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

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 analysis of the efficacy of readthrough 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.

Translational readthrough of ciliopathy genes BBS2 and ALMS1 restores protein, ciliogenesis and function in patient fibroblasts

“…Adenine is most preferred in the 5’ position for highest readthrough whereas cytosine is 3-6 times more effective in the 3’ position such as UGA-C in all TRID studies even though the optimal subsequent order of bases is TRID-dependent [84] , [85] , [86] , [87] . Interestingly, the base immediately following the stop codon exerts the strongest influence on readthrough efficiency due to the interaction of mRNA with release factors to terminate translation [ 84 , 88 ]. Computer modelling has suggested PTC124 has a preference for UGA readthrough due to a more favourable binding energy than UAG or UAA stop codons [89] .…”

Preclinical Research in McArdle Disease: A Review of Research Models and Therapeutic Strategies

“…With regard to gene therapy, although encouraging, positive results have been reported with the ovine and mouse model [ 74 , 75 ], more studies are still needed in order to induce actual increases in the expression of the transgene in the skeletal muscle to further reverse the clinical phenotype before this approach can be proposed for patients. Finally, RTA agents have also been evaluated in vitro both in transiently transfected cells and in skeletal muscle cultures derived from the McArdle mouse model, and the absence of read-through induction with the different tested compounds [ 73 , 91 ], has prevented further studies using in vivo models. This being said, because the number of potential RTA is constantly increasing, it cannot be completely ruled out that a new RTA might be useful for the treatment of McArdle disease.…”

“…Seven years later, RTA were again evaluated as potential therapeutic compounds for McArdle disease, and this time read-through activity was observed in non-muscle cell cultures transiently transfected with p.R50X-green fluorescent protein (GFP) constructs when treated with an aminoglycoside antibiotic, G418 [ 85 ]. Recently, in order to further clarify the potential benefit of these compounds as therapeutic agents, a wider battery of RTAs (including amlexanox, Ataluren, RTC13, RTC14 and G418, among others) were tested again in transiently transfected cells with p.R50*-GFP but also in cells stably expressing these constructs and in skeletal muscle cultures derived from the McArdle mouse model [ 73 ]. In this study, no read-through induction was observed in any of the cells with the different RTA tested.…”

“…In addition, as read-through efficiency is influenced by the stop codon composition and its surrounding sequence, the p.R50* Pygm context sequence was analyzed and compared with 30 different PTC sequences (including 15 nucleotides upstream and downstream from the PTC) with reported positive read-through and 29 PTC sequences with reported negative read-through induction. Compared with these analyzed sequences, the Pygm sequence of McArdle mice showed the TGA stop codon as well as a G nucleotide at position −9, and a C at position −3 (being the first nucleotide of the PTC where the position was defined as +1), which are commonly found in the PTC sequences with positive read-through induction, but also presented a C at −2, more frequently found in PTC sequences with negative read-through induction [ 73 ]. In addition, the Pygm sequence did show a C at +4, which has been described to promote PTC read-through [ 86 ].…”

Preclinical Research in Glycogen Storage Diseases: A Comprehensive Review of Current Animal Models

“…Different RTAs were tested in different McArdle disease cell cultures models: (1) transiently transfected cells with p.R50X plasmid constructs, (2) cells stably expressing p.R50X plasmid constructs and (3) skeletal muscle cells derived from the Pygm R50X/R50X mouse model. Even though it was a promising therapy, no read-through induction was observed in any of these cells cultures with any of the RTAs tested, but further studies are needed to provide a better understanding of these drugs [ 241 ].…”