Muscle Glycogen Phosphorylase Gene Research Articles

PYGM mRNA expression in McArdle disease: Demographic, clinical, morphological and genetic features.

McArdle disease presents clinical and genetic heterogeneity. There is no obvious association between genotype and phenotype. PYGM (muscle glycogen phosphorylase gene) mRNA expression and its association with clinical, morphological, and genetic aspects of the disease as a set have not been studied previously. We investigated genetic variation in PYGM considering the number of PTCs (premature termination codon) per sample and compared mRNA expression in skeletal muscle samples from 15 patients with McArdle disease and 16 controls to PTCs number and different aspects of the disease. The main variant found was c.148C>T (PTC-premature termination codon). Patients with two PTCs showed 42% mRNA expression compared to the control group. Most cases showed an inversely proportional relation among PTCs and mRNA expression. Association between mRNA expression and other aspects of the disease showed no statistically significant difference (p> 0.05). mRNA expression is not useful as a predictor factor for the prognosis and severity of the disease. Different mechanisms as post-transcriptional events, epigenetics factors or protein function may be involved.

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

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.

High frequency of missense mutations in glycogen storage disease type VI

Deficiency of liver glycogen phosphorylase in glycogen storage disease (GSD) type VI results in a reduced ability to mobilize glucose from glycogen. Six mutations of the PYGL gene, which encodes the liver isoform of the enzyme, have been identified in the literature. We have characterized eight patients from seven families with GSD type VI and identified 11 novel PYGL gene defects. The majority of the mutations were missense, resulting in the substitution of highly conserved residues. These could be grouped into those that were predicted to affect substrate binding (p.V456M, p.E673K, p.S675L, p.S675T), pyridoxal phosphate binding (p.R491C, p.K681T), or activation of glycogen phosphorylase (p.Q13P) or that had an unknown effect (p.N632I and p.D634H). Two mutations were predicted to result in null alleles, p.R399X and [c.1964_1969inv6;c.1969+1_+4delGTAC]. Only 7 of the 23 (30%) reported PYGL alleles carry nonsense, splice site or frameshift mutations compared to 68-80% of affected alleles of the highly homologous muscle glycogen phosphorylase gene, PYGM, that underlie McArdle disease. There was heterogeneity in the clinical symptoms observed in affected individuals. These varied from hepatomegaly and subclinical hypoglycaemia, to severe hepatomegaly with recurrent severe hypoglycaemia and postprandial lactic acidosis. We conclude that deficiency of liver glycogen phosphorylase is predominantly the result of missense mutations affecting enzyme activity. There are no common mutations and the severity of clinical symptoms varies significantly.

Variable presentation of the clinical phenotype of McArdle's disease in a kindred harbouring a novel compound genotype in the muscle glycogen phosphorylase gene

We report a Spanish family with muscle glycogen phosphorylase (PYGM) deficiency (McArdle's disease) harbouring a novel compound genotype (A659D/L586P). Four individuals who had the same genotype for PYGM, showed a wide variability in the presentation of the clinical phenotype, including one patient with a restrictive respiratory pattern, which is unusual in McArdle's disease. Moreover, these patients were studied for the insertion/deletion (I/D) trait in the angiotensin converting enzyme (ACE) which has been suggested to be a strong modulator of severity in McArdle's disease. Our results indicate no association of the I/D ACE trait in this family, suggesting that other factors would be more relevant in determining the severity of the clinical presentation.

Chicken ovalbumin upstream promoter-transcription factor I represses the transcriptional activity of the human muscle glycogen phosphorylase promoter in C2C12 cells

The responsiveness of the 1.13 kb proximal human muscle glycogen phosphorylase (MGP) gene promoter to the chicken ovalbumin upstream promoter-transcription factor (COUP-TF) repressor, known to be ablated during muscle cell differentiation, was examined. Constitutive expression of COUP-TFI repressed the activity of the promoter in C2C12 muscle cells and sequential deletion analysis mapped the sensitive region between nucleotides −362 and −185, which included a putative consensus COUP-TF binding half-site at −198/−193. Mutation of this site abolished transcriptional response to COUP-TFI of the −362 construct. A −209/−180 probe bound in vitro to COUP-TFI and to protein extracts from proliferating but not fusing myoblasts. Thus, COUP-TF may be involved in repression of the human MGP gene promoter at the myoblast stage.

Chicken ovalbumin upstream promoter-transcription factor I represses the transcriptional activity of the human muscle glycogen phosphorylase promoter in C2C12 cells

The responsiveness of the 1.13 kb proximal human muscle glycogen phosphorylase (MGP) gene promoter to the chicken ovalbumin upstream promoter-transcription factor (COUP-TF) repressor, known to be ablated during muscle cell differentiation, was examined. Constitutive expression of COUP-TFI repressed the activity of the promoter in C2C12 muscle cells and sequential deletion analysis mapped the sensitive region between nucleotides −362 and −185, which included a putative consensus COUP-TF binding half-site at −198/−193. Mutation of this site abolished transcriptional response to COUP-TFI of the −362 construct. A −209/−180 probe bound in vitro to COUP-TFI and to protein extracts from proliferating but not fusing myoblasts. Thus, COUP-TF may be involved in repression of the human MGP gene promoter at the myoblast stage.

