To develop a screening kit for detecting mutation hotspots of the PAH (phenylalanine hydroxylase) gene. Thirteen exons of the PAH gene were sequenced in 84 cases with PKU (phenylketonuria )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 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%). 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 5 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.

中文翻译：

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ARMS-PCR结合荧光探针技术开发苯丙氨酸羟化酶基因突变热点检测试剂盒。

开发检测PAH（苯丙氨酸羟化酶）基因突变热点的筛选试剂盒。对陕西省新生儿遗传代谢病筛查中诊断出的84例苯丙酮尿症患者PAH基因的13个外显子进行测序，并分析其突变情况。我们设计并开发了一种筛查试剂盒，可检测 9 个突变位点，涵盖陕西省发现的 PAH 突变的 50% 以上（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 和 c.158G>A) 通过使用扩增难治性突变系统-聚合酶链反应 (ARMS-PCR) 结合荧光探针技术。来自 PKU 家族的外周血和干血样本用于新开发试剂盒的临床验证。在 84 名诊断为 PKU 的儿童中检测到 PAH 基因突变。共鉴定出 159 个突变等位基因，包括 100 个错义突变、28 个剪切突变、24 个无义突变和 7 个缺失突变。外显子 7 的突变频率最高（32.08%）。其中，p.r243q的突变频率最高，占全部突变的20.13%，其次是p.r111x、ivs4-1g>A、ex6-96a>G、p.r413p；这5个位点占所有突变的47.17%（75/159）。此外，我们确定了三个以前未报告的 PAH 基因突变（p.c334x、p.g46d 和 p.g256d）。新一代测序（NGS）鉴定的47个PAH携带者共鉴定出15个突变位点，并通过新开发的试剂盒验证，一致率为100%。