A novel missense mutation of the CBFA1 gene in a family with cleidocranial dysplasia (CCD) and variable expressivity

The aim of this study was to analyze the CBFA1 gene in a phenotypically variable family with autosomal dominant cleidocranial dysplasia (CCD). Five members of a family with CCD were characterized clinically. X-rays and photographs of the two clinically affected family members were taken. The genotype of all five affected family members was determined with the use of single strand conformation polymorphism (SSCP) and direct sequencing. A point-mutation in exon 2 (R148G) was detected in a patient with the full-blown clinical phenotype. His son, demonstrating the same mutation, showed only the dental CCD characteristics. No mutation could be found in the three clinically healthy family members. To conclude, a missense mutation in the CBFA1 gene was detected in a family with variably expressed CCD syndrome. A detailed clinical examination is necessary to detect minimally affected gene mutation carriers.     

PEBP2alphaA/CBFA1 mutations in Japanese cleidocranial dysplasia patients

Cleidocranial dysplasia (CCD) is an autosomal dominant human bone disease whose genetic locus has been located on chromosome 6p21, where the PEBP2alphaA/CBFA1 gene essential for osteogenesis also maps. Previously, several heterozygous mutations in PEBP2alphaA/CBFA1 were found in CCD patients. In this study, we identified six different types of mutations in PEBP2alphaA/CBFA1 in Japanese CCD patients. Four cases were similar to those reported previously: two were nonsense mutations in the Runt domain, one was a hemizygous deletion, and the other was a missense mutation in the Runt domain which abolished the DNA-binding activity of Runx2/PEBP2alphaA/CBFA1. The remaining two mutations were novel: one had a heterozygous gt-to-tt mutation at the splice donor site (gt) between the exon3-intron junction, which resulted in abnormal exon3 skipping, and the other had a mutation in exon7, which led to the introduction of a translational stop codon in the middle of the transactivation domain. Thus, defects in either the DNA-binding domain or transactivation domain of Runx2/PEBP2alphaA/CBFA1 can cause CCD. The results not only provide a strong genetic evidence that mutations involving in PEBP2alphaA/CBFA1 contribute to CCD, but also provide a useful tool to study how Runx2/PEBP2alphaA/CBFA1 plays its pivotal role during osteoblastic differentiation.     

A novel Alu-mediated microdeletion in the <Emphasis Type="BoldItalic">RUNX2</Emphasis> gene in a Chinese patient with cleidocranial dysplasia

Cleidocranial dysplasia (CCD; OMIM: 119600) is a rare autosomal dominant skeletal dysplasia caused by RUNX2 gene mutations. The present study described a sporadic case with CCD. The clinical data of the proband with CCD was reported and genetic analysis was performed. The proband presented with typical CCD features including supernumerary impacted teeth, bilateral clavicle dysplasia, delayed closure of cranial sutures, and short stature; while his hands were normal. Sequencing analysis of the entire coding region of the RUNX2 gene revealed no pathogenic changes; however, copy-number analysis with the Affymetrix HD array found \(\sim \)500 kb genomic microdeletion. Real-time quantitative PCR validated this microdeletion in the 1–4 exons of the RUNX2 gene. The junction point of the breaking DNA was located in the directly oriented AluSz6 and AluSx repetitive elements, indicating that this microdeletion might be generated through an Alu–Alu mediated mechanism. In addition, this microdeletion existed in 21.8% of the asymptomatic mother’s peripheral blood cells, demonstrating that the mosaicism was not associated with CCD phenotypes. In summary, a pathogenic microdeletion in the RUNX2 gene located on chromosome 6 was responsible for CCD.     

RUNX2 mutations in Taiwanese patients with cleidocranial dysplasia

Cleidocranial dysplasia (CCD) is an autosomal dominant human skeletal disorder comprising hypoplastic clavicles, wide cranial sutures, supernumerary teeth, short stature, and other skeletal abnormalities. It is known that mutations in the human RUNX2 gene mapped at 6p21 are responsible for CCD. We analyzed the mutation patterns of the RUNX2 gene by direct sequencing in six Taiwanese index cases with typical CCD. One of the patients was a familial case and the others were sporadic cases. Sequencing identified four mutations. Three were caused by single nucleotide substitutions, which created a nonsense (p.R391X), two were missense mutations (p.R190W, p.R225Q), and the forth was a novel mutation (c.1119delC), a one-base deletion. Real time quantitative PCR adapted to determine copy numbers of the promoter, all exons and the 3'UTR region of the RUNX2 gene detected the deletion of a single allele in a sporadic case. The results extend the spectrum of RUNX2 mutations in CCD patients and indicate that complete deletions of the RUNX2 gene should be considered in those CCD patients lacking a point mutation detected by direct sequencing.     

