应用二代测序平台筛查高苯丙氨酸血症相关基因突变  

Screening for genetic mutations in hyperphenylalaninemia using Ion Torrent PGM sequencing

作　　者：曹延延[1] 瞿宇晋[1] 宋昉[1] 白晋丽[1] 金煜炜[1] 王红[1] 

机构地区：[1]首都儿科研究所遗传室,北京100020

出　　处：《中华医学遗传学杂志》2015年第1期16-20,共5页Chinese Journal of Medical Genetics

基　　金：首都儿科研究所科研基金（所-13年-A02）

摘　　要：目的建立基于IonTorrentPGM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Ampliseq^TM多重PCR的方法扩增PAH、GCH1、PTS、QDPR和PCBDl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IonOneTouch^TM系统富集扩增子，最后应用IonTorrentPGM测序仪对目标区域进行检测。所得数据采用TorrentSuitev2．2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94．22Mb，约99．5％的读长位于目标区域。Ion Torrent 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，IonTorrent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。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, GCHI, PTS, QDPR, and PCBD1 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.

关 键 词：二代测序 高苯丙氨酸血症 代谢通路 

分 类 号：R589.3[医药卫生—内分泌]