CAJ | 학술논문

目的应用Ion Torrent半导体测序仪及Ion AmpliSeqTM Inherited Disease Panel检测1例甲基丙二酸血症(methylmalonic acidemia,MMA)患儿的致病基因突变,探讨新一代Ion Torrent测序平台用于复杂单基因病检测的可行性.方法采集患儿外周血,提取基因组DNA,经多重PCR扩增富集目的基因片段,对样品加上序列标签及测序接头,制备成平均片段大小为200 bp左右的文库,然后应用Ion One Touch系统进行模板制备、乳化PCR及磁珠颗粒富集,318半导体测序芯片进行高通量测序,最后采用Ion Torrent Suite v3.0软件进行Ion Torrent数据提取、序列比对及MMA致病基因MMAA、MMAB、MMACHC和MUT的SNVs和Indels提取,经dbSNP 137数据库过滤后,可疑突变经Sanger法测序验证.结果检测到患儿MMAA基因编码区2个无义突变并通过Sanger测序验证,突变分别是第4外显子的c.586C＞T(p.R196X)和第6外显子的c.898C＞T(p.R300X),后者为未见报道的新突变.结论发现甲基丙二酸血症患儿MMAA基因双重杂合突变;半导体测序技术低成本、高通量、高灵敏度,适用于遗传性疾病的基因诊断.
목적 응용Ion Torrent반도체측서의급Ion AmpliSeqTM Inherited Disease Panel검측1례갑기병이산혈증(methylmalonic acidemia,MMA)환인적치병기인돌변,탐토신일대Ion Torrent측서평태용우복잡단기인병검측적가행성.방법 채집환인외주혈,제취기인조DNA,경다중PCR확증부집목적기인편단,대양품가상서렬표첨급측서접두,제비성평균편단대소위200 bp좌우적문고,연후응용Ion One Touch계통진행모판제비、유화PCR급자주과립부집,318반도체측서심편진행고통량측서,최후채용Ion Torrent Suite v3.0연건진행Ion Torrent수거제취、서렬비대급MMA치병기인MMAA、MMAB、MMACHC화MUT적SNVs화Indels제취,경dbSNP 137수거고과려후,가의돌변경Sanger법측서험증.결과 검측도환인MMAA기인편마구2개무의돌변병통과Sanger측서험증,돌변분별시제4외현자적c.586C＞T(p.R196X)화제6외현자적c.898C＞T(p.R300X),후자위미견보도적신돌변.결론 발현갑기병이산혈증환인MMAA기인쌍중잡합돌변;반도체측서기술저성본、고통량、고령민도,괄용우유전성질병적기인진단.
Objective To detect the pathogenic mutation in a patient with methylmalonic acidemia using IonTorrent Personal Genome Machine (PGM) and assess the feasibility of such technology for analyzing complex monogenic diseases.Methods Peripheral blood sample was collected from the patient.Genomic DNA was isolated using a standard method and subjected to targeted sequencing using an Ion AmpliseqTM Inherited Disease Panel.DNA fragment was ligated with a barcoded sequencing adaptor.Template preparation,emulsion PCR,and Ion Sphere Particles enrichment were carried out using the Ion One Touch system.Data from the PGM runs were processed using Ion Torrent Suite 3.2 software to generate sequence reads.All variants were filtered against dbSNPl37.DNA sequences were visualized with an Integrated Genomics Viewer.Results After data analysis and database filtering,a previously reported nonsense mutation,c.586C＞T (p.R196X),and a novel mutation c.898C＞T (p.R300X) were identified in the MMAA gene in this patient.Both mutations were verified by conventional Sanger sequencing.Conclusion Pathogenic MMAA mutations have been identified in a patient with methylmalonic acidemia.This new-generation targeted sequencing on the PGM sequencers can be applied for genetic diagnosis of hereditary diseases.