A novel mutation of adenomatous polyposis coli (APC) gene results in the formation of supernumerary teeth

Supernumerary teeth are teeth that are present in addition to normal teeth. Although several hypotheses and some molecular signalling pathways explain the formation of supernumerary teeth, but their exact disease pathogenesis is unknown. To study the molecular mechanisms of supernumerary tooth‐related syndrome (Gardner syndrome), a deeper understanding of the aetiology of supernumerary teeth and the associated syndrome is needed, with the goal of inhibiting disease inheritance via prenatal diagnosis. We recruited a Chinese family with Gardner syndrome. Haematoxylin and eosin staining of supernumerary teeth and colonic polyp lesion biopsies revealed that these patients exhibited significant pathological characteristics. APC gene mutations were detected by PCR and direct sequencing. We revealed the pathological pathway involved in human supernumerary tooth development and the mouse tooth germ development expression profile by RNA sequencing (RNA‐seq). Sequencing analysis revealed that an APC gene mutation in exon 15, namely 4292‐4293‐Del GA, caused Gardner syndrome in this family. This mutation not only initiated the various manifestations typical of Gardner syndrome but also resulted in odontoma and supernumerary teeth in this case. Furthermore, RNA‐seq analysis of human supernumerary teeth suggests that the APC gene is the key gene involved in the development of supernumerary teeth in humans. The mouse tooth germ development expression profile shows that the APC gene plays an important role in tooth germ development. We identified a new mutation in the APC gene that results in supernumerary teeth in association with Gardner syndrome. This information may shed light on the molecular pathogenesis of supernumerary teeth. Gene‐based diagnosis and gene therapy for supernumerary teeth may become available in the future, and our study provides a high‐resolution reference for treating other syndromes associated with supernumerary teeth.     

A gene for cleidocranial dysplasia maps to the short arm of chromosome 6.

Cleidocranial dysplasia (CCD) is an autosomal dominant generalized bone dysplasia characterized by mild-to-moderate short stature, clavicular aplasia or hypoplasia, supernumerary and ectopic teeth, delayed eruption of secondary teeth, a characteristic craniofacial appearance, and a variety of other skeletal anomalies. We have performed linkage studies in five families with CCD, with 24 affected and 20 unaffected individuals, using microsatellite markers spanning two candidate regions on chromosomes 8q and 6. The strongest support for linkage was with chromosome 6p microsatellite marker D6S282 with a two-point lod score of 4.84 (theta = .03). Furthermore, the multipoint lod score was 5.70 in the interval between D6S282 and D6S291. These data show that the gene for autosomal dominant CCD is located within a 19-cM interval on the short arm of chromosome 6, between D6S282 and D6S291.     

A Novel RUNX2 Mutation in Cleidocranial Dysplasia Patients

Cleidocranial dysplasia (CCD) is an autosomal-dominant heritable skeletal disease caused by heterozygous mutations in the RUNX2 gene. Here, the RUNX2 gene was analyzed within a CCD family from China, and a novel missense mutation (c. 475G --> C [p.G159R]) was identified. Normal and mutant RUNX2 expression vectors were then constructed and expressed transiently in NIH3T3 cells. Immunofluorescent staining and Western blotting showed that wild-type RUNX2 protein was localized exclusively in the nucleus; however, the mutant protein was found in both the nucleus and the cytoplasm, which demonstrated that transport of the RUNX2 mutant into the nucleus was disturbed by the G159R mutation. Therefore, we suggest that G159 is very important to promote RUNX2 nuclear localization. According to clinical analysis, the patient displays severe dysplasia of bones and relatively low-grade craniofacial abnormality, and we infer that G159 may be vital for normal skeletal development, other than control of tooth number. These findings confirm that mutations in the RUNX2 gene are associated with the pathogenesis of CCD across different ethnic backgrounds.     

