Mutation Analysis Of RUNX2 Gene In Chinese Patients With Cleidocranial Dysplasia And Related Function Analysis

Cleidocranial dysplasia(CCD;MIM # 119600) is a skeletal disorder with autosomal-dominant inheritance.The clinical hallmarks of CCD are clavicle rudiment or absence,delayed closure of cranial fontanels and sutures,Wormian bones,frontal bossing,supernumerary and late erupting teeth,wide pubic symphysis,and other skeletal anomalies.The locus for CCD has been mapped to chromosome 6p21 where the responsible runt-related gene 2(RUNX2) has been located,RUNX2 also refers to as polyomavirus enhancer binding protein 2(PEBP2) or murine leukemia virus enhancer core-binding factor(CBF).So far,there is few investigations about Chinese cases with CCD.The prevalence and range of RUNX2 mutations in Chinese eases with CCD were rarely reported.Here,5 different types of heterozygous mutations in the RUNX2 gene in 5 unrelated CCD cases from China who clearly display variable clinical manifestations were reported,the molecular basis of their dysfunction were studied.The lower permanent teeth from one CCD case were collected and the dental pulp cells were cultured,and characterization and gene expression of the cells were analysed in comparison with corresponding cells from normal control.The study was divided into three parts described as followings.1.Clinical examination and analysis of patients with cleidocranial dysplasia 5 unrelated families with the clinical diagnosis of CCD were investigated in the present study,and the unaffected parents and siblings were included.There was no parental consanguinity.Radiological examination regarding osseous malformations was carried out over the entire body.A CCD phenotype was defined by the presence of hypoplastic clavicles and delayed closure of the anterior fontanelle in addition to the observation of classic craniofacial features.The skeletal anomalies as well as the oral manifestations of the syndrome were variable within the affected patients,the case 2 had extra teeth in the molar region(at least four molars),which was rarely reported in the literatures.Moreover,both impacted molars and extra molars had abnormal roots with delayed development,and the erupted first molars had relatively normal roots.It is possible that the abnormal root development may be one of the causes of eruption failure of permanent teeth in CCD cases.Interestingly,in case 3, both the ulna and radius leaned to the radialis so that the humeroulnar joints and humeroradial joints were abnormal,and the elbow looked like a triangle,which was rarely reported in the literatures.All the cases in the present study showed malformation of tarsometatarsal joints to a certain extent.Thus we may infer that RUNX2 may have a role in joint formation by affecting some of chondrocyte and osteoblast differentiation pathways.We have analyzed histologically the structure of teeth obtained from CCD patients.Enamel structure was illegible,dentin tubules appeared obturated, Peritubular dentin had insufficiency mineralization,and typical continuous arc structures were virtually lacking on the enamel-dentinal junction.In deciduous teeth, more enamel lamella and enamel tuft were found,and dentin appeared irregular,size and distribution appeared ununiformed;cellular cementum was virtually lacking in deciduous and permanent teeth.Spectrum analysis of enamel and dentin of teeth from CCD patients and normal subjects showed that the proportion of Ca and P in permanent teeth from CCD was significantly lower than that from normals,so did the enamel of deciduous teeth from CCD patients,however,this did not appear to be the case for dentin of the deciduous teeth. 2.Mutation analysis of RUNX2 and its effect on subcelluar localization of mutantsTo identify mutations in the RUNX2 gene in CCD cases,genomic DNA.from 5 CCD cases and their families were analysed,totally different mutations for each CCD case were detected.Novel c.475 G＞C(p G159R) missense and c.1096 G＞T nonsense mutations were included,and their healthy parents didn't carry the same mutation.Although the other three mutations were reported in the literatures,R225W and R391X mutations were reported in Chinese cases with CCD for the first time.To detect the effect of mutations on nuclear localization of RUNX2 protein, fusion proteins were constructed between green fluorescent protein and RUNX2.The constructs were transiently transfected into mouse fibroblast NIH 3T3 cells.The wild-type RUNX2 protein was detected exclusively in the nucleus.However,R225Q and R225W mutants showed dual localization to both the cytoplasm and the nucleus. 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.These results indicate that G159 is very important to promote RUNX2 nuclear-localization, therefore,G159 may be one of new elements of RUNX2 gene nuclear localization signals.3.Characterization and gene expression analysis of dental pulp cells of CCD patientTo identify morphological and molecular alteration associated with CCD dental tissues,human permanent dental pulp cell cultures were established from age-and sex-matched CCD patient and normal subject.Dental pulp cells were compared for general morphology,proliferation rate,and gene expression profiles microarray technology.CCD pulp cells were in some sort flatter than the normal ones,however, the normal pulp proliferation rates were greater at time points tested than the cells from CCD patient.The flow cytometric analysis showed that the progression from the G1 to S phase in the CCD pulp cell cycle was impeded.The ultrastructure of CCD pulp cells showed that the rough endoplasmic reticulum was expended,and more lamellar body was found in cytoplasma.Partial ribosomes fell off the rough endoplasmic reticulum,and this change might impede the ability to synthesize protein. The CCD pulp cells were induced with mineralization inducer,and no mineralized nodule was found until seven weeks passed,although partial cells showed single pole process of cytoplasma and overlapping growth.The weakening of mineralization ability showed that its potential ability of differentiation might weaken.The 96 genes of Human TGF-β/BMP signaling pathway were analysed by PCR array,and 18 genes displayed significant up-regulated at least two-fold in expression levels,and 14 genes down-regulated.Cdc25A,TGFβ2,Smad3 and EVI1 were down-regulated,and COL3A1,BMP2,4,7 were up-regulated.The proliferation and differentiation were modulated by multi-gene,some genes cooperated with each other and determine the final fate of cells.It was inferred that the potential differentiation of CCD pulp cells into odontoblasts was impeded to some extent by the cooperation between down-regulation of TGFβ2,Smad3 and EVI1 and up-regulation of COL3A1. According to the disfunction of R391X reported in the literatures,E366X mutation may have an effect on the interaction of RUNX2 mutant and Smads,and impede the function of BMP to cell differentiation.The up-regulation expression of BMP2,4,7 might be caused by other negative feedback because of breakoff of the normal signaling pathways.