A Novel PAX9 Mutation Identification And Functional Verification Of A Patient With Congenital Non-syndromic Hypodontia

ObjectiveTo search for possible genetic causes,whole exome sequencing was performed on patients with non-syndromic congenital missing tooth.Through later experiments,the molecular pathway mechanism was studied.This study explored the possible pathogenic mechanism of the mutated genes and laid a theoretical foundation for further genetic research on congenital tooth agenesis.Methods1.The collection of patients with non-syndromic congenital tooth agenesis and data analysis4002 college students recruited in one Tianjin University were all given detailed dental examinations by experienced dentists.The data were analyzed by chi-square test to calculate the prevalence rate of congenital missing tooth and tooth malformation and realize the characteristics of diseases.Patients diagnosed with non-syndromic congenital tooth agenesis were collected and four millilitres of peripheral blood from each participant was obtained and placed in a tube containing EDTA.Peripheral blood samples(4 ml)from each subject were stored at-80?.The study was approved by the ethics committee of Tianjin Medical University and was conducted with the consent of all participants.2.Whole exome sequencing(WES)and generation sequencing verification of peripheral blood in patients with congenital tooth agenesisThe exome regions were efficiently enriched from 1.0?g genomic DNA using the Agilent liquid capture system according to the manufacturer's protocol.All the sequenced mutants were analyzed by Phenolyzer software,and the top 10 genes were further analyzed.The mutation in the base allele frequency of top 10 genes which less than 0.01 was recorded as a meaningful mutation in all populations compared with the data from the 1000 genome project.Meaningful mutations in the genes responsible for congenital tooth agenesis will be further validated by generation sequencing.3.Bioinformatics analysisThe mutations were manipulated using the MutationTaster,CADD,dbNSFP and SIFT to predict its function.The secondary structures of the wild-type protein and the mutant protein of PAX9 were also designed using Protean software(DNASTAR),based on the primary sequences conservation and known protein structures.4.Functional analysis of novel mutation in pathogenic genes in peripheral blood leukocytesTotal RNA was isolated from peripheral blood leukocytes,and cDNA synthesis was performed.The relative PAX9 and BMP4 mRNA expression levels were measured using qRT-PCR.5.Functional analysis of novel mutation in pathogenic genes in vitroThe expression vectors pIRES2-EGFP-PAX9 and pIRES2-EGFP-V111M which verified by sequencing were transiently transfected into the NIH3T3 cells.Total RNA was isolated from cells after 24 hours and 48 hours of instantaneous transfection.cDNA was synthesized from total RNA and the relative PAX9 mRNA expression levels were measured using qRT-PCR.The PAX9 expression plasmid(pIRES2-EGFP-PAX9 or pIRES2-EGFP-PAX9MUT)was co-transfected with pGL3-PAX9 reporter plasmid(which constructed by subcloning the 2.3-kb BMP4promoter sequence into the Bgl?/Hind?sites of the pGL3-Basic luciferase reporter vector).pRL-TK Renilla luciferase vector was also co-transfected for normalisation.Sequencing,the luciferase activity was measured.Results1.The prevalence of hypodontia was 3.92%.Hypodontia in mandibular and female was significant familiar(?~2=4.40 p=0.04;?~2=6.49 P=0.01).The prevalence of morphology anomaly was 4.97%.The prevalence of morphology anomaly in maxillary and Han nationality was significant higher(?~2=43.60 P=0.00;?~2=4.92P=0.03).There was no significant different among different regions about hypodontia and morphology anomaly.Mandibular lateral incisors and maxillary lateral incisors were the most common sites of hypodontia and morphology anomaly,respectively.The severity of tooth agenesis was slight and mild.2.WES found that the a novel significant mutation(c.G1057A p.V111M)and a reported SNP(c.G1444C p.A240P)in PAX9 gene in a sporadic case with non-syndromic congenital tooth agenesis,which were further verified by generation sequencing.3.Both SIFT and MutationTaster showed that the novel mutation on PAX9(c.G1057A p.V111M)was pathogenic mutation;CADD identified it as a harmful mutation;The dbNSFP software showed that the mutant site was found to be highly conserved among different species.Secondary structural analysis by Protean predicted that the?-region close to position of the 111th amino acid was shorter than in the wild-type protein.4.The functional experiments showed that the PAX9 mRNA expression level was upregulated significantly in the proband's peripheral blood cells compared to the wild-type.BMP4 mRNA expression level was downregulated notably in the proband's peripheral blood cells.5.In vitro functional experiments,the mRNA expression level of mutant PAX9 was higher than wildtype PAX9,and the BMP4-promoter luciferase assay experiment showed that the missense mutant exhibited significantly lower activity in transactivation of luciferase expression than the wild-type.Conclusions1?According to the epidemiological investigation,the prevalence of hypodontia and dental malformation in a Tianjin college was 3.92%and 4.87%,respectively.The higher prevalence and distinct characteristics of congenital dental abnormality,which should be paid great attention to by clinicians.2?A novel mutation of PAX9 gene(c.G1057A p.V111M)was discovered and identified through Whole exome sequencing and Sanger sequencing.By changing the normal structure and expression of PAX9 and the ability of transactivation of the downstream gene BMP4 promoter,the novel mutation of PAX9 gene(c.G1057A p.V111M)in a patient with non-syndromes congenital tooth agenesis could down-regulate the expression of the downstream gene BMP4.This mechanism which may be the etiology of tooth agenesis,caused the clinical symptoms of congenital hypodontia in this patient.The results of this study extended the mutant spectrum of PAX9 gene and verified the relationship between PAX9 and BMP4.