Dental treatment strategies in a 40-year-old patient with cleidocranial dysplasia

Oral anomalies and dental treatment in a patient with cleidocranial dysplasia (referred to the dental clinic at the age of 40 years) are presented. Five supernumerary teeth were found in the patient: three in the maxilla in the area of molars and two in the mandibula in the area of premolars. Therapy included surgical exposure of impacted teeth in combination with removal of supernumerary teeth.     

The activation level of the TNF family receptor, Edar, determines cusp number and tooth number during tooth development

Mutations in members of the ectodysplasin (TNF-related) signalling pathway, EDA, EDAR, and EDARADD in mice and humans produce an ectodermal dysplasia phenotype that includes missing teeth and smaller teeth with reduced cusps. Using the keratin 14 promoter to target expression of an activated form of Edar in transgenic mice, we show that expression of this transgene is able to rescue the tooth phenotype in Tabby (Eda) and Sleek (Edar) mutant mice. High levels of expression of the transgene in wild-type mice result in molar teeth with extra cusps, and in some cases supernumerary teeth, the opposite of the mutant phenotype. The level of activation of Edar thus determines cusp number and tooth number during tooth development.     

Inactivation of IL11 signaling causes craniosynostosis, delayed tooth eruption, and supernumerary teeth

Craniosynostosis and supernumerary teeth most often occur as isolated developmental anomalies, but they are also separately manifested in several malformation syndromes. Here, we describe a human syndrome featuring craniosynostosis, maxillary hypoplasia, delayed tooth eruption, and supernumerary teeth. We performed homozygosity mapping in three unrelated consanguineous Pakistani families and localized the syndrome to a region in chromosome 9. Mutational analysis of candidate genes in the region revealed that all affected children harbored homozygous missense mutations (c.662C>G [p.Pro221Arg], c.734C>G [p.Ser245Cys], or c.886C>T [p.Arg296Trp]) in IL11RA (encoding interleukin 11 receptor, alpha) on chromosome 9p13.3. In addition, a homozygous nonsense mutation, c.475C>T (p.Gln159X), and a homozygous duplication, c.916_924dup (p.Thr306_Ser308dup), were observed in two north European families. In cell-transfection experiments, the p.Arg296Trp mutation rendered the receptor unable to mediate the IL11 signal, indicating that the mutation causes loss of IL11RA function. We also observed disturbed cranial growth and suture activity in the Il11ra null mutant mice, in which reduced size and remodeling of limb bones has been previously described. We conclude that IL11 signaling is essential for the normal development of craniofacial bones and teeth and that its function is to restrict suture fusion and tooth number. The results open up the possibility of modulation of IL11 signaling for the treatment of craniosynostosis.     

Effect of cleidocranial dysplasia‐related novel mutation of RUNX2 on characteristics of dental pulp cells and tooth development

Cleidocranial dysplasia (CCD) is an autosomal‐dominant disorder caused by a lack of function of one or more alleles of the RUNX2 gene. Mutations of the RUNX2 gene were analyzed in a family with CCD, and a novel nonsense mutation was identified, c. 1096G > T, p.E366X, which was predicted to cause a number of potential dysfunctions. Western blot analysis showed that the novel mutation created a shortened protein product, which lost 155 aa in the C‐terminal domain. The mutant protein was detected to be localized mostly in the cytoplasm, not in the nucleus, which demonstrated that transport of the RUNX2 protein into the nucleus was disturbed by the p.E366X mutation. For the first time, RUNX2^+/m dental pulp cells (DPCs) were isolated from two permanent incisors of the CCD patient. Compared to RUNX2^+/+ controls, RUNX2^+/m DPCs presented an impeded progression from the G1 to the S phase in the cell cycle, a lower rate of proliferation, weaker ability of calcification, and distinct ultrastructure. More interestingly, the ultrastructural analysis and energy dispersive X‐ray spectrometry (EDS) analysis showed that the CCD tooth exhibited insufficient mineralization of enamel and dentin. This study suggests that the truncated RUNX2 mutant protein may be responsible for the alterations of RUNX2^+/m DPCs, and RUNX2 gene may be involved in dental development by affecting the cell growth and differentiation, which provides new insights into understanding of dental abnormalities in CCD patients. J. Cell. Biochem. 111: 1473–1481, 2010. © 2010 Wiley‐Liss, Inc.