Mutation Spectrum of Six Genes in Chinese Phenylketonuria Patients Obtained through Next-Generation Sequencing

Li Yu,Zhong Cen,Guanghui Yang,Kangmo Lu,Lin Lin,Jing Hao,Zhigang Yang,Jian Huang,Ying Gu,Cees Oudejans,Shujian Cui,Jiabao Peng

doi:10.1371/journal.pone.0094100

Abstract

BackgroundThe identification of gene variants plays an important role in the diagnosis of genetic diseases.Methodology/Principal FindingsTo develop a rapid method for the diagnosis of phenylketonuria (PKU) and tetrahydrobiopterin (BH4) deficiency, we designed a multiplex, PCR-based primer panel to amplify all the exons and flanking regions (50 bp average) of six PKU-associated genes (PAH, PTS, GCH1, QDPR, PCBD1 and GFRP). The Ion Torrent Personal Genome Machine (PGM) System was used to detect mutations in all the exons of these six genes. We tested 93 DNA samples from blood specimens from 35 patients and their parents (32 families) and 26 healthy adults. Using strict bioinformatic criteria, this sequencing data provided, on average, 99.14% coverage of the 39 exons at more than 70-fold mean depth of coverage. We found 23 previously documented variants in the PAH gene and six novel mutations in the PAH and PTS genes. A detailed analysis of the mutation spectrum of these patients is described in this study.Conclusions/SignificanceThese results were confirmed by Sanger sequencing. In conclusion, benchtop next-generation sequencing technology can be used to detect mutations in monogenic diseases and can detect both point mutations and indels with high sensitivity, fidelity and throughput at a lower cost than conventional methods in clinical applications.

Highlights

Phenylketonuria (PKU), or hyperphenylalaninemia (HPA), is the most common metabolic disease in China due to autosomal recessive, inborn errors of phenylalanine metabolism
To quickly discover DNA variants of the causal genes involved in genetic disorders and to sequence all the bases in the exons and intron-exon boundaries of the six genes (PAH, PTS, GCH1, QDPR, PCBD1 and GTP cyclohydrolase I feedback regulatory protein (GFRP)) involved in PKU and BH4 deficiency, 108 primer pairs were designed for multiplex PCR to amplify 39 exons containing 9,967 base pairs
The enriched DNA was used for library construction and sequenced at single-base resolution using the Ion Torrent system

Summary

Introduction

Phenylketonuria (PKU), or hyperphenylalaninemia (HPA), is the most common metabolic disease in China due to autosomal recessive, inborn errors of phenylalanine metabolism. The worldwide regional and ethnic variation of PKU prevalence is apparent, ranging from 1/100,000 in the African population to 1/2,600 in the Turkish population [8]. Patients with PKU usually present with HPA and severe mental retardation unless a low phenylalanine diet is introduced early in life. PKU can be classified as classic PKU, moderate PKU, mild PKU or mild HPA, depending on the enzyme defect, genotype and severity of the disease [8]. Genetic analysis has revealed that mutations in the PAH gene, which encodes phenylalanine hydroxylase, result in PKU. The identification of gene variants plays an important role in the diagnosis of genetic diseases

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