Mutation Screening And Genetical Research Of Congenital Tooth Agenesis

Tooth development is a complex process caused by interactions between epitheliumand mesenchyme, and many transcription factors as well as signaling molecules areengaged in this process. Hypoplasia of teeth is the most common of human congenitalcraniofacial defect, including agenesis of teeth, tooth shape and structural abnormalities.Agenesis of teeth, referring to the unusual number of teeth, is congenital tooth agenesis.Congenital tooth agenesis is the most common human teeth dysplasia. Occurrence rate ofsingle missing tooth could reach20%. And occurrence rate of permanent teeth missingcould also reach1.6%to9.6%except the third molar, while the occurrence rate ofdeciduous teeth is lower. The second premolar and lateral incisor are most easily affectedthan other teeth except the third molar. Congenital tooth agenesis is divided into a few teeth missing, most congenital all missing teeth and mouth without teeth. Most tooth lossrefers to loss of six or more teeth besides the third molar. Congenital tooth agenesis couldbe caused by environmental or genetic factors. Sporadic cases and families’ heredity areoften presented clinically; the latter can be divided into autosomal dominant, autosomalrecessive or X-linked hereditary. Congenital tooth agenesis could affect the chewing,pronunciation, appearance and mental health. And it can be divided into syndromic andnonsyndromic type. Congenital ectodermal dysplasia syndrome is the most commonsyndromic congenital tooth agenesis, which can be divided into two types: little sweat andno sweat, and often accompanies with other tissues developmental disorders, includingnail developmental disorders, disorders of dry skin and thin hair, sweat, clinical severityobstacles in perspiration.Researches have demonstrated that more than200genes are engaged in thedevelopment of teeth dysplasia. It is generally believed that PAX9, MSX1, AXIN2andEDA are closely related to the nonsyndromic congenital tooth agenesis. While EDA,EDARADD and EDAR gene are closely related to congenital ectodermal dysplasiasyndrome, which is the most common syndromic congenital tooth agenesis.The objectives of this study are to screen the relevant candidate genes and discussthe mechanisms of related diseases through collecting clinical data from congenital toothagenesis families, and initial functional prediction.Part I: Clinical investigation and samples collection from congenital toothagenesis families1. Materials and methods: In this study, we had collected7congenital toothagenesis families, which constituted patients with more than6teeth loss. These7congenital tooth agenesis families are divided into nonsyndromic congenital toothagenesis and syndromic congenital tooth agenesis according to whether with hair andcongenital absence of sweat glands dysplasia. Genealogy mapping of congenital toothagenesis families was analyzed. Clinical investigation and samples collection fromcongenital tooth agenesis families’members were conducted.2. Results: Seven congenital tooth agenesis families, including5cases of nonsyndromic congenital tooth agenesis and2cases of syndromic congenital toothagenesis, are Han crowd. Each member of the families showed no other congenitaldevelopmental defects. Families’history showed no closed relatives in the wedding and noabortion. The second premolar and the lateral incisor are the most easily affected teeth innonsyndromic congenital tooth agenesis. Maxillary lateral incisor is more easily affectedthan mandible teeth, while there is no difference between maxillary second premolar andmandible second premolar. Congenital ectodermal dysplasia syndrome showed moreserious symptoms than other families.In addition to the proband of family2deciduous teeth and permanent teeth aremissing, is the less sweat sweat/no type does not contain all the patient’s teeth missingembryonic significantly heavier. Samples collection is based on the strict quality, laying agood foundation for subsequent experiments. In those7congenital tooth agenesis families,family1conform autosomal dominant (AD) mode, family2and6conform X-linkeddominant (XD) mode, family4conform XD mode or AD mode.3. Conclusion: Congenital tooth agenesis may be X-linked dominant or autosomaldominant disease. For the nonsyndromic type, the second premolar and lateral incisor aremore likely to be affected. Lateral incisor missing is more likely to happen in maxillary,while incisor missing is more likely to happen in mandible.Part II: Candidate genes screening for congenital tooth agenesis families1. Materials and methods: Reviewing literatures combined with DNAStar andPremier5.0software design primers for candidate genes (PAX9, MSX1, AXIN2, EDA,EDAR and EDARADD). In this study, PAX9, MSX1, AXIN2and EDA genes were ascandidate genes for non-syndromic hypodontia families. Then EDA, EDAR andEDARADD were as candidate genes for ED families. PCR products were purified andsequenced. All sequencing results aligned with GENEBANK related sequences, thenquery the database to rule out common SNPs.2. Results: Two novel EDA missense mutations (p.G257R and p.V309L), one novelintron splice site mutation (IVS1-2A>T) in MSX1and one reported PAX9missensemutation (p.R47W) were identified in nonsyndromic congenital tooth agenesis families. Three novel EDA missense mutations (p.L296V, p.E308D and p.E308K) were identifiedin congenital ectodermal dysplasia syndrome patients. Those five EDA missenses arelocated in the TNF region.3. Conclusion: All the results above indicated that PAX9, MSX1as well as EDA areclosely related with nonsyndromic type congenital tooth agenesis. Furthermore, EDA isdemonstrated to be the disease causing gene for the syndromic type congenital toothagenesis. Our finding broadens the spectrum of MSX1and EDA mutations.Part III: Genetics analysis and structural function prediction of congenitaltooth agenesis families1. Materials and methods: Design MSX1primers according to mentioned MSX1intron splice site mutation (IVS1-2A>T) and the wild type MSX1gene sequence. PCRamplification of MSX1cDNA samples and attempted to discuss the effects caused by the3’ end MSX1introns splice site mutation at the molecular level. In addition, this studyidentified five novel EDA gene mutations and simulated its protein product trimerthree-dimensional structure. Compare with wild type EDA gene, analysis of proteinconformation and molecular surface energy differences to discuss relationships betweengenotype and phenotype.2. Results: We found the intron splice site mutation of MSX1(IVS1-2A>T) mayaffect the function of MSX1gene by intron splice site prediction. This study demonstratedthat five novel EDA gene mutations were highly conservative. Mutant EDA gene showedsignificant changes of protein conformation product surface and the surface energy of theelectric field, indicating that five novel EDA mutations affected the normal function of theEDA trimer to an extent and causing4families showed tooth agenesis in different clinicalmanifestations.3. Conclusion: Congenital tooth agenesis in certain family may be caused byfunctional or structural changes of certain protein due to mutation of EDA and MSX1.