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. 2002 Oct;71(4):724-38.
doi: 10.1086/342717. Epub 2002 Aug 26.

Functional analysis of RUNX2 mutations in Japanese patients with cleidocranial dysplasia demonstrates novel genotype-phenotype correlations

Taketoshi Yoshida  1 , Hirokazu Kanegane, Motomi Osato, Masatoshi Yanagida, Toshio Miyawaki, Yoshiaki Ito, Katsuya Shigesada
Affiliations

Functional analysis of RUNX2 mutations in Japanese patients with cleidocranial dysplasia demonstrates novel genotype-phenotype correlations

Taketoshi Yoshida et al. Am J Hum Genet. 2002 Oct.

Abstract

Cleidocranial dysplasia (CCD) is an autosomal dominant heritable skeletal disease caused by heterozygous mutations in the osteoblast-specific transcription factor RUNX2. We have performed mutational analysis of RUNX2 on 24 unrelated patients with CCD. In 17 patients, 16 distinct mutations were detected in the coding region of RUNX2: 4 frameshift, 3 nonsense, 6 missense, and 2 splicing mutations, in addition to 1 polymorphism. The missense mutations were all clustered within the Runt domain, and their protein products were severely impaired in DNA binding and transactivation. In contrast, two RUNX2 mutants had the Runt domain intact and remained partially competent for transactivation. One criterion of CCD, short stature, was much milder in the patients with the intact Runt domain than in those without. Furthermore, a significant correlation was found between short stature and the number of supernumerary teeth. On the one hand, these genotype-phenotype correlations highlight a general, quantitative dependency, by skeleto-dental developments, on the gene dosage of RUNX2, which has hitherto been obscured by extreme clinical diversities of CCD; this gene-dosage effect is presumed to manifest on small reductions in the total RUNX2 activity, by approximately one-fourth of the normal level at minimum. On the other hand, the classic CCD phenotype, hypoplastic clavicles or open fontanelles, was invariably observed in all patients, including those with normal height. Thus, the cleidocranial bone formation, as mediated by intramembranous ossification, may require a higher level of RUNX2 than does skeletogenesis (mediated by endochondral ossification), as well as odontogenesis (involving still different complex processes). Overall, these results suggest that CCD could result from much smaller losses in the RUNX2 function than has been envisioned on the basis of the conventional haploinsufficiency model.

