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Annals of Clinical & Laboratory Science 37:115-120 (2007)
© 2007 Association of Clinical Scientists

A Novel Mutation of Gene CBFA1/RUNX2 in Cleidocranial Dysplasia

Lorenzo Lo Muzio1, Stefano Tetè2, Filiberto Mastrangelo2, Angela Pia Cazzolla3, Maria Grazia Lacaita3, Maurizio Margaglione4,5 and Giuseppina Campisi6
1 Department of Surgical Sciences, University of Foggia, Foggia; 2 Department of Oral Sciences, University of Chieti, Chieti; 3 Department of Dentistry and Surgery, University of Bari, Bari; 4 Department of Biomedical Sciences, University of Foggia, Foggia; 5 IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo; and 6 Department of Dental Sciences, University of Palermo, Palermo, Italy

Address correspondence to Professor Lorenzo Lo Muzio, Via Carelli 28, 71100 Foggia, Italy; fax 39 881 685809; e-mail lomuziol{at}tin.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Cleidocranial dysplasia (CCD) is an autosomal dominant skeletal dysplasia characterised by abnormal clavicles, patent sutures and fontanelles, supernumerary teeth, short stature, and a variety of other skeletal changes. The disease gene is CBFA1/RUNX2, which is mapped to chromosome 6p21. Inactivation of the CBFA1/RUNX2 gene by mutations is involved in the skeletal defects that occur in patients with CCD. CBFA1/RUNX2 controls the differentiation of precursor cells into osteoblasts and is essential for membranous as well as endochondral bone formation. In this study of a 14-yr-old boy with typical CCD phenotype, the authors found a novel CBFA1/RUNX2 gene mutation. All of the amplified segments from the patient’s CBFA1/RUNX2 gene were identical to those obtained in controls, except for the one spanning the exon 7 and intron/exon boundary regions. Direct sequencing of the PCR product showed a heterozygous T-to-A transition mutation at nucleotide 1182 in exon 7, leading to Y394X mutation. The predicted protein product lacks 128 amino acids, including part of the PST domain. Identification of this novel mutation constitutes a further step in elucidating the pathogenesis of this autosomal disorder.

Keywords: cleidocranial dysplasia, CBFA1/RUNX2 gene, CBFA1/RUNX2 nonsense mutation


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Cleidocranial dysplasia (CCD), also known as cleidocranial dysostosis or Marie-Sainton disease, is characterized by defective development of the cranial bones, complete or partial absence of the clavicles, and other skeletal changes; CCD is often associated with immunodeficiency [1–7].

CCD is an autosomal dominant disorder of high penetrance, affecting skeletal ossification and tooth development. It has a prevalence of <1 per million persons and is caused by heterozygous mutations in the osteoblast-specific transcription factor CBFA1/RUNX2. This gene, which is mapped to chromosome 6p21, is a member of the runt family of transcription factors and encodes a nuclear protein with a runt-DNA binding domain. This protein is essential for osteoblastic differentiation, chondrocyte maturation, and skeletal morphogenesis, acting as a scaffold for nucleic acid and regulatory factors involved in skeletal gene expression. The protein is essential for both membranous and endochondral bone formation.

CCD was formerly believed to involve only bones of membranous origin. However, recent clinical investigations have shown that CCD is a generalised skeletal dysplasia affecting not only the clavicles and skull but the entire skeleton. CCD is therefore considered to be a dysplasia rather than a dysostosis. The disorder is characterized by delayed closure of fontanelles and by hypoplastic clavicles, which result from defective intramembranous ossification. Additional features, such as short stature and cone epiphyses, suggest an underlying defect in endochondral ossification. Normal skeletal development requires the coordinated activities of bone-forming osteoblasts and bone-resorbing osteoclasts. The activities of both cell types are likely to be regulated by TGF-beta, which is abundant in bone matrix, and by other cytokines involved in innate and adaptive immunity.

The phenotypic spectrum of CCD patients ranges from mildly affected individuals with only dental abnormalities to severely affected patients with generalized osteoporosis that is increased by immunosuppressive drugs. Affected individuals typically have short stature, growth retardation, delayed closure of sutures and fontanelles, wormian bones (particularly around the lamboid suture), delayed ossification of the pubic bone, and various skeletal abnormalities (eg, hypoplastic or aplastic clavicles, cone shaped thorax, coxa vara, hypoplasia of distal phalanges, and pes planus).

The characteristic facial features of CCD patients include brachycephalic head, hypertelorism with exophthalmia, depressed nasal bridge, small maxilla, and mandibular prognatism. Intraoral manifestations include multiple supernumerary teeth, failure of secondary dentition eruption, and delayed maturation of the permanent teeth.

