A novel single-base deletion of the RUNX Family Transcription Factor 2 gene associated with cleidocranial dysplasia.

Cleidocranial dysplasia (CCD) is a rare, autosomal dominant hereditary disorder characterized by skeletal malformations and dental abnormalities. The purpose of this study was to explore the functional role of a novel mutation in the pathogenesis of CCD. Genomic DNA was extracted from peripheral blood mononuclear cells collected from family members of a Chinese patient with CCD. An analysis of their RUNX Family Transcription Factor 2 (RUNX2) gene sequences was performed by PCR amplification and Sanger sequencing. The function of the mutant RUNX2 was studied by bioinformatics, real-time PCR, western blotting, and subcellular localization analysis. Sanger sequencing identified a novel single-base deletion (NM_001024630.4:c.132delG;NP_001019801.3: Val45Trpfs* 99) in the RUNX2 gene present in the Chinese patient with CCD. In vitro, functional studies showed altered protein localization and increased expression of mutant RUNX2 mRNA and mutant Runt-related transcription factor 2 (RUNX2). Luciferase reporter assay demonstrated that the novel RUNX2 mutations significantly increased the transactivation activity of RUNX2 on the osteocalcin gene promoter. In conclusion, we identified a patient with sporadic CCD carrying a novel deletion/frameshift mutation of the RUNX2 gene and performed screening and functional analyses to determine the cause of the CCD phenotype. This study provides new insights into the pathogenesis of CCD.3.

Heterozygous pathogenic variants involving CBFB cause a new skeletal disorder resembling cleidocranial dysplasia.

BACKGROUND: Cleidocranial dysplasia (CCD) is a rare skeletal dysplasia with significant clinical variability. Patients with CCD typically present with delayed closure of fontanels and cranial sutures, dental anomalies, clavicular hypoplasia or aplasia and short stature. Runt-related transcription factor 2 (RUNX2) is currently the only known disease-causing gene for CCD, but several studies have suggested locus heterogeneity. METHODS: The cohort consists of eight subjects from five unrelated families partially identified through GeneMatcher. Exome or genome sequencing was applied and in two subjects the effect of the variant was investigated at RNA level. RESULTS: In each subject a heterozygous pathogenic variant in CBFB was detected, whereas no genomic alteration involving RUNX2 was found. Three CBFB variants (one splice site alteration, one nonsense variant, one 2 bp duplication) were shown to result in a premature stop codon. A large intragenic deletion was found to delete exon 4, without affecting CBFB expression. The effect of a second splice site variant could not be determined but most likely results in a shortened or absent protein. Affected individuals showed similarities with RUNX2-related CCD, including dental and clavicular abnormalities. Normal stature and neurocognitive problems were however distinguishing features. CBFB encodes the core-binding factor ß subunit, which can interact with all RUNX proteins (RUNX1, RUNX2, RUNX3) to form heterodimeric transcription factors. This may explain the phenotypic differences between CBFB-related and RUNX2-related CCD. CONCLUSION: We confirm the previously suggested locus heterogeneity for CCD by identifying five pathogenic variants in CBFB in a cohort of eight individuals with clinical and radiographic features reminiscent of CCD.

A RUNX2 stabilization pathway mediates physiologic and pathologic bone formation.

The osteoblast differentiation capacity of skeletal stem cells (SSCs) must be tightly regulated, as inadequate bone formation results in low bone mass and skeletal fragility, and over-exuberant osteogenesis results in heterotopic ossification (HO) of soft tissues. RUNX2 is essential for tuning this balance, but the mechanisms of posttranslational control of RUNX2 remain to be fully elucidated. Here, we identify that a CK2/HAUSP pathway is a key regulator of RUNX2 stability, as Casein kinase 2 (CK2) phosphorylates RUNX2, recruiting the deubiquitinase herpesvirus-associated ubiquitin-specific protease (HAUSP), which stabilizes RUNX2 by diverting it away from ubiquitin-dependent proteasomal degradation. This pathway is important for both the commitment of SSCs to osteoprogenitors and their subsequent maturation. This CK2/HAUSP/RUNX2 pathway is also necessary for HO, as its inhibition blocked HO in multiple models. Collectively, active deubiquitination of RUNX2 is required for bone formation and this CK2/HAUSP deubiquitination pathway offers therapeutic opportunities for disorders of inappropriate mineralization.

