Cleidocranial Dysplasia Patients Research Articles

Fixed Prosthodontic Rehabilitation of a Patient with Cleidocranial Dysplasia: A Case Report

Introduction Cleidocranial Dysplasia (CCD) is a rare congenital disease characterized by skeletal and dental anomalies. Clinical findings of CCD patients include low facial height, pseudoprognathism, unerupted teeth, an excessive deep bite, chewing difficulties, and unsatisfied dentofacial appearance. These patients’ dental treatments present a substantial challenge. Case Representation This case report describes the prosthodontic treatment of a 29-year-old male CCD patient using porcelain-fused-to-metal restorations. The avoidance of a surgical procedure serves to minimize the potential for complications and expedites the attainment of outcomes with greater celerity. Throughout the follow-up period of 1 year, the patient maintained good periodontal health. The restoration of masticatory function and enhancement of facial esthetics were successfully achieved and the patient expressed a high degree of satisfaction with the outcome of the treatment. Conclusion The use of fixed prostheses in CCD patients is a treatment modality that resolves many of the issues caused by the surgical approach.

Impact of Mechanical Strain and Nicotinamide on RUNX2-Deficient Osteoblast Mimicking Cleidocranial Dysplasia

Cleidocranial dysplasia (CCD) is a rare genetic defect caused by a heterozygous mutation of runt-related transcription factor 2 (RUNX2), which is important for osteoblast and skeletal development. RUNX2-deficiency causes extra- and intra-oral malformations that often require orthodontic treatment. Nicotinamide (NAM) affects bone remodelling processes. As these are crucial for orthodontic therapy, NAM could improve orthodontic treatment in CCD patients. This study investigates the effect of NAM in control and RUNX2-deficient osteoblasts under mechanical strain mimicking orthodontic treatment. First, the optimal NAM concentration and the differences in the expression profile of control and RUNX2-deficient osteoblasts were determined. Subsequently, osteoblasts were exposed to tensile and compressive strain with and without NAM, and the expression of genes critically involved in bone remodelling was investigated. NAM increased the expression of bone remodelling genes. RUNX2-deficient osteoblasts expressed more receptor activator of NFkB ligand (RANKL) and interleukin-6 (IL6), but less colony-stimulating factor-1 (CSF1). Most of the positive effects of NAM on bone remodelling genes were impaired by mechanical loading. In conclusion, NAM stimulated osteoblast differentiation by increasing the expression of RUNX2 and regulated the expression of osteoclastogenic factors. However, the positive effects of NAM on bone metabolism were impaired by mechanical loading and RUNX2 deficiency.

Functional consequences of C-terminal mutations in RUNX2

Cleidocranial dysplasia (CCD) is a genetic disorder caused by mutations in the RUNX2 gene, affecting bone and teeth development. Previous studies focused on mutations in the RUNX2 RHD domain, with limited investigation of mutations in the C-terminal domain. This study aimed to investigate the functional consequences of C-terminal mutations in RUNX2. Eight mutations were analyzed, and their effects on transactivation activity, protein expression, subcellular localization, and osteogenic potential were studied. Truncating mutations in the PST region and a missense mutation in the NMTS region resulted in increased transactivation activity, while missense mutations in the PST showed activity comparable to the control. Truncating mutations produced truncated proteins, while missense mutations produced normal-sized proteins. Mutant proteins were mislocalized, with six mutant proteins detected in both the nucleus and cytoplasm. CCD patient bone cells exhibited mislocalization of RUNX2, similar to the generated mutant. Mislocalization of RUNX2 and reduced expression of downstream genes were observed in MSCs from a CCD patient with the p.Ser247Valfs*3 mutation, leading to compromised osteogenic potential. This study provides insight into the functional consequences of C-terminal mutations in RUNX2, including reduced expression, mislocalization, and aberrant transactivation of downstream genes, contributing to the compromised osteogenic potential observed in CCD.

RUNX2 mutation inhibits the cellular senescence of dental follicle cells via ERK signalling pathway.

