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Once experimentally prohibitive, structural studies of individual missense variants in proteins are increasingly feasible, and can provide a new level of insight into human genetic disease. One example of this is the recently identified inborn error of metabolism known as phosphoglucomutase-1 (PGM1) deficiency. Just as different variants of a protein can produce different patient phenotypes, they may also produce distinct biochemical phenotypes, affecting properties such as catalytic activity, protein stability, or 3D structure/dynamics. Experimental studies of missense variants, and particularly structural characterization, can reveal details of the underlying biochemical pathomechanisms of missense variants. Here, we review four examples of enzyme dysfunction observed in disease-related variants of PGM1. These studies are based on 11 crystal structures of wild-type (WT) and mutant enzymes, and multiple biochemical assays. Lessons learned include the value of comparing mutant and WT structures, synergy between structural and biochemical studies, and the rich understanding of molecular pathomechanism provided by experimental characterization relative to the use of predictive algorithms. We further note functional insights into the WT enzyme that can be gained from the study of pathogenic variants., Competing Interests: Declaration of competing interest The author declares no conflict of interest., (Copyright © 2020. Published by Elsevier B.V.)

Phosphoglucomutase 1 (PGM1) deficiency is a rare genetic disorder that affects glycogen metabolism, glycolysis, and protein glycosylation. Previously known as GSD XIV, it was recently reclassified as a congenital disorder of glycosylation, PGM1-CDG. PGM1-CDG usually manifests as a multisystem disease. Most patients present as infants with cleft palate, liver function abnormalities and hypoglycemia, but some patients present in adulthood with isolated muscle involvement. Some patients develop life-threatening cardiomyopathy. Unlike most other CDG, PGM1-CDG has an effective treatment option, d-galactose, which has been shown to improve many of the patients' symptoms. Therefore, early diagnosis and initiation of treatment for PGM1-CDG patients are crucial decisions. In this article, our group of international experts suggests diagnostic, follow-up, and management guidelines for PGM1-CDG. These guidelines are based on the best available evidence-based data and experts' opinions aiming to provide a practical resource for health care providers to facilitate successful diagnosis and optimal management of PGM1-CDG patients., (© 2020 SSIEM.)

GSD are a group of disorders characterized by a defect in gene expression of specific enzymes involved in glycogen breakdown or synthesis, commonly resulting in the accumulation of glycogen in various tissues (primarily the liver and skeletal muscle). Several different GSD animal models have been found to naturally present spontaneous mutations and others have been developed and characterized in order to further understand the physiopathology of these diseases and as a useful tool to evaluate potential therapeutic strategies. In the present work we have reviewed a total of 42 different animal models of GSD, including 26 genetically modified mouse models, 15 naturally occurring models (encompassing quails, cats, dogs, sheep, cattle and horses), and one genetically modified zebrafish model. To our knowledge, this is the most complete list of GSD animal models ever reviewed. Importantly, when all these animal models are analyzed together, we can observe some common traits, as well as model specific differences, that would be overlooked if each model was only studied in the context of a given GSD.