Clinical and radiological characterization of novel FIG4-related combined system disease with neuropathy.

Variants in the FIG4 gene, which encodes a phosphatidylinositol-3,5-bisphosphatase lead to obstruction of endocytic trafficking, causing accumulation of enlarged vesicles in murine peripheral neurons and fibroblasts. Bi-allelic pathogenic variants in FIG4 are associated with neurological disorders including Charcot-Marie-Tooth disease type-4J (CMT4J) and Yunis-Varón syndrome (YVS). We present four probands from three unrelated families, all homozygous for a recurrent FIG4 missense variant c.506A>C p.(Tyr169Ser), with a novel phenotype involving features of both CMT4J and YVS. Three presented with infant-onset dystonia and one with hypotonia. All have depressed lower limb reflexes and distal muscle weakness, two have nerve conduction studies (NCS) consistent with severe sensorimotor demyelinating peripheral neuropathy and one had NCS showing patchy intermediate/mildly reduced motor conduction velocities. All have cognitive impairment and three have swallowing difficulties. MRI showed cerebellar atrophy and bilateral T2 hyperintense medullary swellings in all patients. These children represent a novel clinicoradiological phenotype and suggest that phenotypes associated with FIG4 missense variants do not neatly fall into previously described diagnoses but can present with variable features. Analysis of this gene should be considered in patients with central and peripheral neurological signs and medullary radiological changes, providing earlier diagnosis and informing reproductive choices.

New Function of RUNX2 in Regulating Osteoclast Differentiation via the AKT/NFATc1/CTSK Axis.

Cleidocranial dysplasia is an autosomal dominant skeletal disorder resulting from RUNX2 mutations. The influence of RUNX2 mutations on osteoclastogenesis and bone resorption have not been reported. To investigate the role of RUNX2 in osteoclast, RUNX2 expression in macrophages (RAW 264.7 cells) was detected. Stable RAW 264.7 cell lines expressing wild-type RUNX2 or mutated RUNX2 (c.514delT, p.172 fs) were established, and their functions in osteoclasts were investigated. Wild-type RUNX2 promoted osteoclast differentiation, formation of F-actin ring, and bone resorption, while mutant RUNX2 attenuated the positive differentiation effect. Wild-type RUNX2 increased the expression and activity of mTORC2. Subsequently, mTORC2 specifically promoted phosphorylation of AKT at the serine 473 residue. Activated AKT improved the nuclear translocation of NFATc1 and increased the expression of downstream genes, including CTSK. Inhibition of AKT phosphorylation abrogated the osteoclast formation of wild-type macrophages, whereas constitutively activated AKT rescued the osteoclast formation of mutant macrophages. The present study suggested that RUNX2 promotes osteoclastogenesis and bone resorption through the AKT/NFATc1/CTSK axis. Mutant RUNX2 lost the function of regulating osteoclast differentiation and bone remodeling, resulting in the defective formation of the tooth eruption pathway and impaction of permanent teeth in cleidocranial dysplasia. This study, for the first time, verifies the effect of RUNX2 on osteoclast differentiation and bone resorption and provides new insight for the explanation of cleidocranial dysplasia.

Three-dimensional evaluation of morphology and position of impacted supernumerary teeth in cases of cleidocranial dysplasia.

Cleidocranial dysplasia (CCD) is a congenital anomaly characterized by the presence of impacted supernumerary teeth and delayed eruption of permanent teeth. However, there has been no detailed investigation on supernumerary teeth in patients with CCD using three-dimensional (3D) imaging techniques. The purpose of this study was to elucidate the morphology and position of supernumerary teeth using 3D images reconstructed from cone-beam computed tomography (CBCT) data in a group of five Japanese subjects (male, 3; female, 2; age, 15.0-25.4 years) with CCD. All five subjects exhibited supernumerary teeth (39 in total; average, 7.8; range, 1-15). All supernumerary teeth were impacted and existed as pairs with adjacent permanent teeth. Comparison of the size (the crown and dental-root lengths, the crown mesiodistal and buccolingual diameters), the number of cusps and dental roots, the position, and direction of supernumerary teeth in relation to the adjacent permanent teeth was analyzed. The results of relationship analyses revealed that, at sites other than the molar region, supernumerary teeth were positioned on the lingual and distal sides and supernumerary teeth resembled the morphology of their adjacent permanent teeth in terms of the number of cusps but were smaller than the adjacent permanent teeth. In the molar region, supernumerary teeth were microdontia, which were apparently small and obscure morphologically. In addition, while all adjacent permanent teeth exhibited normal direction, five supernumerary teeth exhibited inverse direction. The findings of this study will improve our understanding of the characteristics of CCD and provide important information for the pathophysiology and clinical treatment.

