Functional impact of pathogenic Runt domain mutations in Runx2 on skeletal and dental development in cleidocranial dysplasia

Runt-related transcription factor 2 (RUNX2) is essential for skeletogenesis, and mutations in its gene cause cleidocranial dysplasia (CCD), an autosomal dominant skeletal disorder. The evolutionarily conserved 128-amino acid Runt homology domain (RHD) of human RUNX2 is essential for DNA binding and heterodimerization, and serves as a mutation hotspot associated with severe CCD phenotypes. To elucidate the functional impact of pathogenic RHD mutations in vivo, we generated two novel mouse lines: one carrying a missense mutation, c.695G>A (p.R232Q) (Runx2m/+), corresponding to the human RUNX2 c.674G>A (p.R225Q), and the other harboring a frameshift mutation, c.697_698delGA (p.E233TfsTer9) (Runx2112/+), causing a premature stop codon. Homozygous Runx2m/mand Runx2112/112 mice lacked membranous ossification, whereas heterozygous Runx2m/+ and Runx2112/+ mice displayed typical CCD-like skeletal features, including an open anterior fontanelle and clavicle hypoplasia. Unexpectedly, heterozygotes carrying pathogenic mutations in RHD developed an accessory root-like protrusion at the furcation of three-rooted maxillary first molars, representing a previously unrecognized dental phenotype during root development. Dual luciferase assays revealed impaired transactivation of the p.R232Q mutant Runx2 on the osteocalcin enhancer/promoter. Wild-type Runx2 was robustly expressed in osteoblasts and hypertrophic chondrocytes during bone formation, but the mutant Runx2 exhibited reduced expression in hypertrophic chondrocytes and partially impaired nuclear localization, resulting in arrested osteoblast and chondrocyte maturation. Our mutant mouse model provides a valuable in vivo platform to study CCD pathogenesis, mechanisms of tooth root furcation, and therapeutic interventions targeting dysfunctional RHD.