Phosphoglucomutase 1 deficiency is a congenital disorder of glycosylation (CDG) with multiorgan involvement affecting carbohydrate metabolism, N-glycosylation and energy production. The metabolic management consists of dietary D-galactose supplementation that ameliorates hypoglycemia, hepatic dysfunction, endocrine anomalies and growth delay. Previous studies suggest that D-galactose administration in juvenile patients leads to more significant and long-lasting effects, stressing the urge of neonatal diagnosis (0-6 months of age). Here, we detail the early clinical presentation of PGM1-CDG in eleven infantile patients, and applied the modified Beutler test for screening of PGM1-CDG in neonatal dried blood spots (DBSs). All eleven infants presented episodic hypoglycemia and elevated transaminases, along with cleft palate and growth delay (10/11), muscle involvement (8/11), neurologic involvement (5/11), cardiac defects (2/11). Standard dietary measures for suspected lactose intolerance in four patients prior to diagnosis led to worsening of hypoglycemia, hepatic failure and recurrent diarrhea, which resolved upon D-galactose supplementation. To investigate possible differences in early vs. late clinical presentation, we performed the first systematic literature review for PGM1-CDG, which highlighted respiratory and gastrointestinal symptoms as significantly more diagnosed in neonatal age. The modified Butler-test successfully identified PGM1-CDG in DBSs from seven patients, including for the first time Guthrie cards from newborn screening, confirming the possibility of future inclusion of PGM1-CDG in neonatal screening programs. In conclusion, severe infantile morbidity of PGM1-CDG due to delayed diagnosis could be prevented by raising awareness on its early presentation and by inclusion in newborn screening programs, enabling early treatments and galactose-based metabolic management., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to disclose., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Although the discovery of glycogen in the liver, attributed to Claude Bernard, happened more than 160 years ago, the mechanism involved in the initiation of glucose polymerization remained unknown. The discovery of glycogenin at the core of glycogen's structure and the initiation of its glucopolymerization is among one of the most exciting and relatively recent findings in Biochemistry. This review focuses on the initial steps leading to the seminal discoveries of proteoglycogen and glycogenin at the beginning of the 1980s, which paved the way for subsequent foundational breakthroughs that propelled forward this new research field. We also explore the current, as well as potential, impact this research field is having on human health and disease from the perspective of glycogen storage diseases. Important new questions arising from recent studies, their links to basic mechanisms involved in the de novo glycogen biogenesis, and the pervading presence of glycogenin across the evolutionary scale, fueled by high throughput -omics technologies, are also addressed., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Background The phosphoglucomutase 1 (PGM1) enzyme plays a central role in glucose homeostasis by catalyzing the inter-conversion of glucose 1-phosphate and glucose 6-phosphate. Recently, PGM1 deficiency has been recognized as a cause of the congenital disorders of glycosylation (CDGs). Methods Two Chinese Han pediatric patients with recurrent hypoglycemia, hepatopathy and growth retardation are described in this study. Targeted gene sequencing (TGS) was performed to screen for causal genetic variants in the genome of the patients and their parents to determine the genetic basis of the phenotype. Results DNA sequencing identified three variations of the PGM1 gene (NM_002633.2). Patient 1 had a novel homozygous mutation (c.119delT, p.Ile40Thrfs*28). In patient 2, we found a compound heterozygous mutation of c.1172G>T(p.Gly391Val) (novel) and c.1507C>T(p.Arg503*) (known pathogenic). Conclusions This report deepens our understanding of the clinical features of PGM1 mutation. The early molecular genetic analysis and multisystem assessment were here found to be essential to the diagnosis of PGM1-CDG and the provision of timely and proper treatment.

Background: PHKG2 gene mutation can lead to liver phosphorylase kinase (PhK) deficiency, which is related to glycogen storage disease type IX (GSD IX). GSD IXc due to PHKG2 mutation is the second most common GSD IX., Methods: We identified a novel mutation (c.553C>T, p.Arg185X) in PHKG2 in a Chinese family and verified it by next-generation and Sanger sequencing. The mutation spectrum of the PHKG2 gene was summarized based on 25 GSD IXc patients with PHKG2 mutations., Results: We found that missense mutation (39%) was the most common type of mutation, followed by nonsense mutation (23%). Mutations were more prevalent in Asian (12/25) and European (9/25) populations than in populations from elsewhere. The exons had more sites of mutation than the introns, and exons 3 and 6 were the most frequent sites of mutations., Conclusions: This study expands our knowledge of the PHKG2 gene mutation spectrum, providing a molecular basis for GSD IXc.

Glycogen storage disorders (GSDs) are caused by excessive accumulation of glycogen. Some GSDs [adult polyglucosan (PG) body disease (APBD), and Tarui and Lafora diseases] are caused by intracellular accumulation of insoluble inclusions, called PG bodies (PBs), which are chiefly composed of malconstructed glycogen. We developed an APBD patient skin fibroblast cell-based assay for PB identification, where the bodies are identified as amylase-resistant periodic acid-Schiff's-stained structures, and quantified. We screened the DIVERSet CL 10 084 compound library using this assay in high-throughput format and discovered 11 dose-dependent and 8 non-dose-dependent PB-reducing hits. Approximately 70% of the hits appear to act through reducing glycogen synthase (GS) activity, which can elongate glycogen chains and presumably promote PB generation. Some of these GS inhibiting hits were also computationally predicted to be similar to drugs interacting with the GS activator protein phosphatase 1. Our work paves the way to discovering medications for the treatment of PB-involving GSD, which are extremely severe or fatal disorders., (© 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Glycogen phosphorylase (GP) is the key enzyme that regulates glycogen mobilization in cells. GP is a complex allosteric enzyme that comprises a family of three isozymes: muscle GP (mGP), liver GP (lGP), and brain GP (bGP). Although the three isozymes display high similarity and catalyze the same reaction, they differ in their sensitivity to the allosteric activator adenosine monophosphate (AMP). Moreover, inactivating mutations in mGP and lGP have been known to be associated with glycogen storage diseases (McArdle and Hers disease, respectively). The determination, decades ago, of the structure of mGP and lGP have allowed to better understand the allosteric regulation of these two isoforms and the development of specific inhibitors. Despite its important role in brain glycogen metabolism, the structure of the brain GP had remained elusive. Here, we provide an overview of the human brain GP structure and its relationship with the two other members of this key family of the metabolic enzymes. We also summarize how this structure provides valuable information to understand the regulation of bGP and to design specific ligands of potential pharmacological interest., (© 2016 Federation of European Biochemical Societies.)

