Diagnosis and genetic analysis of a case with glycogen storage disease type V caused by compound heterozygous mutations in the PYGM gene.

Glycogen storage disease type V is an autosomal recessive genetic disorder caused by muscle glycogen phosphorylase (PYGM) deficiency, which is characterized by exercise intolerance, second wind phenomena and high level of serum creatine kinase. In this study, we reported a Chinese young man with glycogen storage disease type V, with lower extremity weakness after exercise, increased creatine kinase, and slight fat infiltration in the posterior group of thigh muscle by magnetic resonance imaging (MRI). The proband had complex heterozygous PYGM disease-causing mutations, including c.308T>C (p.L103P) variant transmitted from the mother and c.260_261delCT (p.S87Ffs*23) from the father, of which the former was a novel PYGM mutation. This study enriched the PYGM pathogenic gene mutation spectrum, contributed to improve clinicians' understanding of glycogen storage disease type V and provided a reference for further genetic study of the disease.

High prevalence of paraspinal muscle involvement in adults with McArdle disease.

INTRODUCTION/AIMS: Very few studies analyzing the pattern of muscle involvement in magnetic resonance imaging (MRI) of patients with McArdle disease have been reported to date. We aimed to examine the pattern of muscle fat replacement in patients with McArdle disease. METHODS: We performed a retrospective study including all patients with genetically confirmed McArdle disease followed in our center from January 2010 to March 2021. Clinical data were collected from the medical record. Whole-body MRI was performed as part of the diagnostic evaluation. The distribution of muscle fat replacement and its severity were analyzed. RESULTS: Nine patients were included. Median age at onset was 7 y (range, 5-58) and median age at the time when MRI was performed was 57.3 y (range, 37.2-72.8). At physical examination, four patients had permanent weakness: in three the weakness was limited to paraspinal muscles, whereas in one the weakness involved the paraspinal and proximal upper limb muscles. Muscle MRI showed abnormalities in six of the seven studied patients. In all of them, fat replacement of paravertebral muscles was found. Other muscles frequently affected were the tongue in three, subscapularis in three, and long head of biceps femoris and semimembranosus in two. DISCUSSION: Our findings suggest that paraspinal muscle involvement is common in McArdle disease and support the need to include this disease in the differential diagnosis of the causes of paraspinal muscle weakness. Involvement of the tongue and subscapularis are also frequent in McArdle disease.

Muscle Glycogen Phosphorylase and Its Functional Partners in Health and Disease.

Glycogen phosphorylase (PG) is a key enzyme taking part in the first step of glycogenolysis. Muscle glycogen phosphorylase (PYGM) differs from other PG isoforms in expression pattern and biochemical properties. The main role of PYGM is providing sufficient energy for muscle contraction. However, it is expressed in tissues other than muscle, such as the brain, lymphoid tissues, and blood. PYGM is important not only in glycogen metabolism, but also in such diverse processes as the insulin and glucagon signaling pathway, insulin resistance, necroptosis, immune response, and phototransduction. PYGM is implicated in several pathological states, such as muscle glycogen phosphorylase deficiency (McArdle disease), schizophrenia, and cancer. Here we attempt to analyze the available data regarding the protein partners of PYGM to shed light on its possible interactions and functions. We also underline the potential for zebrafish to become a convenient and applicable model to study PYGM functions, especially because of its unique features that can complement data obtained from other approaches.

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

McArdle disease is an autosomal recessive disorder of muscle glycogen metabolism caused by pathogenic mutations in the PYGM gene, which encodes the skeletal muscle-specific isoform of glycogen phosphorylase. Clinical symptoms are mainly characterized by transient acute "crises" of early fatigue, myalgia and contractures, which can be accompanied by rhabdomyolysis. Owing to the difficulty of performing mechanistic studies in patients that often rely on invasive techniques, preclinical models have been used for decades, thereby contributing to gain insight into the pathophysiology and pathobiology of human diseases. In the present work, we describe the existing in vitro and in vivo preclinical models for McArdle disease and review the insights these models have provided. In addition, despite presenting some differences with the typical patient's phenotype, these models allow for a deep study of the different features of the disease while representing a necessary preclinical step to assess the efficacy and safety of possible treatments before they are tested in patients.

