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

Hyo Jin Kim, Soon Hyeun Nam, Hyun Jung Kim, Hyo Sang Park, Hyun Mo Ryoo, Shin Yoon Kim, Tae Joon Cho, Seung Gon Kim, Suk Chul Bae, In San Kim, Janet L. Stein, Andre J van Wijnen, Gary S. Stein, Jane B. Lian, Je Yong Choi

Research output: Contribution to journalArticle

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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 inatranslational 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 (RUNX2Δe1) and expressed in Chinese hamster ovary (CHO) and HeLa cells. Functional analysis of RUNX2Δe1 was performed with respect to protein stability, nuclear localization, DNA binding, and transactivation activity of a downstream RUNX2 target gene. Protein stability of RUNX2Δe1 is similar to wild-type RUNX2 as determined by Western blot analysis. Subcellular localization of RUNX2Δe1, 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 RUNX2Δe1 in gel shift assay. Finally, RUNX2Δe1 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.

Original languageEnglish (US)
Pages (from-to)114-122
Number of pages9
JournalJournal of Cellular Physiology
Volume207
Issue number1
DOIs
StatePublished - Apr 2006
Externally publishedYes

Fingerprint

Cleidocranial Dysplasia
RNA Splice Sites
Exons
Functional analysis
Genes
Phenotype
Mutation
Assays
DNA
Transcriptional Activation
Protein Stability
Osteocalcin
Glycine
Introns
Arginine
Proteins
Complementary DNA
Gels
Haploinsufficiency
Amino Acids

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Cell Biology
  • Physiology

Cite this

Kim, H. J., Nam, S. H., Kim, H. J., Park, H. S., Ryoo, H. M., Kim, S. Y., ... Choi, J. Y. (2006). Four novel RUNX2 mutations including a splice donor site result in the cleidocranial dysplasia phenotype. Journal of Cellular Physiology, 207(1), 114-122. https://doi.org/10.1002/jcp.20552

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

In: Journal of Cellular Physiology, Vol. 207, No. 1, 04.2006, p. 114-122.

Research output: Contribution to journalArticle

Kim, HJ, Nam, SH, Kim, HJ, Park, HS, Ryoo, HM, Kim, SY, Cho, TJ, Kim, SG, Bae, SC, Kim, IS, Stein, JL, van Wijnen, AJ, Stein, GS, Lian, JB & Choi, JY 2006, 'Four novel RUNX2 mutations including a splice donor site result in the cleidocranial dysplasia phenotype', Journal of Cellular Physiology, vol. 207, no. 1, pp. 114-122. https://doi.org/10.1002/jcp.20552
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. / Four novel RUNX2 mutations including a splice donor site result in the cleidocranial dysplasia phenotype. In: Journal of Cellular Physiology. 2006 ; Vol. 207, No. 1. pp. 114-122.
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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 inatranslational 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 (RUNX2Δe1) and expressed in Chinese hamster ovary (CHO) and HeLa cells. Functional analysis of RUNX2Δe1 was performed with respect to protein stability, nuclear localization, DNA binding, and transactivation activity of a downstream RUNX2 target gene. Protein stability of RUNX2Δe1 is similar to wild-type RUNX2 as determined by Western blot analysis. Subcellular localization of RUNX2Δe1, 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 RUNX2Δe1 in gel shift assay. Finally, RUNX2Δe1 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.",
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AU - Ryoo, Hyun Mo

AU - Kim, Shin Yoon

AU - Cho, Tae Joon

AU - Kim, Seung Gon

AU - Bae, Suk Chul

AU - Kim, In San

AU - Stein, Janet L.

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