Generation And Phenoty Pe Analyses Of A Mouse Model With Vps4b Gene Knockout Analyses On DSPP Gene Mutation For A Chinese Pedigree With Hereditary Dentinogenesis Imperfecta

Background and purposeThe development of teeth is a long-term and complex biological process.It is closely related to the migration and directional differentiation of cranial nerve cells.It can be divided into four stages namely initiation stage,bud stage,cap stage and secretion stage.In the process of tooth development,the interaction between the epithelium and mesenchymal exists.Meantime,the orderly expression and interaction of some biological molecules determine the process of tooth development.As the hardest organ in the human body,teeth is composed by the enamel,dentine,cementum of the hard tissue and pulp tissue inside of the dental pulp cavity.It can seriously affect the patient’s oral beauty and chewing function,any part of the tooth is damaged or stunted.Moreover,it brings great harm to the physical and mental health of the patient.It also imposes a heavy financial burden on families and society.Therefore,it is of great theoretical and clinical significance to define the pathogenic genes of such diseases.Hereditary dentine disease is a common human dentin hereditary disease.It is mainly confined to mesoderm of the teeth and dental papilla dysplastic lesions.Back in the 1970s,Shields divided it into two categories:hereditary dentin dysplasia and dentinogenesis imperfect,and it can be subdivided into 5 small classes(2 types of dentin dysplasia and 3 types of dentinogenesis imperfect).Dentin dysplasia is an rare dominant genetic disease,current studies indicated that mutations on SSUH2 and SMOC2 can cause such diseases.In our previous work,we found a new human dentin dysplasia type Ⅰ(DD-Ⅰ)causative gene VPS4B.In this study,we established a mouse model with Vps4b gene knockout(Vps4b KO),afterwards,the teeth and bone phenotypes of mice were analyzed.Materials and MethodsCRISPR-Cas9 systems is used to build Vps4b KO model,and PCR and sanger sequencing and agarose gel electrophoresis are applied to identify the genotype of mice.Hematoxylin-eosin(HE)staining is used to observe the width of the pre-dentin zone and the change of the pulp.In addition,HE staining of the femur was conducted to observe whether there were abnormal changes in the distribution of bone trabecular structure.Immunohistochemical staining was used to monitor the changes in the expression of Vps4b.The changes of tooth abrasion and appearance were observed by stereoscopic microscope.Observing the skeletal morphology of mice by X-ray.The teeth and femur of the degree of mineralization and the change of the bone trabecular volume and separation are quantitatively analyzed by Micro-CT.Scanning electron microscopy(SEM)was used to observe the number and arrangement of dentinal tubules.Alcian blue-Alizarin red staining reaction shows mineralization changes in bone and cartilage.ResultsImmunohistochemistry showed gene KO was successful and there was decreased expression of Vps4b in heterozygous mice.Knockout Vps4b gene results in the embryonic lethality of Vps4b-/-mice.Hematoxylin and eosin staining indicated that the width of the pre-dentin zone was increased in heterozygous mice,although the arrangement of the odontoblast and bone trabecular were not significantly different from wild-type(WT)mice.Moreover,stereomicroscopic and X-ray radiography results indicated no abnormal manifestations in teeth and bones.Furthermore,statistical analysis for volume and density of dentin and enamel,as well as the skeletal analysis,including the volume and separation of bone trabecular showed no significant differences in Vps4b heterozygous mice.The scanning electron microscopy showed that there was no abnormal change in the arrangement and density of dentinal tubule.Also,there were no significant differences in bone or cartilage mineralization using Alcian blue-Alizarin red staining.ConclusionIn conclusion,the heterozygous Vps4b KO mice do not develop tooth defects that replicated human DD-I.Compared with wild type mice,heterozygous mice teeth show that the width of the pre-dentin zone increased and the Vps4b expression decreased in odontoblasts.It showed that the ability of tooth mineralization in mice was decreased.However,there were no abnormal changes in the skeleton,and no significant difference in mineralization of teeth and bone was found.The internal structure of the