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

INTRODUCTION: 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. METHODS: 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. RESULTS: 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). DISCUSSION: 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.

Longitudinal case study and phenotypic multimodal characterization of McArdle disease-linked retinopathy: insight into pathomechanisms.

Background: We present a longitudinal clinical characterization of PYGM-linked pattern dystrophy in an adult male patient.Materials and Methods: A patient affected by McArdle disease (glycogen storage disease type V) and homozygous for the nonsense variant PYGM c.148C>T p.(Arg50*) underwent ophthalmic examinations over a 9-year-interval, including fundus photography, fundus autofluorescence, optical coherence tomography (OCT), OCT-angiography and electroretinography (ERG).Results: At age 52, the patient was asymptomatic but yellow flecks were first observed in the macula of both eyes. This yellow flecks at the posterior pole progressed towards a pattern-like dystrophy over a 5-year-period. By fundus autofluorescence imaging the appearance of new hyperautofluorescent flecks and the extension of existing ones was observed over time. Concomitantly, a slow progression of the size of atrophic areas was seen at the posterior pole. Scotopic ERGs were within normal limits, but photopic Flicker responses were decreased, indicating reduced cone function.Conclusions: This additional case of PYGM-linked pattern dystrophy further confirms retinopathy as a clinical phenotype associated with McArdle disease. PYGM expression pattern suggests a disease mechanism involving impaired glycogen metabolism both in the retinal pigment epithelium and in cone photoreceptors.

Single-centre experience on genotypic and phenotypic features of southern Brazilian patients with McArdle disease.

McArdle disease (MD) is a metabolic myopathy caused by deficiency of the myophosphorylase enzyme. The aim of our study was to analyse a series of MD patients in Brazil and the correlation between clinical findings, laboratory data, electromyography, muscle biopsy and genetic features. The PYGM gene was analysed by PCR/RLFP and Sanger sequencing. The sample included 12 patients, aged 18-57 years, from unrelated families. Exercise intolerance was present in all cases. Serum creatine kinase levels at rest were increased in all patients. Forearm ischaemic exercise testing in five patients revealed no increase in venous lactate. Needle electromyography presented 'myopathic pattern' in six patients. Muscle biopsy showed vacuolar myopathy in 10 patients and deficiency of myophosphorylase enzyme in all patients. The genetic analysis showed p.R50X as the most common mutation (allelic frequency: 56.25%), other known mutations (p.Y574X, p.G205S, p.W798R, IVS14 + 1G > A and IVS19-1G > A) and a new mutation (p.Asn168Lysfs*15) were also identified. Several features of the disorder were similar to the vast majority of patients worldwide. The genetic findings of this study revealed a range of mutations that are quite similar to the European cohort. The discovery of one novel mutation increases the genotypic heterogeneity of PYGM gene.

The effect of muscle glycogen phosphorylase (Pygm) knockdown on zebrafish morphology.

Muscle glycogen phosphorylase (PYGM) is a key enzyme in the first step of glycogenolysis. Mutation in the PYGM gene leads to autosomal recessive McArdle disease. Patients suffer from exercise intolerance with premature fatigue, muscle cramps and myalgia due to lack of available glucose in muscles. So far, no efficient treatment has been found. The zebrafish has many experimental advantages, and was successfully implemented as an animal model of human myopathies. Since zebrafish skeletal muscles share high similarity with human skeletal muscles, it is our animal of choice to investigate the impact of Pygm knockdown on skeletal muscle tissue. The two forms of the zebrafish enzyme, Pygma and Pygmb, share more than 80% amino acid sequence identity with human PYGM. We show that the Pygm level varies at both the mRNA and protein level in distinct stages of zebrafish development, which is correlated with glycogen level. The Pygm distribution in muscles varies from dispersed to highly organized at 72 hpf. The pygma and pygmb morpholino knockdown resulted in a reduced Pygm level in zebrafish morphants, which exhibited altered, disintegrated muscle structure and accumulation of glycogen granules in the subsarcolemmal region. Thus, lowering the Pygm level in zebrafish larvae leads to an elevated glycogen level and to morphological muscle changes mimicking the symptoms of human McArdle disease. The zebrafish model of this human disease might contribute to further understanding of its molecular mechanisms and to the development of appropriate treatment.