The aim of this study was to explore the regulatory effect of RUNX2 mutation on dental follicle cells (DFCs) senescence and clarify the underlying mechanism. This study aimed to explore the basis for a novel mechanism of delayed permanent tooth eruption in cleidocranial dysplasia (CCD) patients. Dental follicles were collected from a CCD patient and healthy controls. Senescence-associated β-galactosidase (SA-β-gal) staining, Ki67 staining, cell cycle assays, and senescence-related gene and protein expression assays were performed to assess DFCs senescence. Western blotting was performed to detect the activation of mitogen-activated protein kinase (MAPK) signalling pathways, and the molecular mechanism underlying RUNX2 regulating in DFCs senescence was explored. RUNX2 mutation inhibited the cellular senescence of DFCs from the CCD patient compared with healthy controls. Ki67 staining showed that mutant RUNX2 promoted DFCs proliferation, and cell cycle assays revealed that the healthy control-derived DFCs arrested at G1 phase. RUNX2 mutation significantly downregulated senescence-associated gene and protein expression. RUNX2 mutation suppressed ERK signalling pathway activation, an ERK inhibitor decreased healthy control-derived DFCs senescence, and an ERK activator promoted CCD patient-derived DFCs senescence. RUNX2 mutation delayed DFCs senescence through the ERK signalling pathway, which may be responsible for delayed permanent tooth eruption in CCD patients.

Amyloid-mediated remineralization for tooth hypoplasia of cleidocranial dysplasia.

Cleidocranial dysplasia (CCD) is an autosomal-dominant, heritable skeletal and dental disease, involving hypoplastic clavicles, defective ossification of the anterior fontanelle, dentin and enamel hypoplasia, and supernumerary teeth, which can seriously affect the oral and mental health of patients. Amyloid-like protein aggregation, which is established by lysozyme conjugated with polyethylene glycol (Lyso-PEG), forms a mineralized nanofilm layer on a healthy enamel surface. However, whether it can form a remineralization layer in dental tissues from CCD remains unclear. This study evaluated deciduous teeth from healthy individuals and a patient with CCD. Because pulp and dentin are functionally closely related, stem cells from human exfoliated deciduous teeth (SHED) from CCD patients and healthy individuals were collected to compare their biological properties. The results found that deciduous teeth from patients with CCD exhibited dentin hypoplasia. In addition, the proliferative ability and osteogenic potential of SHED from patients with CCD were lower than those of control individuals. Finally, Lyso-PEG was applied to dentin from the CCD and control groups, showing a similar remineralization-induced effect on the dentin surfaces of the two groups. These results extend our understanding of the dentin and SHED of patients with CCD, exhibiting good caries-preventive capacity and good biocompatibility of Lyso-PEG, thus providing a novel dental therapy for CCD and patients with tooth hypoplasia.

Inhibition of miR338 rescues cleidocranial dysplasia in Runx2 mutant mice partially via the Hif1a-Vegfa axis

Haploinsufficiency of Runt-related transcription factor-2 (RUNX2) is responsible for cleidocranial dysplasia (CCD), a rare hereditary disease with a range of defects, including delayed closure of the cranial sutures and short stature. Symptom-based treatments, such as a combined surgical-orthodontic approach, are commonly used to treat CCD patients. However, there have been few reports of treatments based on Runx2-specific regulation targeting dwarfism symptoms. Previously, we found that the miR338 cluster, a potential diagnostic and therapeutic target for postmenopausal osteoporosis, could directly target Runx2 during osteoblast differentiation in vitro. Here, we generated miR338−/−;Runx2+/− mice to investigate whether inhibition of miR338 could rescue CCD defects caused by Runx2 mutation in vivo. We found that the dwarfism phenotype caused by Runx2 haploinsufficiency was recovered in miR338−/−;Runx2+/− mice, with complete bone density restoration and quicker closure of fontanels. Single-cell RNA-seq analysis revealed that knockout of miR338 specifically rescued the osteoblast lineage priming ability of bone marrow stromal cells in Runx2+/− femurs, which was further confirmed by Osterix-specific conditional knockout of miR338 in Runx2+/− mice (OsxCre; miR338fl/fl;Runx2+/−). Mechanistically, ablation of the miR338 cluster in Runx2+/− femurs directly rescued the Hif1a-Vegfa pathway in Runx2+/− osteoblasts, as proven by gene expression profiles and ChIP and Re-ChIP assays. Collectively, our data revealed the genetic interaction between Runx2 and the miR338 cluster during osteoblast differentiation and implied that the miR338 cluster could be a potential therapeutic target for CCD.

