Ion Torrent PGMTM平台在儿童遗传性疾病诊断中应用初探

摘要

目的 在PCR产物定量混合构建文库方法的基础上,探讨Ion Torrent PGMTM平台二代测序应用于儿科常见遗传性疾病诊断的价值.方法 采集临床诊断的2例肌营养不良、1例胆汁酸合成障碍和1例甲基丙二酸血症患儿的外周血,提取基因组DNA,分别针对DMD基因、HSD3B7 基因、AMACR基因及MUT基因采用PCR反应进行编码区扩增,扩增产物定量混合制备成文库,在PGM上完成测序.使用NextGENe软件进行数据分析.同时采用Sanger测序方法,对上述基因进行全基因测序;肌营养不良病例采用多重连接探针扩增(MLPA )进行基因外显子拷贝数变异的检测.结果 例1 肌营养不良患儿DMD基因检测到6个变异,其中c.998C>A,p.333S>X为已知致病突变位点,4个为SNP(rs228406、rs1801187、rs1801188、rs1800280).PGM检测到1个假阳性,为6个连续的T后面插入了1个T.例2 肌营养不良患儿检测到DMD基因 g.2788933943-2790543577共5个外显子的缺失,MLPA检测结果与二代测序结果相符合.例3 胆汁酸合成障碍患儿HSD3B7 基因和AMACR基因共检测到7个变异,其中HSD3B7 基因复合杂合突变c.45-46delAG,FS;c.262G>G/C,p.88G>RG,5个为SNP(rs9938550、rs3195676、rs10941112、rs2278008、rs2287939).例4 甲基丙二酸血症患儿MUT基因检测到3个变异,1个为已知致病突变位点杂合突变c.728-729het-insTT,FS;2个为SNP( rs2229384、rs8589).PGM检测到2个假阳性.结论 基于PGMTM平台、采用PCR产物定量混合构建文库测序的方法,具有低成本、高通量、高灵敏度以及可灵活设计的特点,适合用于儿科临床常见遗传性疾病的检测.但由于二代测序技术可能带来的假阳性,对于检测到的变异需要Sanger直接测序法进一步验证.%Objective Our study aimed to assess the efficiency and reliability of clinical genetic diagnosis as a new approach for genetic diseases using the next-generation Ion Torrent PGM sequencing platform in pediatrics. Methods Four unrelated patients with clinical signs of Duchenne/Becker muscular dystrophy ( case 1 and case 2 ), bile acid synthesis defect ( case 3 ) and methylmalonic academia ( case 4 ) were recruited. DNA from the patients was screened using Ion Torrent PGM platform, and the results were compared with those obtained by dideoxy sequencing or multiplex ligation-dependent probe amplification analysis. Results Case 1: Six variants were identified by Ion Torrent PGM in the coding region of DMD gene, including 4 SNPs ( rs228406, rsl801187, rsl801188, rsl800280 ), one causal mutation ( c. 998C > A, p. 333S > X ) and one insertion ( c. 10127insT, FS ). All variants were confirmed by dideoxy sequencing except the insertion ( c. 10127insT ). Case 2: A five-exonic deletion was detected by Ion Torrent PGM in the DMD gene and confirmed by multiplex ligation-dependent probe amplification. Case 3: Seven variants were identified by Ion Torrent PGM? in the coding region of HSD3B7 and AMACR genes, including 2 compound heterozygous mutations ( c. 45-46delAG, FS; c. 262G > G/C, p. 88G > RG ), 5 SNPs ( rs9938550, rs3195676, rslO941112, rs2278008, rs2287939 ). All variants were confirmed by dideoxy sequencing. Case 4: Three variants were identified by Ion Torrent PGM? in the coding region of MUT gene, including one heterozygous mutation ( c. 729-730het-insTT, FS ), 2 SNPs ( rs2229384, rs8589 ). These variants were confirmed by dideoxy sequencing. Two variants ( c. 1595C > CT, p. 532R > RH; c. 1540G > GT, p. 514Q > KQ ) were detected by Ion Torrent only. Conclusions Here the implementation of Next-generation Ion Torrent Sequencing is discussed. This platform can be readily adopted by clinical molecular testing laboratories and represents a rapid, cost-effective, high throughput approach for screening common genetic diseases.