In the present study the authors investigated the DNA of a patient with typical CCD phenotype, identifying a novel CBFA1/RUNX2 mutation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
DNA extraction and analysis.  By use of standard protocols, DNA was extracted from peripheral blood leukocytes of a 14-yr-old boy with a typical CCD phenotype, who was referred to our institution for study. Amplifications of all coding regions of the CBFA1/RUNX2 gene and its intron/exon boundaries, of the 5’-untranslated region (UTR), and of the 3’UTR were achieved using sense and antisense oligonucleotides designed on the basis of the known sequences of the CBFA1/RUNX2 gene locus (Genbank accession number AL358135 [GenBank] ) (Table 1Go). Oligonucleotide custom syntheses were performed by Life Technologies, Ltd., (Paisley, UK). PCR was carried out on 50 µl samples in a thermal cycler (Perkin-Elmer Cetus, Norwalk, CT). Each sample contained 0.1 µg of genomic DNA, 10 pmoles of each primer, 125 µM of dNTP, 5 mM Tris HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, and 1 U of Taq polymerase. The solution was overlaid with 50 µl of mineral oil. After an initial denaturation step (3 min at 95 °C), each sample was carried through 30 cycles, consisting of 1 min at 95°C, 1 min at 56 to 60°C, and 2 min at 72°C. Then 5 µl samples of the amplification products were separated by electrophoresis in a 2% agarose-gel with TAE buffer (40 mM TRIS-acetate, 1 mM EDTA pH 7.7) containing 0.5 µg/ml ethidium bromide; the gel was visualised under UV light. Finally, amplified DNA fragments were subjected to direct cycle sequence analysis using the Taq dye-deoxy terminator method and ABI PRISM 3100 Avant Genetic Analyzer (Perkin-Elmer Biosystems, Norwalk, CT). To exclude the possibility that the gene replacement found could be a polymorphism, DNA samples from 100 healthy subjects were investigated; the gene variation was found in note.


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  		Table 1. Primer sequences used for PCR.
 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
Clinical findings.  The patient was a 14-yr-old boy who presented with short stature (140 cm, third percentile), high weight (50 kg, fiftieth percentile), 59 cm head circumference, hypoplastic clavicles, coxa vara, hypoplastic distal phalanges, and pes planus (Figs. 1Go,2GoGo), without any evidence of immunodeficiency. During infancy, he had open sutures of the skull, patent fontanelles, and delayed ossification of the pubic bone. The boy’s facial appearance was typical of CCD, with broad forehead, frontal bossing, hypertelorism, exophthalmia, small maxilla, flattened face, and protruding mandible. His nose was reduced in length, increased in width and protrusion, and the nasal bridge was low. He had primary dentition with the first molar erupted; the palate was highly arched, but without a pseudo-cleft. A radiograph showed multiple supernumerary teeth in the maxilla and mandible (Fig. 3Go).


Figure 1
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  		Fig. 1. Photographs of the patient to show the characteristic phenotypic features of cleidocranial dystrophy (CCD).

 

Figure 2A
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  		Fig. 2A. Oral manifestations of CCD to show that the patient was in primary dentition with the first molar erupted.

 

Figure 2B
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  		Fig. 2B. Photograph of the CCD patient’s left hand to show the hypoplastic distal phalanges.

 

Figure 3
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  		Fig. 3. Radiograph of the CCD patient’s mouth to show multiple supernumerary teeth in the maxilla and mandible.

 
The patient attended school with good performance. His mother, age 40 yr, had short stature (150 cm), body weight of 60 kg, hypoplastic clavicles, hypoplastic distal phalanges, and pelvic abnormalities. Her facial appearance resembled her son; she had a removable dental prosthesis because her teeth had all been extracted. The patient’s father did not show phenotypical signs of CCD (Fig. 4Go).


Figure 4
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  		Fig. 4. Genetic pedigree of the CCD patient’s family to show the involvement of the mother and son.

 
Genetic characterization.  Fragments covering the entire coding region of the CBFA1/RUNX2 gene were amplified from the genomic DNA of the patient. The PCR products were subjected to direct sequencing. All of the amplified segments from the CBFA1/RUNX2 gene were identical to those in controls, except for the one spanning exon 7 and its intron/exon boundary regions. Direct sequencing of PCR product of the patient showed a heterozygous T-to-A transition mutation at nucleotide 1182 in exon 7 (Fig. 5Go), leading to Y394X nonsense mutation of the RUNX2 gene, according to the approved nomenclature for mammalian runt-related genes. The predicted protein product lacks 128 amino acids, including part of the PST domain.


Figure 5
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  		Fig. 5. Direct sequencing of the PCR product of the CCD patient to show a heterozygos T-to-A transition mutation at nucleotide 1182 (N) within exon 7 of the CBFA1/RUNX2 gene, leading to Y394X mutation according to the approved nomenclature. The predicted protein lacks 128 amino acids, including part of the PST domain.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
In our patient with typical CCD phenotype, we identified a new determinant nonsense mutation (Y394X) in exon 7 of the CBFA1/RUNX2 gene. It is likely that this mutation gives rise to a null allele resulting in the lack of functional protein. Taking into account that the patient’s parents were clinically evaluated and his father did not show any phenotypic characteristic of CCD, the patient’s Y394X mutation was probably inherited from his mother.

