Mutation spectrum of six genes in Chinese phenylketonuria patients obtained through next-generation sequencing.
BACKGROUND
The identification of gene variants plays an important role in the diagnosis of genetic diseases.


RESULTS
To 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
These 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.
Prenatal Diagnosis Center, Maternal and Child Health Care Hospital, Lianyungang, Jiangsu, China.
Ying
Y
Gu
Prenatal Diagnosis Center, Chengdu Women's and Children's Central Hospital, Chengdu, Sichuan, China.
Kangmo
K
Lu
Shanghai Find Bio-Tech Co., Ltd., Shanghai, China.
Guanghui
G
Yang
Shanghai Find Bio-Tech Co., Ltd., Shanghai, China.
Zhong
Z
Cen
Life Technologies Company, Shanghai, China.
Li
L
Yu
Shanghai Find Bio-Tech Co., Ltd., Shanghai, China.
Lin
L
Lin
Shanghai Find Bio-Tech Co., Ltd., Shanghai, China.
Jing
J
Hao
Shanghai Find Bio-Tech Co., Ltd., Shanghai, China.
Zhigang
Z
Yang
Shanghai Find Bio-Tech Co., Ltd., Shanghai, China.
Jiabao
J
Peng
Shanghai Find Bio-Tech Co., Ltd., Shanghai, China.
Shujian
S
Cui
National Engineering Center for Biochip at Shanghai, Shanghai, China; Shanghai-MOST Key Laboratory for Disease and Health Genomics, Chinese National Human Genome Center at Shanghai, Shanghai, China.
Jian
J
Huang
D044466:Asian People / Q000235:genetics*; D001708:Biopterins / Q000031:analogs & derivatives / Q000172:deficiency; D002681:China; D019295:Computational Biology / Q000379:methods; D004252:DNA Mutational Analysis; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D054643:INDEL Mutation; D060885:Multiplex Polymerase Chain Reaction; D009154:Mutation; D010375:Pedigree; D010661:Phenylketonurias / Q000175:diagnosis / Q000235:genetics*
D001708:Biopterins; C003402:sapropterin