title: 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.
authors.affiliation: Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Daegu, Korea.
authors.forename: Hyo-Jin
authors.initials: HJ
authors.lastname: Kim
authors.forename: Soon-Hyeun
authors.initials: SH
authors.lastname: Nam
authors.forename: Hyun-Jung
authors.initials: HJ
authors.lastname: Kim
authors.forename: Hyo-Sang
authors.initials: HS
authors.lastname: Park
authors.forename: Hyun-Mo
authors.initials: HM
authors.lastname: Ryoo
authors.forename: Shin-Yoon
authors.initials: SY
authors.lastname: Kim
authors.forename: Tae-Joon
authors.initials: TJ
authors.lastname: Cho
authors.forename: Seung-Gon
authors.initials: SG
authors.lastname: Kim
authors.forename: Suk-Chul
authors.initials: SC
authors.lastname: Bae
authors.forename: In-San
authors.initials: IS
authors.lastname: Kim
authors.forename: Janet L
authors.initials: JL
authors.lastname: Stein
authors.forename: Andre J
authors.initials: AJ
authors.lastname: van Wijnen
authors.forename: Gary S
authors.initials: GS
authors.lastname: Stein
authors.forename: Jane B
authors.initials: JB
authors.lastname: Lian
authors.forename: Je-Yong
authors.initials: JY
authors.lastname: Choi
mesh_terms: D000293:Adolescent; D000328:Adult; D000818:Animals; D016466:CHO Cells; D002467:Cell Nucleus / Q000378:metabolism; D002648:Child; D002973:Cleidocranial Dysplasia / Q000235:genetics* / Q000473:pathology; D018389:Codon, Nonsense; D050659:Core Binding Factor Alpha 1 Subunit / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D050658:Core Binding Factor beta Subunit / Q000235:genetics; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D005091:Exons / Q000235:genetics; D005260:Female; D006367:HeLa Cells; D006579:Heterozygote; D006801:Humans; D008297:Male; D009154:Mutation; D020125:Mutation, Missense; D019913:Nuclear Localization Signals / Q000235:genetics; D015675:Osteocalcin / Q000235:genetics; D010641:Phenotype; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D021381:Protein Transport / Q000235:genetics; D022821:RNA Splice Sites / Q000235:genetics*; D015533:Transcriptional Activation / Q000235:genetics; D014162:Transfection
chemical_list: D018389:Codon, Nonsense; D050659:Core Binding Factor Alpha 1 Subunit; D050658:Core Binding Factor beta Subunit; D019913:Nuclear Localization Signals; D022821:RNA Splice Sites; C493709:RUNX2 protein, human; D015675:Osteocalcin; D004247:DNA