Pathogenicity Analysis Of A Severe Nonsyndromic Tooth Agenesis Case

Objective The clinical cases of nonsyndromic tooth agenesis were collected.Whole Exome Sequencing(WES)and pathogenicity prediction of mutant genes were performed on patients with severe congenital tooth missing.Sanger sequencing was used to identify the potential pathogenic mutation.Based on the above results,genetic analysis of genotype-phenotype correlation was carried out.Methods1.Clinical data collection of patients The study was approved by the Ethical Committee of Stomatology hospital of Tianjin Medical University and all operations conformed to the ethical claims.After introducing the purpose of the experiment and the confidentiality of personal privacy to the patients(parents of the children)and signing the informed consent.Routine oral examination was conducted on our patients to screen out those who might have congenital tooth missing.Panoramic radiographs was used to make a clear diagnosis.The medical history was carefully asked to exclude the cases of tooth extraction due to trauma,periodontal disease,pulp disease and periapical disease or with other systemic diseases.To understand the prevalence of tooth agenesis in the family,the family history of patients with the disease was inquired.In this study,proband and normal phenotypic parents were included.The family diagram was drawn and the venous peripheral blood was stored in the refrigerator at-80℃.2.Screening related mutation sites by WES The DNA quality of blood samples was tested.The samples(DNA content > 0.6 μ g)that met the sequencing standard were highly enriched in the whole exon region by Agilent system,and the library was subjected to the quality inspection.The qualified DNA was sequenced on the platform of Illumina.The experiment was carried out in strict accordance with the latest and optimized standards.Remove polymorphism sites(>1%)and synonymous SNP.The SNP and In Del,which are located in exons and affect the cleavage are retained.The mutation of conserved domain may have a great influence on the function of protein and gerp++gt2 was used to predict the conservation of mutation sites.Dbsc SNV was used to predict whether the mutation site affected splicing.The pathogenic prediction of candidate mutation sites were analyzed by SIFT,Polyphen,Mutation Taster and CADD softwares.According to the result of Phenolyzer analysis of genotype-phenotype correlation,the top 15 candidate genes were selected and the most relevant genes were identified for further analysis.The mutation sites that were predicted harmful,located in conserved area and affected cleavage were verified by Sanger sequencing.3.Sanger sequencing The forward and reverse primers of the mutant gene for sequencing was designed by Prime 5 software for sequencing.4.Analysis of allele frequency of target site mutations As an effective tool to analyze BAM data,IGV generated sequence alignment map to display the mutated base frequency changes of target allele directly and quantitatively.5.Protein secondary structure prediction Protean software was used to predict the secondary structure of the mutant protein and compare it with the normal protein so as to analyze the pathogenicity of the mutated protein related to the severe congenital missing tooth phenotype.Results1.Three samples were included in this family.Only the proband had the disease and her parents were normal.MSX1 gene score was 1 in genotype-phenotype correlation ranking,which was most closely associated with tooth agenesis in the proband.The proband and her mother carry two mutations on MSX1.One is an unreported missense mutation at exon2(c.C667 G,p.R223G)and the other is a synonymous mutation at exon1(c.C348 T,p.G116 G,rs34165410).2.Two mutations located in MSX1 were identified by Sanger sequencing.And the result indicated that the peak of missense mutation(c.C667 G,p.R223G)in exon2 of the mother was significantly lower,suggesting the possibility of mosaic mutation.Again,Sanger sequencing confirmed the presence of mosaicism in the mother.3.IGV analysis demonstrated that the G base ratio of the mother was 14% on c.C667 G site of MSX1,which was consistent with the Sanger sequencing results.4.Protean prediction showed that the α helix of mutant protein of MSX1 was obviously truncated near amino acid 223.Conclusions1.The novel mutation(c.C667 G,p.R223G)in MSX1 is located in the highly conservative region,which may be the main reason for tooth agenesis in the proband.2.The maternal mosaic mutation of the c.C667 G explained the reason why the mother did not have congenital missing teeth.The missense mutation of the proband was maternally inherited and resulted in severe tooth agenesis probably.3.For patients with suspected de novo mutations,family history should be inquired carefully to consider whether the mutation was inherited from the maternal/paternal mosaicism.For asymptomatic individuals carrying a mosaic mutation,their offspring are at underlying risk.