Functional impact of pathogenic mutations in the runt homology domain of mouse Runx2 on skeletal and dental phenotypes 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 2 novel mouse lines: one carrying a missense mutation, c.695G>A, p.Arg232Gln (p.R232Q), corresponding to the human RUNX2 c.674G>A, p.Arg225Gln (p.R225Q), and the other harboring a frameshift mutation, c.697_698delGA, p.Glu233Thrfs*9 (p.E233Tfs*9), causing a premature stop codon. Homozygous Runx2R232Q/R232Q and Runx2E233Tfs*9/E233Tfs*9 mice lacked membranous ossification, whereas heterozygous Runx2R232Q/+ and Runx2E233Tfs*9/+ mice displayed typical CCD-like skeletal features, including an open anterior fontanelle and clavicle hypoplasia. Unexpectedly, heterozygotes carrying pathogenic RHD mutations developed small root-like protrusions, mostly 1 but rarely 2, at the pulp chamber floor of 3-rooted maxillary first molars during furcation, revealing a previously unrecognized dental phenotype. Dual luciferase assays showed that p.R232Q almost completely lost transactivation of the osteocalcin enhancer/promoter. Immunostaining showed that wild-type Runx2 was robustly expressed in osteoblasts and hypertrophic chondrocytes during bone formation, while the p.R232Q mutant Runx2 in Runx2R232Q/R232Q mice exhibited reduced expression in hypertrophic chondrocytes and partially impaired nuclear localization. These abnormalities led to defective osteoblast differentiation and chondrocyte maturation. Thus, 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.