Lee Seung-Ah, Ogawa Megumu, Saito Yoshihiko, Shimazaki Rui, Awaya Tomonari, Hosokawa Motoyasu, Kurosawa Ryo, Ohara Hiroaki, Takeuchi Akihide, Hayashi Shinichiro, Goto Yu-Ichi, Hagiwara Masatoshi, Nishino Ichizo, Noguchi Satoru
Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan; Department of Neurology, Ewha Woman's University Mokdong Hospital, Ewha Woman's University College of Medicine, Seoul, Republic of Korea.
Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan.
Genet Med. 2025 Jul;27(7):101431. doi: 10.1016/j.gim.2025.101431. Epub 2025 Apr 8.
Collagen VI-related muscular dystrophies, characterized by proximal muscle weakness and joint contractures, are caused by pathogenic variants in the genes, COL6A1 to COL6A3. A monoallelic variant at the last nucleotide of a COL6A1 exon was initially classified as a missense variant but acted as a splicing variant, resulting in exon skipping. Here, we evaluated whether single-nucleotide variants at the 3'-ends of COL6A1 to COL6A3 exons cause aberrant splicing.
Ten relevant variants were identified in patients from our repository or public databases, and their muscle COL6A1 to COL6A3 transcripts were analyzed. The effects of the variants on splicing were also analyzed by minigene assay and SpliceAI in silico prediction.
Transcripts from muscles of individuals with suspected collagen VI-related phenotypes showed exon skipping (skipping rate >12%). Findings of minigene assay and in silico prediction experiments supported these findings. Two therapeutic approaches, splicing correction of pre-messenger RNA or gene silencing of mature messenger RNA were assessed. Among them, gene silencing using short interfering RNAs targeting the skipped transcripts proved to be effective in restoring collagen VI in cells containing the pathogenic variant.
Single-nucleotide variants at the 3'-ends of exons can lead to aberrant splicing, and allele-specific gene silencing targeting such variants is a promising therapeutic strategy.
以近端肌无力和关节挛缩为特征的VI型胶原蛋白相关肌营养不良症，由COL6A1至COL6A3基因中的致病变异引起。COL6A1外显子最后一个核苷酸处的单等位基因变异最初被归类为错义变异，但实际上是一个剪接变异，导致外显子跳跃。在此，我们评估了COL6A1至COL6A3外显子3'端的单核苷酸变异是否会导致异常剪接。
在我们的数据库或公共数据库中的患者中鉴定出10个相关变异，并分析了他们肌肉中的COL6A1至COL6A3转录本。还通过小基因分析和SpliceAI计算机模拟预测分析了这些变异对剪接的影响。
疑似VI型胶原蛋白相关表型个体肌肉中的转录本显示出外显子跳跃（跳跃率>12%）。小基因分析和计算机模拟预测实验的结果支持了这些发现。评估了两种治疗方法，即对信使前体RNA进行剪接校正或对成熟信使RNA进行基因沉默。其中，使用靶向跳跃转录本的短干扰RNA进行基因沉默被证明可有效恢复含有致病变异的细胞中的VI型胶原蛋白。
外显子3'端的单核苷酸变异可导致异常剪接，针对此类变异的等位基因特异性基因沉默是一种有前景的治疗策略。
