Laboratory of Biochemistry, Chulaborn Research Institute, Bangkok, Thailand.
Division of Medical Genetics, Department of Pediatrics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.
Biochem Biophys Res Commun. 2022 Dec 25;636(Pt 1):147-154. doi: 10.1016/j.bbrc.2022.10.081. Epub 2022 Oct 27.
Mucopolysaccharidosis type I Hurler syndrome (MPS IH) is a severe lysosomal storage disorder caused by alpha-l-iduronidase (IDUA) deficiency. Premature truncation mutations (PTC) are the most common (50%-70%) type of IDUA mutations and correlate with MPS IH. Nonsense suppression therapy is a therapeutic approach that aims to induce stop codon readthrough. The different ability of gentamicin to bind mutant mRNA in readthrough is determined by nucleotide sequence (PTC context: UGA > UAG > UAA) and inserted amino acid including the nucleotide position +4 of the PTC, as well as the mRNA secondary structure. We used COS-7 cells to investigate the functional characteristics of p.Q500X and p.R619X, IDUA variants and the effects of gentamicin in inducing stop codon readthrough of seven IDUA variants including p.Q500X, p.R619X, p.Q70X, p.E299X, p.W312X, p.Q380X, and p.W402X. Moreover, we performed prediction of RNA secondary structure using the online tool RNAfold. We found that cells treated with gentamicin showed significantly enhanced full-length IDUA expression and restored IDUA activity, in a dose-dependent manner, only in cells expressing cDNA with W312X, Q380X, W402X, and R619X. Among the readthrough-responsive variants, we observed UGA PTC in W312X, W402X and R619X; and UAG PTC with C at nucleotide +4 in Q380X. Changes of RNA secondary structure were noted only in mutants with readthrough-responsive variants including W312X, Q380X, W402X, and R619X. Additional preclinical studies of selected PTCs with potential readthrough, using drugs with less oto-nephrotoxicity, in patient's skin fibroblasts and animal model are necessary for the premise of personalized medicine.
黏多糖贮积症 I 型 Hurler 综合征（MPS IH）是一种由α-L-艾杜糖苷酸酶（IDUA）缺乏引起的严重溶酶体贮积症。提前终止密码突变（PTC）是最常见的（50%-70%）IDUA 突变类型，与 MPS IH 相关。无义抑制治疗是一种旨在诱导终止密码通读的治疗方法。不同的庆大霉素结合通读突变 mRNA 的能力取决于核苷酸序列（PTC 上下文：UGA>UAG>UAA）和插入的氨基酸，包括 PTC 的核苷酸位置+4，以及 mRNA 二级结构。我们使用 COS-7 细胞研究了 IDUA 变体 p.Q500X 和 p.R619X 的功能特征以及庆大霉素在诱导包括 p.Q500X、p.R619X、p.Q70X、p.E299X、p.W312X、p.Q380X 和 p.W402X 在内的七种 IDUA 变体终止密码通读中的作用。此外，我们使用在线工具 RNAfold 进行了 RNA 二级结构预测。我们发现，只有在表达 cDNA 为 W312X、Q380X、W402X 和 R619X 的细胞中，庆大霉素处理的细胞以剂量依赖性方式显著增强全长 IDUA 的表达并恢复 IDUA 活性。在通读反应变体中，我们观察到 W312X、W402X 和 R619X 中的 UGA PTC；以及 Q380X 中核苷酸+4 位的 C 引起的 UAG PTC。只有在具有通读反应变体的突变体中才观察到 RNA 二级结构的变化，包括 W312X、Q380X、W402X 和 R619X。在个性化医疗的前提下，有必要在患者皮肤成纤维细胞和动物模型中对具有潜在通读潜力的选定 PTC 进行药物筛选，这些药物的耳肾毒性较小。
