| Cleidocranial dysplasia (CCD) is a rare genetic disease of the skeletal system withautosomal dominant mode. The locus for CCD has been mapped to chromosome6p21where the osteoblast-specific transcription factor (RUNX2/CBFA1) has been located. Themolecular etiology of this syndrome is mutations of the base, large deletions,chromosome ectopic, and variation of submicroscopic structure. CCD can affect eitherthe ossification of membrane and cartilage, or the development of tooth. The mainclinical manifestations of CCD are absent or hypoplastic clavicles, persistently open ordelayed closure of sutures, wormian bones, delayed eruption of permanent dentition,supernumerary teeth, short stature, and other skeletal changes. Of all the clinicalphenotypes, craniofacial and dental abnormities which lead to chewing dysfunction andfacial deformity are the most conspicuous symptoms.At present, early diagnosis of the disease and correction of tooth abnormities are themost important for clinicians; to explore the molecular etiology of the disease is the mostinteresting for researchers. In order to establish early diagnosis and to guide the clinical sequence therapy, retrospective study of eight cases of CCD was conducted. In order toinvestigate the pathogenesis of CCD and to carry out genetic counseling/geneticdiagnosis, the studies of RUNX2gene mutation and the function of the protein structurewere carried out for4unrelated pedigrees. This study consists of3parts, summarized asfollows:1. Clinical phenotype and sequencing treatment of patients with cleidoeranialdysplasiaDetailed clinical examination and X-ray examination were undertaken for4unrelated pedigrees with cleidoeranial dysplasia. The results have shown that all patientshave typical "triad-performance", namely skull, clavicle, and teeth dysplasia, so thediagnosis of cleidoeranial dysplasia can be made. On the other hand, the abnormalities ofsternum, pelvis, hands, and feet also present in all of the patients with CCD. The skeletalabnormalities and oral manifestations of the syndrome are variable greatly betweendifferent families and different patients of the same family.In order to fully understand craniofacial characteristics of the syndrome, the clinicaldata (facial photographs, lateral cephalometric radiographs, and panoramic radiographs)of eight cases with cleidoeranial dysplasia were retrospectively analyzed. To analysis theskeletal age of all the patients by lateral cephalometric radiographs, the results found that4patients were at the pre peak age (CS-2) or peak age (CS-3) of the growth anddevelopment;4patients finished the growth and development of skeleton (CS-5, orCS-6). The analysis of facial proportion, craniofacial characteristic has founded that8cases have inclined eye fissure, and7cases have wide orbital distances;8cases presentwith bulge forehead, and2cases have depression in the middle of the forehead; theunderdevelopment of middle faces of4pre-peak-age patients were more obvious thanthat of4post-development patients. Panoramic radiographs and lateral radiographs havefound that coracoids of all patients were pointed up or backwards; mandible angle,cranial base, and nasal bone of most cases are significantly abnormal. The maxillaryhypoplasia are not evident in the patients at the early growth and development, and other parts of abnormalities such as supernumerary teeth, occipital Worm’s bone, nasal bonehypoplasia, and deformed coracoids may be presented in all/most of the patients.The patients with CCD were affected with tooth eruption abnormalities, and severemalocclusion, so the oral treatment is very complicated and difficult. It is needed thecooperation of various disciplines to restore the chewing function and to improve thefacial appearance. The treatment of CCD can be divided into the following four stages:1)Approximately at7-10years old, it is needed to remove barriers to help the impactedteeth to erupt spontaneously;2) Approximately at13-14years old, it is needed to initiateorthodontic treatment to tract the impacted anterior tooth, and to provide a goodenvironment for the subsequent permanent teeth eruption;3) Approximately at13-14years old, for the purpose to restore tooth function, canines and premolars are neededorthodontic treatment;4) At18-20years old, orthognathic surgery is needed to correctfacial deformities for the purpose to restore the oral function and facial esthetic.2. Genetic diagnosis of patients with cleidocranial dysplasiaTo identify mutations in the RUNX2gene, polymerase chain reaction and directsequencing of DNA were conducted for CCD cases came from4unrelated pedigrees.Beside one case of pedigree Ⅳ, three different mutations of RUNX2gene were detectedin CCD cases of the other pedigrees. Deletion mutation c.243-260del18(p.81-86del6)which was identified in pedigreeⅠis resided in Q/A domain of exon1; nonsensemutation c.1200C> A (p.stop400) which was identified in pedigreeⅡwas in the NMTSdomain of exon7; Mission mutant c.674G> T (p.R225L) which was identified inpedigree Ⅲ was located in the NLS domain of exon3. The mutations (p.81-86del6andp.stop400) of this study were detected for the first time, and the results of this studyfurther prove that RUNX2gene mutation is molecular etiology of CCD.To identify copy number variation in RUNX2gene, real-time PCR was carried outin CCD case of pedigree Ⅳ. The results found that the copy number of all the exons ofRUNX2gene in this pedigree significantly decreased. This study has proved that copynumber variation is another molecular etiology of CCD; real-time PCR is a simple,convenient, sensitive and reliable method for the detection of copy number variation. Both PCR-DNA sequencing and real-time PCR can be conventionally used together tomake genetic diagnosis of patients with CCD.3. The effect of mutations on three-dimensional structure and subcellularlocalization of RUNX2gene mutantsTo analysis the protein structure of RUNX2gene mutants, bioinformatics softwarewere used to predict three-dimensional structure of the mutants. The results ofSwiss-Model homology modeling showed that the missense mutant c.674G> T (p.R225L)had no significant change in secondary structure; owning to the substitution of aminoacids, some intramolecular hydrogen bonding are lost, and the molecular electrostaticpotential energy is decreased. The results of I-TASSER modeling have showed that thesecondary and tertiary conformations of mutants (p.81-86del6and p.stop400) changesignificantly; parts of the helix and the folded structure miss. The experiment has provedthat the change of amino acid sequence affects the spatial structure of mutant proteins.To ascertain the effect of mutations on subcellular localization of RUNX2protein,eukaryotic expression vectors containing the mutants and wild-type RUNX2wereconstructed. To observe the fluorescence of the pEGFP-Cl-RUNX2vector after celltransfection, the results found that green fluorescent protein expression of wild-type, andmutant(p.81-86del6, p.stop400) were completely expressed in the nucleus; mutantP.R225L was localized in both the cytoplasm and the nucleus. This experimentdemonstrated that nuclear localization of RUNX2protein was not affected by thep.81-86del6and p.stop400mutation. |
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