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Dis Model Mech. 2019 Dec 17. pii: dmm.043281. doi: 10.1242/dmm.043281. [Epub ahead of print]

Absence of p.R50X Pygm read-through in McArdle disease cellular models.

Author information

1
Mitochondrial and Neuromuscular Disorders Unit, Vall d'Hebron Institut de Recerca, Universitat Autònoma de Barcelona, Barcelona, Spain.
2
Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain.
3
Gene and Cell Therapy Unit, Vall d'Hebron Institut de Recerca, Universitat Autònoma de Barcelona, Barcelona, Spain.
4
Laboratori de Malalties Neuromusculars, Institut de Recerca Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, Spain.
5
Sorbonne Université, INSERM UMRS_974, Center of Research in Myology, 75013 Paris, France.
6
Mitochondrial and Neuromuscular Diseases Laboratory, 12 de Octubre Hospital Research Institute (i+12), Madrid, Spain.
7
Faculty of Sport Sciences, European University, Madrid, Spain.
8
Copenhagen Neuromuscular Center, Department of Neurology, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark.
9
Mitochondrial and Neuromuscular Disorders Unit, Vall d'Hebron Institut de Recerca, Universitat Autònoma de Barcelona, Barcelona, Spain tomas.pinos@vhir.org.

Abstract

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 the analysis of the efficacy of read-through agents with no concomitant nonsense-mediated decay interference. The third model consisted on skeletal muscle cultures derived from the McArdle mouse model (knock-in for the p.R50X mutation in the Pygm gene). We found no evidences of read-through at detectable levels in any of the models evaluated. Besides, we performed a literature search and compared the premature termination codon context sequences with reported positive and negative read-through induction and identified a potential role for nucleotide positions -9, -8, -3, -2, +13 and +14 (first nucleotide of the stop codon is assigned as +1). The Pygm p.R50X mutation presents TGA as a stop codon, G nucleotide at positions -1 and -9 and a C at -3, which potentially generate a good context for read-through induction, counteracted by the presence of a C at -2 and its absence at +4.

KEYWORDS:

Cellular models; McArdle disease; Metabolic myopathy; Premature termination codon; Read-through

PMID:
31848135
DOI:
10.1242/dmm.043281
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