Background: Equine type 1 polysaccharide storage myopathy (PSSM1) is associated with a missense mutation (R309H) in the glycogen synthase (GYS1) gene, enhanced glycogen synthase (GS) activity and excessive glycogen and amylopectate inclusions in muscle., Methods: Equine muscle biochemical and recombinant enzyme kinetic assays in vitro and homology modelling in silico, were used to investigate the hypothesis that higher GS activity in affected horse muscle is caused by higher GS expression, dysregulation, or constitutive activation via a conformational change., Results: PSSM1-affected horse muscle had significantly higher glycogen content than control horse muscle despite no difference in GS expression. GS activity was significantly higher in muscle from homozygous mutants than from heterozygote and control horses, in the absence and presence of the allosteric regulator, glucose 6 phosphate (G6P). Muscle from homozygous mutant horses also had significantly increased GS phosphorylation at sites 2+2a and significantly higher AMPKα1 (an upstream kinase) expression than controls, likely reflecting a physiological attempt to reduce GS enzyme activity. Recombinant mutant GS was highly active with a considerably lower K m for UDP-glucose, in the presence and absence of G6P, when compared to wild type GS, and despite its phosphorylation., Conclusions: Elevated activity of the mutant enzyme is associated with ineffective regulation via phosphorylation rendering it constitutively active. Modelling suggested that the mutation disrupts a salt bridge that normally stabilises the basal state, shifting the equilibrium to the enzyme's active state., General Significance: This study explains the gain of function pathogenesis in this highly prevalent polyglucosan myopathy., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Objective and Importance: Phosphoglucomutase 1 (PGM1) deficiency, first described as a glycogenosis (type XIV) is also a congenital disorder of glycosylation (CDG). We want to illustrate the wide clinical spectrum of PGM1 deficiency and in particular the associated disturbance in glucose metabolism and the endocrine dysfunction. Treatment with d-galactose is experimental., Case Presentation: PGM1 deficiency was diagnosed in an 8-year-old boy, who was referred because of an unexplained complex syndrome, including recurrent hypoglycaemia and low IGF-1 mediated growth failure., Conclusion: The timely diagnosis of this disorder is particularly important, because d-galactose treatment can improve the latter symptoms.

Objective: To define phenotypic groups and identify predictors of disease severity in patients with phosphoglucomutase-1 deficiency (PGM1-CDG)., Study Design: We evaluated 27 patients with PGM1-CDG who were divided into 3 phenotypic groups, and group assignment was validated by a scoring system, the Tulane PGM1-CDG Rating Scale (TPCRS). This scale evaluates measurable clinical features of PGM1-CDG. We examined the relationship between genotype, enzyme activity, and TPCRS score by using regression analysis. Associations between the most common clinical features and disease severity were evaluated by principal component analysis., Results: We found a statistically significant stratification of the TPCRS scores among the phenotypic groups (P < .001). Regression analysis showed that there is no significant correlation between genotype, enzyme activity, and TPCRS score. Principal component analysis identified 5 variables that contributed to 54% variance in the cohort and are predictive of disease severity: congenital malformation, cardiac involvement, endocrine deficiency, myopathy, and growth., Conclusions: We established a scoring algorithm to reliably evaluate disease severity in patients with PGM1-CDG on the basis of their clinical history and presentation. We also identified 5 clinical features that are predictors of disease severity; 2 of these features can be evaluated by physical examination, without the need for specific diagnostic testing and thus allow for rapid assessment and initiation of therapy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Skeletal muscle disorders of glycogenolysis and glycolysis account for most of the conditions collectively termed glycogen storage diseases (GSDs). These disorders are rare (incidence 1 in 20,000-43,000 live births), and are caused by autosomal or X-linked recessive mutations that result in a specific enzyme deficiency, leading to the inability to utilize muscle glycogen as an energy substrate. McArdle disease (GSD V) is the most common of these disorders, and is caused by mutations in the gene encoding muscle glycogen phosphorylase. Symptoms of McArdle disease and most other related GSDs include exercise intolerance, muscle contracture, acute rhabdomyolysis, and risk of acute renal failure. Older patients may exhibit muscle wasting and weakness involving the paraspinal muscles and shoulder girdle. For patients with these conditions, engaging with exercise is likely to be beneficial. Diagnosis is frequently delayed owing to the rarity of the conditions and lack of access to appropriate investigations. A few randomized clinical trials have been conducted, some focusing on dietary modification, although the quality of the evidence is low and no specific recommendations can yet be made. The development of EUROMAC, an international registry for these disorders, should improve our knowledge of their natural histories and provide a platform for future clinical trials.

