Northcott Neuroscience Laboratory, ANZAC Research Institute, Concord, NSW, Australia.
Sydney Medical School, University of Sydney, Camperdown, NSW, Australia.
Neurogenetics. 2021 Jul;22(3):149-160. doi: 10.1007/s10048-021-00650-9. Epub 2021 Jun 5.
The second most common form of Charcot-Marie-Tooth neuropathy (CMT), X-linked CMT type X1 (CMTX1), is caused by coding and non-coding mutations in the gap junction beta 1 (GJB1) gene. The non-coding GJB1 c.-103C > T mutation (NM_000166.5) has been reported to cause CMTX1 in multiple families. This study assessed the internal ribosomal entry site (IRES) activity previously reported for the rat Gjb1 P2 5' untranslated region (UTR). Using a bicistronic assay and transfecting RT4 Schwann cells, IRES activity of the human GJB1 P2 5' UTR was compared to the GJB1 P2 5' UTR containing either the c.-103C > T mutation or the non-pathogenic c.-102G > A variant. No differences in GJB1 P2 5' UTR IRES activity were observed between the negative control, the wild-type P2 5' UTR, the c.-103C > T 5' UTR or the c.-102G > A 5' UTR, irrespective of the GJB1 intron being present (p = .429 with intron, and p = .865 without). A theoretical c.-131A > G variant was predicted to result in the same RNA secondary structure as the GJB1 c.-103C > T P2 5' UTR. However, no significant difference was observed between expression from the wild-type GJB1 P2 5' UTR and the GJB1 c.-131A > G variant (p = .688). Deletion of the conserved region surrounding the c.-103C > T mutation (c.-108_-103del) resulted in significantly higher expression than the c.-103C > T mutation alone (p = .019), suggesting that the conserved c.-108_-103 region was not essential for translation. The reporter assays in this study do not recapitulate the previously reported GJB1 IRES activity and suggest an alternate pathogenic mechanism for the c.-103C > T CMTX1 non-coding mutation.
X 连锁遗传性运动感觉神经病（CMTX）是第二常见的夏科-马里-图思神经病（Charcot-Marie-Tooth neuropathy，CMT）类型，其病因是连接蛋白β 1（GJB1）基因突变，包括编码区和非编码区突变。非编码区 GJB1c.-103C>T 突变（NM_000166.5）已在多个家族中报道可导致 CMTX1。本研究评估了先前报道的大鼠 Gjb1 P2 5'UTR 的内部核糖体进入位点（IRES）活性。采用双顺反子测定法和 RT4 施万细胞转染，比较了人类 GJB1 P2 5'UTR 的 IRES 活性与包含 c.-103C>T 突变或非致病性 c.-102G>A 变体的 GJB1 P2 5'UTR。无论是否存在 GJB1 内含子，阴性对照、野生型 P2 5'UTR、c.-103C>T5'UTR 或 c.-102G>A5'UTR 之间的 GJB1 P2 5'UTR IRES 活性均无差异（存在内含子时为 p=0.429，不存在内含子时为 p=0.865）。理论上 c.-131A>G 变体预计会导致与 GJB1 c.-103C>T P2 5'UTR 相同的 RNA 二级结构。然而，野生型 GJB1 P2 5'UTR 和 GJB1 c.-131A>G 变体之间的表达无显著差异（p=0.688）。与单独的 c.-103C>T 突变相比，删除 c.-103C>T 突变周围的保守区域（c.-108_-103del）导致表达显著升高（p=0.019），这表明保守的 c.-108_-103 区域对于翻译并非必需。本研究中的报告基因测定未能重现先前报道的 GJB1 IRES 活性，并提示 c.-103C>T CMTX1 非编码突变的另一种致病机制。
