An Analysis Of The Phenotype And Genotype And Pathogenic Mechanism Of Non-syndromic Tooth Agenesis Patients

Objective:1.Whole-exome sequencing(WES)identified mutation genes from two probands with non-syndrome tooth agenesis(NSTA)and Sanger sequencing was verified,to explore the pathology of selective tooth agenesis.Analysis of the conservative of multi-species and the structure predict of wild type and mutations of the gene.Exploring the possible pathogenic mechanism of the mutated gene for further genetic research on non-syndrome tooth agenesis2.Analysis of the molecular mechanism of six novel mutations from non-syndrome tooth agenesis patients verified by Whole-exome sequencing,improving the comprehension of the pathology of this disease.Methods:1.The patients’ clinical manifestations were collected based on complete oral examinations,panoramic radiographs and pedigree information.Peripheral venous blood was taken from probands and their parents,then DNA was extracted.Whole-exome sequencing(WES)and Sanger sequencing were performed to identify the causal gene variants.Aligned multi sequencing was conducted to predict the conservation of LRP6 and predict the structure change of LRP6 mutation.2.Six novel mutations of EVC2 were identified by WES from six probands with NSTA.Constructed wild type(WT)and six mutation plasmids of EVC2 by homologous recombination then transiently transfected into the 293 T cells.Total RNA and proteins were isolated from cells after 36 hours.The WT and mutants EVC2 m RNA expression levels were measured by RT-q PCR and the protein expression levels were measured by western blotting(WB).Through immunofluorescence assay to confirm the wild type and mutants EVC2 protein locating in U2 OS cells.Results:1.We discovered two probands with sporadic or heredity diagnosed non-syndrome tooth agenesis,proband 1 had a nonsense mutation(c.C1573 T,p.R525X)of LRP6 and proband 2 had a frameshift mutation(c.4611 del T,p.C1537fs)of LRP6.The predicting structure of LRP6 and LRP6 mutation illustrated that the mutation altered protein structure and created a premature stop codon.Aligned multi sequencing showed the LRP6 protein sequence highly conservative,suggesting that the mutations were hazardous.2.We discovered six novel missense mutations of EVC2(c.C1117 G,p.P373A;c.G1888 A,p.A630T;c.G2322 C,p.Q774H;c.C3055 T,p.R1019W;c.G2615 A,p.R872Q;c.G3526 A,p.G1176R)from non-syndrome tooth agenesis patients.Through q PCR and Western blotting experiments showed that m RNA stability and protein expression were not significantly different with wild type.The location of mutants’ EVC2 protein was not dominantly different from the wild type.Conclusion:1.The study revealed two novel mutations of LRP6 of selective tooth agenesis which is helpful in prenatal diagnosis and genetic counseling.2.The six novel mutations of EVC2 from non-syndrome tooth agenesis were identified by whole-exome sequencing.These mutations did not alter the m RNA stability and protein expression levels,the location of EVC2 protein in cells was not seriously different between wild type and mutants.More experiments are needed to identify the pathogenic mechanism of the patients.