity of pancreatic β cells during embryonic development and into adulthood. In this review, we will highlight major findings identifying PERK as a crucial player in β cell physiology and in diabetes., (© 2017 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.)

Diagnosis of bone marrow failure (BMF) disorders is challenging but essential for optimal patient management. Here, we report a young adult from nonconsanguineous parents with progressive pancytopenia since childhood, bone pain, increased bone density, and haphazard ossification replacing hematopoiesis within the bone marrow. Sequencing revealed two novel biallelic variants of unknown significance within the thromboxane A synthase gene, TBXAS1 (c.266T > C; c.989T > C), bioinformatically predicted to disrupt the protein. TBXAS1 mutations result in Ghosal hematodiaphyseal dysplasia (OMIM 231095), the autosomal recessive syndrome associated with abnormal bone structure and BMF. Identification of the genetic defect prompted steroid therapy leading to resolution of symptoms., (© 2017 Wiley Periodicals, Inc.)

The SH2 domain containing inositol phosphatase 2 (SHIP2) dephosphorylates PI(3,4,5)P3 to generate PI(3,4)P2, a lipid involved in the control of cell migration and adhesion. The INPPL1 gene that encodes SHIP2 has been found to be mutated in several cases of opsismodysplasia (OPS), a rare autosomal recessive chondrodysplasia characterized by growth plate defects and delayed bone maturation. Reported mutations often result in premature stop codons or missense mutations in SHIP2 catalytic domain. SHIP2 biochemical properties are known from studies in cancer cells; its role in endochondral ossification is unknown. Here, we report two novel mutations in the INPPL1 gene and show that cell migration is very much decreased in fibroblasts derived from three OPS patients as compared with control individuals. In contrast, cell adhesion on fibronectin is increased in OPS fibroblasts. An inhibitory effect on migration was also observed when normal fibroblasts were incubated in the presence of a SHIP2 competitive inhibitor. We conclude that both migration and adhesion are very much disrupted in OPS-derived fibroblasts. It is suggested that signaling events linked to migration and particularly to adhesion, which are lost in OPS patients, would prevent normal endochondral ossification., (© 2017 Wiley Periodicals, Inc.)

Type 2 acrodysostosis (ACRDYS2), a rare developmental skeletal dysplasia characterized by short stature, severe brachydactyly and facial dysostosis, is caused by mutations in the phosphodiesterase (PDE) 4D (PDE4D) gene. Several arguments suggest that the mutations should result in inappropriately increased PDE4D activity, however, no direct evidence supporting this hypothesis has been presented, and the functional consequences of the mutations remain unclear. We evaluated the impact of four different PDE4D mutations causing ACRDYS2 located in different functional domains on the activity of PDE4D3 expressed in Chinese hamster ovary cells. Three independent approaches were used: the direct measurement of PDE activity in cell lysates, the evaluation of intracellular cAMP levels using an EPAC-based (exchange factor directly activated by cAMP) bioluminescence resonance energy transfer sensor , and the assessment of PDE4D3 activation based on electrophoretic mobility. Our findings indicate that PDE4D3s carrying the ACRDYS2 mutations are more easily activated by protein kinase A-induced phosphorylation than WT PDE4D3. This occurs over a wide range of intracellular cAMP concentrations, including basal conditions, and result in increased hydrolytic activity. Our results provide new information concerning the mechanism whereby the mutations identified in the ACRDYS2 dysregulate PDE4D activity, and give insights into rare diseases involving the cAMP signaling pathway. These findings may offer new perspectives into the selection of specific PDE inhibitors and possible therapeutic intervention for these patients., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

In humans, activating mutations in the PRKAR1A gene cause acrodysostosis 1 (ACRDYS1). These mutations result in a reduction in PKA activation caused by an impaired ability of cAMP to dissociate mutant PRKAR1A from catalytic PKA subunits. Two striking features of this rare developmental disease are renal resistance to PTH and chondrodysplasia resulting from the constitutive inhibition of PTHR1/Gsa/AC/cAMP/PKA signaling. We developed a knock-in of the recurrent ACRDYS1 R368X PRKAR1A mutation in the mouse. No litters were obtained from [R368X]/[+] females (thus no homozygous [R368X]/[R368X] mice). In [R368X]/[+] mice, Western blot analysis confirmed mutant allele heterozygous expression. Growth retardation, peripheral acrodysostosis (including brachydactyly affecting all digits), and facial dysostosis were shown in [R368X]/[+] mice by weight curves and skeletal measurements (μCT scan) as a function of time. [R368X]/[+] male and female mice were similarly affected. Unexpected, however, whole-mount skeletal preparations revealed a striking delay in mineralization in newborn mutant mice, accompanied by a decrease in the height of terminal hypertrophic chondrocyte layer, an increase in the height of columnar proliferative prehypertrophic chondrocyte layer, and changes in the number and spatial arrangement of proliferating cell nuclear antigen (PCNA)-positive chondrocytes. Plasma PTH and basal urinary cAMP were significantly higher in [R368X]/[+] compared to WT mice. PTH injection increased urinary cAMP similarly in [R368X]/[+] and WT mice. PRKACA expression was regulated in a tissue (kidney not bone and liver) manner. This model, the first describing the germline expression of a PRKAR1A mutation causing dominant repression of cAMP-dependent PKA, reproduced the main features of ACRDYS1 in humans. It should help decipher the specificity of the cAMP/PKA signaling pathway, crucial for numerous stimuli. In addition, our results indicate that PRKAR1A, by tempering intracellular cAMP levels, is a molecular switch at the crossroads of signaling pathways regulating chondrocyte proliferation and differentiation. © 2016 American Society for Bone and Mineral Research., (© 2016 American Society for Bone and Mineral Research.)

