Identification of a Novel Splice Site Mutation in RUNX2 Gene in a Family with Rare Autosomal Dominant Cleidocranial Dysplasia

Abstract

Background: Pathogenic variants of RUNX2, a gene that encodes an osteoblast-specific transcription factor, have been shown as the cause of Cleidocranial dysplasia (CCD), which is a rare hereditary skeletal and dental disorder with dominant mode of inheritance and a broad range of clinical variability. Due to the relative lack of clinical complications resulting in CCD, the medical diagnosis of this disorder is challenging, which leaves it underdiagnosed.

Methods: : In this study, nine healthy and affected members of an Iranian family were investigated. PCR and sequencing of all exons and exon-intron boundaries of runt-related transcription factor 2 (RUNX2; NM_001024630) gene was performed on proband. Co-segregation analysis was conducted in the other family members for the identified variant. Additionally, a cohort of 100 Iranian ethnicity-matched healthy controls was screened by Amplification Refractory Mutation System PCR method.

Results: The novel splice site variant (c.860-2A>G), which was identified in the intron 6 of RUNX2 gene, co-segregated with the disease in the family, and it was absent in healthy controls. Pathogenicity of this variant was determined by several software, including , human splicing finder, which predicts the formation or disruption of splice donor sites, splice acceptor sites, exonic splicing silencer sites, and exonic splicing enhancer sites. In silico analysis predicted this novel variant to be disease causing.

Conclusion: The identified variant is predicted to have an effect on splicing, which leads to exon skipping and producing a truncated protein via introducing a premature stop codon.

Keywords: Cleidocranial dysplasia; RUNX2; Splice site.

Fig. 1

(A) The pedigree of the family with CCD and genotype of the studied samples for the c.860-2A>G variant in RUNX2. (B) Left hand X-ray of IV1 showing shortened middle phalange, hypoplastic distal phalanges, brachydactyly (especially the fifth digit), and mildly longed second metacarpus. (C) Panoramic mandibular X-ray of IV1 demonstrating dental caries, crowded, irregular and wide spaced teeth, and supernumerary teeth, occult, and unruptured multiple permanent teeth. (D) Sequencing chromatographs for the region of the c.860-2A>G variant in the RUNX2 gene in one healthy (II-1) and one affected member (III-4) of the family. “R” designates purine (A or G) ribonucleotides