In patients with CCD, craniofacial growth is affected in many ways [8,9]. Head circumference is usually at the upper limit without being macrocephalic; there is frontal bossing and some hypertelorism; the midfrontal area is poorly developed and shows a frontal groove owing to incomplete ossification of the metopic suture [1]. Closure of the anterior fontanelle, sagittal, and metopic sutures is delayed, often for life. In infants, generalised delay in ossification of the skull can be observed and, in extreme cases, the parietal bones are absent at birth [1]. With increasing age the unossified areas become smaller and true wormian bones develop, particularly around the lambdoid suture [1]. Frontal and paranasal sinuses are frequently absent or reduced in size. Other changes of the skull include small or absent nasal bones, segmental calvarial thickening, underdevelopment of the maxilla, delayed union of the mandibular symphysis, a small cranial base with reduced sagittal diameter, and a large foramen magnum [1]. The skeletal changes include a large, brachycephalic head with parietal and marked frontal bosses separated by a metopic groove, a depressed nasal bridge, hypertelorism with possible exophthalmia, and a small maxilla, which gives the face a small, flattened appearance with mandible prognatism [1]. The CDD phenotype is linked to CBFA1 gene mutations [2,3,5,7,10–15].

CBFA1 (also called PEBP2{alpha}A, AML3, and OSF2) is one of the 3 mammalian genes that encode the {alpha} subunit of the heterodimeric transcription factor PEBP2/CBF, which is composed of 2 structurally unrelated subunits, {alpha} and ß. The {alpha} subunit is characterized by a highly conserved 128-amino-acid region termed the "runt domain," which shares a high degree of homology with the products of the Drosophila genes, runt and lozenge. This gene is also named RUNX2, according to the recently introduced standard nomenclature for mammalian runt-related genes. The other two {alpha}-subunit–encoding genes are RUNX1/AML1/{alpha}B/ CBFA2 and RUNX3/PEBP2{alpha}C/AML2/CBFA3. The runt domain is responsible for DNA binding and heterodimerization with the ß subunit. The runt domain also contains a nuclear-localization signal (NLS) on its C-terminal border. The C-terminus of the gene product, Runx2, is rich in proline, serine, and threonine, which are needed for Runx2-mediated transcriptional regulation and are involved in functional interactions with other transcription factors, coactivators, and corepressors [16–21].

Mundlos et al [2] established that the RUNX2 gene is the site of mutations responsible for CCD. They found heterozygous deletions in some families, and insertion, deletion, or missense mutations in other families; these mutations lead to a translational stop codon in the DNA-binding domain or in the C-terminal transactivating region [2]. Lee et al [7] described the first missense mutations in the CBFA1 gene and identified 2 nonsense mutations that interfere with the function of the putative DNA binding domain. Quack et al [22] analyzed the CBFA1 gene in 42 unrelated patients and detected mutations in the coding region (8 frameshift, 2 nonsense, 9 missense mutations, as well as novel polymorphisms) in 18 patents. A cluster of missense mutations at arginine-225 identified this residue as crucial for CBFA1 function. Otto et al [6] tabulated a large number of mutations and stated that mutations affecting arginine-225 inhibit the nuclear accumulation of Runx2 protein.

Runx2 is a well-characterized critical transcriptional regulator of osteoblastic differentiation [3]. Heterozygosity for mutations in RUNX2 causes CCD [2,7]. Homozygous Runx2-deficient mice have no bone formation, whereas the heterozygotic mice display abnormalities similar to those seen in individuals with CCD [23]. Runx2 heterozygotic mice have fewer osteoblasts and bone-specific proteins, and their alkaline phosphatase activity is low [23]. Molecular and mouse genetic studies demonstrate that the abnormal bone formation is mainly due to disturbance of Runx2 transcriptional regulation of its target genes during osteoblastic differentiation. Beyond the skeleton, studies suggest that Runx2 regulates the expression of molecules in mesenchymal tissues that act reciprocally on the dental epithelium to control its growth and differentiation, and this partly explains the dental abnormalities found in patients with CCD and in Runx2 heterozygotic mice [24].

The CCD characteristics of delayed closure of fontanelles and hypoplasia of the clavicles reflect defective intramembranous ossification. However, the significantly hypoplastic or absent clavicles point to an effect on both intramembranous and endochondral ossification, because the clavicle ossifies embryologically by both routes [25–27]. Reports that emphasize short stature as a prominent feature in CCD provide supporting evidence for RUNX2 effects on endochondral ossification [4].

In the present study, identification of a novel mutation in a patient with CCD represents a further step in elucidating the pathogenesis of this disabling autosomal dysplasia.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
The authors thank Mario Sarno and Roberto Santacroce (Department of Biomedical Sciences, University of Foggia) for technical help and they thank Pio Conti (Immunology Division, University of Chieti) for useful suggestions.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

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D. Xuan, S. Li, X. Zhang, F. Hu, L. Lin, C. Wang, and J. Zhang
Mutations in the RUNX2 Gene in Chinese Patients with Cleidocranial Dysplasia
Ann. Clin. Lab. Sci., January 1, 2008; 38(1): 15 - 24.
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