Lon protease is a nuclear-encoded, mitochondrial ATP-dependent protease highly conserved throughout the evolution, crucial for the maintenance of mitochondrial homeostasis. Lon acts as a chaperone of misfolded proteins, and is necessary for maintaining mitochondrial DNA. The impairment of these functions has a deep impact on mitochondrial functionality and morphology. An altered expression of Lon leads to a profound reprogramming of cell metabolism, with a switch from respiration to glycolysis, which is often observed in cancer cells. Mutations of Lon, which likely impair its chaperone properties, are at the basis of a genetic inherited disease named of the cerebral, ocular, dental, auricular, skeletal (CODAS) syndrome. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The main target of cAMP is PKA, the main regulatory subunit of which (PRKAR1A) presents mutations in two genetic disorders: acrodysostosis and Carney complex. In addition to the initial recurrent mutation (R368X) of the PRKAR1A gene, several missense and nonsense mutations have been observed recently in acrodysostosis with hormonal resistance. These mutations are located in one of the two cAMP-binding domains of the protein, and their functional characterization is presented here. Expression of each of the PRKAR1A mutants results in a reduction of forskolin-induced PKA activation (measured by a reporter assay) and an impaired ability of cAMP to dissociate PRKAR1A from the catalytic PKA subunits by BRET assay. Modeling studies and sensitivity to cAMP analogs specific for domain A (8-piperidinoadenosine 3',5'-cyclic monophosphate) or domain B (8-(6-aminohexyl)aminoadenosine-3',5'-cyclic monophosphate) indicate that the mutations impair cAMP binding locally in the domain containing the mutation. Interestingly, two of these mutations affect amino acids for which alternative amino acid substitutions have been reported to cause the Carney complex phenotype. To decipher the molecular mechanism through which homologous substitutions can produce such strikingly different clinical phenotypes, we studied these mutations using the same approaches. Interestingly, the Carney mutants also demonstrated resistance to cAMP, but they expressed additional functional defects, including accelerated PRKAR1A protein degradation. These data demonstrate that a cAMP binding defect is the common molecular mechanism for resistance of PKA activation in acrodysosotosis and that several distinct mechanisms lead to constitutive PKA activation in Carney complex., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Collagen prolyl 4-hydroxylases (C-P4H-I, C-P4H-II, and C-P4H-III) catalyze formation of 4-hydroxyproline residues required to form triple-helical collagen molecules. Vertebrate C-P4Hs are α2β2 tetramers differing in their catalytic α subunits. C-P4H-I is the major isoenzyme in most cells, and inactivation of its catalytic subunit (P4ha1(-/-)) leads to embryonic lethality in mouse, whereas P4ha1(+/-) mice have no abnormalities. To study the role of C-P4H-II, which predominates in chondrocytes, we generated P4ha2(-/-) mice. Surprisingly, they had no apparent phenotypic abnormalities. To assess possible functional complementarity, we established P4ha1(+/-);P4ha2(-/-) mice. They were smaller than their littermates, had moderate chondrodysplasia, and developed kyphosis. A transient inner cell death phenotype was detected in their developing growth plates. The columnar arrangement of proliferative chondrocytes was impaired, the amount of 4-hydroxyproline and the Tm of collagen II were reduced, and the extracellular matrix was softer in the growth plates of newborn P4ha1(+/-);P4ha2(-/-) mice. No signs of uncompensated ER stress were detected in the mutant growth plate chondrocytes. Some of these defects were also found in P4ha2(-/-) mice, although in a much milder form. Our data show that C-P4H-I can to a large extent compensate for the lack of C-P4H-II in proper endochondral bone development, but their combined partial and complete inactivation, respectively, leads to biomechanically impaired extracellular matrix, moderate chondrodysplasia, and kyphosis. Our mouse data suggest that inactivating mutations in human P4HA2 are not likely to lead to skeletal disorders, and a simultaneous decrease in P4HA1 function would most probably be required to generate such a disease phenotype., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Phosphodiesterase 4 (PDE4) is an essential contributor to intracellular signaling and an important drug target. The four members of this enzyme family (PDE4A to -D) are functional dimers in which each subunit contains two upstream conserved regions (UCR), UCR1 and -2, which precede the C-terminal catalytic domain. Alternative promoters, transcriptional start sites, and mRNA splicing lead to the existence of over 25 variants of PDE4, broadly classified as long, short, and supershort forms. We report the X-ray crystal structure of long form PDE4B containing UCR1, UCR2, and the catalytic domain, crystallized as a dimer in which a disulfide bond cross-links cysteines engineered into UCR2 and the catalytic domain. Biochemical and mass spectrometric analyses showed that the UCR2-catalytic domain interaction occurs in trans, and established that this interaction regulates the catalytic activity of PDE4. By elucidating the key structural determinants of dimerization, we show that only long forms of PDE4 can be regulated by this mechanism. The results also provide a structural basis for the long-standing observation of high- and low-affinity binding sites for the prototypic inhibitor rolipram.

Glycosaminoglycans, including chondroitin, dermatan, and heparan sulfate, have various roles in a wide range of biological events such as cell signaling, cell proliferation, tissue morphogenesis, and interactions with various growth factors. Their polysaccharides covalently attach to the serine residues on specific core proteins through the common linker region tetrasaccharide, -xylose-galactose-galactose-glucuronic acid, which is produced through the stepwise addition of respective monosaccharides by four distinct glycosyltransferases. Mutations in the human genes encoding the glycosyltransferases responsible for the biosynthesis of the linker region tetrasaccharide cause a number of genetic disorders, called glycosaminoglycan linkeropathies, including Desbuquois dysplasia type 2, spondyloepimetaphyseal dysplasia, Ehlers-Danlos syndrome, and Larsen syndrome. This review focused on recent studies on genetic diseases caused by defects in the biosynthesis of the common linker region tetrasaccharide.

