Development of a mutation hotspot detection kit for the phenylalanine
hydroxylase gene by ARMS-PCR combined with fluorescent probe technology
Abstract
To develop a screening kit for detecting mutation hotspots of the PAH
gene.. We designed and developed a screening kit to detect 9 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%).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 5 loci accounted for 47.17%
(75/159) of all mutations. Fifteen mutation sites were identified in the
47 PAH carriers identified by NCS, 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.