RNA-seq analysis, targeted long-read sequencing and in silico prediction to unravel pathogenic intronic events and complicated splicing abnormalities in dystrophinopathy
RNA-seq analysis, targeted long-read sequencing and in silico prediction to unravel pathogenic intronic events and complicated splicing abnormalities in dystrophinopathy
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DOI：
10.1007/s00439-022-02485-2
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发表时间：
2022-09-01
期刊：
影响因子：
5.3
通讯作者：
Nishino, Ichizo
中科院分区：
文献类型：
--
作者：
Okubo, Mariko;Noguchi, Satoru;Nishino, Ichizo
肌营养不良症是由DMD的改变引起的。大约1%的患者在基因上仍未被诊断，因为标准方法无法检测到内含子变异。在这里，我们结合实验室和计算机分析来鉴定遗传未确诊患者的致病基因组变异，并确定异常DMD转录物生成的调控机制。通过转录组测序分析了20例基因未确诊的肌营养不良症患者的DMD转录本，尽管肌肉活检显示肌营养不良蛋白缺乏，但未发现外显子变异。基因组测序捕获的内含子变异及其影响使用硅工具进行解释。靶向长读测序应用于疑似结构基因组异常的病例。20例中有19例检测到DMD转录本异常；内含子序列外显子化15例，外显子跳跃1例，转录物异常剪接和聚腺苷化2例，转录终止1例。内含子单核苷酸变异、染色体重排和核苷酸重复扩增是DMD基因转录改变的致病原因。我们的联合分析方法成功地鉴定了致病事件。检测DMD转录本中的致病机制可以为肌营养不良症患者的治疗选择提供信息。
Dystrophinopathy is caused by alterations in DMD. Approximately 1% of patients remain genetically undiagnosed, because intronic variations are not detected by standard methods. Here, we combined laboratory and in silico analyses to identify disease-causing genomic variants in genetically undiagnosed patients and determine the regulatory mechanisms underlying abnormal DMD transcript generation. DMD transcripts from 20 genetically undiagnosed dystrophinopathy patients in whom no exon variants were identified, despite dystrophin deficiency on muscle biopsy, were analyzed by transcriptome sequencing. Genome sequencing captured intronic variants and their effects were interpreted using in silico tools. Targeted long-read sequencing was applied in cases with suspected structural genomic abnormalities. Abnormal DMD transcripts were detected in 19 of 20 cases; Exonization of intronic sequences in 15 cases, exon skipping in one case, aberrantly spliced and polyadenylated transcripts in two cases and transcription termination in one case. Intronic single nucleotide variants, chromosomal rearrangements and nucleotide repeat expansion were identified in DMD gene as pathogenic causes of transcript alteration. Our combined analysis approach successfully identified pathogenic events. Detection of diseasing-causing mechanisms in DMD transcripts could inform the therapeutic options for patients with dystrophinopathy.