An overview of the intraoral features and craniofacial morphology of growing and adult Japanese cleidocranial dysplasia subjects.

Orthodontic treatment for cleidocranial dysplasia (CCD) requires an understanding of the nature of the retained deciduous teeth, supernumerary teeth, delayed eruption of the permanent teeth, and craniofacial morphology from childhood to adulthood. This study aimed to provide an overview of the intraoral and craniofacial characteristics of growing and adult Japanese CCD subjects. We assessed cross-sectionally the intraoral features of 28 CCD subjects (males, 15.3 ± 7.0 years; females, 15.2 ± 5.1 years) using orthopantomograms and photographs. Mean facial diagrams (profilograms) of 3 age groups (5-10 years, 11-14 years, over 15 years: adult) were constructed, and linear and angular measurements of 2 age groups (under 15 years, adult) were performed by using cephalograms. The data were compared with Japanese standards. A mean of 11.7 and 8.4 retained deciduous teeth, 10.4 and 15.8 erupted permanent teeth were observed in the adult males and females, and a mean of 6.8 and 5.3 supernumerary teeth were observed in all males and females, respectively. A positive correlation was found between the number of supernumerary teeth and the age at initial visit. Cephalometric analysis showed an average to anteriorly positioned maxilla, a tendency for counter-clockwise rotation of the ramus, and a prognathic mandible in all groups. The number of supernumerary teeth increased with age. The maxilla was average to anteriorly positioned, and the mandible was counter-clockwise rotated and prognathic for all groups. These characteristic craniofacial morphologies and changes of intraoral conditions at different ages in CCD patients should be considered when proposing rational orthodontic treatment plans.

RUNX2 Regulates Osteoblast Differentiation via the BMP4 Signaling Pathway

RUNX2 is a master osteogenic transcription factor, and mutations in RUNX2 cause the inherited skeletal disorder cleidocranial dysplasia (CCD). Studies have revealed that RUNX2 is not only a downstream target of the bone morphogenetic protein (BMP) pathway but can also regulate the expression of BMPs. However, the underlying mechanism of the regulation of BMPs by RUNX2 remains unknown. In this project, we diagnosed a CCD patient with a 7.86-Mb heterozygous deletion on chromosome 6 containing all exons of RUNX2 by multiplex ligation-dependent probe amplification (MLPA) and whole-genome sequencing (WGS). Bone marrow mesenchymal stem cells (BMSCs) were further extracted from patient alveolar bone fragments (CCD-BMSCs), an excellent natural model to explore the possible mechanism. The osteogenic differentiation ability of CCD-BMSCs was severely affected by RUNX2 heterozygous deletion. Also, BMP4 decreased most in BMP ligands, and CHRDL1, a BMP antagonist, was abnormally elevated in CCD-BMSCs. Furthermore, BMP4 treatment essentially rescued the osteogenic capacity of CCD-BMSCs, and RUNX2 overexpression reversed the abnormal expression of BMP4 and CHRDL1. Notably, we constructed CRISPR/Cas9 Runx2+/m MC3T3-E1 cells, which simulated a variant in CCD-BMSCs, to exclude the interference of other gene deletions and the heterogeneity of the genetic background of primary cells, and verified all findings from the CCD-BMSCs. Moreover, the luciferase reporter experiment showed that RUNX2 could inhibit the transcription of CHRDL1. Through immunofluorescence, the inhibitory effect of CHRDL1 on BMP4/Smad signaling was confirmed in MC3T3-E1 cells. These results revealed that RUNX2 regulated the BMP4 pathway by inhibiting CHRDL1 transcription. We collectively identified a novel RUNX2/CHRDL1/BMP4 axis to regulate osteogenic differentiation and noted that BMP4 might be a valuable therapeutic option for treating bone diseases.