Functional analysis of novel RUNX2 mutations identified in patients with cleidocranial dysplasia.

RUNX2 (Runt-related transcription factor 2) is a master regulator of osteoblast differentiation, cartilage and bone development. Pathogenic variants in RUNX2 have been linked to the Cleidocranial dysplasia (CCD), which is characterized by hypoplasia or aplasia of clavicles, delayed fontanelle closure, and dental anomalies. Here, we report 11 unrelated Polish patients with CCD caused by pathogenic alterations located in the Runt domain of RUNX2. In total, we identified eight different intragenic variants, including seven missense and one splicing mutation. Three of them are novel: c.407T>A p.(Leu136Gln), c.480C>G p.(Asn160Lys), c.659C>G p.(Thr220Arg), additional three were not functionally tested: c.391C>T p.(Arg131Cys), c.580+1G>T p.(Lys195_Arg229del), c.652A>G p.(Lys218Glu), and the remaining two: c.568C>T p.(Arg190Trp), c.673C>T p.(Arg225Trp) were previously reported and characterized. The performed transactivation and localization studies provide evidence of decreased transcriptional activity of RUNX2 due to mutations targeting the Runt domain and prove that impairment of nuclear localization signal (NLS) affects the subcellular localization of the protein. Presented data show that pathogenic variants discovered in our patients have a detrimental effect on RUNX2, triggering the CCD phenotype.

The ERK MAPK Pathway Is Essential for Skeletal Development and Homeostasis.

Mitogen-activated protein kinases (MAPKs) are a family of protein kinases that function as key signal transducers of a wide spectrum of extracellular stimuli, including growth factors and pro-inflammatory cytokines. Dysregulation of the extracellular signal-regulated kinase (ERK) MAPK pathway is associated with human skeletal abnormalities including Noonan syndrome, neurofibromatosis type 1, and cardiofaciocutaneous syndrome. Here, we demonstrate that ERK activation in osteoprogenitors is required for bone formation during skeletal development and homeostasis. Deletion of Mek1 and Mek2, kinases upstream of ERK MAPK, in osteoprogenitors (Mek1OsxMek2-/-), resulted in severe osteopenia and cleidocranial dysplasia (CCD), similar to that seen in humans and mice with impaired RUNX2 function. Additionally, tamoxifen-induced deletion of Mek1 and Mek2 in osteoprogenitors in adult mice (Mek1Osx-ERTMek2-/-) significantly reduced bone mass. Mechanistically, this corresponded to decreased activation of osteoblast master regulators, including RUNX2, ATF4, and ß-catenin. Finally, we identified potential regulators of osteoblast differentiation in the ERK MAPK pathway using unbiased phospho-mass spectrometry. These observations demonstrate essential roles of ERK activation in osteogenesis and bone formation.

Sphenoid bone hypoplasia is a skeletal phenotype of cleidocranial dysplasia in a mouse model and patients.

