CAJ | 학술논문

目的建立基于Ion Torrent PGM平台的高苯丙氨酸血症(hyperphenylalaninemia,HPA)相关基因二代测序方法,以达到HPA早期病因诊断和鉴别诊断的目的.方法选取3例致病突变已知的HPA患儿和1名正常对照个体为研究对象用于方法的建立;选取10例致病突变已知的HPA患儿用于方法的验证;通过AmpliseqTM多重PCR的方法扩增PAH、GCH1、PTS、QDPR和PCBD1基因的5 '和3'端非翻译区和编码区以及外显子与内含子的交界区域,经Ion OneTouchTM系统富集扩增子,最后应用Ion Torrent PGM测序仪对目标区域进行检测.所得数据采用Torrent Suite v2.2软件包进行分析处理.所有变异位点经Sanger测序验证.结果用于方法建立的4份样本数据输出总量为94.22 Mb,约99.5％的读长位于目标区域.IonTorrent PGM共检测到变异位点74个(28种),其中已知的6个致病突变全部检出;其余变异位点经数据库检索和Sanger验证55个(18种)为多态位点,13个(4种)为假阳性位点.用于验证此方法的10例HPA样本中的所有已知致病突变均成功检出.结论与常规Sanger测序技术相比,Ion Torrent技术从代谢通路层面筛查高苯丙氨酸血症的相关基因变异,可以满足个体化诊断和治疗的医疗需求.
목적 건립기우Ion Torrent PGM평태적고분병안산혈증(hyperphenylalaninemia,HPA)상관기인이대측서방법,이체도HPA조기병인진단화감별진단적목적.방법 선취3례치병돌변이지적HPA환인화1명정상대조개체위연구대상용우방법적건립;선취10례치병돌변이지적HPA환인용우방법적험증;통과AmpliseqTM다중PCR적방법확증PAH、GCH1、PTS、QDPR화PCBD1기인적5 '화3'단비번역구화편마구이급외현자여내함자적교계구역,경Ion OneTouchTM계통부집확증자,최후응용Ion Torrent PGM측서의대목표구역진행검측.소득수거채용Torrent Suite v2.2연건포진행분석처리.소유변이위점경Sanger측서험증.결과 용우방법건립적4빈양본수거수출총량위94.22 Mb,약99.5％적독장위우목표구역.IonTorrent PGM공검측도변이위점74개(28충),기중이지적6개치병돌변전부검출;기여변이위점경수거고검색화Sanger험증55개(18충)위다태위점,13개(4충)위가양성위점.용우험증차방법적10례HPA양본중적소유이지치병돌변균성공검출.결론 여상규Sanger측서기술상비,Ion Torrent기술종대사통로층면사사고분병안산혈증적상관기인변이,가이만족개체화진단화치료적의료수구.
Objective To establish a hyperphenylalaninemia-related genes screening method using Ion Torrent Personal Genome Machine (PGM) for early detection and differential diagnosis of hyperphenylalaninemia (HPA).Methods Three children with known HPA mutations and a healthy control were used for setting up the method.Ten children with HPA with known mutations were recruited for validating the method.Ion AmpliseqTM PCR was used to amplify the 5' and 3'untranslated region,coding sequence,and flanking introns ofPAH,GCH1,PTS,QDPR,andPCBD1 genes.After the enrichment with the Ion OneTouchTM system,the products were sequenced by PGM.Data from the PGM were processed with Torrent Suite v2.2 software package.All variations were confirmed by Sanger sequencing.Results For the 4 samples,the PGM output was 94.22 Mb,with approximately 99.5％ of reads mapping to the target regions.Among these samples,we detected 74 variations (28 positions) including 6 known mutations.Compared with database and results of Sanger sequencing,55 (18 positions) polymorphisms and 13 (4 positions) false positive calls were confirmed.For the 10 samples,all the known mutations were successfully identified.Conclusion Ion Torrent PGM sequencing is suitable for screening genetic mutation underlying HPA from the perspective of metabolic pathways,which can meet the clinical demand for individualized diagnosis and treatment.