应用新一代半导体靶向测序技术检测枫糖尿症致病基因突变的研究

目的应用Ion Torrent半导体靶向测序技术检测1例枫糖尿症(MSUD)患儿的致病基因突变,明确其致病突变,并探讨该技术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,构建平均片段大小为300bp左右的文库,经乳液PCR及磁珠颗粒富集,最后采用318半导体测序芯片进行高通量测序,应用Ion Torrent Suite v3.0软件进行数据提取、序列比对及SNPs和Indels提取,再用dbSNP 137数据库过滤得到SNPs和Indels,可疑突变经Sanger法测序验证。结果检出患儿1个新发错义点突变并通过Sanger测序验证,突变是BCKDHB基因第6外显子的NM183050:c.586CT(p.His196Tyr)。结论 Ion Torrent半导体靶向测序技术可对复杂单基因遗传病进行快速、准确地基因诊断。

Study on detection of pathogenic gene mutations in maple syrup urine disease by new-generation semiconductor targeted sequencing

Objective To detect the pathogenic gene mutations of maple syrup urine disease(MSUD) by using the Ion Torrent new-generation semiconductor targeted sequencing for validating the pathogenic mutation and to in-vestigate the feasibility of application of this technique in the detection of complex monogenic diseases .Methods Pe-ripheral blood sample was collected from the MSUD patient ,genomic DNA was extracted ,the target gene fragments were amplified and enriched by multiple PCR for constructing the library with the mean fragment size of 300 bp , which was enriched by microemulsion PCR and magnetic bead ,finally the 318 semiconductor sequencing chip was a-dopted to conduct the high-throughput sequencing .The Ion Torrent Suite v3 .2 software was applied to conduct the data extract ,sequence alignment and SNPs and Indels extraction .Then the SNPs and Indels were filtered by the db-SNP l37 database .The suspected mutation was sequenced and verified by the Sanger method .Results One novel mis-sense mutation in this patient was detected and verified by the Sanger sequencing ,the mutation was NM183050 :c . 586C> T(p .His196Tgr) in exon 6 of the BCKDHB gene .Conclusion The Ion Torrent semiconductor targeted se-quencing can conduct the rapid and accurate gene diagnosis of complex single gene inheritance diseases .