Targeted massively parallel sequencing and histological assessment of skeletal muscles for the molecular diagnosis of inherited muscle disorders
Targeted massively parallel sequencing and histological assessment of skeletal muscles for the molecular diagnosis of inherited muscle disorders
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DOI：
10.1136/jmedgenet-2016-104073
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发表时间：
2017-02-01
影响因子：
4
通讯作者：
Nishino, Ichizo
中科院分区：
文献类型：
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作者：
Nishikawa, Atsuko;Mitsuhashi, Satomi;Nishino, Ichizo
背景遗传性骨骼肌疾病是由150多个基因突变引起的遗传异质性疾病。在本研究中，我们开发了一种有用的方法来筛选与骨骼肌疾病相关的基因突变。方法我们建立了四个靶基因小组，每个小组覆盖了与下列肌肉疾病相关的基因的所有外显子和侧翼区域：(1)肌营养不良(MD)，(2)先天性肌病/先天性肌无力综合征，(3)代谢性肌病和(4)肌肉病蛋白聚集/边缘空泡。根据活检肌肉样本的临床和组织学分析结果，我们为每个患者分配了一个小组，并使用Ion PGM下一代测序仪进行高通量测序。我们还进行了蛋白质分析，以确认患有主要肌营养不良症的患者存在蛋白质缺陷。结果在纳入研究的33%(62/188)的患者中，我们发现了可能的致病基因突变。结论建立了一种诊断遗传性肌肉疾病的高通量测序技术。这项技术与组织学和蛋白质分析一起用于遗传性骨骼肌疾病患者的突变筛查可能是有用的，也是经济有效的。
Background Inherited skeletal muscle diseases are genetically heterogeneous diseases caused by mutations in more than 150 genes. This has made it challenging to establish a high-throughput screening method for identifying causative gene mutations in clinical practice.Aim In the present study, we developed a useful method for screening gene mutations associated with the pathogenesis of skeletal muscle diseases.Methods We established four target gene panels, each covering all exonic and flanking regions of genes involved in the pathogenesis of the following muscle diseases: (1) muscular dystrophy (MD), (2) congenital myopathy/congenital myasthenic syndrome, (3) metabolic myopathy and (4) myopathy with protein aggregations/rimmed vacuoles. We assigned one panel to each patient based on the results of clinical and histological analyses of biopsied muscle samples and performed high-throughput sequencing by using Ion PGM next-generation sequencer. We also performed protein analysis to confirm defective proteins in patients with major muscular dystrophies. Further, we performed muscle-derived cDNA analysis to identify splice-site mutations.Results We identified possible causative gene mutations in 33% of patients (62/188) included in this study. Our results showed that the MD panel was the most useful, with a diagnostic rate of 46.2%.Conclusions Thus, we developed a high-throughput sequencing technique for diagnosing inherited muscle diseases. The use of this technique along with histological and protein analyses may be useful and cost-effective for screening mutations in patients with inherited skeletal muscle diseases.