Acrodysostosis without hormone resistance is a rare skeletal disorder characterized by brachydactyly, nasal hypoplasia, mental retardation and occasionally developmental delay. Recently, loss-of-function mutations in the gene encoding cAMP-hydrolyzing phosphodiesterase-4D (PDE4D) have been reported to cause this rare condition but the pathomechanism has not been fully elucidated. To understand the pathogenetic mechanism of PDE4D mutations, we conducted 3D modeling studies to predict changes in the binding efficacy of cAMP to the catalytic pocket in PDE4D mutants. Our results indicated diminished enzyme activity in the two mutants we analyzed (Gly673Asp and Ile678Thr; based on PDE4D4 residue numbering). Ectopic expression of PDE4D mutants in HEK293 cells demonstrated this reduction in activity, which was identified by increased cAMP levels. However, the cells from an acrodysostosis patient showed low cAMP accumulation, which resulted in a decrease in the phosphorylated cAMP Response Element-Binding Protein (pCREB)/CREB ratio. The reason for this discrepancy was due to a compensatory increase in expression levels of PDE4A and PDE4B isoforms, which accounted for the paradoxical decrease in cAMP levels in the patient cells expressing mutant isoforms with a lowered PDE4D activity. Skeletal radiographs of 10-week-old knockout (KO) rats showed that the distal part of the forelimb was shorter than in wild-type (WT) rats and that all the metacarpals and phalanges were also shorter in KO, as the name acrodysostosis implies. Like the G-protein α-stimulatory subunit and PRKAR1A, PDE4D critically regulates the cAMP signal transduction pathway and influences bone formation in a way that activity-compromising PDE4D mutations can result in skeletal dysplasia. We propose that specific inhibitory PDE4D mutations can lead to the molecular pathology of acrodysostosis without hormone resistance but that the pathological phenotype may well be dependent on an over-compensatory induction of other PDE4 isoforms that can be expected to be targeted to different signaling complexes and exert distinct effects on compartmentalized cAMP signaling., (Copyright © 2014. Published by Elsevier Inc.)

Background: Sedaghatian-type spondylometaphyseal dysplasia (SSMD) is a neonatal lethal form of spondylometaphyseal dysplasia characterised by severe metaphyseal chondrodysplasia with mild limb shortening, platyspondyly, cardiac conduction defects, and central nervous system abnormalities. As part of the FORGE Canada Consortium we studied two unrelated families to identify the genetic aetiology of this rare disease., Methods and Results: Whole exome sequencing of a child affected with SSMD and her unaffected parents identified two rare variants in GPX4. The first (c.587+5G>A) was inherited from the mother, and the second (c.588-8&#95;588-4del) was de novo (NM&#95;001039848.1); both were predicted to impact splicing of GPX4. In vitro studies confirmed the mutations spliced out part of exon 4 and skipped exon 5, respectively, with both resulting in a frameshift and premature truncation of GPX4. Subsequently, a second child with SSMD was identified; although DNA from the child was not available, the two unaffected parents were found by Sanger sequencing to each carry the same heterozygous stop mutation in exon 3 of GPX4, c.381C>A, p.Tyr127* (NM&#95;001039848.1)., Conclusions: Our identification of truncating mutations in GPX4 in two families affected with SSMD supports the pathogenic role of mutated GPX4 in this very rare disease. GPX4 is a member of the glutathione peroxidase family of antioxidant defence enzymes and protects cells against membrane lipid peroxidation. GPX4 is essential for early embryo development, regulating anti-oxidative and anti-apoptotic activities. Our findings highlight the importance of this enzyme in development of the cardiac, nervous, and skeletal systems., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Proteoglycans (PGs) are a major component of the extracellular matrix in many tissues and function as structural and regulatory molecules. PGs are composed of core proteins and glycosaminoglycan (GAG) side chains. The biosynthesis of GAGs starts with the linker region that consists of four sugar residues and is followed by repeating disaccharide units. By exome sequencing, we found that B3GALT6 encoding an enzyme involved in the biosynthesis of the GAG linker region is responsible for a severe skeletal dysplasia, spondyloepimetaphyseal dysplasia with joint laxity type 1 (SEMD-JL1). B3GALT6 loss-of-function mutations were found in individuals with SEMD-JL1 from seven families. In a subsequent candidate gene study based on the phenotypic similarity, we found that B3GALT6 is also responsible for a connective tissue disease, Ehlers-Danlos syndrome (progeroid form). Recessive loss-of-function mutations in B3GALT6 result in a spectrum of disorders affecting a broad range of skeletal and connective tissues characterized by lax skin, muscle hypotonia, joint dislocation, and spinal deformity. The pleiotropic phenotypes of the disorders indicate that B3GALT6 plays a critical role in a wide range of biological processes in various tissues, including skin, bone, cartilage, tendon, and ligament., (Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Nasu-Hakola disease (NHD) is a rare autosomal recessive disorder, characterized by progressive presenile dementia and formation of multifocal bone cysts, caused by a loss-of-function mutation of DNAX-activation protein 12 (DAP12) or triggering receptor expressed on myeloid cells 2 (TREM2). TREM2 and DAP12 constitute a receptor/adaptor complex on myeloid cells. The post-receptor signals are transmitted via rapid phosphorylation of the immunoreceptor tyrosine-based activating motif (ITAM) of DAP12, mediated by Src protein tyrosine kinases, followed by binding of phosphorylated ITAM to Src homology 2 (SH2) domains of spleen tyrosine kinase (Syk), resulting in autophosphorylation of the activation loop of Syk. To elucidate the molecular mechanism underlying the pathogenesis of NHD, we investigated Syk expression and activation in the frontal cortex and the hippocampus of three NHD and eight control brains by immunohistochemistry. In NHD brains, the majority of neurons expressed intense immunoreactivities for Syk and Y525/Y526-phosphorylated Syk (pSyk) chiefly located in the cytoplasm, while more limited populations of neurons expressed Src. The levels of pSyk expression were elevated significantly in NHD brains compared with control brains. In both NHD and control brains, substantial populations of microglia and macrophages expressed pSyk, while the great majority of reactive astrocytes and myelinating oligodendrocytes did not express pSyk, Syk or Src. These observations indicate that neuronal expression of pSyk was greatly enhanced in the cerebral cortex and the hippocampus of NHD brains, possibly via non-TREM2/DAP12 signaling pathways involved in Syk activation., (© 2011 Japanese Society of Neuropathology.)

