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

• Published in: Disease models & mechanisms 2020-01, Vol.13 (1)
• Main Authors: Tarrasó, Guillermo, Real-Martinez, Alberto, Parés, Marta, Romero-Cortadellas, Lídia, Puigros, Laura, Moya, Laura, de Luna, Noemí, Brull, Astrid, Martín, Miguel Angel, Arenas, Joaquin, Lucia, Alejandro, Andreu, Antoni L, Barquinero, Jordi, Vissing, John, Krag, Thomas O, Pinós, Tomàs
• Format: Article
• Language: English
• Subjects: Biochemistry, Molecular Biology; Cell growth; cellular models; Human health and pathology; Life Sciences; Localization; mcardle disease; metabolic myopathy; Musculoskeletal system; Mutation; Plasmids; premature termination codon; Proteins; read-through; Sheep; White people
• ISSN: 1754-8403; 1754-8411
• DOI: 10.1242/dmm.043281

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- constructs presenting the p.R50X mutation (GFP- p.R50X and Ex1-GFP p.R50X). The second cellular model was based on the creation of HEK293T cell lines stably expressing the Ex1-GFP p.R50X construct. As these plasmids encode murine 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 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 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.