Insights into the molecular pathogenesis of cardiospondylocarpofacial syndrome: MAP3K7 c.737-7A > G variant alters the TGFß-mediated α-SMA cytoskeleton assembly and autophagy.

Transforming growth factor beta-activated kinase 1 (TAK1) is a highly conserved kinase protein encoded by MAP3K7, and activated by multiple extracellular stimuli, growth factors and cytokines. Heterozygous variants in MAP3K7 cause the cardiospondylocarpofacial syndrome (CSCFS) which is characterized by short stature, dysmorphic facial features, cardiac septal defects with valve dysplasia, and skeletal anomalies. CSCFS has been described in seven patients to date and its molecular pathogenesis is only partially understood. Here, the functional effects of the MAP3K7 c.737-7A > G variant, previously identified in a girl with CSCFS and additional soft connective tissue features, were explored. This splice variant generates an in-frame insertion of 2 amino acid residues in the kinase domain of TAK1. Computational analysis revealed that this in-frame insertion alters protein dynamics in the kinase activation loop responsible for TAK1 autophosphorylation after binding with its interactor TAB1. Co-immunoprecipitation studies demonstrate that the ectopic expression of TAK1-mutated protein impairs its ability to physically bind TAB1. In patient's fibroblasts, MAP3K7 c.737-7A > G variant results in reduced TAK1 autophosphorylation and dysregulation of the downstream TAK1-dependent signaling pathway. TAK1 loss-of-function is associated with an impaired TGFß-mediated α-SMA cytoskeleton assembly and cell migration, and defective autophagy process. These findings contribute to our understanding of the molecular pathogenesis of CSCFS and might offer the rationale for the design of novel therapeutic targets.

A novel FAM20C mutation causes a rare form of neonatal lethal Raine syndrome.

Raine syndrome is a rare, autosomal recessive, osteosclerotic bone dysplasia due to pathogenic variants in FAM20C. The clinical phenotype is characterized by generalized osteosclerosis affecting all bones, cerebral calcifications, and craniofacial dysmorphism. Most cases present during the neonatal period with early lethality due to pulmonary hypoplasia and respiratory compromise while only few affected individuals have been reported to survive into adulthood. FAM20C is a ubiquitously expressed protein kinase that contains five functional domains including a catalytic domain, a binding pocket for FAM20A and three distinct N-glycosylation sites. We report a newborn infant with a history of prenatal onset fractures, generalized osteosclerosis, and craniofacial dysmorphism and early lethality. The clinical presentation was highly suggestive of Raine syndrome. A homozygous, novel missense variant in exon 5 of FAM20C (c.1007T>G; p.Met336Arg) was identified by targeted Sanger sequencing. Following in silico analysis and mapping of the variant on a three-dimensional (3D) model of FAM20C it is predicted to be deleterious and to affect N-glycosylation, protein folding, and subsequent secretion of FAM20C. In addition, we reviewed all published FAM20C mutations and observed that most pathogenic variants affect functional regions within the protein establishing evidence for an emerging genotype-phenotype correlation.

A novel FAM20C mutation causing hypophosphatemic osteomalacia with osteosclerosis (mild Raine syndrome) in an elderly man with spontaneous osteonecrosis of the knee.

Raine syndrome is characterized by FGF23-mediated hypophosphatemic osteomalacia with osteosclerosis caused by mutations in the FAM20C gene. We report a case of a 72-year-old man who presented with rapid progressive spontaneous osteonecrosis of the knee (SONK). A full osteologic assessment including dual energy X-ray absorptiometry (DXA), high-resolution peripheral quantitative computed tomography (HR-pQCT), and serum analyses revealed a high bone mass in the lumbar spine and hip (DXA T-score + 7.5 and + 4.7/+4.2) with increased bone microstructural parameters in the distal radius and tibia (BV/TV 127%, 140% of the age-matched mean, respectively), as well as a low bone turnover state. Phosphate levels were low due to renal phosphate wasting and high FGF23 levels (126.5 pg/ml, reference range 23.2-95.4 pg/ml). Using gene panel sequencing, we identified a novel FAM20C heterozygous missense mutation in combination with a homozygous duplication that potentially alters splicing. Taken together, this is the first case of mild Raine syndrome with spontaneous osteonecrosis of the knee, phosphate wasting, and a pronounced trabecular high bone mass phenotype.