McArdle Disease: New Insights into Its Underlying Molecular Mechanisms.

McArdle disease, also known as glycogen storage disease type V (GSDV), is characterized by exercise intolerance, the second wind phenomenon, and high serum creatine kinase activity. Here, we recapitulate PYGM mutations in the population responsible for this disease. Traditionally, McArdle disease has been considered a metabolic myopathy caused by the lack of expression of the muscle isoform of the glycogen phosphorylase (PYGM). However, recent findings challenge this view, since it has been shown that PYGM is present in other tissues than the skeletal muscle. We review the latest studies about the molecular mechanism involved in glycogen phosphorylase activity regulation. Further, we summarize the expression and functional significance of PYGM in other tissues than skeletal muscle both in health and McArdle disease. Furthermore, we examine the different animal models that have served as the knowledge base for better understanding of McArdle disease. Finally, we give an overview of the latest state-of-the-art clinical trials currently being carried out and present an updated view of the current therapies.

Can hyperuricemia predict glycogen storage disease (McArdle's disease) in rheumatology practice? (Myogenic hyperuricemia).

Gout disease is an inflammatory arthritis that arises due to the accumulation of monosodium urate crystals (MSU) around the joints and in tissues. Clinical manifestation of metabolic diseases leading to secondary hyperuricemia most predominantly occurs in the form of gouty arthritis. Hyperuricemia and gout may develop during the course of glycogen storage diseases (GSD), particularly in GSD type I, which involves the liver. On the other hand, during the course of GSD type V (GSDV, McArdle's disease), which merely affects the muscle tissue due to the deficiency of the enzyme myophosphorylase, hyperuricemia and/or gout is rarely an expected symptom. These patients may mistakenly be diagnosed as having idiopathic hyperuricemia and associated gout, leading to the underlying secondary causes be overlooked and thus, diagnostic delays may occur. In this case report, we present a premenopausal female patient who experienced flare-ups of chronic arthritis while on disease-modifying antirheumatic drugs and intraarticular steroids due to a diagnosis of undifferentiated arthritis. The patient was initially suspected of having gouty arthritis because elevated concentrations of uric acid were incidentally detected, but then, a diagnosis of asymptomatic GSDV was made owing to elevated concentrations of muscle enzymes during colchicine use. Our aims were to remind rheumatologists of the phenomenon of "myogenic hyperuricemia" and to discuss the potential causes of hyperuricemia that develop during GSD along with the available literature.

Low survival rate and muscle fiber-dependent aging effects in the McArdle disease mouse model.

McArdle disease is an autosomal recessive disorder caused by the absence of the muscle glycogen phosphorylase, which leads to impairment of glycogen breakdown. The McArdle mouse, a model heavily affected by glycogen accumulation and exercise intolerance, was used to characterize disease progression at three different ages. The molecular and histopathological consequences of the disease were analyzed in five different hind-limb muscles (soleus, extensor digitorum longus, tibialis anterior, gastrocnemius and quadriceps) of young (8-week-old), adult (35-week-old) and old (70-week-old) mice. We found that McArdle mice have a high perinatal and post-weaning mortality. We also observed a progressive muscle degeneration, fibrosis and inflammation process that was not associated with an increase in muscle glycogen content during aging. Additionally, this progressive degeneration varied among muscle and fiber types. Finally, the lack of glycogen content increase was associated with the inactivation of glycogen synthase and not with compensatory expression of the Pygl and/or Pygb genes in mature muscle.

Muscle diffusion tensor imaging in glycogen storage disease V (McArdle disease).

