Expression of the muscle glycogen phosphorylase gene in patients with McArdle disease: the role of nonsense-mediated mRNA decay

Abstract

Nearly 35% of all mutations identified in the muscle glycogen phosphorylase gene (PYGM) in patients with McArdle disease result in premature termination codons (PTCs), particularly the p.R50X mutation. The latter accounts for more than 50% of the mutated alleles in most Caucasian patient populations. Mutations resulting in PTC could trigger the degradation of mRNA through a mechanism known as nonsense mediated decay (NMD). To investigate if NMD affects the levels of transcripts containing PYGM mutations, 28 Spanish patients with McArdle disease, harboring 17 different mutations with PTCs in 77% of their alleles, were studied. Transcripts levels of PYGM were measured and sequenced. We assessed that 92% of patients showed NMD. The most frequent mutation (p.R50X) elicited decay in all the genotypes tested. Other PTC producing mutations resulting in NMD were: p.L5VfsX22, p.Q73HfsX7, p.E125X, p.N134KfsX161, p.W388SfsX34, p.R491AfsX7, and p.D534VfsX5. Located in the last exon, the mutation p.E797VfsX19 was not affected by NMD. Missense mutations did not appear to be affected by NMD. In the cDNA sequences they appeared as homozygous, despite being heterozygous in the genomic DNA sequences. Exceptions to the rules governing NMD were found in the mutations p.A704 V and p.K754NfsX49.

Novel mutations in patients with McArdle disease by analysis of skeletal muscle mRNA

“…Thereafter, PYGM cDNA was amplified in two overlapping fragments by the high capacity cDNA archive kit (Applied Biosystems) as described elsewhere,9 using a fragment in the porphobilinogen deaminase gene (PBGD) as a control. The PYGM cDNA and PBGD fragments were run in 1.5% agarose gel.…”

“…In particular the p.R50X and most of frameshift mutations resulting in premature termination codons (PTCs) undergo mRNA nonsense mediated decay,9 and an exonic synonymous variant (p.K609K) leads to abnormal mRNA splicing species 10…”

Loss of exon identity is a common mechanism of human inherited disease

“…Of the three different disease-causing mutations we selected-OPA1, PYGM, and TFR2-there was no a priori evidence for any effect on splicing from in vitro analysis (Bruno et al 1999;Camaschella et al 2000;Schimpf et al 2008). However, aberrant splicing of OPA1, PYGM, and TFR2 is observed in patients carrying coding and non-coding mutations at other positions in these genes (Schimpf et al 2006;Biasiotto et al 2008;Nogales-Gadea et al 2008). Hexamers corresponding to exonic splicing silencers were obtained from the FAS-hex2 database and scored for loss or gain as described in A.…”

Clinical utility gene card for McArdle disease

“…The majority of variants are located within exons and all result in severely reduced or absent myophosphorylase enzyme activity, often as a result of nonsense-mediated decay [4].…”