The Pathology of Type II Modic Changes: Fat Deposition or Osteosclerosis? A Study Using CT Scan.

STUDY DESIGN: A retrospective cross-sectional study of type II Modic changes (MCs). OBJECTIVE: To evaluate the CT values of type II MCs and determine their relationship with disc degeneration. METHODS: 124 type II MCs from 66 patients' MRI and CT were included and analyzed. Disc degeneration adjacent to MCs was evaluated based on Pfirrmann classification. CT values of bone marrow area and endplate from MC regions, adjacent non-MC regions, and L1 vertebra were measured. CT value changes (ΔCT value) were defined as MCs' CT value minus non-MCs'. According to the types of variables, paired t-test, signed-rank test, two-way ANOVA, and Friedman test were used. RESULTS: The CT value of MCs was significantly higher than that of non-MCs at both bone marrow area and endplate (P<0.0001, resp.). Good consistency was found between non-MCs and L1 vertebra in the CT value of bone marrow area and endplate (P=0.2129, P=0.2272, resp.), suggesting the control group was valid. Adjacent to type II MCs, there were 8 (6.4%) discs with grade III degeneration, 58 (46.8%) with grade IV, and 58 (46.8%) with grade V. The ΔCT value of grade V disc degeneration was larger than that of grade IV at the endplate (P=0.0150). CONCLUSIONS: Osteosclerosis may exist in type II MCs. The more severe the degeneration of the adjacent disc, the greater the degree of osteosclerosis.

A Review of the Clinical, Radiological and Biochemical Characteristics and Genetic Causes of High Bone Mass Disorders.

BACKGROUND: High bone mass (HBM) disorders are a group of clinically and genetically heterogeneous bone diseases characterized by increased bone density on radiographs, due to progressive bone overgrowth or impaired bone resorption, or both. Some HBM cases are secondary to other diseases, such as chronic hepatitis C virus infection. Despite the great advance in gene diagnostic technology, the majority of HBM individuals remain undiagnosed. OBJECTIVE: In this review, we will summarize the clinical, radiological and biochemical characteristics of HBM cases due to varying etiologies, since these features are helpful in the differential diagnosis of HBM. RESULTS: Each subgroup of HBM cases shows distinctive clinical, radiological and biochemical characteristics. HBM, due to bone overgrowth, was designated as sclerosteosis, as a result of mutations located in genes critically involved in the Wnt/beta-catenin signal pathway. Mutations in genes encoding factors relevant to the differentiation and maturation of osteoclasts, or critical for the acidification and resorption of osteoclasts may lead to osteopetrosis. Hepatitis C associated osteosclerosis is characterized by a generalized increase in bone mass and markedly elevated serum levels of bone specific alkaline phosphatase. CONCLUSION: The clarification of the etiologies of HBM may have a breakthrough role in understanding the molecular mechanisms involved in bone metabolism and may provide new pathways for the intervention of osteoporosis.

Specific ablation of mouse Fam20C in cells expressing type I collagen leads to skeletal defects and hypophosphatemia.

FAM20C mutations in humans cause Raine syndrome and our previous studies showed that global inactivation of mouse Fam20C led to bone and dental defects. By crossbreeding 2.3 kb Col 1a1-Cre mice with Fam20C flox/flox mice, we created 2.3 kb Col 1a1-Cre;Fam20C foxl/flox (cKO) mice, in which Fam20C was inactivated in cells expressing Type I collagen. This study showed that the long bones of cKO mice were shorter and had a lower level of mineralization compared to the normal mice. The collagen fibrils in Fam20C-deficient bone were disorganized and thicker while the growth plate cartilage in cKO mice was disorganized and wider compared to the normal mice. The Fam20C-deficient bone had a lower level of dentin matrix protein 1, and higher levels of osteopontin and bone sialoprotein than the normal. The blood of cKO mice had an elevated level of fibroblast growth factor 23 and reduced level of phosphorus. These findings indicate that inactivation of Fam20C in cells expressing type I collagen led to skeletal defects and hypophosphatemia. The altered levels of dentin matrix protein 1 and osteopontin in Fam20C-deficient bone may be significant contributors to the mineralized tissue defects in human patients and animals suffering from the functional loss of FAM20C.