dentin is also normal.We speculate that is likely caused by the differences in tooth development between the two species.Of course,we cannot exclude the possibility that other genes and regulatory mechanisms will influence the development of mouse teeth and bones.Then,the phenotype is remedied.In addition,we only obtained the heterozygous mice,so we think the more likely reason is that the phenotype changes in heterozygous mice were not obvious,so we could not see the phenotypic changes.Background and purposeDentinogenesis Imperfect(DGI)is a common type of human dentine disorders,which is inherited by autosomal dominant mode.DGI is divided into three types:DGI-Ⅰ,which is incomplete in the dentin formation and is accompanied by osteogenesis imperfection;DGI-Ⅱ,incidence of a disease is approximately 1/8000,also known as hereditary opalescence dentine;DGI-Ⅲ,also known as brandy type dentin dysplasia,is a special type of hereditary opalescence dentine,which is isolated in three separate ethnic groups in Maryland,USA.The mutation of DSPP gene has been proved to be the main morbigenous genes of dentin developmental defect.And it has been determined that the mutation of DSPP gene is the pathogenic cause of DGI-II and DGI-III.In this study,we conducted a clinical phenotype of a Chinese pedigree affected by Hereditary Dentinogenesis imperfect and isolating the associated pathogenic gene DSPP.Meanwhile,prenatal diagnosis and genetic counselling is urgent can be made in future for second pregnancy.It can prevent the birth of affected infants,which can improve the population quality.Materials and methods1.Subject:a pedigree affected with hereditary dentinogenesis imperfect from Henan zhengzhou.The proband is a seven-year-old girl.Due to abnormal color and serious wear of tooth,she receives the treatment at the affiliated dental hospital of Zhengzhou University.2.Methods:Investigation on the basic situation and oral examination for affected members using intraoral photograph,dental film and panoramic piece.Genomic DNA was extracted from peripheral venous blood.We designed 7 pairs of primers covered the DSPP exons and promoter by Primer-BLAST on NCBI and oligo7.DSPP gene was amplified by PCR.PCR products was performed Sanger sequencing.The sequencing results were compared and analyzed by Seqman online software to screen the mutations.PolyPhen-2 and SIFT were performed to predict the harmfulness of mutations.The structure of the wild type and mutant proteins were predicted by SOPMA and Swiss-Prot.Predicting the splicing site by NNSPLICEversion 0.9(http://www.fruitfly.org/seq-tools/splice.html).Mutation frequency screening was performed on 200 local random samples by PCR-RFLP technique.Results1.Pedigree and phenotype analysis:Investigation on the oral examination of the patient,we diagnosed patients with DGI-Ⅱ.The disease is a autosomal dominant genetic disease,pathogenic genes of DSPP.2.Mutation at DSPP identification:A heterozygous mutation c.50C>T in exon 2 of DSPP gene was identified in the proband and proband’s father.After the mutation,the amino acid of DSPP protein 17 was changed from proline into leucine(p.Pro17Leu).3.Bioinformatics analysis:SIFT predicted that the mutation is likely to be harmful to human.The score was 0.We think that will affect the phenotype.Also,the mutation was predicted to be harmful by PolyPhen-2.The score was 0.998(The score was 1 which was the most likely to be harmful).The Pro-17 residues and its surrounding positions in DSPP were highly conserved across species.The program yielded did not change in the prediction value suggesting the mutation did not affect the splicing event of mRNA(0.98→0.98).In 2013,there was a report on the missense mutation of the DSPP gene(c.50C>T)in foreign country.The mutation did not influence pre-mRNA splicing but cause ER retention and defective protein secretion.It shows that the prediction result is very reliable.In addition,3D structure predicted by Swiss-Prot showed that the mutation cause the structure of protein to change greatly.4.Mutation screening frequency:The random screening of 200 samples was wild type,and no mutation was found.ConclusionIn this research,there are heritable heterozygous missense mutation c.50C>T in DSPP of patient in this DGI-Ⅱ family.The mutation was the pathogenesis of this family.The results extend the spectrum of DSPP gene mutations and provides the theory basis for genetic counseling and prenatal diagnosis in the family.