A Research On The Pathogenic Mechanism Of A Family With Cleidocranial Dysplasia

Objective Cleidocranial dysplasia(OMIM: 119600),referred as CCD,is a rare congenital autosomal dominant inherited skeletal dysplasia disease,with a prevalence of about1/1000000.About 2/3 of CCD cases are related to the mutation of RUNX2 gene,however,the pathogenic mechanism is still unclear.Therefore,the purpose of this study is to find new causative gene mutations,explore its pathogenesis through a series of experiments,enrich the mutational spectrum of CCD disease,and provide reference for disease diagnosis and prenatal examination of pregnant women in the near future.Methods1.Collect clinical information of patientsOn the premise of signing the informed consent form of all participants,the clinical information of the patients in the CCD family were collected,the oral and systemic health examination forms were filled out,the pedigree map was drawn,the intraoral and facial photos were taken.Approximately 4 ml of peripheral blood from the proband,her mother,and normal controls were stored in EDTA tubes at-80℃.The protocol was approved by the Ethical Committee of Tianjin Medical University.2.Preliminary screening and locking of mutant genesWhole-exome sequencing was performed using the Agilent Liquid Capture System in strict accordance with the experimental procedure.Under the premise of strict quality control,the data was finely filtered,and the location,mutation type,conservation,and other information of SNP and In Del were annotated with ANNOVAR software.Suspicious gene mutations and their information were screened according to the characteristics of the disease and their mutation sites were verified by was verified by Sanger sequencing.3.Prediction and functional verification of mutation3.1 Prediction of structure by bioinformatics toolsThe corresponding base sequence and amino acid sequence of RUNX2 werefound on NCBI,and the secondary and three-dimensional structures of mutant and wild type were predicted by Protean and SWISS MODEL software,respectively.The effect of mutation on protein structure was analyzed,which was a clear direction for the next step of experiment.3.2 Mutation sites verification by RT-PCR direct sequencingUsing Primer3 Plus software to design bidirectional primers for the extracted c DNA.PCR sequencing experiment was carried out to verify the mutation site.3.3 Real-time fluorescence quantitative PCR reaction(q RT-PCR)To explore the effect of this new mutation on the function of RUNX2,the relative expression of RUNX2 and its downstream gene IBSP in peripheral blood was measured by q RT-PCR.Results1.There are 5 patients in this Chinese CCD family.Intraoral examination of the proband revealed that only four first permanent molars,two lower anterior teeth,and the lower right second permanent molar had partially erupted,while the rest of the permanent teeth had not yet erupted.The panoramic radiograph showed most of the deciduous teeth in the proband’s mouth remained,the permanent teeth were impacted,and more than 20 supernumerary teeth could be seen.Systemic examination showed that the proband had typical CCD features,including short stature,delayed closure of fontanel,protrusion of forehead,widening of eye distance,collapse of bridge of nose,maxillary retraction / mandibular extension,right clavicle hypoplasia,scoliosis and so on.2.Whole exome sequencing revealed a new splice site mutation(NM＿001015051:exon5: c.581-9 T> G)in the RUNX2 gene,and Sanger sequencing confirmed an amino acid changes at position 581-9.3.The three-dimensional structure of mutant RUNX2 protein was predicted by SWISS MODEL software.the results showed that the three-dimensional structure of mutant RUNX2 protein was significantly shorter than that of wild type.The secondary structure of the mutant protein was predicted by Protean software.Compared with the wild type,the mutant RUNX2 protein had an alpha helix structure at the 200 amino acid position.4.The mutation was further verified by RT-PCR sequencing.The new splicing site mutation cause an insertion of an 8-bp fragment within the terminal exon 5splice-acceptor site.5.The q RT-PCR results showed that the expression level of RUNX2 gene in blood was relatively down-regulated,and that of IBSP gene was relatively up-regulated.Conclusions1.Whole-exome sequencing result showed that there was a new splice site mutation of RUNX2 gene(NM＿001015051: c.581-9 T> G)shared by the proband and her mother,which was confirmed by Sanger sequencing2.The structure of mutant RUNX2 protein was predicted by bioinformatics software.It was found that the new mutation caused the occurrence of termination codon in advance,resulting in truncation of RUNX2 protein and the change of α-helix structure,which affected the normal structure of RUNX2 protein.RT-PCR direct sequencing further confirmed that the mutation resulted in the insertion of 8bp(ATCTGCAG)at the beginning of exon 5.3.The q RT-PCR results showed that the expression level of RUNX2 gene in blood was relatively down-regulated,and that of IBSP gene was relatively up-regulated.It is suggested that this new splice site mutation has a certain effect on the function of RUNX2,further confirming that the new mutation of RUNX2 is closely related to the occurrence of this family CCD.This result extends the mutation spectrum of the RUNX2 gene.