First case of osteopathia striata with cranial sclerosis in an adult male with Klinefelter syndrome.

Osteopathia striata with cranial sclerosis is a rare X-linked disorder. It is often lethal in male patients, and is considered X-linked dominant since affected females exhibit clinical signs, although milder than males. We describe here an adult male patient, with clinical and radiological signs similar to those described in female patients. Diagnosis was confirmed by the identification of an AMER1 mutation. The presence of long bones striation and the clinical phenotype of the patient also led to the diagnosis of non-mosaic Klinefelter syndrome, probably explaining the non-lethal and even rather minor phenotype compared to the rare affected males already described.

A Novel ANO5 Mutation Causing Gnathodiaphyseal Dysplasia With High Bone Turnover Osteosclerosis.

Gnathodiaphyseal dysplasia (GDD) is a rare skeletal syndrome that involves an osteopetrosis-like sclerosis of the long bones and fibrous dysplasia-like cemento-osseous lesions of the jawbone. Although the genetic analysis of the respective patients has revealed mutations in the ANO5 gene as an underlying cause, there is still no established consensus regarding the bone status of GDD patients. We report a new case of GDD in a 13-year-old boy with recurrent diaphyseal fractures of the femur, in whom we identified a novel de novo missense mutation in the ANO5 gene, causing a p.Ser500Phe substitution at the protein level. After confirming the presence of GDD-characteristic abnormalities within the jaw bones, we focused on a full osteologic assessment using dual-energy X-ray absorptiometry (DXA), high-resolution peripheral quantitative computed tomography (HR-pQCT), and serum analyses. We thereby identified increased trabecular bone mass accompanied by elevated serum markers of bone formation and bone resorption. The high turnover bone pathology was further confirmed through the analysis of an iliac crest biopsy, where osteoblast and osteoclast indices were remarkably increased. Taken together, our findings provide evidence for a critical and generalized role of anoctamin-5 (the protein encoded by the ANO5 gene) in skeletal biology. As it is reasonable to speculate that modifying the function of anoctamin-5 might be useful for therapeutically activating bone remodeling, it is now required to analyze its function at a molecular level, for instance in mouse models. © 2016 American Society for Bone and Mineral Research.

Non lethal Raine syndrome and differential diagnosis.

Raine syndrome is a rare autosomal recessive bone dysplasia characterized by characteristic facial features with exophthalmos and generalized osteosclerosis. Amelogenesis imperfecta, hearing loss, seizures, and intracerebral calcification are apparent in some affected individuals. Originally, Raine syndrome was originally reported as a lethal syndrome. However, recently a milder phenotype, compatible with life, has been described. Biallelic variants inFAM20C, encoding aGolgi casein kinase involved in biomineralisation, have been identified in affected individuals. We report here a consanguineous Moroccan family with two affected siblingsa girl aged 18 and a boy of 15years. Clinical features, including learning disability, seizures and amelogenesis imperfecta, initially suggested a diagnosis of Kohlschutter-Tonz syndrome. However,a novel homozygous FAM20Cvariantc.676T > A, p.(Trp226Arg) was identified in the affected siblings. Our report reinforces that Raine syndrome is compatible with life, and that mild hypophosphatemia and amelogenesis imperfecta are key features of the attenuated form.

Role of Endothelin-1 in a Syndrome of Myelofibrosis and Osteosclerosis.

CONTEXT: Primary myelofibrosis is one of the chronic myeloproliferative disorders characterized by bone marrow fibrosis associated with extramedullary hematopoiesis and osteosclerosis. Endothelin-1 (ET1) is a potent vasoconstrictor that is also a key mediator of osteoblastic bone metastases by stimulating osteoblast proliferation and new bone formation. CASE DESCRIPTION: We report laboratory, radiographic, bone densitometry, and bone histology data of a patient presenting with newly diagnosed, biopsy-proven myelofibrosis and osteosclerosis. We were able to demonstrate abundant ET1 signaling in the bones of our patient. CONCLUSIONS: We believe that ET1 is responsible for the osteosclerosis that develops with advanced myelofibrosis and suggest that ET1 signaling may play a role in other osteosclerotic settings as well.