Linkage analysis with subsequent candidate gene sequencing is typically used to diagnose novel inherited syndromes. It is now possible to expedite diagnosis through the sequencing of all coding regions of the genome (the exome) or full genomes. We sequenced the exomes of four members of a family presenting with spondylo-epiphyseal dysplasia and retinitis pigmentosa and identified a six-base-pair (6-bp) deletion in GNPTG, the gene implicated in mucolipidosis type IIIγ. The diagnosis was confirmed by biochemical studies and both broadens the mucolipidosis type III phenotype and demonstrates the clinical utility of next-generation sequencing to diagnose rare genetic diseases., (Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Site-1 protease (S1P) is a proprotein convertase with essential functions in lipid homeostasis and unfolded protein response pathways. We previously studied a mouse model of cartilage-specific knock-out of S1P in chondroprogenitor cells. These mice exhibited a defective cartilage matrix devoid of type II collagen protein (Col II) and displayed chondrodysplasia with no endochondral bone formation even though the molecular program for endochondral bone development appeared intact. To gain insights into S1P function, we generated and studied a mouse model in which S1P is ablated in postnatal chondrocytes. Postnatal ablation of S1P results in chondrodysplasia. However, unlike early embryonic ablations, the growth plates of these mice exhibit a lack of Ihh, PTHrP-R, and Col10 expression indicating a loss of chondrocyte hypertrophic differentiation and thus disruption of the molecular program required for endochondral bone development. S1P ablation results in rapid growth plate disruption due to intracellular Col II entrapment concomitant with loss of chondrocyte hypertrophy suggesting that these two processes are related. Entrapment of Col II in the chondrocytes of the prospective secondary ossification center precludes its development. Trabecular bone formation is dramatically diminished in the primary spongiosa and is eventually lost. The primary growth plate is eradicated by apoptosis but is gradually replaced by a fully functional new growth plate from progenitor stem cells capable of supporting new bone growth. Our study thus demonstrates that S1P has fundamental roles in the preservation of postnatal growth plate through chondrocyte differentiation and Col II deposition and functions to couple growth plate maturation to trabecular bone development in growing mice.

Vertebral and metaphyseal dysplasia, spasticity with cerebral calcifications, and strong predisposition to autoimmune diseases are the hallmarks of the genetic disorder spondyloenchondrodysplasia. We mapped a locus in five consanguineous families to chromosome 19p13 and identified mutations in ACP5, which encodes tartrate-resistant phosphatase (TRAP), in 14 affected individuals and showed that these mutations abolish enzyme function in the serum and cells of affected individuals. Phosphorylated osteopontin, a protein involved in bone reabsorption and in immune regulation, accumulates in serum, urine and cells cultured from TRAP-deficient individuals. Case-derived dendritic cells exhibit an altered cytokine profile and are more potent than matched control cells in stimulating allogeneic T cell proliferation in mixed lymphocyte reactions. These findings shed new light on the role of osteopontin and its regulation by TRAP in the pathogenesis of common autoimmune disorders.

A family with dominant X-linked chondrodysplasia was previously described. The disease locus was ascribed to a 24 Mb interval in Xp11.3-q13.1. We have identified a variant (c.*281A>T) in the 3' untranslated region (UTR) of the HDAC6 gene that totally segregates with the disease. The variant is located in the seed sequence of hsa-miR-433. Our data showed that, in MG63 osteosarcoma cells, hsa-miR-433 (miR433) down-regulated both the expression of endogenous HDAC6 and that of an enhanced green fluorescent protein-reporter mRNA bearing the wild-type 3'-UTR of HDAC6. This effect was totally abrogated when the reporter mRNA bore the mutated HDAC6 3'-UTR. The HDAC6 protein was found to be over-expressed in thymus from an affected male fetus. Concomitantly, the level of total alpha-tubulin, a target of HDAC6, was found to be increased in the affected fetal thymus, whereas the level of acetylated alpha-tubulin was found to be profoundly decreased. Skin biopsies were obtained from a female patient who presented a striking body asymmetry with hypotrophy of the left limbs. The mutated HDAC6 allele was expressed in 31% of left arm-derived fibroblasts, whereas it was not expressed in the right arm. Overexpression of HDAC6 was observed in left arm-derived fibroblasts. Altogether these results strongly suggest that this HDAC6 3'-UTR variant suppressed hsa-miR-433-mediated post-transcriptional regulation causing the overexpression of HDAC6. This variant is likely to constitute the molecular cause of this new form of X-linked chondrodysplasia. This represents to our knowledge the first example of a skeletal disease caused by the loss of a miRNA-mediated post-transcriptional regulation on its target mRNA.

The SNF2 family of ATPases acts in the context of chromatin to regulate transcription, replication, repair and recombination. Defects in SNF2 genes cause many human diseases. For example, mutations in SMARCAL1 (also named HARP) cause Schimke immuno-osseous dysplasia (SIOD); a multi-system disorder characterized by growth defects, immune deficiencies, renal failure and other complex phenotypes. Several groups including ours recently identified SMARCAL1 as a replication stress response protein. Importantly, SMARCAL1 localizes to stalled replication forks and this localization of SMARCAL1 activity prevents DNA damage accumulation during DNA replication. We determined that SIOD-related SMARCAL1 mutants could not prevent replication-associated DNA damage in cells in which endogenous SMARCAL1 was silenced, establishing the first link between SIOD and a defect in a specific biological activity. Here, we also report that cells from patients with SIOD exhibit elevated levels of DNA damage that can be rescued by re-introduction of wild-type SMARCAL1. Our data suggest that loss of SMARCAL1 function in patients may cause DNA replication-associated genome instability that contributes to the pleiotropic phenotypes of SIOD.