Two novel mutations in the muscle glycogen phosphorylase gene in McArdle's disease.

We report on a Spanish family with myophosphorylase (EC 2.4.1.1) deficiency (McArdle's disease). The proband and his symptomatic sister were compound heterozygous for two novel mutations: a T-to-G transversion in exon 14 (c1722 T>G) that changes a tyrosine to a stop codon (Y573X), and a G-to-A transition in exon 15 (c1827 G>A) that disrupts the consensus signal at the donor splicing site. These findings further expand knowledge of the genetic bases of muscle glycogen phosphorylase deficiency.

Nerve-dependent factors regulating transcript levels of glycogen phosphorylase in skeletal muscle.

1. Muscle glycogen phosphorylase (MGP), the rate-limiting enzyme for glycogen metabolism in skeletal muscle, is neurally regulated. Steady-state transcript levels of the skeletal muscle isozyme of MGP decrease significantly following muscle denervation and after prolonged muscle inactivity with an intact motor nerve. These data suggest that muscle activity has an important influence on MGP gene expression. The evidence to this point, however, does not preclude the possibility that MGP is also regulated by motor neuron-derived trophic factors. This study attempts to distinguish between regulation provided by nerve-evoked muscle contractile activity and that provided by the delivery of neurotrophic factors. 2. Steady-state MGP transcript levels were determined in rat tibialis anterior (TA) muscles following controlled interventions aimed at separating the contributions of contractile activity from axonally transported trophic factors. The innervated TA was rendered inactive by daily epineural injections of tetrodotoxin (TTX) into the sciatic nerve. Sustained inhibition of axonal transport was accomplished by applying one of three different concentrations of the antimicrotubule agent, vinblastine (VIN), to the proximal sciatic nerve for 1 hr. The axonal transport of acetylcholinesterase (AChE) was assessed 7, 14, and 28 days after the single application of VIN. 3. MGP transcript levels normalized to total RNA were reduced by 67% in rat TA, 7 days after nerve section. Daily injection of 2 microg TTX into the sciatic nerve for 7 days eliminated muscle contractile activity and reduced MGP transcript levels by 60%. 4. A single, 1-hr application of 0.10% (w/v) VIN to the sciatic nerve reduced axonal transport but did not alter MGP transcript levels in the associated TA, 7 days after treatment. Application of 0.10% VIN to the sciatic nerve also did not affect IA sensory or motor nerve conduction velocities or TA contractile function. 5. Treatment of the sciatic nerve with 0.40% (w/v) VIN for 1 hr reduced axonal transport and decreased MGP transcript levels by 50% within 7 days, but also reduced sensory and motor nerve conduction velocities and depressed TA contractile function. 6. Myogenin, a member of a family of regulatory factors shown to influence the transcription of many muscle genes, including MGP, was used as a molecular marker for muscle inactivity. Myogenin transcript levels were increased following denervation and after treatment with TTX or 0.40% VIN but not after treatment with 0.10% VIN. 7. The results suggest that MGP transcript levels in TA are regulated predominantly by muscle activity, rather than by the delivery of neurotrophic factors. Intrinsic myogenic factors, however, also play a role in MGP expression, since denervation did not reduce MGP transcript levels below 30% of control TA. The dominant influence of activity in the regulation of MGP contrasts with the proposed regulation of oxidative enzyme expression, which appears to depend on both activity and trophic factor influences.

McArdle's disease: a rare frameshift mutation in exon 1 of the muscle glycogen phosphorylase gene

We have previously discovered a common nonsense mutation in exon 1 of the myophosphorylase gene in patients with McArdle's disease, but this failed to explain all cases. We now report a second mutation (G → TT) in one patient, also in exon 1. This mutation causes a shift in the reading frame which results in the replacement of Val 15 by Phe. A further 10 amino acids of mis-sense protein are synthesised before a stop codon is reached.

Assignment of the Human FAU Gene to a Subregion of Chromosome 11q13

The FAU gene is the cellular homologue of the fox sequence in the Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV). FAU (for FBR-MuSV a ssociated ubiquitously expressed gene) encodes the ribosomal protein S30 fused to a ubiquitin-like protein. A cosmid clone containing the human FAU gene was used for fluorescence in situ hybridization to metaphase chromosomes. The obtained localization to 11q13 was confirmed by hybridization against a panel of somatic cell hybrids containing different parts of chromosome 11 on a hamster background. FAU was then further mapped, both on a panel of radiation-reduced somatic cell hybrids designed to carry different parts of the 11q13 region and by pulsed-field gel electrophoresis. This fine mapping assigned FAU close to the skeletal muscle glycogen phosphorylase gene (PYGM), in a region that contains several oncogenes as well as the putative tumor suppressor genes MEN1 and ST3.