Hepatitis C-associated osteosclerosis: a new case with long-term follow-up and a review of the literature.

Hepatitis C-associated osteosclerosis (HCAO) is an uncommon condition characterized by increased bone density, skeletal pain and elevated bone formation markers. Since 1992, only 17 cases have been reported. We herein describe the case of a 61-year-old woman affected by severe pain involving the lower limbs. The laboratory data showed an elevated serum alkaline phosphatase level, and the patient was found to be seropositive for hepatitis C virus infection. In addition, an X-ray skeleton survey showed marked cortical thickening of both femurs and tibias, and a whole-body bone scan revealed an increased cortical radionuclide uptake in the involved bones. These findings were consistent with a diagnosis of HCAO. In this report, we discuss the patient's clinical course over 16 years and, for the first time, show a normalized radioisotope uptake on bone scanning 10 years after the diagnosis.

Bayesian belief network analysis applied to determine the progression of temporomandibular disorders using MRI.

OBJECTIVES: This study investigated the applicability of a Bayesian belief network (BBN) to MR images to diagnose temporomandibular disorders (TMDs). Our aim was to determine the progression of TMDs, focusing on how each finding affects the other. METHODS: We selected 1.5-T MRI findings (33 variables) and diagnoses (bone changes and disc displacement) of patients with TMD from 2007 to 2008. There were a total of 295 cases with 590 sides of temporomandibular joints (TMJs). The data were modified according to the research diagnostic criteria of TMD. We compared the accuracy of the BBN using 11 algorithms (necessary path condition, path condition, greedy search-and-score with Bayesian information criterion, Chow-Liu tree, Rebane-Pearl poly tree, tree augmented naïve Bayes model, maximum log likelihood, Akaike information criterion, minimum description length, K2 and C4.5), a multiple regression analysis and an artificial neural network using resubstitution validation and 10-fold cross-validation. RESULTS: There were 191 TMJs (32.4%) with bone changes and 340 (57.6%) with articular disc displacement. The BBN path condition algorithm using resubstitution validation and 10-fold cross-validation was >99% accurate. However, the main advantage of a BBN is that it can represent the causal relationships between different findings and assign conditional probabilities, which can then be used to interpret the progression of TMD. CONCLUSIONS: Osteoarthritic bone changes progressed from condyle to articular fossa and finally to mandibular bone contours. Disc displacement was directly related to severe bone changes. Early bone changes were not directly related to disc displacement. TMJ functional factors (condylar translation, bony space and disc form) and age mediated between bone changes and disc displacement.

Rapid skeletal turnover in a radiographic mimic of osteopetrosis.

Among the high bone mass disorders, the osteopetroses reflect osteoclast failure that prevents skeletal resorption and turnover, leading to reduced bone growth and modeling and characteristic histopathological and radiographic findings. We report an 11-year-old boy with a new syndrome that radiographically mimics osteopetrosis (OPT), but features rapid skeletal turnover. He presented at age 21 months with a parasellar, osteoclast-rich giant cell granuloma. Radiographs showed a dense skull, generalized osteosclerosis and cortical thickening, medullary cavity narrowing, and diminished modeling of tubular bones. His serum alkaline phosphatase was >5000 IU/L (normal <850 IU/L). After partial resection, the granuloma re-grew but then regressed and stabilized during 3 years of uncomplicated pamidronate treatment. His hyperphosphatasemia transiently diminished, but all bone turnover markers, especially those of apposition, remained elevated. Two years after pamidronate therapy stopped, bone mineral density (BMD) Z-scores reached +9.1 and +5.8 in the lumbar spine and hip, respectively, and iliac crest histopathology confirmed rapid bone remodeling. Serum multiplex biomarker profiling was striking for low sclerostin. Mutation analysis was negative for activation of lipoprotein receptor-related protein 4 (LRP4), LRP5, or TGFß1, and for defective sclerostin (SOST), osteoprotegerin (OPG), RANKL, RANK, SQSTM1, or sFRP1. Microarray showed no notable copy number variation. Studies of his nonconsanguineous parents were unremarkable. The etiology and pathogenesis of this unique syndrome are unknown.

Diffuse osteosclerosis in a patient with prostate cancer.