Glycogen branching enzyme 1 (GBE1) plays an essential role in glycogen biosynthesis by generating α-1,6-glucosidic branches from α-1,4-linked glucose chains, to increase solubility of the glycogen polymer. Mutations in the GBE1 gene lead to the heterogeneous early-onset glycogen storage disorder type IV (GSDIV) or the late-onset adult polyglucosan body disease (APBD). To better understand this essential enzyme, we crystallized human GBE1 in the apo form, and in complex with a tetra- or hepta-saccharide. The GBE1 structure reveals a conserved amylase core that houses the active centre for the branching reaction and harbours almost all GSDIV and APBD mutations. A non-catalytic binding cleft, proximal to the site of the common APBD mutation p.Y329S, was found to bind the tetra- and hepta-saccharides and may represent a higher-affinity site employed to anchor the complex glycogen substrate for the branching reaction. Expression of recombinant GBE1-p.Y329S resulted in drastically reduced protein yield and solubility compared with wild type, suggesting this disease allele causes protein misfolding and may be amenable to small molecule stabilization. To explore this, we generated a structural model of GBE1-p.Y329S and designed peptides ab initio to stabilize the mutation. As proof-of-principle, we evaluated treatment of one tetra-peptide, Leu-Thr-Lys-Glu, in APBD patient cells. We demonstrate intracellular transport of this peptide, its binding and stabilization of GBE1-p.Y329S, and 2-fold increased mutant enzymatic activity compared with untreated patient cells. Together, our data provide the rationale and starting point for the screening of small molecule chaperones, which could become novel therapies for this disease., (© The Author 2015. Published by Oxford University Press.)

Metabolic myopathies are disorders of utilization of carbohydrates or fat in muscles. The acute nature of energy failure is manifested either by a metabolic crisis with weakness, sometimes associated with respiratory failure, or by myoglobinuria. A typical disorder where permanent weakness occurs is glycogenosis type II (GSDII or Pompe disease) both in infantile and late-onset forms, where respiratory insufficiency is manifested by a large number of cases. In GSDII the pathogenetic mechanism is still poorly understood, and has to be attributed more to structural muscle alterations, possibly in correlation to macro-autophagy, rather than to energetic failure. This review is focused on recent advances about GSDII and its treatment, and the most recent notions about the management and treatment of other metabolic myopathies will be briefly reviewed, including glycogenosis type V (McArdle disease), glycogenosis type III (debrancher enzyme deficiency or Cori disease), CPT-II deficiency, and ETF-dehydrogenase deficiency (also known as riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency or RR-MADD). The discovery of the genetic defect in ETF dehydrogenase confirms the etiology of this syndrome. Other metabolic myopathies with massive lipid storage and weakness are carnitine deficiency, neutral lipid storage-myopathy (NLSD-M), besides RR-MADD. Enzyme replacement therapy is presented with critical consideration and for each of the lipid storage disorders, representative cases and their response to therapy is included. This article is part of a Special Issue entitled: Neuromuscular Diseases: Pathology and Molecular Pathogenesis., (Copyright © 2014. Published by Elsevier B.V.)

Recent studies have identified phosphoglucomutase 1 (PGM1) deficiency as an inherited metabolic disorder in humans. PGM1 deficiency is classified as both a muscle glycogenosis (type XIV) and a congenital disorder of glycosylation of types I and II. Affected patients show multiple disease phenotypes, reflecting the central role of the enzyme in glucose homeostasis, where it catalyzes the interconversion of glucose 1-phosphate and glucose 6-phosphate. The influence of PGM1 deficiency on protein glycosylation patterns is also widespread, affecting both biosynthesis and processing of glycans and their precursors. To date, 21 different mutations involved in PGM1 deficiency have been identified, including 13 missense mutations resulting in single amino acid changes. Growing clinical interest in PGM1 deficiency prompts a review of the molecular context of these mutations in the three-dimensional structure of the protein. Here the known crystal structure of PGM from rabbit (97 % sequence identity to human) is used to analyze the mutations associated with disease and find that many map to regions with clear significance to enzyme function. In particular, amino acids in and around the active site cleft are frequently involved, including regions responsible for catalysis, binding of the metal ion required for activity, and interactions with the phosphosugar substrate. Several of the known mutations, however, are distant from the active site and appear to manifest their effects indirectly. An understanding of how the different mutations that cause PGM1 deficiency affect enzyme structure and function is foundational to providing clinical prognosis and the development of effective treatment strategies.