PURPOSE: To evaluate differences in diffusion parameters in thigh muscles in patients with glycogen storage disease type V (McArdle disease) using muscle diffusion tensor imaging (mDTI) compared to healthy controls METHODS: In this prospective study, we evaluated thigh muscles from hip to knee of 10 McArdle patients (5 female, mean age 33.7 ± 14.4 years) and 10 healthy age- and gender-matched volunteers. MRI scans were performed at 3 T and comprised mDTI, T1-weighted and T2-weighted imaging between May 2015 and May 2017. Needle biopsy of the vastus lateralis muscle was performed in three McArdle patients. The muscle tissue was analyzed by using histochemical and enzyme-histochemical techniques for glycogen content and histopathological changes. Mean values of the eigenvalues (λ1-λ3), fractional anisotropy (FA), and mean diffusivity (MD) were obtained for the vastus lateralis, vastus medialis, rectus femoris, biceps femoris, semitendinosus, and semimembranosus and compared between groups using Student's t tests, as well as ANCOVA; significance level was set at p < 0.05. RESULTS: Needle biopsy showed intracellular glycogen accumulation in skeletal muscle fibers of three McArdle patients. Extracellular histopathological changes were not found. Muscle DTI analysis did not show statistically significant differences between patients and controls for any of the muscles. CONCLUSION: Despite intracellular glycogen accumulation in the three biopsy samples, mDTI parameters were not altered in McArdle patients compared to controls. We conclude that the currently used mDTI acquisition and processing lack the sensitivity to detect intracellular changes due to accumulated glycogen in this cohort of McArdle patients. KEY POINTS: • Despite intracellular glycogen accumulation in three examined biopsy samples, mDTI parameters were not altered in McArdle patients compared to controls. • In its current form, diffusion MR does not provide additional information in quantifying intracellular glycogen accumulations within skeletal muscle fibers in McArdle patients.

Establishment of a human iPSC line (IISHDOi001-A) from a patient with McArdle disease.

Human iPSC line IISHDOi001-A was generated from fibroblasts of a patient with McArdle disease harbouring the mutation, c.148C>T; p.Arg50Ter, in the PYGM gene. Reprogramming factors Oct3/4, Sox2, Klf4, and c-Myc were delivered using Sendai virus.

Metabolic profiles of exercise in patients with McArdle disease or mitochondrial myopathy.

McArdle disease and mitochondrial myopathy impair muscle oxidative phosphorylation (OXPHOS) by distinct mechanisms: the former by restricting oxidative substrate availability caused by blocked glycogen breakdown, the latter because of intrinsic respiratory chain defects. We applied metabolic profiling to systematically interrogate these disorders at rest, when muscle symptoms are typically minimal, and with exercise, when symptoms of premature fatigue and potential muscle injury are unmasked. At rest, patients with mitochondrial disease exhibit elevated lactate and reduced uridine; in McArdle disease purine nucleotide metabolites, including xanthine, hypoxanthine, and inosine are elevated. During exercise, glycolytic intermediates, TCA cycle intermediates, and pantothenate expand dramatically in both mitochondrial disease and control subjects. In contrast, in McArdle disease, these metabolites remain unchanged from rest; but urea cycle intermediates are increased, likely attributable to increased ammonia production as a result of exaggerated purine degradation. Our results establish skeletal muscle glycogen as the source of TCA cycle expansion that normally accompanies exercise and imply that impaired TCA cycle flux is a central mechanism of restricted oxidative capacity in this disorder. Finally, we report that resting levels of long-chain triacylglycerols in mitochondrial myopathy correlate with the severity of OXPHOS dysfunction, as indicated by the level of impaired O2 extraction from arterial blood during peak exercise. Our integrated analysis of exercise and metabolism provides unique insights into the biochemical basis of these muscle oxidative defects, with potential implications for their clinical management.

Muscle fiber type proportion and size is not altered in mcardle disease.

INTRODUCTION: McArdle disease is a metabolic myopathy that presents with exercise intolerance and episodic rhabdomyolysis. Excessive muscle recruitment has also been shown to be present during strenuous exercise, suggesting decreased power output. These findings could potentially be explained by either impaired contractility, decreased fiber size, or altered fiber type proportion. However, there is a paucity of data on the morphological features seen on muscle histology. METHODS: We examined muscle biopsies of patients with McArdle disease from a Spanish cohort and compared the findings with healthy controls. RESULTS: We found no significant difference in the fiber type proportion or mean fiber size between McArdle patients and controls in the biceps brachii or vastus lateralis muscles. CONCLUSIONS: No alterations in muscle fiber type proportion or size were found on muscle histology of patients with McArdle disease. Future research should focus on assessment of muscle fiber contractility to investigate the functional impairment. Muscle Nerve 55: 916-918, 2017.

