Identification And Functional Study Of A Novel Mutation Of PAX9 In A Non-syndromic Tooth Agenesis Family

BACKGROUND:Non-syndromic Tooth Agenesis(NSTA)refers to a congenital reduction in the number of teeth without abnormalities in the development of other organs in the body.NSTA has adverse effects on maxillofacial development,aesthetics and masticatory function.Therefore,it is very important to find the etiology of NSTA.OBJECTIVE: To search for possible pathogenic genes andmutation sites,and to explore the possible pathogenic mechanism of NSTA through in vitro cell experiments.METHODS:(1)Collect hereditary tooth agenesis family and complete clinical and radiological examination data.(2)Extracting patients’ DNA samples.Whole-exome sequencing technology and co-segregation analysis of family members were performed to screen and confirm the pathogenic gene.(3)The harmfulness of mutant protein was predicted by using bioinformatics websites and softwares.(4)Cycloheximide chase assay was used to analyze the degr adation-rate of mutant protein.(5)The localization of mutated protein in cells was detected by immunofluorescence assay.(6)Dual-luciferase reporter assay was used to detect the effect of mutant protein on downstream target genes.RESULTS:(1)A NSTA family was collected in this study,including 12 family members,and 7 DNA samples were collected.Four patients who wereborn with 2,3,7 and 12 permanent teeth missing respectively.(2)A novel missense mutation(c.296C>A:p.A99D)of PAX9 gene was found in this family,and the inheritance mode is autosomal dominant inheritance.(3)Mutation Taster and Polyphen-2 predicted that this mutation was harmful.SOPMA and Protean showed that α-helix and extended chain structure increased,while β-rotation and random curl structure decreased.(4)Mutation of PAX9 protein did not affect its localization in the nucleus,but the degradation rate of the mutant protein in the cell was accelerated(P <0.05).(5)PAX9 mutation inhibited the activation of downstream target gene BMP4(P < 0.05).CONCLUSION:(1)In this study,a novel missense mutation of PAX9 gene(c.296C>A:p.A99D)was found in a NSTA family,which expanded the pathogenic gene spectrum.(2)The changes in protein structure and degradation rate of PAX9 mutation,as well as the inhibition of downstream BMP4,may be the molecular mechanism of congenital tooth agenesis in this family.