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

Abstract

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. K E Y W O R D S

Characterization of dental phenotype in patients with cleidocranial dysplasia using longitudinal data

“…Cleidocranial dysplasia (CCD; OMIM 119600) is known to be caused by mutations in the RUNX2 gene and inherited in an autosomal dominant pattern. 1,2 The RUNX2 gene encodes a transcription factor required in osteoblast differentiation, chondrocyte maturation, and skeletal morphogenesis. [1][2][3] CCD is characterized by skeletal abnormalities including dysplasia (aplasia or hypoplasia) of the clavicles, patent sutures and fontanelles, formation of wormian bone, short stature, brachycephaly, or a depressed nasal bridge.…”

“…1,2 The RUNX2 gene encodes a transcription factor required in osteoblast differentiation, chondrocyte maturation, and skeletal morphogenesis. [1][2][3] CCD is characterized by skeletal abnormalities including dysplasia (aplasia or hypoplasia) of the clavicles, patent sutures and fontanelles, formation of wormian bone, short stature, brachycephaly, or a depressed nasal bridge. 2,4 In addition to the skeletal abnormalities, CCD typically involves dental complications such as multiple supernumerary teeth and impaction or delayed eruption of permanent teeth.…”

“…[1][2][3] CCD is characterized by skeletal abnormalities including dysplasia (aplasia or hypoplasia) of the clavicles, patent sutures and fontanelles, formation of wormian bone, short stature, brachycephaly, or a depressed nasal bridge. 2,4 In addition to the skeletal abnormalities, CCD typically involves dental complications such as multiple supernumerary teeth and impaction or delayed eruption of permanent teeth. 2,4 However, the spectrum of CCD phenotypes ranges from mild dental issues to severe skeletal malformation.…”

“…2,4 In addition to the skeletal abnormalities, CCD typically involves dental complications such as multiple supernumerary teeth and impaction or delayed eruption of permanent teeth. 2,4 However, the spectrum of CCD phenotypes ranges from mild dental issues to severe skeletal malformation. [2][3][4][5][6] Therefore, the severity of skeletal abnormalities is not necessarily directly related to that of the dental complications.…”

“…2,4 However, the spectrum of CCD phenotypes ranges from mild dental issues to severe skeletal malformation. [2][3][4][5][6] Therefore, the severity of skeletal abnormalities is not necessarily directly related to that of the dental complications. As a result, Baumert et al 7 described four groups of CCD patients showing significant differences in phenotypic expression.…”