Fisetin: An Integrated Approach to Identify a Strategy Promoting Osteogenesis.

Flavonoids may modulate the bone formation process. Among flavonoids, fisetin is known to counteract tumor growth, osteoarthritis, and rheumatoid arthritis. In addition, fisetin prevents inflammation-induced bone loss. In order to evaluate its favorable use in osteogenesis, we assayed fisetin supplementation in both in vitro and in vivo models and gathered information on nanoparticle-mediated delivery of fisetin in vitro and in a microfluidic system. Real-time RT-PCR, Western blotting, and nanoparticle synthesis were performed to evaluate the effects of fisetin in vitro, in the zebrafish model, and in ex vivo samples. Our results demonstrated that fisetin at 2.5 µM concentration promotes bone formation in vitro and mineralization in the zebrafish model. In addition, we found that fisetin stimulates osteoblast maturation in cell cultures obtained from cleidocranial dysplasia patients. Remarkably, PLGA nanoparticles increased fisetin stability and, consequently, its stimulating effects on RUNX2 and its downstream gene SP7 expression. Therefore, our findings demonstrated the positive effects of fisetin on osteogenesis and suggest that patients affected by skeletal diseases, both of genetic and metabolic origins, may actually benefit from fisetin supplementation.

Induced pluripotent stem cells from homozygous Runx2-deficient mice show poor response to vitamin D during osteoblastic differentiation.

Cleidocranial dysplasia (CCD) is a hereditary disorder associated with skeletal dysplasia and dental abnormalities. CCD arises from heterozygous loss of function mutations in the Runt-related transcription factor 2 (RUNX2) gene. Osteoporosis is often observed in CCD patients and conventional vitamin D supplementation is recommended. However, sufficient evidences have not been presented yet. This study investigated the role of RUNX2 in osteoblastic differentiation and sought to identify potential target genes for the treatment of osteoporosis associated with CCD, using induced pluripotent stem cell (iPSC) technology. We successfully established Runx2-/-, Runx2+/- and wild-type miPSCs from litter-matched mice and found poor Vdr expression in Runx2-/-cells. Significant down-regulation of osteoblastic differentiation in Runx2-/- miPSCs was observed. Gene expression array revealed unexpected results such as remarkable increase of Rankl expression and decrease of Vdr in Runx2-/- cells. Insufficient response to vitamin D in Runx2-/- cells was also observed. Our results suggest that RUNX2 functions as a regulator of Rankl and Vdr and thereby controls bone density. These findings also suggest that conventional vitamin D supplementation may not be as effective as previously expected, in the treatment of osteoporosis associated with CCD, and that inhibiting RANKL function might be worth considering as an alternative treatment strategy.

Clinical characteristics and comprehensive treatment of patients with cleidocranial dysplasia

