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

Cited 43 time in Web of Science Cited 51 time in Scopus
Authors
Kim, Hyo-Jin; Nam, Soon-Hyeun; Kim, Hyun-Jung; Park, Hyo-Sang; Ryoo, Hyun-Mo; Kim, Shin-Yoon; Cho, Tae-Joon; Kim, Seung-Gon; Bae, Suk-Chul; Kim, In-San; Stein, Janet L.; van Wijnen, Andre J.; Stein, Gary S.; Lian, Jane B.; Choi, Je-Yong
Issue Date
2006
Publisher
John Wiley & Sons
Citation
J Cell Physiol 207:114-122
Keywords
CHO CellsCell Nucleus/metabolismCleidocranial Dysplasia/*genetics/pathologyCodon, NonsenseCore Binding Factor Alpha 1 Subunit/chemistry/*genetics/metabolismCore Binding Factor beta Subunit/geneticsCricetinaeCytoplasm/metabolismDNA/metabolismExons/geneticsHela CellsHeterozygote*MutationMutation, MissenseNuclear Localization Signals/geneticsOsteocalcin/geneticsPhenotypePromoter Regions, Genetic/geneticsProtein BindingProtein Transport/geneticsRNA Splice Sites/*geneticsTranscriptional Activation/geneticsTransfection
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.
ISSN
0021-9541 (Print)
Language
English
URI
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16270353

http://hdl.handle.net/10371/10869
DOI
https://doi.org/10.1002/jcp.20552
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College of Medicine/School of Medicine (의과대학/대학원)Orthopedic Surgery (정형외과학전공)Journal Papers (저널논문_정형외과학전공)
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