使用高通量测序平台对肌张力障碍患者进行基因筛查

发布时间：2018-03-19 08:15

本文选题：高通量测序　切入点：肌张力障碍　出处：《北京协和医学院》2016年硕士论文　论文类型：学位论文

【摘要】：背景肌张力障碍(dystonia)是一种运动障碍性疾病,通常以持续或偶发的肌肉收缩而引起的异常运动或姿势为特征。肌张力障碍的临床表现根据受累肌肉的部位、收缩强度和不同肌肉的组合而有不同表现。现阶段对肌张力障碍进行临床诊断多按照临床表现和病史依据医生的临床经验进行诊断,难以满足临床准确诊断的要求。而各种辅助检查也做不到准确快速。临床诊断中正急需一种快速准确的诊断方法目的应用Ion AmliSeq多重PCR定制序列捕获系统结合Ion Torrent PGM高通量测序平台检测肌张力障碍患者的目标基因突变,在找寻肌张力障碍致病基因新突变的基础上评估高通量测序技术在肌张力障碍临床诊断上应用的可能性。方法使用的个性化定制panel包括25个肌张力障碍明确致病基因及相关易感基因的全部外显子区域,应用Ion AmliSeq多重PCR定制序列捕获系统对病人的目的基因区域进行捕获,通过Ion Torrent PGM高通量测序平台进行靶向重测序,对得到的变异数据进行肌张力障碍候选基因突变分析。结果本研究共使用Ion Chip 318芯片6张,318V2芯片12张,平均测序深度为134±23,覆盖度为96.12%±0.71%,单张芯片最高产生数据达到900MB(IonChip 318V2)。经测序后数据分析和Sanger测序验证,在肌张力障碍的致病基因上共检测到15(60%,15/25)个突变,其中4种为已知变异,11种为之前未报道的新突变。结论应用Ion AmliSeq多重PCR定制序列捕获系统结合Ion Torrent PGM高通量测序平台可以完成对肌张力障碍的基因筛查工作。
[Abstract]:Background dystonia is a dystonia, usually characterized by abnormal movement or posture caused by continuous or occasional muscle contraction. The clinical manifestations of dystonia are based on the site of the affected muscle. The contractile intensity and the combination of different muscles have different manifestations. At this stage, the clinical diagnosis of dystonia is mostly based on the clinical manifestations and the history of the disease according to the doctor's clinical experience. It is difficult to meet the requirement of clinical accurate diagnosis, and all kinds of auxiliary examination can not be done accurately and quickly. In clinical diagnosis, a fast and accurate diagnostic method is urgently needed; to apply Ion AmliSeq multiple PCR custom sequence capture system combined with Ion Torrent. PGM high-throughput sequencing platform was used to detect target gene mutations in patients with dystonia. On the basis of searching for new mutations in dystonia gene, the possibility of high throughput sequencing in clinical diagnosis of dystonia was evaluated. The personalized panel used by the method includes 25 specific pathogenicity of dystonia. All exon regions of genes and associated susceptibility genes, The target gene region of patients was captured by Ion AmliSeq multiplex PCR customized sequence capture system, and targeted resequencing was carried out by Ion Torrent PGM high-throughput sequencing platform. The mutational data were analyzed by candidate gene mutation analysis of dystonia. Results in this study, Ion Chip 318 chip 6 pieces of 318V2 chip was used to analyze the mutation. The average depth of sequencing was 134 卤23, the coverage was 96.12% 卤0.71, and the maximum data produced by single chip was 900MB IonChip 318V2. After sequencing and Sanger sequencing, a total of 1560g / 1525) mutations were detected in the pathogenic gene of dystonia. Four of them are known mutations and 11 are new mutations that have not been reported before. Conclusion Gene screening for dystonia can be completed by using Ion AmliSeq multiplex PCR custom sequence capture system and Ion Torrent PGM high-throughput sequencing platform.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R746

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