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Figures

Figure  1
Figure  1
Schematic representation of RUNX2, showing the localization of mutations in patients with CCD. The seven exons of RUNX2 are represented by boxes, and introns are represented by lines; hatched boxes indicate the Runt domain. The mutations identified in the present study and previous studies are indicated above and below the diagrammatic structure of RUNX2 (the 507-amino-acid isoform starting with MRIPVD).
Figure  2
Figure  2
Sequence analysis of splicing mutations. A, Point mutation with eventual 4-base skipping in patient 15. The boundary sequences of exons 6 and 7 in the wild-type cDNA are shown in the lower boxes. The mutated nucleotide is underlined and annotated as indicated by arrows. The resultant cDNA sequence causing frameshift is shown in the upper box. B, Exon-skipping mutations found in patients 16 and 17. The boundary sequences of exons 2–4 in the wild-type cDNA are shown in the lower boxes. The mutated nucleotide(s) at the upstream boundary of intron 3 are underlined and annotated as indicated by arrows. The resultant cDNA sequence lacking exon 3 is shown in the upper box. RT-PCR was performed as described elsewhere (Zhang et al. 2000). Wt = wild type.
Figure  3
Figure  3
Mutational alterations in DNA-binding and heterodimerization activities of the Runt domain. β = PEBP2β; Wt = wild type. a, Partial RUNX2 proteins (indicated at top) produced in E. coli and subjected to EMSA. The “+” and “−” symbols signify the presence and absence, respectively, of PEBP2β. b, Partial RUNX2 proteins subjected to affinity assay. A = input RUNX2 protein; W = unbound proteins in the third wash; E = bound proteins eluted at 250 mmol/liter imidazole.
Figure  4
Figure  4
Transactivation of the osteocalcin promoter by exogenously expressed RUNX2 proteins in NIH3T3 cells. Cells were transfected with a reporter plasmid (0.5 μg), indicated RUNX2 expression constructs (0.5 μg), and PEBP2β (0.2 μg). Luciferase activities were measured and presented as the fold increase relative to the control mock-transfected with the backbone expression vector. The mean values from three separate measurements are given with SDs (thin vertical bars). Results obtained with and without coexpression of PEBP2β are denoted by solid and shaded bars, respectively. WT = wild type.
Figure  5
Figure  5
Subcellular localization of RUNX2. The indicated RUNX2 proteins were overexpressed in NIH3T3 cells and were made visible by immunofluorescence staining with an anti-PEBP2αA antibody: wild type (a), R176W (b), F183S (c), Q266X (d), 348fs (e), 151fs (f), R179X (g), K204N (h), T206I (i), R211Q (j), and R211W (k). For each mutant, at least 100 well-stained cells were counted and were classified into a few subpopulations according to their pattern of staining. A typical image of each distinct subpopulation is presented together with its fractional percentage.
Figure  6
Figure  6
Colocalization of RUNX2 and PEBP2β. PEBP2β was coexpressed with either the wild-type RUNX2 (a and c) or T206I RUNX2 (b and d) in NIH3T3 cells, and the two subunits were visualized by double staining with anti-RUNX and anti-β antibodies, respectively. a, Wild-type RUNX2. b, T206I RUNX2. c, PEBP2β coexpressed with wild-type RUNX2. d, PEBP2β coexpressed with T206I RUNX2.
Figure  7
Figure  7
Comparison of height SD scores between groups of patients with different genotypes or phenotypic variations. ○ = Void; ● = impaired in the Runt domain; ▴ = intact in the Runt domain; ●+ = 151fs with 47,XXX; ●− = 151fs with 46,XX. a, Mutational effects on the Runt domain, impaired (n=27) and intact (n=4) (P<.01). b, Fontanelles, normal (none) and delayed (n=16). c, Clavicles. AA = aplastic on both sides (n=4); AH = aplastic on one side and hyopoplastic on the other side (n=3); HH = hypoplastic on both sides (n=16).
Figure  8
Figure  8
Correlation between height SD score and transactivation ability of each RUNX2 mutant (% wild-type control), as determined in figure 4 (n=16; r=0.71; P<.01). Symbols are as defined in figure 7.
Figure 9
Figure 9
Correlation between number of supernumerary teeth and height SD score (n=9; r=0.96; P<.0005). Symbols are as defined in figure 7.
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References

Electronic-Database Information

    1. GenBank, http://www.ncbi.nlm.nih.gov/Genbank/ (for RUNX2 [accession number AF001450])
    1. Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/ (for CCD [MIM 119600])

References

    1. Adya N, Stacy T, Speck NA, Liu PP (1998) The leukemic protein core binding factor β (CBFβ)-smooth-muscle myosin heavy chain sequesters CBFα2 into cytoskeletal filaments and aggregates. Mol Cell Biol 18:7432–7443 - PMC - PubMed
    1. Bae SC, Takahashi E, Zhang YW, Ogawa E, Shigesada K, Namba Y, Satake M, Ito Y (1995) Cloning, mapping and expression of PEBP2αC, a third gene encoding the mammalian Runt domain. Gene 159:245–248 - PubMed
    1. Bae SC, Yamaguchi-Iwai Y, Ogawa E, Maruyama M, Inuzuka M, Kagoshima H, Shigesada K, Satake M, Ito Y (1993) Isolation of PEBP2αB cDNA representing the mouse homolog of human acute myeloid leukemia gene, AML1. Oncogene 8:809–814 - PubMed
    1. Bravo J, Li Z, Speck NA, Warren AJ (2001) The leukemia-associated AML1 (Runx1)–CBFβ complex functions as a DNA-induced molecular clamp. Nat Struct Biol 8:371–378 - PubMed
    1. Chitayat D, Hodgkinson KA, Azouz EM (1992) Intrafamilial variability in cleidocranial dysplasia: a three generation family. Am J Med Genet 42:298–303 - PubMed

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