Cartilage-hair hypoplasia (CHH) is a rare autosomal-recessive disorder characterized by short-limbed dwarfism, sparse hair, and immune deficiency. It is caused by mutations in the RMRP gene, which encodes the RNA component of the mitochondrial RNA-processing ribonuclease (RNase MRP). Several mutations have been identified in its promoter region or transcribed sequence. However, homozygous mutations in the promoter region have been only reported in a patient with primary immunodeficiency without other features of CHH. We report on a Thai girl who first presented with chronic diarrhea, recurrent pneumonia, and severe failure to thrive, without apparently disproportionate dwarfism. The diagnosis of CHH was made after the severe wasting was corrected, and disproportionate growth became noticeable. The patient had the typical features of CHH, including sparse hair and metaphyseal abnormalities. The immunologic profiles were consistent with combined immune deficiency. Mutation analysis identified a novel homozygous mutation, g.-19&#95;-25 dupACTACTC, in the promoter region of the RMRP gene. Identification of the mutation enabled us to provide a prenatal diagnosis in the subsequent pregnancy. This patient is the first CHH case with the characteristic features due to the homozygous mutation in the promoter region of the RMRP gene. The finding of severe immunodeficiency supports that promoter mutations markedly disrupt mRNA cleavage function, which causes cell-cycle impairment.

Tibial dyschondroplasia (TD) is a prevalent skeletal abnormality associated with rapid growth rate in many avian species. It is characterized by the presence of a nonvascularized, nonmineralized lesion that extends from the epiphyseal growth plate into the metaphysis of the proximal tibiotarsal bones. In this study, we examined the expression of 4 members of the matrix metalloproteinase (MMP) family (MMP-2, -3, -9, and -13) in thiram-induced TD lesions and in the process of recovery from TD, by in situ hybridization analysis and quantitative real-time PCR. A model for the induction and recovery of TD was established, consisting of 3 groups of broilers: (1) thiram group, chicks fed a thiram-enriched diet to induce TD; (2) recovery group, chicks fed a thiram-enriched diet during the first week of the experiment and a normal diet from the second week on; and (3) control group, chicks fed a normal diet throughout the experimental period. In agreement with our previous data, the 4 MMP were diminished in the TD lesion (P < 0.05); however, in the current study we show that the growth plate was able to repair itself and that the MMP reappeared during the process of recovery from TD. Our results strengthen the link between MMP expression and growth-plate impairment, and we suggest that gelatinase activity (MMP-2 and 9) facilitates this process.

Osteoblasts and chondrocytes arise from common osteo-chondroprogenitor cells. We show here that inactivation of ERK1 and ERK2 in osteo-chondroprogenitor cells causes a block in osteoblast differentiation and leads to ectopic chondrogenic differentiation in the bone-forming region in the perichondrium. Furthermore, increased mitogen-activated protein kinase signaling in mesenchymal cells enhances osteoblast differentiation and inhibits chondrocyte differentiation. These observations indicate that extracellular signal-regulated kinase 1 (ERK1) and ERK2 play essential roles in the lineage specification of mesenchymal cells. The inactivation of ERK1 and ERK2 resulted in reduced beta-catenin expression, suggesting a role for canonical Wnt signaling in ERK1 and ERK2 regulation of skeletal lineage specification. Furthermore, inactivation of ERK1 and ERK2 significantly reduced RANKL expression, accounting for a delay in osteoclast formation. Thus, our results indicate that ERK1 and ERK2 not only play essential roles in the lineage specification of osteo-chondroprogenitor cells but also support osteoclast formation in vivo.

Cartilage-hair hypoplasia is a short limbed skeletal dysplasia associated with impairments in host-defense. To better understand the clinical heterogeneity of this disorder, we studied 25 Amish patients with homozygous mutations in RMRP (RMRP 70 A>G). Despite mutation homogeneity, eight (32%) patients had severe or recurrent infections, two (8%) of these children underwent bone-marrow transplantation for combined immunodeficiency, and the remainder were healthy. Features distinguishing patients who underwent bone marrow transplantation from others were shorter birth length, and lower serum IgG, undetectable serum IgA, and elevated circulating NK cells before 2 years of age. Irrespective of clinical phenotype, most patients had lymphopenia and reduced lymphocyte proliferation to mitogens in vitro. Our cohort analysis suggests that many patients with cartilage-hair hypoplasia are at risk for infection susceptibility particularly during the first 2 years of life. Gauging this risk is difficult, and thus careful monitoring of all patients with cartilage-hair hypoplasia is warranted.

The spondylo-meta-epiphyseal dysplasia [SMED] short limb-hand type [SMED-SL] is a rare autosomal-recessive disease, first reported by Borochowitz et al. in 1993.(1) Since then, 14 affected patients have been reported.(2-5) We diagnosed 6 patients from 5 different consanguineous Arab Muslim families from the Jerusalem area with SMED-SL. Additionally, we studied two patients from Algerian and Pakistani ancestry and the parents of the first Jewish patients reported.(1) Using a homozygosity mapping strategy, we located a candidate region on chromosome 1q23 spanning 2.4 Mb. The position of the Discoidin Domain Receptor 2 (DDR2) gene within the candidate region and the similarity of the ddr2 knockout mouse to the SMED patients' phenotype prompted us to study this gene(6). We identified three missense mutations c.2254 C > T [R752C], c. 2177 T > G [I726R], c.2138C > T [T713I] and one splice site mutation [IVS17+1g > a] in the conserved sequence encoding the tyrosine kinase domain of the DDR2 gene. The results of this study will permit an accurate early prenatal diagnosis and carrier screening for families at risk.

