Functional Study Of The POU4F3 Gene Mutation On Autosomes Associated With Non-syndromic Deafness

Objectives The POU4F3 protein is a transcription factor of POU family.It has been shown that the targeted deletion of Pou4f3 gene will lead to vestibular dysfunction and severe deafness in mice,but the specific pathogenesis is not clear.So far,30 mutations of human POU4F3 gene have been reported,among which only five have been identified as pathogenic mutations(c.884del8,p.I295fs*5;c.668T>C,p.L223P;c.865C>T,p.L289F;c.593G>A,p.R198H;c.704＿705del,p.T235fs)by detailed functional research.The aim of this study is to investigate whether the remaining POU4F3 mutant proteins are pathogenic.Methods In this paper,first four variant POU4F3 proteins which had not been studied functionally(c.665C>T,p.S222L;c.718A>T,p.N540Y;c.841A>G,p.I281V;c.982A>G,p.K328E)have been obtained by means of structural biology,cell biology and biochemistry.Then we use the principle of Electrophoretic Mobility Shift Assay(EMSA)— if DNA binds to protein,it will form a DNA-protein complex with a larger molecular weight and it will run slower than free DNA on non-denatured polypropylene gel.— and Ethidium bromide(EB)technology for labeling DNA to investigate the affinity of the four variant POU4F3 proteins to DNA,to determine whether the transcriptional activity of the variant protein is affected and whether the mutation is pathogenic by comparing with the wild-type POU4F3 protein,in order to facilitate prenatal diagnosis and treatment interventions.Results After running the gel with 9% polyacrylamide separation gel(Native gel),we can see at the same distance of the DNA band position of the negative control group(the DNA sample without any protein added),almost no DNA bands can be seen in the positive control group(the wild type POU4F3 protein-DNA mixture)and the band position is obviously lagging.On the other side,the experimental group(the variant POU4F3 protein-DNA mixture)shows two clear DNA bands after running the gel and the electrophoresis distance is very close to the above two bands.Conclusions Compared with the wild-type POU4F3 protein,the affinity of four mutants(c.665C>T,p.S222L;c.718A>T,p.N540Y;c.841A>G,p.I281V;c.982A>G,p.K328E)to DNA is obviously decreased,suggesting that the transcriptional activity of the four mutants is significantly affected and could be identified as pathogenic mutations.