Cleidocranial dysplasia (CCD) is an autosomal dominant disorder caused by heterozygous mutations in RUNX2. Affected individuals exhibit delayed maturation or hypoplasia in various bones, mainly including those formed by intramembranous ossification. Although several reports described deformation of the sphenoid bone in CCD patients, details of the associated changes have not been well documented. Most parts of the sphenoid bone are formed by endochondral ossification; however, the medial pterygoid process is formed by intramembranous ossification associated with secondary cartilage. We first investigated histological changes in the medial pterygoid process during different developmental stages in Runx2+/+ and Runx2+/- mice, finding that mesenchymal cell condensation of the anlage of this structure was delayed in Runx2+/- mice as compared with that in Runx2+/+ mice. Additionally, in Runx2+/+ mice, Osterix-positive osteoblastic cells appeared at the upper region of the anlage of the medial pterygoid process, and bone trabeculae appeared to associate with subsequent secondary cartilage formation. By contrast, few Osterix-positive osteoblastic cells appeared at the upper region of the anlage of the medial pterygoid process, and no bone trabeculae appeared thereafter in Runx2+/- mice. At more advanced embryonic stages, endochondral ossification occurred at the lower part of the medial pterygoid process in both Runx2+/+ and Runx2+/- mice. After birth, well-developed bone trabeculae occupied two-thirds of the cranial side of the medial pterygoid process, and cartilage appeared beneath these bones in Runx2+/+ mice, whereas thin trabecular bone appeared at the center of the cartilage of the medial pterygoid process in Runx2+/- mice. In adult mice, the body and medial pterygoid processes of the sphenoid bone comprised mature bones in both Runx2+/+ and Runx2+/- mice, although the axial length of the medial pterygoid processes was apparently lower in Runx2+/-mice as compared with that in Runx2+/+mice based on histological and micro-computed tomography (CT) examinations. Moreover, medical-CT examination revealed that in CCD patients, the medial pterygoid process of sphenoid bone was significantly shorter relative to that in healthy young adults. These results demonstrated that the medial pterygoid process of the sphenoid bone specifically exhibited hypoplasia in CCD.

A Case of Cleidocranial Dysplasia with a Novel Mutation and Growth Velocity Gain with Growth Hormone Treatment

Cleidocranial dysplasia (CCD) is a rare congenital autosomal dominant skeletal disorder that is characterized by hypoplasia or aplasia of clavicles, failure of cranial suture closure, dental anomalies, short stature and other changes in skeletal patterning and growth. The gene responsible for pathogenesis has been mapped to the short arm of chromosome 6p21, core binding factor alpha-1 (CBFA1) or runt related transcription factor-2 (RUNX2). Here we describe a CCD patient with a novel mutation in the RUNX2 gene. A five-and-a-half year old girl presented with severe short stature, dysmorphic facial appearance (hypertelorism, prominent forehead, high palate, midfacial hypoplasia), macrocephaly, large anterior fontanelle, increased anteroposterior chest diameter. Her shoulders were close to each other and her bilateral clavicles appeared short on physical examination. Bilateral hypoplastic clavicles, coxa valga, hypoplasia of iliac bones, wide symphysis pubis and phalangeal dysplastic features were detected on her skeletal X-ray examination. She was diagnosed as having CCD. Molecular analysis detected a novel heterozygous mutation 'NM_001024630.3p.T155P(c.463A>C)' in the RUNX2 gene. At age seven years and two months old, because of her severe short stature, growth hormone (GH) treatment was started and she responded well to GH therapy with no adverse effects. In conclusion, hypoplasia or aplasia of the clavicles, failure of cranial suture closure, dental anomalies and short stature should bring CCD to mind. We present a novel mutation in the RUNX2 gene for CCD. We obtained growth velocity gain with GH treatment in our patient.

An 18 bps in-frame deletion mutation in RUNX2 gene is a population polymorphism rather than a pathogenic variant.

We recruited a family with an affected child exhibiting features of cleidocranial dysplasia with some phenotypic variations from reported cases. Whole exome sequencing data analysis identified an 18-bps heterozygous in-frame deletion variant (c.243-260delGGCGGCTGCGGCGGCGGC) in the RUNX2 gene. Sanger sequencing validated the presence of deletion in affected individual. Initially, we considered this variant as a causal mutation for the patient's phenotype based on previous report(s). However, further analysis of variant revealed that it is present in high frequency in variety of genome variation databases. Moreover, segregation analysis discovered the presence of variant in mother as well. Furthermore, screening of population matched control individuals revealed that the variant is present in apparently healthy individuals as well. Three-dimensional structures of the wild-type and mutant RUNX2 protein (p.Ala82_Ala87del) were analysed and it was found that both wild type and mutant protein show similar secondary structure pattern. Presence of RUNX2 deletion variant (c.243-260delGGCGGCTGCGGCGGCGGC) in control individuals, its high population frequency, benign effect on the overall protein structure lead to the argument that this variant is a population polymorphism and not a pathogenic mutation.

Tooth formation and eruption - lessons learnt from cleidocranial dysplasia.