A 61-year-old man was referred to our outpatient clinic because of severe bilateral upper leg pain for 1 year. On admission, the patient had anemia and a high serum alkaline phosphatase level. Lumbar and femoral neck T-scores were +10.5 and +9.6, respectively. His radius 33 % T-score was -2.8. Plain radiographs of the patient's pelvis, spine, and long bones revealed osteosclerosis. The patient had previously undergone a prostate biopsy, which showed prostate adenocarcinoma (Gleason score 3 + 4). The patient's total and free prostate-specific antigen were very high. According to previous records, the patient did not have anemia, and his serum alkaline phosphatase (ALP) level was normal. An abdominal radiograph taken 2 years earlier revealed a normal spine and pelvic bone. Bone scintigraphy yielded nontypical findings for prostate cancer metastasis. Computed tomography of the patient's thorax and abdomen showed heterogeneous sclerotic areas in all bones consistent with prostate cancer metastasis. A bone marrow biopsy disclosed disseminated carcinomatosis of bone marrow in association with prostate cancer. Clinicians should be aware of the possibility of prostate malignancy as a cause of high bone mineral density (BMD), even in the absence of typical localized findings on plain radiographs.

Hereditary deletion of the entire FAM20C gene in a patient with Raine syndrome.

Raine syndrome is an autosomal recessive disorder caused by mutations in the FAM20C gene that is characterized by generalized osteosclerosis with periosteal new bone formation and distinctive craniofacial dysmorphism. We report on a child who is homozygous for a 487-kb deletion in 7p22.3 that contains FAM20C. Both parents were heterozygous for the deletion. Our patient had the common craniofacial features as well as, uncommon features such as protruding tongue, short stature, and hypoplastic distal phalanges. In addition, he had wormian bones and pyriform aperture stenosis, features that are usually under diagnosed. It is clear that Raine syndrome has a wide range of expression and may not be lethal in the neonatal period. Furthermore, Raine cases due to whole gene deletion do not seem to have a major difference in the phenotype over those caused by various mutations.

Neurophysiologic, audiometric and vestibular function tests in patients with hyperostosis cranialis interna.

OBJECTIVE: Hyperostosis cranialis interna (HCI) is an autosomal dominant sclerosing bone dysplasia affecting the skull base and the calvaria, characterized by cranial nerve deficits due to stenosis of neuroforamina. The aim of this study is to describe the value of several neurophysiological, audiometric and vestibular tests related to the clinical course of the disorder. METHODS: Ten affected subjects and 13 unaffected family members were recruited and tested with visual evoked potentials, masseter reflex, blink reflex, pure tone and speech audiometry, stapedial reflexes, otoacoustic emissions, brainstem evoked response audiometry and electronystagmography. RESULTS: Due to the symmetrical bilateral nature of this disease, the sensitivity of visual evoked potentials (VEPs), masseter reflex and blink reflex is decreased (25-37.5%), therefore reducing the value of single registration. Increased hearing thresholds and increased BERA latency times were found in 60-70%. The inter-peak latency I-V parameter in BERA has the ability to determine nerve encroachment reliably. 50% of the patients had vestibular abnormalities. No patient had disease-related absence of otoacoustic emissions, because the cochlea is not affected. CONCLUSION: In patients with HCI and similar craniofacial sclerosing bone dysplasias we advise monitoring of vestibulocochlear nerve function with tone and speech audiometry, BERA and vestibular tests. VEPs are important to monitor optic nerve function in combination with radiological and ophthalmologic examination. We do not advise the routine use of blink and masseter reflex.

[Computed tomographic criteria for the diagnosis of variable manifestations of Paget's disease in the cerebral cranium and facial bones].

OBJECTIVE: to optimize the diagnosis of different stages of Paget's disease, by determining the extent of bone structural lesions in the cerebral and visceral cranium on the basis of computed tomography data. MATERIAL AND METHODS: Computed tomographic data were assessed by keeping in mind the structure, density, outlines, shadow shapes of the described tumor-like disease and the state of involved bone structures. Twelve patients with histologically verified Paget's disease were examined. RESULTS: The findings allowed the high informative value of computed tomography in diagnosing different stages of Paget's disease to be estimated in bone structural lesions in the cerebral and visceral cranium and skull base. Also, the obtained computed tomography data permitted the tracing of the extent of the lesion in the area under study.