1] Department of Neuromuscular Research, National Institute of Neuroscience, Translational Medical Center, National Center of Neurology and Psychiatry, Tokyo, Japan [2] Department of Clinical Development, Translational Medical Center, National Center of Neurology and Psychiatry, Tokyo, Japan.
Department of Neuromuscular Research, National Institute of Neuroscience, Translational Medical Center, National Center of Neurology and Psychiatry, Tokyo, Japan.
Mol Ther Nucleic Acids. 2014 Jun 24;3(6):e171. doi: 10.1038/mtna.2014.22.
Ullrich congenital muscular dystrophy (UCMD) is an inherited muscle disorder characterized clinically by muscle weakness, distal joint hyperlaxity, and proximal joint contractures. Sporadic and recessive mutations in the three collagen VI genes, COL6A1, COL6A2, and COL6A3, are reported to be causative. In the sporadic forms, a heterozygous point mutation causing glycine substitution in the triple helical domain has been identified in higher rate. In this study, we examined the efficacy of siRNAs, which target point mutation site, on specific knockdown toward transcripts from mutant allele and evaluated consequent cellular phenotype of UCMD fibroblasts. We evaluated the effect of siRNAs targeted to silence-specific COL6A1 alleles in UCMD fibroblasts, where simultaneous expression of both wild-type and mutant collagen VI resulted in defective collagen localization. Addition of mutant-specific siRNAs allowed normal extracellular localization of collagen VI surrounding fibroblasts, suggesting selective inhibition of mutant collagen VI. Targeting the single-nucleotide COL6A1 c.850G>A (p.G284R) mutation responsible a sporadic autosomal dominant form of UCMD can potently and selectively block expression of mutant collagen VI. These results suggest that allele-specific knockdown of the mutant mRNA can potentially be considered as a therapeutic procedure in UCMD due to COL6A1 point mutations.
先天性肌营养不良症（UCMD）是一种遗传性肌肉疾病，临床上表现为肌肉无力、远端关节过度松弛和近端关节挛缩。已报道三型胶原 VI 基因（COL6A1、COL6A2 和 COL6A3）的散发性和隐性突变是致病原因。在散发性形式中，已在更高的比例中鉴定出导致三螺旋结构域中甘氨酸取代的杂合点突变。在这项研究中，我们研究了靶向点突变的 siRNA 对突变等位基因转录物的特异性敲低的功效，并评估了 UCMD 成纤维细胞的相应细胞表型。我们评估了靶向沉默 UCMD 成纤维细胞中特定 COL6A1 等位基因的 siRNAs 的效果，其中野生型和突变型胶原 VI 的同时表达导致胶原定位缺陷。添加突变特异性 siRNAs 允许胶原 VI 正常定位于成纤维细胞周围，表明对突变型胶原 VI 的选择性抑制。针对导致散发性常染色体显性形式 UCMD 的单个核苷酸 COL6A1 c.850G>A（p.G284R）突变的靶向作用可以有效地和选择性地阻断突变型胶原 VI 的表达。这些结果表明，由于 COL6A1 点突变，针对突变型 mRNA 的等位基因特异性敲低可能被认为是 UCMD 的一种治疗方法。
