Revisiting the pathogenic mechanism of the GJB1 5\u2019 UTR c.-103C\u2009>\u2009T mutation causing CMTX1

Bianca R Grosz,Gonzalo Perez-Siles,John Svaren,Garth A Nicholson,Marina L Kennerson

doi:10.1007/s10048-021-00650-9

Abstract

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.

Highlights

Charcot-Marie-Tooth type X1 (CMTX1), the second most common hereditary motor and sensory peripheral neuropathy, is caused by mutations in the gap junction beta 1 (GJB1) gene
Bicistronic vectors were designed in which translation of the first reporter gene was 5’ cap-dependent, and translation of the second reporter gene (NanoLuc luciferase; NLuc) required the human GJB1 P2 5’ untranslated region (UTR) to function as an internal ribosomal entry site (IRES) and initiate translation through a 5’ cap-independent mechanism (Fig. 1)
Previous studies examining the GJB1 c.-103C > T mutation proposed that it abolished an IRES in the GJB1 P2 5’ UTR

Summary

Introduction

Charcot-Marie-Tooth type X1 (CMTX1), the second most common hereditary motor and sensory peripheral neuropathy, is caused by mutations in the gap junction beta 1 (GJB1) gene. Translation commonly utilises a 5’ cap-dependent mechanism in which ribosomal subunits assemble around the 5′-m7G cap end of mature mRNA. Neurogenetics (2021) 22:149–160 cap-independent translation occurs where regions of mRNA, known as an IRES, are able to recruit ribosomal subunits to initiate translation independently of the 5’ cap. It was suggested that the c.-103C > T mutation caused dysfunction of an IRES, as a luciferase reporter assay for the mutation showed that transcription and splicing were not affected, but the translation was abolished [5]. This study suggested that an IRES in the P2 5’ UTR could allow the ribosome to bypass two upstream open reading frames (uORFs) which slow the rate of translation by causing ribosomal stalling [12]. Whilst the vast majority of viral IRES elements are well validated and supported through multiple experimental approaches [13], cellular IRES elements remain contentious and many that have been reported have not been validated using further stringent assays [14,15,16,17,18,19]

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Conclusion