Whole-exome sequencing identification of a novel splicing mutation of RUNX2 in a Chinese family with cleidocranial dysplasia.

Objectives: Cleidocranial dysplasia (CCD) is a congenital autosomal dominant skeletal disease characterized by multiple craniofacial and dental anomalies. Here, we investigated mutation of the runt-related transcription factor 2 (RUNX2) gene, which is considered responsible for most instances of CCD in patients, in a Chinese family with CCD.

Methods: Genomic DNA was extracted from the peripheral blood lymphocytes of all participants, and mutation analysis was performed using whole-exome and Sanger sequencing. Biophysical predictions of the altered protein were analyzed using various bioinformatics tools, and direct sequencing via reverse transcription polymerase chain reaction (PCR) was performed for functional analysis of the mutation. To determine the function of the mutated protein, expression of RUNX2 and integrin-binding sialoprotein (IBSP) was investigated via quantitative PCR.

Results: We identified a novel splicing mutation (c.581-9 T > G) in all affected members, with this RUNX2 mutation incorporating in a new splice site to replace the canonical splice site, thereby resulting in insertion of an 8-bp fragment within the terminal exon 5 splice-acceptor site and premature translation termination. qPCR results confirmed attenuated RUNX2 expression and IBSP overexpression in the peripheral blood lymphocytes of patients.

Conclusions: These results suggested that the newly identified splice-site mutation (c.581-9 T > G) in RUNX2 was responsible for CCD in this family through its alteration of RUNX2 activity and upregulated IBSP levels. These findings extend the mutational spectrum of the RUNX2 gene and might contribute to genetic diagnosis and counseling of families with CCD.