Background: Cartilage hair hypoplasia is an autosomal recessive type of metaphyseal chondrodysplasia, caused by mutations in the ribonuclease mitochondrial RNA processing (RMRP) gene. Typical features of cartilage hair hypoplasia include short stature, a predisposition to malignancy, and a variable degree of impairment of cellular immunity., Objective: We sought to describe the heterogeneity of clinical and immunologic phenotype in 12 consecutive patients with RMRP mutations who were referred to 2 different institutions for immunologic evaluation., Methods: We have retrospectively analyzed the clinical and laboratory features in 12 patients with molecular defects in the RMRP gene. T-cell repertoire was investigated by quantitating Vbeta families' expression and analyzing their diversity. T-cell receptor excision circle analysis was used to study thymic output., Results: All 12 patients had significant immune abnormalities, leading to severe immune deficiency in 9. CD8 lymphocytopenia was identified as a novel phenotype associated with RMRP mutations. Significant, even intrafamilial, phenotypic heterogeneity was observed. In 3 cases, severe immunodeficiency was the only phenotypic manifestation associated with RMRP mutations, a novel finding. Mutations leading to significant immune defects were most often located in the promoter, and the first case of a compound heterozygote for 2 such mutations is reported., Conclusion: This report broadens the spectrum of phenotypes associated with RMRP mutations and suggests that mutations in this gene should be considered when evaluating patients with combined immune deficiency, regardless of the presence of other manifestations.

Wolcott-Rallison syndrome (WRS) (OMIM 226980) is a rare, autosomal recessive disorder with infancy-onset diabetes mellitus, multiple epiphyseal dysplasia, osteopenia, mental retardation or developmental delay, and hepatic and renal dysfunction as main clinical findings. Patients with WRS have mutations in the EIF2AK3 gene, which encodes the pancreatic eukaryotic translation initiation factor 2-alpha kinase 3. We report a female patient who developed insulin-requiring diabetes at 2.5 months of age. Multiple epiphyseal dysplasia was diagnosed at age 2 years. At age 5.5 years she developed a Reye-like syndrome with hypoketotic hypoglycaemia and renal and hepatic insufficiency and died. A partial autopsy showed fat infiltration in the liver and kidneys. Examination of urine by gas chromatography and mass spectrometry showed large amounts of C(6)-dicarboxylic acid (adipic acid), 3-hydroxy-C(8)-dicarboxylic acid, 3-hydroxy-C(10)-dicarboxylic acid, and 3-hydroxydecenedioic acid. Acetoacetate and 3-hydroxybutyrate were absent. The findings suggested a metabolic block in mitochondrial fatty acid oxidation, but lack of material precluded enzyme analyses. The clinical diagnosis of WRS was suggested in retrospect, and confirmed by sequencing of DNA extracted from stored autopsy material. The patient was compound heterozygous for the novel EIF2AK3 mutations c.1694&#95;1695delAT (Y565X) and c.3044T > C (F1015S). Our data suggest that disruption of the EIF2AK3 gene may lead to defective mitochondrial fatty acid oxidation and hypoglycaemia, thus adding to the heterogeneous phenotype of WRS.

Chondrocytes within the growth plates acclimatize themselves to a variety of stresses that might otherwise disturb cell fate. The tumor suppressor PTEN (phosphatase and tensin homolog deleted from chromosome 10) has been implicated in the maintenance of cell homeostasis. However, the functions of PTEN in regulating chondrocytic adaptation to stresses remain largely unknown. In this study, we have created chondrocyte-specific Pten knockout mice (Pten(co/co);Col2a1-Cre) using the Cre-loxP system. Following AKT activation, Pten mutant mice exhibited dyschondroplasia resembling human enchondroma. Cartilaginous nodules originated from Pten mutant resting chondrocytes that suffered from impaired proliferation and differentiation, and this was coupled with enhanced endoplasmic reticulum (ER) stress. We further found that ER stress in Pten mutant chondrocytes only occurred under hypoxic stress, characterized by an upregulation of unfolded protein response-related genes as well as an engorged and fragmented ER in which collagens were trapped. An upregulation of hypoxia-inducible factor 1alpha (HIF1alpha) and downstream targets followed by ER stress induction was also observed in Pten mutant growth plates and in cultured chondrocytes, suggesting that PI3K/AKT signaling modulates chondrocytic adaptation to hypoxic stress via regulation of the HIF1alpha pathway. These data demonstrate that PTEN function in chondrocytes is essential for their adaptation to stresses and for the inhibition of dyschondroplasia.

Schwartz-Jampel syndrome (SJS) is a recessive neuromyotonia with chondrodysplasia. It results from hypomorphic mutations of the gene encoding perlecan, leading to a decrease in the levels of this heparan sulphate proteoglycan in basement membranes (BMs). It has been suggested that SJS neuromyotonia may result from endplate acetylcholinesterase (AChE) deficiency, but this hypothesis has never been investigated in vivo due to the lack of an animal model for neuromyotonia. We used homologous recombination to generate a knock-in mouse strain with one missense substitution, corresponding to a human familial SJS mutation (p.C1532Y), in the perlecan gene. We derived two lines, one with the p.C1532Y substitution alone and one with p.C1532Y and the selectable marker Neo, to down-regulate perlecan gene activity and to test for a dosage effect of perlecan in mammals. These two lines mimicked SJS neuromyotonia with spontaneous activity on electromyogramm (EMG). An inverse correlation between disease severity and perlecan secretion in the BMs was observed at the macroscopic and microscopic levels, consistent with a dosage effect. Endplate AChE levels were low in both lines, due to synaptic perlecan deficiency rather than major myofibre or neuromuscular junction disorganization. Studies of muscle contractile properties showed muscle fatigability at low frequencies of nerve stimulation and suggested that partial endplate AChE deficiency might contribute to SJS muscle stiffness by potentiating muscle force. However, physiological endplate AChE deficiency was not associated with spontaneous activity at rest on EMG in the diaphragm, suggesting that additional changes are required to generate such activity characteristic of SJS.

