Functional analysis of novel RUNX2 mutations in cleidocranial dysplasia

Cleidocranial dysplasia (CCD) is a rare autosomal dominant skeletal disorder caused by mutation of runt-related transcription factor 2 (RUNX2) gene. The purpose of this study was to explore novel RUNX2 mutations in seven individuals with CCD and investigate the function of the mutant RUNX2 proteins. DNA samples were prepared from the peripheral blood of the CCD individuals, and then subjected to DNA sequencing. Conservation and secondary structure analysis were performed based on RUNX2 sequencing results. pEGFP-C1 plasmids containing GFP-tagged wild-type RUNX2 and three novel RUNX2 mutations expression cassettes were constructed, and then transfected into HEK293T cells. Cell fluorescence, luciferase assay and western blotting were used to analyse the subcellular distribution and function of the mutant RUNX2 proteins. Three novel mutations (R193G, 258fs, Y400X) were found in the seven CCD patients. Conservation and structure analysis show one novel mutation (R193G) in Runt domain and two novel mutations (258fs and Y400X) in PST domain of RUNX2. Western blotting confirmed that the 258fs and Y400X mutations produced truncated proteins. Fluorescence detection showed that the three novel mutants localised exclusively in the nucleus. However, luciferase assay indicated all mutants severely impaired the transactivation activities of RUNX2 on osteocalcin promoter. Our results broaden the spectrum of RUNX2 mutations in CCD individuals and demonstrated that loss of function in RUNX2 is responsible for CCD.