Objective: To summarize the clinical characteristics of patients with cleidocranial dysplasia (CCD) and analyze their treatment methods. Methods: From January 2000 to December 2020, patients with CCD who completed comprehensive treatment in the Department of Orthodontics and the First Dental Clinic, School and Hospital of Stomatology, China Medical University were retrospectively analyzed. A total of 14 CCD patients [7 males and 7 females, aged (16.1±4.5) years] were collected. There were 153 impacted permanent teeth in this study. In addition to the teeth that needed to be extracted due to special conditions, 147 impacted teeth were pulled into the dentition using closed traction. Patients were divided into adolescent group (≥12 years and<18 years, 10 patients) and adult group (≥18 years, 4 patients). Failure rate of traction was compared between the two groups. Factors affecting the success rate of closed traction such as vertical position of teeth (high, middle and low) and horizontal position of the teeth (palatal, median and buccal) were analyzed. Results: The incidence of maxillary impacted teeth [69.3% (97/140)] was higher than that of mandibular impacted teeth [40% (56/140)]. The difference was statistically significant (χ2=24.22, P<0.001). The supernumerary teeth were mainly located in the premolar area 61.4% (21/44), and most of them were in the palatal region of the permanent teeth 95.5% (42/44). They were generally located at the same height or the occlusal side of the corresponding permanent teeth. The success rate of closed traction was 93.9% (138/147). The success rate in the adolescent group [98.2% (108/110)] was higher than that in the adult group [81.1% (30/37)], and the difference was significant (χ2=14.09, P<0.05). Failure after closed traction of 9 teeth was found totally, including 7 second premolars. The success rate of traction in impacted second premolars at different vertical (χ2=11.44, P<0.05) and horizontal (χ2=9.71, P<0.05) positions in alveolar bone was different significantlly. The success rates of the second premolars were high (15/16), middle (12/13), low (2/7), and lingual palatine (10/17), median (19/19), lip-buccal (0/0), respectively. Conclusions: The closed traction of impacted teeth in patients with CCD was effective, and the age was the main variable affecting the outcome. The success rate of traction in impacted second premolars located in low position vertically or in palatal position was low, which required close observation during treatment.

Surgical treatment of multiple impacted supernumerary teeth in a cleidocranial dysplasia patient

AbstractTo report a case of cleidocranial dysplasia (CCD) with 15 supernumerary teeth and review the relevant literature. Diagnose and treat a CCD patient with 15 impacted supernumerary teeth in the maxilla and mandible. When CCD is accompanied by supernumerary teeth ambushed in the jaw and does not affect the health of the body, it can be regarded as a part of normal bone tissue. If X‐ray or computed tomography examination shows that supernumerary teeth affect permanent tooth eruption, the replacement process of adjacent teeth and permanent tooth eruption should be closely observed, and supernumerary teeth should be extracted in time if necessary.

Cleidocranial dysplasia and novel RUNX2 variants: dental, craniofacial, and osseous manifestations.

Cleidocranial dysplasia (CCD) is a skeletal disorder affecting cranial sutures, teeth, and clavicles, and is associated with the RUNX2 mutations. Although numerous patients have been described, a direct genotype–phenotype correlation for RUNX2 has been difficult to establish. Further cases must be studied to understand the clinical and genetic spectra of CCD.ObjectivesTo characterize detailed phenotypes and identify variants causing CCD in five unrelated patients and their family members.MethodologyClinical and radiographic examinations were performed. Genetic variants were identified by exome and Sanger sequencing, data were analyzed by bioinformatics tools.ResultsThree cases were sporadic and two were familial. Exome sequencing successfully detected the heterozygous pathogenic RUNX2 variants in all affected individuals. Three were novel, comprising a frameshift c.739delA (p.(Ser247Valfs*)) in exon 6 (Patient-1), a nonsense c.901C>T (p.(Gln301*)) in exon 7 (Patient-2 and affected mother), and a nonsense c.1081C>T (p.(Gln361*)) in exon 8 (Patient-3). Two previously reported variants were missense: the c.673C>T (p.(Arg225Trp)) (Patient-4) and c.674G>A (p.(Arg225Gln)) (Patient-5) in exon 5 within the Runt homology domain. Patient-1, Patient-2, and Patient-4 with permanent dentition had thirty, nineteen, and twenty unerupted teeth, respectively; whereas Patient-3 and Patient-5, with deciduous dentition, had normally developed teeth. All patients exhibited typical CCD features, but the following uncommon/unreported phenotypes were observed: left fourth ray brachymetatarsia (Patient-1), normal clavicles (Patient-2 and affected mother), phalangeal malformations (Patient-3), and normal primary dentition (Patient-3, Patient-5).ConclusionsThe study shows that exome sequencing is effective to detect mutation across ethnics. The two p.Arg225 variants confirm that the Runt homology domain is vital for RUNX2 function. Here, we report a new CCD feature, unilateral brachymetatarsia, and three novel truncating variants, expanding the phenotypic and genotypic spectra of RUNX2 , as well as show that the CCD patients can have normal deciduous teeth, but must be monitored for permanent teeth anomalies.

