Application Of Next-generation Sequencing In Detection Of Phenylketonuria Gene Mutation

Objective:Mutations of the PAH、GCH1、PTS、QDPR and PCBD1 in 64 PKU patients from Xinjiang were detected by using the next-generation sequencing,and evaluate the application value of next-generation sequencing in the genetic diagnosis of PKU,in order to provide basic information for gene diagnosis and prenatal diagnosis.To analyze the gene mutation of PKU patients in Xinjiang area,master the mutation type and the distribution frequency of PKU gene and establish the common type of gene mutation in this region.Methods:We designed PAH,GCH1,PTS,QDPR,PCBD1 five gene primers for multiplex PCR amplification to obtain target sequences.The product was purified and adding tag sequence enrichment products.Then detected by the next-generation sequencing technology of Next Seq500 Desktop Sequencer.The results were verified by Sanger sequencing.Results:We have successfully established a highly efficient,fast and convenient target area for next-generation sequencing analysis system.We detected 64 samples,124 mutations were detected in 128 PAH alleles.A total of 55 types of mutations,including 41 missense mutations,6 nonsense mutations,7 splice site mutations,1 deletion mutation.Genotype-accurate biochemical phenotype correlation by using the Guldberg AV system revealed consistency rate of 59.26%(16/27 cases),which the consistency rate between accurate biochemical phenotype and predictive phenotype of classical genotype was 60%(3/5 cases),and mild genotype was 57.14%(12/21cases).Conclusion:(1)We established efficient,fast and convenient analysis system for target area sequencing and mutation screening using the next-generation sequencing.All of the exons and promoters of the PAH、GCH1、PTS、QDPR and PCBD1 genes were detected in a single reaction tube.(2)We detected mutations of the PAH,GCH1,PTS,QDPR and PCBD1 gene in 64 PKU patients in Xinjiang.124 PAH mutations were detected in 128 alleles and the mutation detection rate was 96.88%(124/128),and identified three novel mutations:c.673C>T,c.1316-1G>A,c.1200-2A>C.(3)The frequency of PAH mutation compared with other parts of the country,genetic display in Xinjiang area not only has the characteristics of independence and conservative but also has characteristics of intercross and amalgamation.It is the genetic resources for study genetic diversity、PAH gene heterogeneity and human origin and migration.At the same time,it is also an important supplement to the national and regional data of PKU research in China,and provides a scientific basis for the development of targeted PKU prevention strategy in the region.