McArdle's disease: a nonsense mutation in exon 1 of the muscle glycogen phosphorylase gene explains some but not all cases.

McArdle's disease is an inherited disease that results from a lack of functional muscle glycogen phosphorylase. We report here the identification of a C to T transition in exon 1 of the muscle phosphorylase gene found in all patients studied. This base pair mutation results in the substitution of a stop codon (TGA) for the codon (CGA) for Arg49 in the mature protein, and generates a novel restriction site for NlaIII. Of sixteen McArdle's patients, ten are homozygous for this mutation; the remainder are heterozygous. Additional unidentified mutations must lead to the McArdle's phenotype in the latter group of patients.

Adenovirus-mediated transfer of the muscle glycogen phosphorylase gene into hepatocytes confers altered regulation of glycogen metabolism.

The muscle isozyme of glycogen phosphorylase is potently activated by the allosteric ligand AMP, whereas the liver isozyme is not. In this study we have investigated the metabolic impact of expression of muscle phosphorylase in liver cells. To this end, we constructed a replication-defective, recombinant adenovirus containing the muscle glycogen phosphorylase cDNA (termed AdCMV-MGP) and used this system to infect hepatocytes in culture. AMP-activatable glycogen phosphorylase activity was increased 46-fold 6 days after infection of primary liver cells with AdCMV-MGP. Despite large increases in phosphorylase activity, glycogen levels were only slightly reduced in AdCMV-MGP-infected liver cells compared to uninfected cells or cells infected with wild-type adenovirus. The lack of correlation of phosphorylase activity and glycogen content suggests that the liver cell environment can inhibit the muscle phosphorylase isozyme. This inhibition can be overcome, however, by addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP), which increases AMP levels by 30-fold and causes a much larger decrease in glycogen levels in AdCMV-MGP-infected cells than in uninfected or wild-type adenovirus-infected controls. CCCP treatment also caused a preferential decrease in glycogen content relative to glucagon treatment in AdCMV-MGP-infected hepatocytes (74% versus 11%, respectively), even though the two drugs caused equal increases in phosphorylase a activity. Introduction of muscle phosphorylase into hepatocytes therefore confers a capacity for glycogenolytic response to effectors that is not provided by the endogenous liver phosphorylase isozyme. The remarkable efficiency of adenovirus-mediated gene transfer into primary hepatocytes and the demonstration of altered regulation of glycogen metabolism as a consequence of expression of a non-cognate phosphorylase isozyme may have implications for gene therapy of glycogen storage diseases.

Identification of a tissue-specific regulatory element within the human muscle glycogen phosphorylase gene.

Northern blot analysis showed that human muscle glycogen phosphorylase is developmentally regulated in human adult and fetal skeletal muscle. Furthermore, muscle phosphorylase mRNA expression is temporally regulated in the C2C12 mouse muscle cell line. To define regulatory elements that control expression of the human muscle glycogen phosphorylase gene, the structure of the 5' end of the gene was determined, and 1,129 base pairs of the 5'-flanking region were subcloned and sequenced. Primer extension, RNase protection, and S1 nuclease protection experiments mapped the transcription start site to 76 base pairs upstream from the starting methionine. Sequential deletions of the 5'-flanking region were tested for the ability to activate chloramphenicol acetyltransferase (CAT) expression in fused or proliferating C2C12 cells. Inclusion of the 43 base pairs between -612 and -570 led to a 9-fold increase in CAT activity in fused myotubes. No increase was observed in proliferating myoblasts. This region contains a 10-base pair sequence, CTCCAAAAGG, at -592, which is also repeated at -252. Mutation of the sequence at -592 results in a 50% decrease in CAT activity compared with the amount of CAT activity observed with the normal or a control mutation. These results indicate that a regulatory element is found within -612 to -570 of the 5'-flanking DNA of the human muscle glycogen phosphorylase gene which activates transcription only in differentiated muscle cells.

High-Resolution Chromosome Sorting and DNA Spot-Blot Analysis Assign McArdle's Syndrome to Chromosome 11

A rapid gene-mapping system uses a high-resolution, dual-laser sorter to identify genes from separate human chromosomes prepared with a new stain combination. This system was used to sort 21 unique chromosome types onto nitrocellulose filter papers. Several labeled gene probes hybridized to the sorted chromosomal DNA types predicted by their previous chromosome assignments. The skeletal muscle glycogen phosphorylase gene was then mapped to a portion of chromosome 11 by spot blotting normal and translocated chromosomes.