Differential Muscle Involvement in Mice and Humans Affected by McArdle Disease.

McArdle disease (muscle glycogenosis type V) is caused by myophosphorylase deficiency, which leads to impaired glycogen breakdown. We investigated how myophosphorylase deficiency affects muscle physiology, morphology, and glucose metabolism in 20-week-old McArdle mice and compared the findings to those in McArdle disease patients. Muscle contractions in the McArdle mice were affected by structural degeneration due to glycogen accumulation, and glycolytic muscles fatigued prematurely, as occurs in the muscles of McArdle disease patients. Homozygous McArdle mice showed muscle fiber disarray, variations in fiber size, vacuoles, and some internal nuclei associated with cytosolic glycogen accumulation and ongoing regeneration; structural damage was seen only in a minority of human patients. Neither liver nor brain isoforms of glycogen phosphorylase were upregulated in muscles, thus providing no substitution for the missing muscle isoform. In the mice, the tibialis anterior (TA) muscles were invariably more damaged than the quadriceps muscles. This may relate to a 7-fold higher level of myophosphorylase in TA compared to quadriceps in wild-type mice and suggests higher glucose turnover in the TA. Thus, despite differences, the mouse model of McArdle disease shares fundamental physiological and clinical features with the human disease and could be used for studies of pathogenesis and development of therapies.

Taking advantage of an old concept, "illegitimate transcription", for a proposed novel method of genetic diagnosis of McArdle disease.

PURPOSE: McArdle disease is a metabolic disorder caused by pathogenic mutations in the PYGM gene. Timely diagnosis can sometimes be difficult with direct genomic analysis, which requires additional studies of cDNA from muscle transcripts. Although the "nonsense-mediated mRNA decay" (NMD) eliminates tissue-specific aberrant transcripts, there is some residual transcription of tissue-specific genes in virtually all cells, such as peripheral blood mononuclear cells (PBMCs). METHODS: We studied a subset of the main types of PYGM mutations (deletions, missense, nonsense, silent, or splicing mutations) in cDNA from easily accessible cells (PBMCs) in 12 McArdle patients. RESULTS: Analysis of cDNA from PBMCs allowed detection of all mutations. Importantly, the effects of mutations with unknown pathogenicity (silent and splicing mutations) were characterized in PBMCs. Because the NMD mechanism does not seem to operate in nonspecific cells, PBMCs were more suitable than muscle biopsies for detecting the pathogenicity of some PYGM mutations, notably the silent mutation c.645G>A (p.K215=), whose effect in the splicing of intron 6 was unnoticed in previous muscle transcriptomic studies. CONCLUSION: We propose considering the use of PBMCs for detecting mutations that are thought to cause McArdle disease, particularly for studying their actual pathogenicity.Genet Med 18 11, 1128-1135.

Xanthine Oxidase Pathway and Muscle Damage. Insights from McArdle Disease.

The intent of this article is to summarize current body of knowledge on the potential implication of the xanthine oxidase pathway (XO) on skeletal muscle damage. The possible involvement of the XO pathway in muscle damage is exemplified by the role of XO inhibitors (e.g., allopurinol) in attenuating muscle damage. Reliance on this pathway (as well as on the purine nucleotide cycle) could be exacerbated in conditions of low muscle glycogen availability. Thus, we also summarize current hypotheses on the etiology of both baseline and exertional muscle damage in McArdle disease, a condition caused by inherited deficiency of myophosphorylase. Because myophosphorylase catalyzes the first step of muscle glycogen breakdown, patients are unable to obtain energy from their muscle glycogen stores. Finally, we provide preliminary data from our laboratory on the potential implication of the XO pathway in the muscle damage that is commonly experienced by these patients.