Cao Yan-yan, Qu Yu-jin, Song Fang, Zhang Ting, Bai Jin-li, Jin Yu-wei, Wang Hong
Department of Medical Genetics, Capital Institute of Pediatrics, Beijing 100020, China.
Mol Genet Metab. 2014 Dec;113(4):261-6. doi: 10.1016/j.ymgme.2014.10.004. Epub 2014 Oct 12.
Hyperphenylalaninemia (HPA) can be classified into phenylketonuria (PKU) and tetrahydrobiopterin deficiency (BH4D), according to the defect of enzyme activity, both of which vary substantially in severity, treatment, and prognosis of the disease. To set up a fast and comprehensive assay in order to achieve early etiological diagnosis and differential diagnosis for children with HPA, we designed a custom AmpliSeq™ panel for the sequencing of coding DNA sequence (CDS), flanking introns, 5' untranslated region (UTR) and 3' UTR from five HPA-causing genes (PAH, PTS, QDPR, GCH1, and PCBD1) using the Ion Torrent Personal Genome Machine (PGM) Sequencer. A standard group of 15 samples with previously known DNA sequences and a test group of 37 HPA patients with unknown mutations were used for assay validation and application, respectively. All variations were confirmed by Sanger sequencing. In the standard group, all the known mutations were detected and were consistent with the results of previous Sanger sequencing. In the test group, we identified mutations in 71 of 74 alleles, with a mutation detection rate of 95.9%. We also found a frame shift deletion p.Ile25Metfs*13 in PAH that was previously unreported. In addition, 1 of 37 in the test group was inconsistent with either the molecular diagnosis or clinical diagnosis by traditional differential methods. In conclusion, our comprehensive assay based on a custom AmpliSeq™ panel and Ion Torrent PGM sequencing has wider coverage, higher throughput, is much faster, and more efficient when compared with the traditional molecular detection method for HPA patients, which could meet the medical need for individualized diagnosis and treatment.
高苯丙氨酸血症（HPA）可根据酶活性缺陷分为苯丙酮尿症（PKU）和四氢生物蝶呤缺乏症（BH4D），这两种疾病在严重程度、治疗方法和预后方面存在很大差异。为了建立一种快速全面的检测方法，以便对HPA患儿进行早期病因诊断和鉴别诊断，我们设计了一种定制的AmpliSeq™ 检测板，用于对五个导致HPA的基因（PAH、PTS、QDPR、GCH1和PCBD1）的编码DNA序列（CDS）、侧翼内含子、5'非翻译区（UTR）和3'UTR进行测序，使用Ion Torrent个人基因组测序仪（PGM）。分别使用一组15个已知DNA序列的标准样本和一组37例HPA患者的测试样本进行检测验证和应用，所有变异均通过桑格测序进行确认。在标准组中，所有已知突变均被检测到，且与之前的桑格测序结果一致。在测试组中，我们在74个等位基因中的71个中检测到突变，突变检出率为95.9%。我们还发现了PAH基因中一个以前未报道的移码缺失p.Ile25Metfs*13。此外，测试组中的37例患者中有1例在分子诊断或传统鉴别方法的临床诊断方面不一致。总之，我们基于定制的AmpliSeq™ 检测板和Ion Torrent PGM测序的综合检测方法与传统的HPA患者分子检测方法相比，具有更广泛的覆盖范围、更高的通量、更快的速度和更高的效率，能够满足个性化诊断和治疗的医疗需求。
