Four novel RUNX2 mutations including a splice donor site result in the cleidocranial dysplasia phenotype

Abstract

Cleidocranial dysplasia (CCD) is an autosomal dominant disorder caused by haploinsufficiency of the RUNX2 gene. In this study, we analyzed by direct sequencing RUNX2 mutations from eleven CCD patients. Four of seven mutations were novel: two nonsense mutations resulted in a translational stop at codon 50 (Q50X) and 112 (E112X); a missense mutation converted arginine to glycine at codon 131 (R131G); and an exon 1 splice donor site mutation (donor splice site GT/AT, IVS1 + 1G > A) at exon 1-intron junction resulted in the deletion of QA stretch contained in exon 1 of RUNX2. We focused on the functional analysis of the IVS1 + 1G > A mutation. A full-length cDNA of this mutation was cloned (RUNX2Deltae1) and expressed in Chinese hamster ovary (CHO) and HeLa cells. Functional analysis of RUNX2Deltae1 was performed with respect to protein stability, nuclear localization, DNA binding, and transactivation activity of a downstream RUNX2 target gene. Protein stability of RUNX2Deltae1 is similar to wild-type RUNX2 as determined by Western blot analysis. Subcellular localization of RUNX2Deltae1, assessed by in situ immunofluorescent staining, was observed with partial retention in both the nucleus and cytoplasm. This finding is in contrast to RUNX2 wild-type, which is detected exclusively in the nucleus. DNA binding activity was also compromised by the RUNX2Deltae1 in gel shift assay. Finally, RUNX2Deltae1 blocked transactivation of the osteocalcin gene determined by transient transfection assay. Our findings demonstrate for the first time that the CCD phenotype can be caused by a splice site mutation, which results in the deletion of N-terminus amino acids containing the QA stretch in RUNX2 that contains a previously unidentified second nuclear localization signal (NLS). We postulate that the QA sequence unique to RUNX2 contributes to a competent structure of RUNX2 that is required for nuclear localization, DNA binding, and transactivation function.

Interrelationship of Runx2 and estrogen pathway in skeletal tissues

“…Furthermore, many in vitro studies have shown that Runx2 is a positive regulator for the expression of bone matrix genes, including type I collagen, osteopontin, bone sialoprotein, osteocalcin, and matrix metalloproteinase 9 (6-10). The heterozygous mutation of Runx2 results in cleidocranial dysplasia, a dominantly inherited developmental disorder of bone, in both mice and humans (3,4,(11)(12)(13). Moreover, Runx2 accelerates chondrocyte differentiation in response to the upregulation of Runx2 target genes, which include the indian hedgehog (Ihh) and type X collagen (Col10A1) (14,15).…”

RUNX2 mutations in cleidocranial dysplasia

“…RUNX2 is also known as core binding factor A1 (CBFA1) and it is located on chromosome 6p21 (Bae et al, 1993;Ducy et al, 1997). It is generally believed that the haploinsufficiency or loss of function of RUNX2 underlies the mechanism of CCD pathogenesis (Quack et al, 1999;Kim et al, 2006).…”

Intragenic microdeletion of RUNX2 is a novel mechanism for cleidocranial dysplasia

“…Numerous CCD patients without any detectable mutations in RUNX2 by sequencing or FISH have been identified (Kim et al 2006;Otto et al 2002;Quack et al 1999;Yoshida et al 2002). This would indicate a genetic heterogeneity such as mutation in RUNX2 gene's interacting proteins or regulatory elements or due to other mechanism that was not yet reported.…”