Mutation Screening Of Non-syndromic Tooth Agenesis&Case Report

Tooth agenesis is one of the most common developmental anomalies in human.According to the number of missing permanent teeth,it can be divided into three categories: hypodontia referred to absence of less than six teeth excluding third molars.Absence of six teeth or more excluding third molars is described as oligodontia.Complete absence of teeth is categorized as anodontia.Tooth agenesis can also be classified as either non-syndromic,the isolated absence of teeth,or syndromic characterized by the presence of other inherited abnormalities such as nail dysplasia,sparse hair,lack of sweat glands or cleft lip and palate.Tooth agenesis takes place in either a sporadic or hereditary form and can be inherited in autosomal-dominant,autosomal-recessive or X-linked modes.The etiology of tooth agenesis is complex and not yet completely elucidated.Both genetic and environmental factors have effects as pathogenic factors.So far,more than 80 genes have been associated with tooth agenesis.Among those genes,non-syndromic tooth agenesis is mostly associated with mutations in MSX1,PAX9,AXIN2,WNT10 A and EDA.Environmental factors such as trauma,chemotherapeutic drugs,radiotherapy or thalidomide use during pregnancy may be occasionally happened.Objectives: our team assembled families and sporadic cases with tooth agenesis for candidate gene detection,searching for pathogenic genes in candidate genes and performing structural modeling and bioinformatics analysis of the wide-type and mutated MSX1 causing non-syndromic tooth agenesis.Materials and Methods: 1)With the written informed consents of the patients and their families,a detailed investigation,oral and systemic clinical examination and blood sample collection were conducted to construct the pedigree.Tooth agenesis was confirmed by panoramic radiographs.2)Blood samples were collected from the probands,their available family members and 300 unrelated healthy controls.Genomic DNA was extracted from the peripheral blood samples of all available members and controls according to standard salt extraction procedures.Screening of pathogenic mutations by polymerase chain reaction(PCR)amplification and sequencing the exons and exon–intron boundaries of five candidate genes,namely: MSX1,PAX9,EDA,AXIN2 and WNT10 A.3)Based on the crystal structure of the MSX1 homeodomain complex with DNA,the three-dimensional structure of wild-type and mutated MSX1 homeodomain was derived using Swiss-Model with PDB: 1ig7.1.C as a template.Then we built the 3D model by Swiss-Pdb Viewer.Visualization of the threedimensional(3D)structures was performed with Py MOL.Results: 1)Unrelated non-syndromic families and sporadic patients were collected,they were all from Chinese Han families and came from Hubei Province.Only permanent dentition was involved.No abnormalities were found in nails,sweat glands and hair.The patients denied a history of tooth extraction and trauma.No special drugs were taken during pregnancy.2)We identified two novel MSX1 mutations,a frameshift mutation c.590?594 dup TGTCC was found in family 2 and a missense mutation Phe191 Ser was found in sporadic patient 3.Both mutations were occurred in the homeodomain of MSX1.No mutation was detected in PAX9,EDA,AXIN2 and WNT10A.3)MSX1 frameshift insertion c.590?594 dup TGTCC is located at the end of the loop domain between ?-I and ?-II helix,generated a premature stop codon after an unrelated polypeptide sequence consisting of 22 amino acid residues.MSX1 missense mutation Phe191 Ser is located at the end of ?-I helix,and the mutation changes the spatial structure and the number of hydrogen bonds compared to the wide-type MSX1 protein.Conclusion: These two novel mutations in MSX1 enlarged the mutation spectrum.Structural modeling and bioinformatics analysis showed that the missense mutation altered the spatial conformation of the region around the mutation site.Combined with other six missense mutations causing non-syndromic tooth agenesis reported previously,a three-dimensional structural analysis was performed.Thr180 Ile and Leu230 Pro resulted in the loss of fewer than six teeth,neither of which caused hydrogen bonding changes and no significant changes in protein structure,possibly explaining the milder phenotype.The other five missense mutations all changed hydrogen bonds,including the mutation(Phe191Ser)found in our study.These mutations all caused more than 6 missing teeth and had alterations in hydrogen bond formation.These alterations might lead to changes of the helical conformation,ultimately resulting in an alteration of protein folding and decreased stability.The number of missing teeth caused by MSX1 missense mutations with protein structure and hydrogen bond changes is larger,but more studies on MSX1 missense mutations and protein function are needed to further verify this conclusion.The frameshift mutation had a significant impact on the structure of the protein,leading to severe consequences in protein interactions and DNA binding of the gene.It also led to a more serious tooth agenesis phenotype.