Absence of p.R50X Pygm read-through in McArdle disease cellular models
Tarrasó, Guillermo (Hospital Universitari Vall d'Hebron. Institut de Recerca)
Real-Martinez, Alberto (Hospital Universitari Vall d'Hebron. Institut de Recerca)
Parés, Marta (Hospital Universitari Vall d'Hebron. Institut de Recerca)
Romero-Cortadellas, Lídia (Hospital Universitari Vall d'Hebron. Institut de Recerca)
Puigros, Laura (Hospital Universitari Vall d'Hebron. Institut de Recerca)
Moya Borrego, Laura (Hospital Universitari Vall d'Hebron. Institut de Recerca)
de Luna Salva, Noemí (Hospital Universitari Vall d'Hebron. Institut de Recerca)
Brull, Astrid (Sorbonne Université, INSERM UMRS_974, Center of Research in Myology)
Martín, Miguel Angel (Centro de Investigación Biomédica en Red de Enfermedades Raras)
Arenas, Joaquín (Hospital Universitari Vall d'Hebron. Institut de Recerca)
Lucia, Alejandro (European University)
Andreu Périz, Antoni Lluís (Hospital Universitari Vall d'Hebron. Institut de Recerca)
Barquinero, Jordi (Hospital Universitari Vall d'Hebron. Institut de Recerca)
Vissing, John (Copenhagen University Hospital Rigshospitalet)
Krag, Thomas (Copenhagen University Hospital Rigshospitalet)
Pinós Figueras, Tomàs (Hospital Universitari Vall d'Hebron. Institut de Recerca)
Universitat Autònoma de Barcelona

Data:	2020
Resum:	McArdle disease is an autosomal recessive disorder caused by the absence of muscle glycogen phosphorylase, which leads to blocked muscle glycogen breakdown. We used three different cellular models to evaluate the efficiency of different read-through agents (including amlexanox, Ataluren, RTC13 and G418) in McArdle disease. The first model consisted of HeLa cells transfected with two different GFP- PYGM constructs presenting the Pygm p. R50X mutation (GFP- PYGM p. R50X and PYGM Ex1-GFP p. R50X). The second cellular model was based on the creation of HEK293T cell lines stably expressing the PYGM Ex1-GFP p. R50X construct. As these plasmids encode murine Pygm cDNA without any intron sequence, their transfection in cells would allow for analysis of the efficacy of read-through agents with no concomitant nonsense-mediated decay interference. The third model consisted of skeletal muscle cultures derived from the McArdle mouse model (knock-in for the p. R50X mutation in the Pygm gene). We found no evidence of read-through at detectable levels in any of the models evaluated. We performed a literature search and compared the premature termination codon context sequences with reported positive and negative read-through induction, identifying a potential role for nucleotide positions −9, −8, −3, −2, +13 and +14 (the first nucleotide of the stop codon is assigned as +1). The Pygm p. R50X mutation presents TGA as a stop codon, G nucleotides at positions −1 and −9, and a C nucleotide at −3, which potentially generate a good context for read-through induction, counteracted by the presence of C at −2 and its absence at +4. Summary: Here, we evaluated the efficiency of different read-through agents in McArdle disease cell culture models, revealing that read-through compounds do not restore full-length muscle glycogen phosphorylase.
Ajuts:	Instituto de Salud Carlos III PI16/01492 Instituto de Salud Carlos III PI15/00172 Instituto de Salud Carlos III PI15/00558
Drets:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Llengua:	Anglès
Document:	Article ; recerca ; Versió publicada
Matèria:	McArdle disease ; Metabolic myopathy ; Premature termination codon ; Read-through ; Cellular models
Publicat a:	Disease Models & Mechanisms, Vol. 13 (january 2020) , ISSN 1754-8411

DOI: 10.1242/dmm.043281
PMID: 31848135

12 p, 5.8 MB

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