Appropriateness of standard cephalometric norms for the assessment of dentofacial characteristics in patients with cleidocranial dysplasia.

Cleidocranial dysplasia (CCD) is a rare skeletal syndrome affecting craniofacial and dental development. As a consequence, conventional cephalometric landmarks may not be valid for CCD patients, and the appropriateness of norms used for the general population should be critically discussed. Five patients 9- to 22-year-old (three females, two males) with CCD were included. Lateral-cephalograms, orthopantomographies, and intra-oral photos were retrospectively analysed. Lateral-cephalograms of 50 normal controls (ten for each CCD patient) matched for age and sex were selected from an online database. Cephalometric measurements of each CCD patients were compared with average values of matched controls using Wilcoxon signed-rank test for paired values (α = 0.05). In CCD patients, a shortening of the cranial base was present (ΔSN = -17.1 mm, p = 0.043). Thus, the mandible (ΔSNPg = +9.5°, p = 0.043) and the maxilla (ΔSNA = +11.2°, p = 0.043) showed protrusion compared to the cranial base, despite a reduced maxillary (ΔCo-A = -15.1 mm, p = 0.043) and mandibular (ΔCo-Gn = -15.2 mm, p = 0.080) length. The mandibular divergence was reduced (ΔSN/GoGn = -6.4°, p = 0.043), a reduced overbite was present (ΔOverbite = -2.9 mm, p = 0.043), and the interincisal angle was increased (ΔInterincisalAngle = +13.7°, p = 0.043), mainly due to retro-inclination of lower incisors. Standard cephalometric norms for the assessment of horizontal jaw position may not be applicable to CCD patients because of a reduced anterior cranial base length compared to normal subjects. Vertical relationships may not be affected, and mandibular hypodivergency was confirmed.

Identification of a familial cleidocranial dysplasia with a novel RUNX2 mutation and establishment of patient-derived induced pluripotent stem cells

Cleidocranial dysplasia (CCD) is an autosomal dominant hereditary disease associated with the gene RUNX2. Disease-specific induced pluripotent stem cells (iPSCs) have emerged as a useful resource to further study human hereditary diseases such as CCD. In this study, we identified a novel CCD-specific RUNX2 mutation and established iPSCs with this mutation. Biopsies were obtained from familial CCD patients and mutation analyses were performed through Sanger sequencing and next generation sequencing. CCD-specific human iPSCs (CCD-hiPSCs) were established and maintained under completely defined serum, feeder, and integration-free condition using a non-integrating replication-defective Sendai virus vector. We identified the novel mutation RUNX2_c.371C>G and successfully established CCD-hiPSCs. The CCD-hiPSCs inherited the same mutation, possessed pluripotency, and showed the ability to differentiate the three germ layers. We concluded that RUNX2_c.371C>G was likely pathogenic because our results, derived from next generation sequencing, are supported by actual clinical evidence, familial tracing, and genetic data. Thus, we concluded that hiPSCs with a novel CCD-specific RUNX2 mutation are viable as a resource for future studies on CCD.

An Exploration of Mutagenesis in a Family with Cleidocranial Dysplasia without RUNX2 Mutation.

