Development of a mutation hotspot detection kit for the phenylalanine hydroxylase gene by ARMS-PCR combined with fluorescent probe technology.

Content Provider	Europe PMC
Author	Qiang, Rong Wang, Lin He, JinHua Xu, Wei Jie Li, Wei Cai, Na Wang, Xiao Bin Zhang, RuiXue Zhang, Li Ping Ma, Xiao Ping Wei, Chen Song, ChengRong Yu, WenWen Wang, Xiang Li, Xu
Copyright Year	2021
Abstract	AbstractTo develop a screening kit for detecting mutation hotspots of the phenylalanine hydroxylase (PAH) gene. Thirteen exons of the PAH gene were sequenced in 84 cases with phenylketonuria (PKU) diagnosed during neonatal genetic and metabolic disease screening in Shaanxi province, and their mutations were analyzed. We designed and developed a screening kit to detect nine mutation sites covering more than 50% of the PAH mutations found in Shaanxi province (c.728G>A, c.1197A>T, c.331C>T, c.1068C>A, c.611A>G, c.1238G>C, c.721C>T, c.442-1G>A, and c.158G>A) by using amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) combined with fluorescent probe technology. Peripheral blood and dried blood samples from PKU families were used for clinical verification of the newly developed kit. PAH gene mutations were detected in 84 children diagnosed with PKU. A total of 159 mutant alleles were identified, consisting of 100 missense mutations, 28 shear mutations, 24 nonsense mutations, and 7 deletion mutations. Exon 7 had the highest mutation frequency (32.08%). Among them, the mutation frequency of p.R243Q was the highest, accounting for 20.13% of all mutations, followed by p.R111X, IVS4-1G>A, EX6-96A>G, and p.R413P; these five loci accounted for 47.17% (75/159) of all mutations. In addition, we identified three previously unreported PAH gene mutations (p.C334X, p.G46D, and p.G256D). Fifteen mutation sites were identified in the 47 PAH carriers identified by next-generation sequencing (NGS), which were verified by the newly developed kit, with an agreement rate of 100%. This newly developed kit based on ARMS-PCR combined with fluorescent probe technology can be used to detect common PAH gene mutations.
Page Count	9
ISSN	01448463
Volume Number	41
PubMed Central reference number	PMC7897920
Issue Number	2
PubMed reference number	33564846
Journal	Bioscience Reports [Biosci. Rep]
e-ISSN	15734935
DOI	10.1042/BSR20201660
Language	English
Publisher	Portland Press Ltd.
Publisher Date	2021-02-01
Access Restriction	Open
Rights License	This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). © 2021 The Author(s).
Subject Keyword	amplification refractory mutation system PCR gene mutation next-generation sequencing phenylalanine hydroxylase phenylketonuria
Content Type	Text
Resource Type	Article
Subject	Cell Biology Molecular Biology Biochemistry Biophysics