Research On The Clinical Characteristics And Disease-causing Mechanisms In A Chinese Family With Cleidocranial Dysplasia

Background and objectivesCleidocranial dysplasia(CCD),unrelated to the gender and age,is a rare autosomal dominant disease(OMIM:119600),with a prevalence of 1/1,000,000.The most typical clinical features are:unclosed or lingeringly closed fontanel;clavicular agenesis;retained deciduous teeth,impacted teeth,and supernumerary teeth.Runt-related transcription factor 2(RUNX2)was found to be the pathogenic gene of CCD.During the osteoblast differentiation and maturation,this gene plays a significant role.At present,the main challenges in clinical work are the diagnosis and treatment of the CCD.Recently,the hot research topic is about the probing for the pathogenesis of CCD by applying techniques and methods of molecular biology and bioinformatics.Although in the past years,the mutation sites of RUNX2 mutation have been reported bit by bit,the gene screening of pathogenesis is far from enough.This study has analyzed the craniomaxillofacial features of a CCD pedigree,helping with the clinical diagnosis;and we have a functional study of the mutation sites in the RUNX2 to further investigate the CCD aetiological agents,contributing to the genetic counseling and gene diagnosis.Materials and methods1.A CCD pedigree in Guangzhou,China,was selected as the study object and the medical history were taken and recorded from all the sick members(Oral examination,systemically general examination,and radio-graphic examination included),and cephalometry was used to analyze the craniofacial features.2.The phenol/chloroform method was adopted to extract the PB DNA of the CCD family members;Sanger sequencing technique and family coseparation,and other methods were adopted to confirm the pathogenic gene and mutation sites.3.200 blood samples were randomly taken in the area where the family was located,and a high-resolution melting(HRM)technique was used to confirm the mutation frequency of the mutation sites in the RUNX2.4.Peripheral blood cells were extracted to acquire the total RNA by using Trizol reagent,and qRT-PCR was utilized to detect the difference of the total transcription levels of RUNX2 between the normal human peripheral blood and that from the CCD pedigree..5.The software I-TASSER and SOPMA were used to predict the secondary protein structure and the three-dimensional structure of the RUNX2 mutant proteins;6.The cDNA of normal transcripts and aberrant transcripts of the RUNX2 were cloned to the carrier pEGFP-C1,and lipo2000 was used to transfect them into HEK-293T cells,the cells were collected 24 hours after transfection,then total RNA and total protein were extracted.qRT-PCR and Western Blot were conducted to compare the expression levels of the RUNX2 mRNA and its protein between the wild-type and the mutant type after the transfection to the HEK-293T.7.With the well-transfected HEK-293T cells and DAPI nuclear staining,fluorescence microscope was used to observe and photograph to compare the differences of the expression sites between the wild-type protein and the mutant protein.Results1.Clinical features of CCD patientsThe proband manifested such typical CCD features as oral anomaly(deciduous tooth retention,multiple impacted teeth,and supernumerary teeth)and clavicular agenesis.There were changes in the maxillofacial region featuring ocular hypertelorism,flat nose and underdeveloped midface;cephalometrics analysis indicated underdeveloped maxilla and mandibular prognathism.The nasal bone was distinctly underdeveloped,especially the especially the mandibular angle was nearly absent,and the mandibular ramus was nearly parallel.The cephalometrics indicated decreased length and height of the upper mandible.The elder sister of the proband showed the similar maxillofacial features.2.Confirmation of the mutation sitesSanger sequencing in the pedigree indicated there was deletion of the 1271st,the base C(c.1271delC)in the RUNX2,and this mutation was located in the NMTS,subnuclear matrix targeting signal encoded by exon 7,resulting in the deletion of 39 amino acids.Mutation screening indicated that in the local 200 random samples of peripheral blood the same mutation site was not found.3.Analysis of mRNA levels in vivoqRT-PCR indicated that in the patients' peripheral blood the expression level of mRNA in RUNX2 bore no statistic difference.4.Prediction and analysis of bioinformaticsThe predicated result of the protein secondary structure indicated the mutatant protein lost parts of the ? turn,and the ?-pleated sheet and the random coil increased the a spiral structure.The comprehensive modeling of I-TASSER indicated that the deletion mutant(c.1271delC)whose tertiary structure of the protein changed significantly.5.Expression analysis of mRNA and protein in vitroqRT-PCR test indicated there were no remarkable differences of relative transcript level of the RUNX2 mRNA after the wild-type and the mutant-type were transfected to the HEK-293T.Western blot indicated the expression level of RUNX2 fusion protein of the mutation is higher than that of the wild protein(p<0.001).6.The function of subcellular localizationThe subcellular localization results indicated that the fusion protein in the wild-type RUNX2 was only expressed in the nuclei,whereas in the mutant group of RUNX2,green fluorescence could be noted in the cytoplasm and nucleus.Conclusion1.The proband in the study had typical clinical manifestations of CCD,such as oral anomaly(deciduous tooth retention,multiple impacted teeth,and supernumerary teeth)and clavicular agenesis,there were also distinctive maxillofacial changes like ocular hypertelorism,underdeveloped midface,and mandibular prognathism.2.In this study,a novel frameshift mutation,c.1271delC,was found in the RUNX2 gene in a CCD pedigree and the same site was not found in this family's healthy members or other 200 random samples.3.The new mutation site generated a new termination codon in advance,which made RUNX2 protein truncated and significantly changed the secondary and tertiary structure.4.The deletion mutant c.1271delC made RUNX2 protein lost the transcription repression domain and parts of the NMTS region,causing the remarkable increasing of the expression level of the mutant proten and changing the speciality of transcription factor,which was only expressed in the nuclei.