Cleidocranial dysplasia (CCD) is an autosomal dominant inheritable skeletal disorder characterized by cranial dysplasia, clavicle hypoplasia, and dental abnormalities. Mutations involving Runt-related transcription factor 2 (RUNX2) are currently the only known molecular etiology for CCD but are not identified in all CCD patients. No RUNX2 abnormality can be detected in about 20–30% of patients, and the molecular cause remains unknown. The present study includes a family case with typical features of CCD. RUNX2 mutation was first screened by sequencing analysis, and no mutation was detected. Copy number alterations of the RUNX2 gene were then measured by quantitative PCR and multiplex ligation-dependent probe amplification (MLPA). No copy number variation in RUNX2 could be detected. We performed whole-exome sequencing (WES) to identify the underlying genetic mutations. Unexpectedly, no abnormalities could be detected in genes related to the RUNX2 signaling pathway. Therefore, it was supposed that other new unknown gene variations might contribute to the CCD phenotype. We focused on Immunoglobulin superfamily member 10 (IGSF10), a gene related to bone development. An IGSF10 frameshift mutation (c.6001_6002delCT, p.Leu2001Valfs*24) was detected by WES. Sanger sequencing verified that this mutation was only detected in the patient and her affected mother but not in her unaffected father. Bioinformatics studies demonstrated that this mutation could change the 3D structure of the IGSF10 protein and severely damage its function. In addition, alkaline phosphatase (ALP) activity and the ability to form mineralized nodules were inhibited by IGSF10 knockdown compared with normal controls. The expression of bone sialoprotein (BSP) was significantly reduced by IGSF10 knockdown, but not that of other osteogenic markers. Our results provide new genetic evidence that IGSF10 mutation might contribute to CCD.

Two Novel C-Terminus RUNX2 Mutations in Two Cleidocranial Dysplasia (CCD) Patients Impairing p53 Expression.

Cleidocranial dysplasia (CCD), a dominantly inherited skeletal disease, is characterized by a variable phenotype ranging from dental alterations to severe skeletal defects. Either de novo or inherited mutations in the RUNX2 gene have been identified in most CCD patients. Transcription factor RUNX2, the osteogenic master gene, plays a central role in the commitment of mesenchymal stem cells to osteoblast lineage. With the aim to analyse the effects of RUNX2 mutations in CCD patients, we investigated RUNX2 gene expression and the osteogenic potential of two CCD patients’ cells. In addition, with the aim to better understand how RUNX2 mutations interfere with osteogenic differentiation, we performed string analyses to identify proteins interacting with RUNX2 and analysed p53 expression levels. Our findings demonstrated for the first time that, in addition to the alteration of downstream gene expression, RUNX2 mutations impair p53 expression affecting osteogenic maturation. In conclusion, the present work provides new insights into the role of RUNX2 mutations in CCD patients and suggests that an in-depth analysis of the RUNX2-associated gene network may contribute to better understand the complex molecular and phenotypic alterations in mutant subjects.

Skeletal and molecular findings in 51 Cleidocranial dysplasia patients from Turkey

Loss or decrease of function in runt-related transcription factor 2 encoded by RUNX2 is known to cause a rare autosomal-dominant skeletal disorder, cleidocranial dysplasia (CCD). Clinical spectrum and genetic findings in 51 CCD patients from 30 unrelated families are herein presented. In a majority of the patients, facial abnormalities, such as delayed fontanel closure (89%), parietal and frontal bossing (80%), metopic groove (77%), midface hypoplasia (94%), and abnormal mobility of shoulders (90%), were recorded following clinical examination. In approximately one-half of the subjects, wormian bone (51%), short stature (43%), bell-shaped thorax (42%), wide pubic symphysis (50%), hypoplastic iliac wing (59%), and chef's hat sign (44%) presented in available radiological examinations. Scoliosis was identified in 28% of the patients. Investigation of RUNX2 revealed small sequence alterations in 90% and gross deletions in 10% of the patients; collectively, 23 variants including 11 novel changes (c.29_30insT, c.203delAinsCG, c.423 + 2delT, c.443_454delTACCAGATGGGAinsG, c.505C > T, c.594_595delCTinsG, c.636_637insC, c.685 + 5G > A, c.1088G > T, c.1281delC, Exon 6-9 deletion) presented high allelic heterogeneity. Novel c.29_30insT is unique in affecting the P1-driven long isoform of RUNX2, which is expected to disrupt the N-terminal region of RUNX2; this was shown in two unrelated phenotypically discordant patients. The clinical findings highlighted mild intra-familial genotype-phenotype correlation in our CCD cohort.

