A novel RUNX2 mutation in exon 8, G462X, in a patient with Cleidocranial Dysplasia.

To identify a novel mutation of Runx2 gene in Cleidocranial Dysplasia (CCD) patients and to characterize the functional consequences of this mutation. The subjects consisted of 12 Korean CCD patients. After oral epithelial cells were collected using a mouthwash technique, genomic DNA was extracted. Screening for Runx2 mutation was performed using direct sequencing of polymerase chain reaction (PCR) products for exons 1-8. Restriction fragment length polymorphism (RFLP) analysis was performed to confirm the novel mutation. For functional studies, we performed luciferase assay for Runx2 transacting activity, cyclohexamide chase assay for Runx2 protein stability, real-time PCR for mRNA level of Runx2 downstream bone marker genes, and alkaline phosphatase (ALP) staining assay in mesenchymal stem cells for osteoblast differentiation. Of the 12 patients, seven showed Runx2 mutations reported previously and four showed no mutation. A novel mutation, G462X in exon 8, which was located in the C-terminus of proline/serine/threonine-rich (PST) domain, was found in one patient. In the luciferase assay, Runx2 transacting activity was decreased in Runx2-G462X transfected cells. In the cyclohexamide chase assay, Runx2-G462X mutation reduced the stability of Runx2 protein. Expression of the bone marker genes (osteocalcin, ALP, Type I collagen αI, matrix metalloproteinase-13, bone sialoprotein, and osteopontin) decreased in G462X-transfected cells. In the ALP staining assay, osteoblast differentiation was reduced in Runx2-G462X overexpressed cell. The G462X mutation might reduce the Runx2 transacting activity, lower the protein stability, downgrade the expression of bone marker genes, and eventually diminish osteoblast differentiation in CCD patients.