Identification And Functional Study Of New Pathogenic Sites In Hereditary Abnormal Number Of Teeth And Dentin Dysplasia

Research background and objectivesOral genetic diseases refer to genetic diseases mainly affecting oral tissues,such as dentine hypoplasia and tooth loss.Some systemic diseases may also involve oral tissues,such as congenital ectodermal dysplasia,osteogenesis imperfection,clavicular dysplasia,etc.There are various kinds of oral genetic diseases,the main cause of which is genetic factors.In addition,the quality of life of patients suffering from the disease is significantly reduced,so the search for its etiology has become the focus of common concern of patients and many researchers.In this study,four unrelated families with oral genetic diseases of simple type(two cases of congenital tooth loss and one case of dentine hypoplasia type I,DGI-I)and syndrome type(cleidocranial dysplasia,CCD)were collected from all over China.With the help of whole exon sequencing and Sanger sequencing,related candidate genes were detected for each disease and their functional changes were analyzed to explore the pathogenic mechanism of mutated genes,laying a theoretical foundation for in-depth research on oral genetic diseases.Materials and Methods1.Families with oral genetic diseases with congenital abnormal number of teeth,dental dysplasia or syndromic clinical manifestations were collected,and clinical data and peripheral blood were collected.2.The selected patients were subjected to full exon sequencing(WES),and the screening results were analyzed and verified by Sanger sequencing.3.SOPMA,I-TASSER,Polyphen2 and other software were used to analyze the influence of pathogenic gene variation on protein function.4.Expression vectors of wild-type(WT)and mutated(MUT)of the target gene were constructed and transfected into HEK-293T cells or human dental pulp stem cells(hDPSCs).The expression of mutated genes was detected by RT-qPCR and Western Blot.Confocal microscopy was used to observe the localization of mutated protein in cells.5.The molars collected from the patients were analyzed by micro-CT,toluidine staining,scanning electron microscopy and nano indentation.Primary hDPSCs were cultured in patients to explore the changes of odontogenic differentiation ability.6.For X chromosome inherited diseases,androgen receptor gene analysis method was used to detect the X chromosome inactivation type of female diseasecausing gene carriers.Results1.In this study,one known pathogenic gene locus and three new mutation sites were screened and verified by WES in four Chinese Han families with oral genetic diseases.The pathogenic genes involved include COL1A1,KDF1,EDA,and RUNX2.2.A new mutation of COL1A1 was detected in family 3(c.1463G>C,p.G488A),and the patient presented with non-syndromic dentinogenesis imperfecta.The mutation was predicted to be harmful mutation,with changes in protein threedimensional structure and protein expression level.Ultrastructural analysis showed that the number of dentin tubules was sparse and blocked,the odontoblast cell layer was disorganized and polarity disappeared,the hardness of dentin was reduced,and the hDPSCs were over-mineralized.3.A new mutation of KDF1 c.911T>A was detected in family 2,and this missense mutation could cause p.I304N.The clinical manifestations of the patient were severe and simple loss of all permanent teeth embryo.The mutation was predicted by software to be harmful and highly conserved across species.The mutation affected the two-dimensional and three-dimensional structure of KDF1 protein,as well as its expression at mRNA and protein levels.4.A known EDA mutation site C.1013C>T missense mutation was detected in family 1,which can cause p.T338M.The clinical manifestation was that most teeth were missing in male patients while only one tooth was missing in female patients.The mutation affected the expression of EDA in hDPSCs and resulted in the change of NF-κB signaling pathway.On the X chromosome inactivation type test,female carriers were found to have a slight degree of offset inactivation.5.A new truncated coding mutation of RUNX2 c.132delG,was detected in patients with CCD in family 4,resulting in RUNX2 loss of multiple functional domains and production of truncated protein with only 143 amino acid residues(p.va145trPFSX98).The mutation caused significant changes in the secondary and tertiary structures of RUNX2 protein,and the location of RUNX2 protein in the cell was significantly changed,and it clustered in the nucleus as a dot.And affect its mRNA and protein expression level.ConclusionIn this study,we found a known mutation site of EDA and a new mutation site of KDF1 in families with non-syndromic congenital tooth loss,which for the first time explored the possible influence mechanism of EDA in hDPSCs and the effect of X chromosome inactivation in the disease,and also reported for the first time that KDF1 gene mutation can lead to and non-syndromic anodontia.The discovery of a novel mutation of COLIA1 in a rare non-syndromic form of dentinogenesis imperfecta adds to the evidence supporting the association between the two.New deletion frame-shift mutations were found in rare CCD,providing genetic counseling and eugenics guidance for family members of patients.