The principles of formation, renewal, and eruption of teeth are discussed. Numerous genetic aberrations may affect the formation and eruption of teeth. Cleidocranial dysplasia (CCD), caused by mutations in the runt-related transcription factor 2 (RUNX2) gene, is such a condition. The dental phenotype includes problems in both tooth formation (multiple supernumerary permanent teeth) and tooth eruption (lack of shedding of primary teeth and delayed or arrested eruption of permanent teeth). Clinical studies, animal models, and molecular biology studies have documented that RUNX2 is of paramount importance for osteoblast differentiation, for regression of the dental lamina, and for osteoclastogenesis in the dental follicle and the periodontal ligament. Jensen & Kreiborg, 25 yr ago, proposed a treatment strategy to be applied to patients with CCD, focussing on the importance of early treatment to promote spontaneous eruption of permanent teeth through extraction of primary teeth, surgical removal of supernumerary teeth, and removal of bone covering the first formed permanent teeth at the time when root formation of the permanent teeth has reached half or two-thirds of their final length. This strategy still seems valid and seems to lead to reduction in the burden of care for patients compared with the treatment protocols otherwise recommended.

RUNX2 mutation reduces osteogenic differentiation of dental follicle cells in cleidocranial dysplasia.

Disturbed permanent tooth eruption is common in cleidocranial dysplasia (CCD), a skeletal disorder caused by heterozygous mutation of RUNX2, but the mechanism underlying is still unclear. As it is well known that dental follicle cells (DFCs) play a critical role in tooth eruption, the changed biological characteristics of DFCs might give rise to disturbance of permanent tooth eruption in CCD patients. Thus, primary DFCs from one CCD patient and normal controls were collected to investigate the effect of RUNX2 mutation on the bone remodeling activity of DFCs and explore the mechanism of impaired permanent tooth eruption in this disease. Conservation and secondary structure analysis revealed that the RUNX2 mutation (c.514delT, p.172fs) found in the present CCD patient was located in the highly conserved RUNT domain and converted the structure of RUNX2. After osteogenic induction, we found that the mineralised capacity of DFCs and the expression of osteoblast-related genes, including RUNX2, ALP, OSX, OCN and Col Iα1, in DFCs was severely interfered by the RUNX2 mutation found in CCD patients. To investigate whether the osteogenic deficiency of DFCs from the CCD patient can be rescued by RUNX2 restoration, we performed 'rescue' experiments. Surprisingly, the osteogenic deficiency and the abnormal expression of osteoblast-associated genes in DFCs from the CCD patient were almost rescued by overexpression of wild-type RUNX2 using lentivirus. All these findings indicate that RUNX2 mutation can reduce the osteogenic capacity of DFCs through inhibiting osteoblast-associated genes, thereby disturbing alveolar bone formation, which serves as a motive force for tooth eruption. This effect may provide valuable explanations and implications for the mechanism of delayed permanent tooth eruption in CCD patients.

Multiple supernumerary teeth in a likely syndromic individual from prehistoric Illinois.

OBJECTIVE: This paper reports the first published case of a prehistoric human with five or more supernumerary teeth. Such cases are often neglected in paleopathology, in part due to a gap between the medicodental and anthropological literature leading to the view in anthropology that supernumerary teeth are curious anomalies rather than pathologies. DESIGN: Reconstruction and pathological description of the skeletal remains were performed according to standard osteological protocols. Each supernumerary tooth was categorized based on its morphology, location, and orientation. The dental characteristics of the individual were compared to published norms for incidences of syndromic and non-syndromic supernumerary teeth and a differential diagnosis was subsequently performed. RESULTS: Six supernumerary teeth and one deciduous tooth were identified. Additionally, the individual suffered from impacted teeth, dilacerated roots, and extensive sutural anomalies (including retention of the metopic suture into adulthood and an unusually high number of sutural bones). The morphology and location of the supernumerary teeth, in conjunction with the suite of other symptoms, are highly unusual among non-syndromic patients and therefore are indicative of a complex genetic disorder. CONCLUSIONS: The individual reported here almost certainly suffered from a genetic disorder or syndrome resulting in extensive dental and sutural abnormalities. Despite a lack of post-cranial involvement, a tentative diagnosis of Cleidocranial Dysplasia was made on the basis that mutations in the RUNX2 gene may cause the dental symptoms without any pathology of the clavicle.

Abnormal bone remodelling activity of dental follicle cells from a cleidocranial dysplasia patient.

