Mutation Screening And Functional Analysis On The Molecular Pathogenesis Of Non-syndromic Tooth Agenesis

Non-syndromic tooth agenesis is one of the most common oral heditary diseases, which manifested as missing tooth. Non-syndromic tooth agenesis shows a wide phenotypic heterogeneity and is classified as a sporadic or familial form, inherited in an autosomal-dominant (AD), autosomal-recessive (AR) or X-linked mode. Population studies have revealed that the most commonly missing teeth are third molars followed by deficiency of lower second premolars or upper lateral incisors. Agenesis of the first and second molars is very rare. When more than six teeth, excluding third molars, are missing, the condition is referred to as oligodontia. To date, non-syndromic familial and sporadic tooth agenesis has associated with mutations in MSX1, PAX9, AXIN2and EDA. The purpose of the present study is to investigate the mutational characteristics of MSX1,PAX9, AXIN2and EDA genes in Chinese patients with tooth agenesis and further to perform functional analysis on the molecular pathogenesis of non-syndromic tooth agenesis.PART1Pedigree collectionObjectives:Collect non-syndromic tooth agenesis families to lay a basis for candidate gene detection and functional analysis.Materials and Methods:We collected eight tooth agenesis families. Pedigree construction was conducted on the basis of the results of clinical examination and interview of the available family members. Panoramic radiographs were taken to verify tooth agenesis. Blood samples were collected from available members of the family. Results:These families are all Chinese Han families, in which seven families manifested as non-syndromic tooth agenesis except one family had congenital cleft lip as well. The patients denied tooth extraction or medical history that led to tooth falling out. No special medicine was taken as pregnancy. The expressivity varied among patients even in the same family.Conclusions:All families conform to AD mode, while in one family X-linked mode can't be excluded.PART2Mutation detectionObjectives:Detect candidate genes in the eight families to look for mutations.Materials and Methods:Genomic DNA was isolated from the peripheral blood lymphocytes of all the available members and controls, according to a modified technique using sodium dodecyl sulfate, proteinase K, and chloroform. Pairs of primers were designed for amplifying MSX1, PAX9, AXIN2and EDA genes for all families. Mutation analysis was performed by amplifying the coding regions and splicing junctions of these genes, sequencing the products.Results:We identified two novel missense mutations, L27P and I29T, in the paired-domain of PAX9and a novel nonsense mutation, Q189X, in the homeodomain of MSX1. But there is no mutation detected in the other five families.Conclusion:Two novel missense mutations in PAX9and a novel nonsense mutation in MSX1enlarged the mutation spectrum. PART3Fuctional analysis of PAX9and MSX1Objectives:Perform functional analysis of the three mutations causing tooth agenesis.Materials and Methods:We conducted site-directed mutagenesis to generate the mutated vectors. The wild-type and mutated PAX9vectors were then transfected separately to NIH3T3cells. Immunolocalization, electrophoretic mobility shift assay (EMSA) and luciferase reporter assay were performed to analyze the effects of mutations on protein function. As to MSX1, real-time PCR was performed to test the mRNA level of mutated MSX1in transfected COS7cell lines.Results:Analysis of homology PAX proteins indicated that these two substitutions may affect the function of PAX9protein. Results of immunofluorescence and westemblot showed that the mutations did not alter nuclear localization of PAX9. Gel shift assays and luciferase reporter assays indicated that both mutant proteins can't bind DNA or transactivate BMP4promoter. Real-time PCR showed that the expression level of the mutated MSX1was dramatically reduced compared with that of the wild type.Conclusion:Two novel missense mutations in PAX9and a novel nonsense mutation in MSX1have been indentified in Chinese families causing oligodontia.