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. Objectives: To characterize detailed phenotypes and identify variants causing CCD in five unrelated patients and their family members. Methodology: Clinical and radiographic examinations were performed. Genetic variants were identified by exome and Sanger sequencing, data were analyzed by bioinformatics tools. Results: Three 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). Conclusions: The 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

Lrp4 Modulates Extracellular Integration of Cell Signaling Pathways in Development

The extent to which cell signaling is integrated outside the cell is not currently appreciated. We show that a member of the low-density receptor-related protein family, Lrp4 modulates and integrates Bmp and canonical Wnt signalling during tooth morphogenesis by binding the secreted Bmp antagonist protein Wise. Mouse mutants of Lrp4 and Wise exhibit identical tooth phenotypes that include supernumerary incisors and molars, and fused molars. We propose that the Lrp4/Wise interaction acts as an extracellular integrator of epithelial-mesenchymal cell signaling. Wise, secreted from mesenchyme cells binds to BMP's and also to Lrp4 that is expressed on epithelial cells. This binding then results in the modulation of Wnt activity in the epithelial cells. Thus in this context Wise acts as an extracellular signaling molecule linking two signaling pathways. We further show that a downstream mediator of this integration is the Shh signaling pathway

AI-based diagnosis in mandibulofacial dysostosis with microcephaly using external ear shapes

IntroductionMandibulo-Facial Dysostosis with Microcephaly (MFDM) is a rare disease with a broad spectrum of symptoms, characterized by zygomatic and mandibular hypoplasia, microcephaly, and ear abnormalities. Here, we aimed at describing the external ear phenotype of MFDM patients, and train an Artificial Intelligence (AI)-based model to differentiate MFDM ears from non-syndromic control ears (binary classification), and from ears of the main differential diagnoses of this condition (multi-class classification): Treacher Collins (TC), Nager (NAFD) and CHARGE syndromes.MethodsThe training set contained 1,592 ear photographs, corresponding to 550 patients. We extracted 48 patients completely independent of the training set, with only one photograph per ear per patient. After a CNN-(Convolutional Neural Network) based ear detection, the images were automatically landmarked. Generalized Procrustes Analysis was then performed, along with a dimension reduction using PCA (Principal Component Analysis). The principal components were used as inputs in an eXtreme Gradient Boosting (XGBoost) model, optimized using a 5-fold cross-validation. Finally, the model was tested on an independent validation set.ResultsWe trained the model on 1,592 ear photographs, corresponding to 1,296 control ears, 105 MFDM, 33 NAFD, 70 TC and 88 CHARGE syndrome ears. The model detected MFDM with an accuracy of 0.969 [0.838–0.999] (p < 0.001) and an AUC (Area Under the Curve) of 0.975 within controls (binary classification). Balanced accuracies were 0.811 [0.648–0.920] (p = 0.002) in a first multiclass design (MFDM vs. controls and differential diagnoses) and 0.813 [0.544–0.960] (p = 0.003) in a second multiclass design (MFDM vs. differential diagnoses).ConclusionThis is the first AI-based syndrome detection model in dysmorphology based on the external ear, opening promising clinical applications both for local care and referral, and for expert centers

Functional consequences of C-terminal mutations in RUNX2

Abstract 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

Expression of fibroblast growth factors (Fgfs) in murine tooth development

Fgf signalling is known to play critical roles in tooth development. Twenty-two Fgf ligands have been identified in mammals, but expression of only 10 in molars and three in the incisor loop stem cell region have been documented in murine tooth development. Our understanding of Fgf signalling in tooth development thus remains incomplete and we therefore carried out comparative in situ hybridisation analysis of unexamined Fgf ligands (eight in molars and 15 in cervical loops of incisors; Fgf11–Fgf14 were excluded from this analysis because they are not secreted and do not activate Fgf receptors) during tooth development. To identify where Fgf signalling is activated, we also examined the expression of Etv4 and Etv5, considered to be transcriptional targets of the Fgf signalling pathway. In molar tooth development, the expression of Fgf15 and Fgf20 was restricted to the primary enamel knots, whereas Etv4 and Etv5 were expressed in cells surrounding the primary enamel knots. Fgf20 expression was observed in the secondary enamel knots, whereas Fgf15 showed localised expression in the adjacent mesenchyme. Fgf16, Etv4 and Etv5 were strongly expressed in the ameloblasts of molars. In the incisor cervical loop stem cell region, Fgf17, Fgf18, Etv4 and Etv5 showed a restricted expression pattern. These molecules thus show dynamic temporo-spatial expression in murine tooth development. We also analysed teeth in Fgf15(−/−) and Fgf15(−/−);Fgf8(+/−) mutant mice. Neither mutant showed significant abnormalities in tooth development, indicating likely functional redundancy