Construction Of Three Phenylalanine Hydroxylase Gene Mutation In Eukaryotic Cell Model And Study On The Function Of Three Gene Mutations

Object:To date,more than 1000 PAH gene mutations have been reported benefit from development of sequencing technology.It is a hot topic to study the function and phenotype of novel PAH gene mutations.With constructing the eukaryotic expression system in vitro of PAH gene mutants,the nature of mutations can be correctly distinguished,in addition,we can understand more of how mutations affect PAH protein function and the mechanism of mutations of PKU.In this research,we analyzed three novel PAH gene mutation detected in our previous work by constructing the expression systems in vitro,and indentified residual enzyme activities of the gene mutations.Method:Firstly,we predicted the structure of three novel PAH gene variants including c.[59A>C;60G>C](Q20P),c.690-691 ins G(S231fx X51)and c.1119-1120 ins T(I374fx X20)by bioinformatics analysis.Secondly,we used COS-1 cell line of African green monkey kidney cells to built three stable expression cell models by the gene editing technology(CRISPR/Cas9).Functional verification followed by three cell models were successfully constructed,including testing of PAH m RNA,protein and enzyme activity.Finally we combined bioinformatics prediction results to reveal the relationship between genotype and clinical phenotype.Result:(1)Software predicted the structure change of PAH protein caused by three mutant: c.[59A>C;60G>C](Q20P),c.690-691 ins G(S231fx X51),c.1119-1120 ins T(I374fx X20).Bioinformatics analysis may helpful to predict the damage of mutations on protein structures,but there are still a big space to improve on its reliabilities.(2)By CRISPR/Cas9 gene editing technology,we successfully constructed stable cell lines of three PAH gene mutations,which were proven by Sanger sequencing of specific mutation loci.(3)The m RNA expression of the mutants were detected by RT-PCR.The m RNA levels of c.[59A>C;60G>C](Q20P)was 95.4 of the wild type,showed no significant change on PAH gene expression compared with the wild type.The m RNA expression of c.690-691 ins G(S231fx X51)and c.1119-1120 ins T(I374fx X20)were more badly affected,with the expression levels of 87.2% and 82% of the wild type respectively.(4)By enzyme activity detection we found that comparing to wild-type PAH,in vitro residual enzyme activity of missense mutation c.[59A>C;60G>C](Q20P)was 57.3%,the insertion mutation c.690-691 ins G(S231fx X51)was 22.8%,and the insertion mutation c.1119-1120 ins T(I374fx X20)was 20.3%.Conclusion:(1)Three stable expression cell models of PAH gene mutant : c.[59A>C;60G>C](Q20P),c.690-691 ins G(S231fx X51),c.1119-1120 ins T(I374fx X20)were successfully constructed by CRISPR/Cas9 gene editing method.(2)The results of enzyme activity detection of mutant cell lines showed that the expressed enzyme activities of the three mutations in vitro were reduced significantly,and the residual enzyme activities were 53.7%,22.8%,and 20.3% of the wild type respectively.The mutation c.[59A>C;60G>C](Q20P)had little effect on PAH gene m RNA and PAH protein expression.c.[59A>C;60G>C](Q20P)mutant PAH enzyme activity is greatly affected.The mutations of c.690-691 ins G(S231fx X51)and c.1119-1120 ins T(I374fx X20)have more significant effects on the three-dimensional structure and enzyme activity of PAH protein.(3)The reason that c.[59A>C;60G>C](Q20P)mutation causes the decreasion of enzyme activity may be that the mutation leads to the misfolding of PAH protein or the unstable structure of the resulting protein causes its hydrolysis.The reason why c.690-691 ins G(S231fx X51)and c.1119-1120 ins T(I374fx X20)mutations decrease the enzyme activity is that the protein structure is incomplete,and then affects its protein expression and the decreasion of enzyme activity after mutation.(4)Prediction of protein three-dimensional structure by bioinformatics methods combined with the in vitro expression of PAH genes may be helpful to a better understand on the molecular mechanism of gene mutations.