OBJECTIVES: To explore the role of dental follicle cells (DFCs) with a novel cleidocranial dysplasia (CCD) causative gene RUNX2 mutation (DFCsRUNX2+/m ) in delayed permanent tooth eruption. MATERIALS AND METHODS: A CCD patient with typical clinical features was involved in this study. DFCsRUNX2+/m were cultured and DNA was extracted for RUNX2 mutation screening. Measurements of cell proliferation, alkaline phosphatase (ALP) activity, alizarin red staining and osteoblast-specific genes expression were performed to assess osteogenesis of DFCsRUNX2+/m . Co-culture of DFCs and peripheral blood mononuclear cells (PBMCs), followed tartrate-resistant acid phosphatase (TRAP) staining, real-time PCR and western blot were performed to evaluate osteoclast-inductive capacity of DFCsRUNX2+/m . RESULTS: A missense RUNX2 mutation (c. 557G>C) was found in DFCsRUNX2+/m from the CCD patient. Compared with normal controls, this mutation did not affect the proliferation of DFCsRUNX2+/m , but down-regulated the expression of osteogenesis-related genes, leading to a decrease in ALP activity and mineralisation. Co-culture results showed that DFCsRUNX2+/m reduced the formation of TRAP+ multinucleated cells and the expression of osteoclastogenesis-associated genes. Furthermore, the mutation reduced the ratio of RANKL/OPG in DFCsRUNX2+/m . CONCLUSIONS: DFCsRUNX2+/m disturbs bone remodelling activity during tooth eruption through RANK/RANKL/OPG signalling pathway and may thus be responsible for impaired permanent tooth eruption in CCD patients.

Characterization of dental phenotype in patients with cleidocranial dysplasia using longitudinal data.

OBJECTIVE: To investigate the characteristics of the dental phenotype in patients with cleidocranial dysplasia (CCD) using longitudinal data. MATERIALS AND METHODS: Twelve unrelated Korean CCD patients were observed using a longitudinal series of radiographs and clinical photographs. Statistical analysis was performed on the dental phenotypic data. RESULTS: Although dysplasia of the clavicles, open fontanelle, and wormian bone were observed in all 12 patients, delayed fusion of the mandibular symphysis was found in four patients. One patient did not have a supernumerary tooth (ST). However, 62 STs were found in 11 patients (mean, 5.6 per patient; range of ST emergence, 5 years 6 months-14 years 8 months; developing position, occlusal to the permanent incisors, canines, and premolars and distal and apical to the permanent molars). The mandibular premolar region was the most frequent area of ST development (50.0%, P < .001). All 12 patients showed impacted permanent teeth (IPT), including one patient without ST (mean, 17.8 per patient). Impaction occurred most frequently in the mandibular premolar region and least frequently in the maxillary molar region (93.8% vs 39.6%, P < .01). The ratio of spontaneous eruption of IPT after removal of retained deciduous teeth and/or ST was highest for the maxillary and mandibular incisors (all 54.6%) and lowest for the mandibular canines and premolars (26.7% and 28.9%, respectively); however, the difference was not significant. CONCLUSIONS: The emergence time and development position of ST and the root development of IPT should be considered to determine the timing for the removal of ST and forced eruption of IPT.

Mutant Runx2 regulates amelogenesis and osteogenesis through a miR-185-5p-Dlx2 axis.

Regulation of microRNAs (miRNA) has been extensively investigated in diseases; however, little is known about the roles of miRNAs in cleidocranial dysplasia (CCD). The aim of the present study was to investigate the potential involvement of miRNAs in CCD. In vitro site-directed mutagenesis was performed to construct three mutant Runx2 expression vectors, which were then transfected into LS8 cells and MC3T3-E1 cells, to determine the impact on amelogenesis and osteogenesis, respectively. miRCURY LNA miRNA microarray identify miR-185-5p as a miRNA target commonly induced by all three Runx2 mutants. Real-time quantitative PCR was applied to determine the expression of miR-185-5p and Dlx2 in samples. Dual-luciferase reporter assays were conducted to confirm Dlx2 as a legitimate target of miR-185-5p. The suppressive effect of miR-185-5p on amelogenesis and osteogenesis of miR-185-5p was evaluated by RT-PCR and western blot examination of Amelx, Enam, Klk4, and Mmp20 gene and protein expression, and by Alizarin Red stain. We found that mutant Runx2 suppressed amelogenesis and osteogenesis. miR-185-5p, induced by Runx2, suppressed amelogenesis and osteogenesis. Furthermore, we identified Dlx2 as direct target of miR-185-5p. Consistently, Dlx2 expression was inversely correlated with miR-185-5p levels. This study highlights the molecular etiology and significance of miR-185-5p in CCD, and suggests that targeting miR-185-5p may represent a new therapeutic strategy in prevention or intervention of CCD.

mTOR/Raptor signaling is critical for skeletogenesis in mice through the regulation of Runx2 expression.

