CAJ | 학술논문

目的应用Ion Torrent PGM半导体测序仪和Ion AmpliSeqTMInherited Disease Panel对3例马凡综合征(Marfan syndrome,MFS)进行致病基因突变检测,明确其致病突变,并评价下一代半导体靶向测序诊断复杂单基因遗传病的效果.方法在知情同意的基础上采集3例MFS患者及1名正常志愿者外周血,提取基因组DNA,经多重PCR扩增富集目的基因片段.每个样本用特异性序列标签进行标记后,应用Ion One Touch系统进行模板制备、乳化PCR及磁珠颗粒富集；最后用318半导体测序芯片进行高通量测序.用Ion Torrent Suite 3.2软件进行序列比对及SNPs和Indels提取,再用dbSNP 137数据库过滤得到SNPs和indels,剩余的可疑突变经Sanger法测序验证.结果用一张318芯片得到855.80Mb的总数据量,4个样本的平均测序深度均达到100×以上,对目的区域的覆盖度在98％以上.数据经软件分析及数据库过滤后,在3例MFS患者中分别得到3个FBN1基因可疑突变,并经Sanger法测序验证,一个为已报道FBN1基因错义突变(p.E1811K),另外两个为新发现的突变,包括一个无义突变(p.E2264X),1个插入突变(p.L871FfsX23).结论在3例MFS患者中都成功检出FBN1基因致病突变,表明半导体靶向测序可对复杂单基因遗传病进行高效、准确的基因诊断.
목적 응용Ion Torrent PGM반도체측서의화Ion AmpliSeqTMInherited Disease Panel대3례마범종합정(Marfan syndrome,MFS)진행치병기인돌변검측,명학기치병돌변,병평개하일대반도체파향측서진단복잡단기인유전병적효과.방법 재지정동의적기출상채집3례MFS환자급1명정상지원자외주혈,제취기인조DNA,경다중PCR확증부집목적기인편단.매개양본용특이성서렬표첨진행표기후,응용Ion One Touch계통진행모판제비、유화PCR급자주과립부집；최후용318반도체측서심편진행고통량측서.용Ion Torrent Suite 3.2연건진행서렬비대급SNPs화Indels제취,재용dbSNP 137수거고과려득도SNPs화indels,잉여적가의돌변경Sanger법측서험증.결과 용일장318심편득도855.80Mb적총수거량,4개양본적평균측서심도균체도100×이상,대목적구역적복개도재98％이상.수거경연건분석급수거고과려후,재3례MFS환자중분별득도3개FBN1기인가의돌변,병경Sanger법측서험증,일개위이보도FBN1기인착의돌변(p.E1811K),령외량개위신발현적돌변,포괄일개무의돌변(p.E2264X),1개삽입돌변(p.L871FfsX23).결론 재3례MFS환자중도성공검출FBN1기인치병돌변,표명반도체파향측서가대복잡단기인유전병진행고효、준학적기인진단.
Objective To detect pathogenic mutations in Marfan syndrome (MFS) using an Ion Torrent Personal Genome Machine (PGM) and to validate the result of targeted next-generation semiconductor sequencing for the diagnosis of genetic disorders.Methods Peripheral blood samples were collected from three MFS patients and a normal control with informed consent.Genomic DNA was isolated by standard method and then subjected to targeted sequencing using an Ion AmpliseqTM Inherited Disease Panel.Three multiplex PCR reactions were carried out to amplify the coding exons of 328 genes including FBN1,TGFBR1 and TGFBR2.DNA fragments from different samples were ligated with barcoded sequencing adaptors.Template preparation and emulsion PCR,and Ion Sphere Particles enrichment were carried out using an Ion One Touch system.The ion sphere particles were sequenced on a 318 chip using the PGM platform.Data from the PGM runs were processed using an Ion Torrent Suite 3.2 software to generate sequence reads.After sequence alignment and extraction of SNPs and indels,all the variants were filtered against dbSNP137.DNA sequences were visualized with an Integrated Genomics Viewer.The most likely disease-causing variants were analyzed by Sanger sequencing.Results The PGM sequencing has yielded an output of 855.80 Mb,with a ＞100× median sequencing depth and a coverage of ＞ 98％ for the targeted regions in all the four samples.After data analysis and database filtering,one known missense mutation (p.E1811K) and two novel premature termination mutations (p.E2264X and p.L871FfsX23) in the FBN1 gene were identified in the three MFS patients.All mutations were verified by conventional Sanger sequencing.Conclusion Pathogenic FBN1 mutations have been identified in all patients with MFS,indicating that the targeted next-generation sequencing on the PGM sequencers can be applied for accurate and high-throughput testing of genetic disorders.