Characterisation of novel RUNX2 mutation with alanine tract expansion from Japanese cleidocranial dysplasia patient

Cleidocranial dysplasia (CCD; MIM 119600) is an autosomal dominant skeletal dysplasia characterised by hypopalstic and/or aplastic clavicles, midface hypoplasia, absent or delayed closure of cranial sutures, moderately short stature, delayed eruption of permanent dentition and supernumerary teeth. The molecular pathogenesis can be explained in about two-thirds of CCD patients by haploinsufficiency of the RUNX2 gene. In our current study, we identified a novel and rare variant of the RUNX2 gene (c.181_189dupGCGGCGGCT) in a Japanese patient with phenotypic features of CCD. The insertion led an alanine tripeptide expansion (+3Ala) in the polyalanine tract. To date, a RUNX2 variant with alanine decapeptide expansion (+10Ala) is the only example of a causative variant of RUNX2 with polyalanine tract expansion to be reported, whilst RUNX2 (+1Ala) has been isolated from the healthy population. Thus, precise analyses of the RUNX2 (+3Ala) variant were needed to clarify whether the tripeptide expanded RUNX2 is a second disease-causing mutant with alanine tract expansion. We therefore investigated the biochemical properties of the mutant RUNX2 (+3Ala), which contains 20 alanine residues in the polyalanine tract. When transfected in COS7 cells, RUNX2 (+3Ala) formed intracellular ubiquitinated aggregates after 24 h, and exerted a dominant negative effect in vitro. At 24 h after gene transfection, whereas slight reduction was observed in RUNX2 (+10Ala), all of these mutants significantly activated osteoblast-specific element-2, a cis-acting sequence in the promoter of the RUNX2 target gene osteocalcin. The aggregation growth of RUNX2 (+3Ala) was clearly lower and slower than that of RUNX2 (+10Ala). Furthermore, we investigated several other RUNX2 variants with various alanine tract lengths, and found that the threshold for aggregation may be RUNX2 (+3Ala). We conclude that RUNX2 (+3Ala) is the cause of CCD in our current case, and that the accumulation of intracellular aggregates in vitro is related to the length of the alanine tract.