Cell models for McArdle disease and aminoglycoside-induced read-through of a premature termination codon

Abstract

McArdle disease results from mutations in the gene encoding muscle glycogen phosphorylase (PYGM) protein and the two most common mutations are a premature termination codon (R50X) and a missense mutation (G205S). Myoblasts from patients cannot be used to create a cell model of McArdle disease because even normal myoblasts produce little or no PYGM protein in cell culture. We therefore created cell models by expressing wild-type or mutant (R50X or G205S) PYGM from cDNA integrated into the genome of Chinese hamster ovary cells. These cell lines enable the study of McArdle mutations in the absence of nonsense-mediated decay of mRNA transcripts. Although all cell lines produced stable mRNA, only wild-type produced detectable PYGM protein. Our data suggest that the G205S mutation affects PYGM by causing misfolding and accelerated protein turnover. Using the N-terminal region of PYGM containing the R50X mutation fused to green fluorescent protein, we were able to demonstrate both small amounts of truncated protein production and read-through of the R50X premature termination codon induced by the aminoglycoside, G418.