Spondyloepiphyseal dysplasia (SED), Omani type (OMIM 608637) is a recessively inherited skeletal dysplasia previously described in two distantly related families from the Republic of Oman. The phenotype consists of short stature, severe kyphoscoliosis, arthritic joints (elbows, wrists, knees), secondary large joint dislocations, rhizomelia, fusion of carpal bones and mild brachydactyly. Affected individuals were homozygous for a missense mutation, R304Q in CHST3 that encodes the enzyme chondroitin 6-O-sulfotransferase-1 (C6ST-1). This enzyme mediates the sulfation of proteoglycans, particularly chondroitin sulfate (CS), in the extracellular matrix of cartilage. Here we describe the identification of a mutation (857T > C predicting the substitution L286P) in CHST3 in a Turkish family and extend the clinical phenotype of SED-Omani type to include congenital joint dislocation, club feet, ventricular septal defect, deafness, metacarpal shortening and accessory carpal ossification centers. Fibroblasts and urine obtained from affected patients demonstrated negligible levels of 6-O-sulfated GalNAc residue in CS. Furthermore, the 6-O-sulfotransferase activity of cloned C6ST-1 into which the L286P mutation had been introduced was dramatically reduced, confirming the pathogenicity of this substitution. These results indicate that the clinical consequences of a deficiency of 6-O-sulfation in CS can be varied and that a clinical spectrum may exist similar to that seen in other skeletal dysplasias characterized by disorders of proteoglycan sulfation., (Copyright 2008 Wiley-Liss, Inc.)

Enzymes of the matrix metalloproteinase (MMP) family regulate angiogenesis and are involved in the endochondral ossification process. Tibial dyschondroplasia (TD) and rickets are 2 disorders associated with impairments in this process, mainly in the vascularization of the avian growth plate. In this paper, we induced TD and rickets and studied the expression patterns of 4 members of the MMP family known to be important for endochondral ossification, MMP-2, 3, 9, and 13, in normal and impaired avian growth plates. The expression of MMP-3, 9, and 13 was reduced in the lesions and lined up parallel to the expulsion of blood vessels, which was extended up to the border of the lesion, but did not penetrate into it. Matrix metallopro-teinase-2 was not expressed in the TD lesion but was overexpressed in the rachitic lesion. We also studied the differentiation stage of the chondrocytes populating the lesions and found that the rachitic lesions were populated with proliferative chondrocytes, whereas the TD lesions were filled with chondrocytes that presented both proliferative and hypertrophic markers. These results suggest that MMP-3, 9, and 13 play a role in the vascularization and ossification processes, whereas MMP-2 is related to chondrocyte differentiation and may be involved in cartilage remodeling in the avian growth plate.

Deficiency of carbohydrate sulfotransferase 3 (CHST3; also known as chondroitin-6-sulfotransferase) has been reported in a single kindred so far and in association with a phenotype of severe chondrodysplasia with progressive spinal involvement. We report eight CHST3 mutations in six unrelated individuals who presented at birth with congenital joint dislocations. These patients had been given a diagnosis of either Larsen syndrome (three individuals) or humero-spinal dysostosis (three individuals), and their clinical features included congenital dislocation of the knees, elbow joint dysplasia with subluxation and limited extension, hip dysplasia or dislocation, clubfoot, short stature, and kyphoscoliosis developing in late childhood. Analysis of chondroitin sulfate proteoglycans in dermal fibroblasts showed markedly decreased 6-O-sulfation but enhanced 4-O-sulfation, confirming functional impairment of CHST3 and distinguishing them from diastrophic dysplasia sulphate transporter (DTDST)-deficient cells. These observations provide a molecular basis for recessive Larsen syndrome and indicate that recessive Larsen syndrome, humero-spinal dysostosis, and spondyloepiphyseal dysplasia Omani type form a phenotypic spectrum.

Prenatal diagnosis of perinatal lethal hypophosphatasia (PL-HPH) by ultrasonography is difficult as PL-HPH must be differentiated from other skeletal dysplasias with short long bones and poor mineralization of the skeleton, such as osteogenesis imperfecta type II and achondrogenesis/hypochondrogenesis. Here we present a case of molecularly confirmed PL-HPH and illustrate specific ultrasonographic findings that help to distinguish PL-HPH from similar conditions., ((c) 2008 Wiley-Liss, Inc.)

Cartilage hair hypoplasia (CHH) or McKusick type metaphyseal chondrodysplasia (MCD) (OMIM # 250250) is due to either the homozygous or compound heterozygous mutations in the nuclear encoded, non-coding RNA gene RMRP. Twenty-seven CHH patients were referred for molecular evaluation of the clinical diagnosis. RMRP mutations were found in 22 patients. The phenotype in one of the five mutation-negative patients was fully congruent with the adopted case definition of CHH. In a second of these patients, the diagnosis of Schmid type MCD (OMIM # 156500) was made and confirmed by the detection of a mutation in the COL10A1 gene. The remaining patients most likely represent one or more MCDs hitherto not yet delineated. The pattern of cumulative growth in infancy and early childhood in the latter four patients was the single feature with greatest negative predictive power for CHH. Fourteen mutations detected here, had not been reported previously. In this ethnically heterogeneous population, we performed a retrospective study to compare the prevalence of clinical features compared to previous reports based mostly on more ethnically homogenous groups.

Mutations in the EIF2AK3 gene have been identified in patients with Wolcott-Rallison syndrome - a rare autosomal recessive disorder associated with permanent neonatal insulin-dependent diabetes. Despite the fact that different mutations have been observed in every single unrelated case reported so far, most patients presented with similar characteristics, such as osteopenia, epiphyseal dysplasia as well as hepatic and/or renal dysfunction. The EIF2AK3 gene was analyzed using a PCR-based sequencing approach in two Wolcott-Rallison patients and their parents. We report two cases from different families carrying the same and novel truncating nonsense mutation in the EIF2AK3 gene that encodes the pancreatic eukaryotic initiation factor 2alpha kinase 3. This mutation clearly displays different clinical characteristics in the two patients we examined. Remarkably, the onset of diabetes was different for the two patients, and there was also heterogeneity in other clinical manifestations. These cases illustrate the important role of alternative pathways that could, to some extent, take over or supplement a defective metabolic pathway. This supports the idea that there is no simple relationship among clinical manifestations and EIF2AK3 mutations.