Radiographic features of cleidocranial dysplasia on panoramic radiographs.

PurposeThis study aimed to investigate the panoramic imaging features of cleidocranial dysplasia (CCD) with a relatively large sample.Materials and MethodsThe panoramic radiographs of 40 CCD patients who visited Seoul National University Dental Hospital between 2004 and 2018 were analyzed. Imaging features were recorded based on the consensus of 2 radiologists according to the following criteria: the number of supernumerary teeth and impacted teeth; the shape of the ascending ramus, condyle, coronoid process, sigmoid notch, antegonial notch, and hard palate; the mandibular midline suture; and the gonial angle.ResultsThe mean number of supernumerary teeth and impacted teeth were 6.1 and 8.3, respectively, and the supernumerary teeth and impacted teeth were concentrated in the anterior and premolar regions. Ramus parallelism was dominant (32 patients, 80.0%) and 5 patients (12.5%) showed a mandibular midline suture. The majority of mandibular condyles showed a rounded shape (61.2%), and most coronoid processes were triangular (43.8%) or round (37.5%). The mean gonial angle measured on panoramic radiographs was 122.6°.ConclusionPanoramic radiographs were valuable for identifying the features of CCD and confirming the diagnosis. The presence of numerous supernumerary teeth and impacted teeth, especially in the anterior and premolar regions, and the characteristic shapes of the ramus, condyle, and coronoid process on panoramic radiographs may help to diagnose CCD.

Delayed tooth movement in Runx2+/− mice associated with mTORC2 in stretch-induced bone formation

Runt-related transcription factor 2 (Runx2) is an essential transcription factor for osteoblast differentiation, and is activated by mechanical stress to promote osteoblast function. Cleidocranial dysplasia (CCD) is caused by mutations of RUNX2, and CCD patients exhibit malocclusion and often need orthodontic treatment. However, treatment is difficult because of impaired tooth movement, the reason of which has not been clarified. We examined the amount of experimental tooth movement in Runx2+/− mice, the animal model of CCD, and investigated bone formation on the tension side of experimental tooth movement in vivo. Continuous stretch was conducted to bone marrow stromal cells (BMSCs) as an in vitro model of the tension side of tooth movement. Compared to wild-type littermates the Runx2+/− mice exhibited delayed experimental tooth movement, and osteoid formation and osteocalcin (OSC) mRNA expression were impaired in osteoblasts on the tension side of tooth movement. Runx2 heterozygous deficiency delayed stretch-induced increase of DNA content in BMSCs, and also delayed and reduced stretch-induced alkaline phosphatase (ALP) activity, OSC mRNA expression, and calcium content of BMSCs in osteogenic medium. Furthermore Runx2+/− mice exhibited delayed and suppressed expression of mammalian target of rapamycin (mTOR) and rapamycin-insensitive companion of mTOR (Rictor), essential factors of mTORC2, which is regulated by Runx2 to phosphorylate Akt to regulate cell proliferation and differentiation, in osteoblasts on the tension side of tooth movement in vivo and in vitro. Loss of half Runx2 gene dosage inhibited stretch-induced PI3K dependent mTORC2/Akt activity to promote BMSCs proliferation. Furthermore, Runx2+/− BMSCs in osteogenic medium exhibited delayed and suppressed stretch-induced expression of mTOR and Rictor. mTORC2 regulated stretch-elevated Runx2 and ALP mRNA expression in BMSCs in osteogenic medium. We conclude that Runx2+/− mice present a useful model of CCD patients for elucidation of the molecular mechanisms in bone remodeling during tooth movement, and that Runx2 plays a role in stretch-induced proliferation and osteogenesis in BMSCs via mTORC2 activation.