Mapping Of Pathogenic Gene And Studying Of Morphologic In One DGI-Ⅱ Familie

Background Dentinogenesis Imperfect type Ⅱ is a kind of autosomal dominant inherited disease,characterized by dentin development defects.Dentinogenesis Imperfect type Ⅱ,causing severe tooth discoloration,enamel stripping and severe tooth wear,will seriously affect the patient’s chewing function and aesthetics,bring greater harm to the patient’s physical and mental health,and bring heavy burden to family and society.Since pathogenic gene of DGI-Ⅱ was firstly reported by Chinese scholars in 2001,it has been the focus of research in this field to find and explore the genetic and molecular pathogenesis of hereditary dentin defects.At present,the DGI-Ⅱ gene is DSPP gene,and it is found that different pathogenic sites can cause the same symptoms,but the pathogenesis of DGI-Ⅱ is not fully understood.In previous studies,our research group found a hereditary dentin dysplasia type I family in the north area of Henan Province,through intensive study on this family,finally determining the mutation gene and pathogenesis.Through the above studies,we understood some characteristics of abnormal hereditary dentinogenesis and the mechanism of dentinogenesis,which is the foundation of further research on abnormal hereditary dentinogenesis.Then we found a DGI-Ⅱ family in the same area,in order to determine the pathogenic gene and pathological characteristics of this family,our group collected the clinical data of the DGI-Ⅱ family,observated the pathological structure of the affected teeth and sequenced pathogenic genes.Method1 collection data of pedigrees with hereditary dentin dysplasia Our research group collected the pedigree samples of the eastern Henan family affected by DGI-Ⅱ disease,existing 4 generation with a total of 34 people(33 people survived)and 12 people with disease.In this study,we observed the clinical and the systemic condition of the family members,diagnosed as DGI-Ⅱ disease.Then,the clinical data of the family(oral photos and X-ray)were collected,the pedigree map was drawn,and in order to complete the follow-up biochemical detection and gene detection,the genomic DNA was extracted from the peripheral blood of 20 members of this pedigree.2,Observation on the organization structure of teeth affected by Dentinogenesis imperfecta type Ⅱ via the atomic force microscopy and two-photon microscopy Two impacted teeth of patients with DGI-Ⅱ were collected,and the two healthy teeth were collected from person with the same sex and similar age,used as controls.Scanning electron microscopy,atomic force microscopy and multiphoton microscopy were used to observe the histological structure of DGI-Ⅱ and normal controls.3,Detection pathogenic genes of Dentinogenesis imperfecta type Ⅱ To further screen mutation gene of the Henan family with DGI-Ⅱ,promoter,introns and exons of DSPP were amplified by PCR,and then detected by Sanger sequencing.Thtough screening DSPP,we did find no mutation in the DSPP.Then we used linkage analysis and whole genome exome analysis technology to find candidate genes,and the candidate genes were detected.Result(1)It is found that the patients have typical DGI-Ⅱ features,such as discoloration of the teeth,the peeling of the enamel and the severe abrasion of the teeth.The pedigree map shows that the family is in line with the characteristics of autosomal dominant inheritance: passed down from generation to generation;the patient with one affected parent;normal parents having normal children.(2)Through the atomic force microscopy(AFM)and two-photon microscopy(MPM),we found that: the larger surface roughness of enamel samples,enamel hydroxyapatite crystal becomes sparse;the smaller dentin hydroxyapatite particles,denser hydroxyapatite crystal,dentinal tubules filled with messy irregular crystal, and larger surface roughness;enhanced two-photon excited fluorescence and the two harmonic signal appeared in the area without tubule,wave enhanced two-photon excited fluorescenc appears,corresponding to the area with disappeared two harmonic signals;the enamel crystals adjacenting to dentoenamel junction becoming sparse,a microporous,denser crystals in dentin,smaller particle size,and the surface roughness becomes larger;increasing the content of organic matter in the dentoenamel junction.(3)There were no specific changes in DSPP promoter,exons and introns region of DSPP in this DGI-Ⅱ family,only finding 3 SNP.Linkage analysis showed that the mutation gene of this family most likely to be in the range of: 4 chromosome? ~88612414bp(GRCh38 version).Whole genome exome analysis technology detected25 genes as candidate genes,and then the candidate genes were further studied by bioinformatics analysis.The results showed that the candidate genes had no relation with DGI-Ⅱ.Conclusion Atomic force microscopy and multiphoton microscopy revealed that the enamel and mantle dentin were affected,which was helpful to deeply understand of the ultrastructure of DGI-Ⅱ.Genetic screening did not find a pathogenic mutation,so a wide range of information technology should be used to detect the mutation gene.