Okubo Mariko, Noguchi Satoru, Awaya Tomonari, Hosokawa Motoyasu, Tsukui Nobue, Ogawa Megumu, Hayashi Shinichiro, Komaki Hirofumi, Mori-Yoshimura Madoka, Oya Yasushi, Takahashi Yuji, Fukuyama Tetsuhiro, Funato Michinori, Hosokawa Yousuke, Kinoshita Satoru, Matsumura Tsuyoshi, Nakamura Sadao, Oshiro Azusa, Terashima Hiroshi, Nagasawa Tetsuro, Sato Tatsuharu, Shimada Yumi, Tokita Yasuko, Hagiwara Masatoshi, Ogata Katsuhisa, Nishino Ichizo
Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawa-Higashi, Kodaira, Tokyo, 187-8502, Japan.
Department of Anatomy and Developmental Biology, Graduate School of Medicine and Faculty of Medicine, The University of Kyoto, Kyoto, Japan.
Hum Genet. 2023 Jan;142(1):59-71. doi: 10.1007/s00439-022-02485-2. Epub 2022 Sep 1.
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.
肌营养不良蛋白病由DMD基因的改变引起。约1%的患者在基因层面仍无法确诊，因为标准方法无法检测到内含子变异。在此，我们结合实验室分析和计算机模拟分析，以识别基因未确诊患者中导致疾病的基因组变异，并确定异常DMD转录本产生的调控机制。对20例基因未确诊的肌营养不良蛋白病患者的DMD转录本进行了分析，这些患者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。通过转录组测序对这些转录本进行分析。基因组测序捕获内含子变异，并使用计算机模拟工具解释其影响。对于怀疑存在结构基因组异常的病例，应用靶向长读长测序。20例病例中有19例检测到异常DMD转录本；15例为内含子序列外显子化，1例为外显子跳跃，2例为异常剪接和多聚腺苷酸化转录本，1例为转录终止。在DMD基因中鉴定出内含子单核苷酸变异、染色体重排和核苷酸重复扩增是转录本改变的致病原因。我们的联合分析方法成功识别了致病事件。检测DMD转录本中的致病机制可为肌营养不良蛋白病患者的治疗选择提供依据。