The mammalian target of rapamycin (mTOR)/regulatory-associated protein of mTOR (Raptor) pathway transmits and integrates different signals including growth factors, nutrients, and energy metabolism. Nearly all these signals have been found to play roles in skeletal biology. However, the contribution of mTOR/Raptor to osteoblast biology in vivo remains to be elucidated as the conclusions of recent studies are controversial. Here we report that mice with a deficiency of either mTOR or Raptor in preosteoblasts exhibited clavicular hypoplasia and delayed fontanelle fusion, similar to those found in human patients with cleidocranial dysplasia (CCD) haploinsufficient for the transcription factor runt-related transcription factor 2 (Runx2) or those identified in Runx2+/- mice. Mechanistic analysis revealed that the mTOR-Raptor-S6K1 axis regulates Runx2 expression through phosphorylation of estrogen receptor α, which binds to Distal-less homeobox 5 (DLX5) and augments the activity of Runx2 enhancer. Moreover, heterozygous mutation of raptor in osteoblasts aggravates the bone defects observed in Runx2+/- mice, indicating a genetic interaction between Raptor and Runx2. Collectively, these findings reveal that mTOR/Raptor signaling is essential for bone formation in vivo through the regulation of Runx2 expression. These results also suggest that a selective mTOR/Raptor antagonist, which has been developed for treatment of many diseases, may have the side effect of causing bone loss.

Functional analysis of novel RUNX2 mutations in cleidocranial dysplasia.

Cleidocranial dysplasia (CCD) is a rare autosomal dominant skeletal disorder caused by mutation of runt-related transcription factor 2 (RUNX2) gene. The purpose of this study was to explore novel RUNX2 mutations in seven individuals with CCD and investigate the function of the mutant RUNX2 proteins. DNA samples were prepared from the peripheral blood of the CCD individuals, and then subjected to DNA sequencing. Conservation and secondary structure analysis were performed based on RUNX2 sequencing results. pEGFP-C1 plasmids containing GFP-tagged wild-type RUNX2 and three novel RUNX2 mutations expression cassettes were constructed, and then transfected into HEK293T cells. Cell fluorescence, luciferase assay and western blotting were used to analyse the subcellular distribution and function of the mutant RUNX2 proteins. Three novel mutations (R193G, 258fs, Y400X) were found in the seven CCD patients. Conservation and structure analysis show one novel mutation (R193G) in Runt domain and two novel mutations (258fs and Y400X) in PST domain of RUNX2. Western blotting confirmed that the 258fs and Y400X mutations produced truncated proteins. Fluorescence detection showed that the three novel mutants localised exclusively in the nucleus. However, luciferase assay indicated all mutants severely impaired the transactivation activities of RUNX2 on osteocalcin promoter. Our results broaden the spectrum of RUNX2 mutations in CCD individuals and demonstrated that loss of function in RUNX2 is responsible for CCD.

Cleidocranial dysplasia: Clinical, endocrinologic and molecular findings in 15 patients from 11 families.

Cleidocranial dysplasia (CCD) is an autosomal dominant disorder characterized by skeletal anomalies such as delayed closure of the cranial sutures, underdeveloped or absent clavicles, multiple dental abnormalities, short stature and osteoporosis. RUNX2, encoding Runt DNA-binding domain protein important in osteoblast differentiation, is the only known gene related to the disease and identified as responsible in 70% of the cases. Our clinical evaluations revealed that short stature present at a rate of 28.6%, osteoporosis at a rate of 57.1% and osteopenia at 21.4%. In this study, RUNX2 sequencing revealed nine different variations in 11 families, eight being pathogenic of which one was novel gross insertion (c.1271_1272ins20) and one other being predicted benign in frame gross deletion (c.241_258del).