Goals: The aim of this study is to evaluate the benefits of early intervention in two major spondyloepiphyseal dysplasias of Saudi Arabia, namely, multiple sulfatase deficiency (MSD, Austin's disease) and Morquio's disease. The MSD is encountered frequently in the Kingdom and poses significant health risk to the child because of cord compression. The clinics of this hospital have several Austin's patients., Results: This study indicates that early intervention before serious irreversible damage to the cervical cord occurs improves the neurological course of the patient; no patient had a worse outcome. On the other hand, neurosurgical intervention after the neurological symptoms of cord compression occurs was not as rewarding., Conclusion: Morquio's disease is more common outside the Kingdom. The results in this study also confirm that early intervention in this disease is beneficial. Other surgeons make a similar recommendation for Morquio's disease. However, their experience with Austin's disease is not reported due to the rarity of this disease elsewhere.

MMPs, which degrade components of the ECM, have roles in embryonic development, tissue repair, cancer, arthritis, and cardiovascular disease. We show that a missense mutation of MMP13 causes the Missouri type of human spondyloepimetaphyseal dysplasia (SEMD(MO)), an autosomal dominant disorder characterized by defective growth and modeling of vertebrae and long bones. Genome-wide linkage analysis mapped SEMD(MO) to a 17-cM region on chromosome 11q14.3-23.2 that contains a cluster of 9 MMP genes. Among these, MMP13 represented the best candidate for SEMD(MO), since it preferentially degrades collagen type II, abnormalities of which cause skeletal dysplasias that include Strudwick type SEMD. DNA sequence analysis revealed a missense mutation, F56S, that substituted an evolutionarily conserved phenylalanine residue for a serine in the proregion domain of MMP13. We predicted, by modeling MMP13 structure, that this F56S mutation would result in a hydrophobic cavity with misfolding, autoactivation, and degradation of mutant protein intracellularly. Expression of wild-type and mutant MMP13s in human embryonic kidney cells confirmed abnormal intracellular autoactivation and autodegradation of F56S MMP13 such that only enzymatically inactive, small fragments were secreted. Thus, the F56S mutation results in deficiency of MMP13, which leads to the human skeletal developmental anomaly of SEMD(MO).

The rare osteosclerotic disease, pycnodysostosis, is characterized by decreased osteoclastic bone collagen degradation due to the absence of active cathepsin K. Although this enzyme is primarily expressed by osteoclasts, there is increasing evidence that it may also be present in other cells, including fibroblasts. Since fibroblasts are known to degrade collagen intracellularly following phagocytosis, we analyzed various soft connective tissues (periosteum, perichondrium, tendon, and synovial membrane) from a 13-week-old human fetus with pycnodysostosis for changes in this collagen digestion pathway. In addition, the same tissues from cathepsin K-deficient and control mice were analyzed. Microscopic examination of the human fetal tissues showed that cross-banded collagen fibrils had accumulated in lysosomal vacuoles of fibroblasts. Morphometric analysis of periosteal fibroblasts revealed that the volume density of collagen-containing vacuoles was 18 times higher than in fibroblasts of control patients. A similar accumulation was seen in periosteal fibroblasts of three children with pycnodysostosis. In contrast to the findings in humans, an accumulation of internalized collagen was not apparent in fibroblasts of mice with cathepsin K deficiency. Our observations indicate that the intracellular digestion of phagocytosed collagen by fibroblasts is inhibited in humans with pycnodysostosis, but probably not in the mouse model mimicking this disease. The data strongly suggest that cathepsin K is a crucial protease for this process in human fibroblasts. Murine fibroblasts may have other proteolytic activities that are expressed constitutively or up regulated in response to a deficiency of cathepsin K. This may explain why cathepsin K-deficient mice lack the dysostotic features that are prominent in patients with pycnodysostosis.

Normal physeal and dyschondroplastic cartilage of broiler chickens was examined for six enzymes by isoelectric focusing in thin-layer polyacrylamide slab gels. Acid phosphatase (ACP), esterase (EST), malate dehydrogenase (MDH), and peroxidase (PRX) were present in the normal physeal cartilage but not in the dyschondroplastic cartilage. Staining intensity of glucose-6-phosphate isomerase (GPI) and triose-phosphate isomerase (TPI) was reduced in the dyschondroplastic cartilage compared with that of the physeal cartilage. Differences in the presence of these enzymes possibly demonstrated their roles in processes of bone formation, cartilage resorption, and calcification. ACP could be involved in calcification. Lack of EST and PRX may be related to the failure of vascular invasion in dyschondroplastic cartilage of afflicted birds. A deficiency of MDH and reduced GPI and TPI in dyschondroplastic cartilage may reflect a reduction in the activity of energetic metabolism, causing the dissipation of energy and necrotic cells.

Tibial dyschondroplasia (TD) in poultry is a disorder of growth plate cartilage that fails to resorb and consequently prevents bone formation. Matrix metalloproteinases (MMP) contribute to the process of resorption through the degradation of extracellular matrices and facilitating vascularization, growth plate remodeling, and maturation. In order to understand the cause of the failure of cartilage degradation in TD, the gelatinase and collagenase activities, and the levels of collagen and glycosaminoglycans of conditioned media derived from cartilage-explant cultures of normal and TD growth plates were measured. Substrate zymography exhibited two prominent gelatinolytic and collagenolytic bands corresponding to MW 63, 59, and a broad but fuzzy band of activity between 100 and 200 kDa. On treatment with 4-aminophenylmercuric acetate, a compound that converts proenzyme forms of MMP, the 63 kDa MW gelatinolytic band migrated as a approximately 60 kDa band and contributed to the broadening of the 59 kDa band. The TD-growth plate-conditioned media had significantly lower collagenolytic-gelatinolytic activities. The sulfated glycosaminoglycans, but not the collagen contents of the conditioned media from TD-explant cultures, were also reduced significantly. It is likely that the decreased matrix metalloproteinase activities of growth plate chondrocyte may contribute to a reduced turnover of extracellular matrices (ECM), leading to the retention of cartilage and its lack of vascularity in TD-affected growth plates.