Recent studies have identified phosphoglucomutase 1 (PGM1) deficiency as an inherited metabolic disorder in humans. Affected patients show multiple disease phenotypes, including dilated cardiomyopathy, exercise intolerance, and hepatopathy, reflecting the central role of the enzyme in glucose metabolism. We present here the first in vitro biochemical characterization of 13 missense mutations involved in PGM1 deficiency. The biochemical phenotypes of the PGM1 mutants cluster into two groups: those with compromised catalysis and those with possible folding defects. Relative to the recombinant wild-type enzyme, certain missense mutants show greatly decreased expression of soluble protein and/or increased aggregation. In contrast, other missense variants are well behaved in solution, but show dramatic reductions in enzyme activity, with kcat/Km often <1.5% of wild-type. Modest changes in protein conformation and flexibility are also apparent in some of the catalytically impaired variants. In the case of the G291R mutant, severely compromised activity is linked to the inability of a key active site serine to be phosphorylated, a prerequisite for catalysis. Our results complement previous in vivo studies, which suggest that both protein misfolding and catalytic impairment may play a role in PGM1 deficiency., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Branched polymers of glucose are universally used for energy storage in cells, taking the form of glycogen in animals, fungi, Bacteria, and Archaea, and of amylopectin in plants. Some enzymes involved in glycogen and amylopectin metabolism are similarly conserved in all forms of life, but some, interestingly, are not. In this paper we focus on the phylogeny of glycogen branching and debranching enzymes, respectively involved in introducing and removing of the α(1-6) bonds in glucose polymers, bonds that provide the unique branching structure to glucose polymers., Results: We performed a large-scale phylogenomic analysis of branching and debranching enzymes in over 400 completely sequenced genomes, including more than 200 from eukaryotes. We show that branching and debranching enzymes can be found in all kingdoms of life, including all major groups of eukaryotes, and thus were likely to have been present in the last universal common ancestor (LUCA) but have been lost in seemingly random fashion in numerous single-celled eukaryotes. We also show how animal branching and debranching enzymes evolved from their LUCA ancestors by acquiring additional domains. Furthermore, we show that enzymes commonly perceived as orthologous, such as human branching enzyme GBE1 and E. coli branching enzyme GlgB, are in fact related by a gene duplication and consequently paralogous., Conclusions: Despite being usually associated with animal liver glycogen and plant starch, energy storage in the form of branched glucose polymers is clearly an ancient process and has probably been present in the last universal common ancestor of all present life. The evolution of the enzymes enabling this form of energy storage is more complex than previously thought and illustrates the need for explicit phylogenomic analysis in the study of even seemingly "simple" metabolic enzymes. Patterns of conservation in the evolution of the glycogen/starch branching and debranching enzymes hint at some as yet unknown mechanisms, as mutations disrupting these patterns lead to a variety of genetic diseases in humans and other mammals.

Background: Bladder cancer is the most common malignancy of the urinary system, yet our molecular understanding of this disease is incomplete, hampering therapeutic advances., Methods: Here we used a genome-wide functional short-hairpin RNA (shRNA) screen to identify suppressors of in vivo bladder tumor xenograft growth (n = 50) using bladder cancer UMUC3 cells. Next-generation sequencing was used to identify the most frequently occurring shRNAs in tumors. Genes so identified were studied in 561 patients with bladder cancer for their association with stratification of clinical outcome by Kaplan-Meier analysis. The best prognostic marker was studied to determine its mechanism in tumor suppression using anchorage-dependent and -independent growth, xenograft (n = 20), and metabolomic assays. Statistical significance was determined using two-sided Student t test and repeated-measures statistical analysis., Results: We identified the glycogen debranching enzyme AGL as a prognostic indicator of patient survival (P = .04) and as a novel regulator of bladder cancer anchorage-dependent (P < .001), anchorage-independent (mean ± standard deviation, 180 ± 23.1 colonies vs 20±9.5 in control, P < .001), and xenograft growth (P < .001). Rescue experiments using catalytically dead AGL variants revealed that this effect is independent of AGL enzymatic functions. We demonstrated that reduced AGL enhances tumor growth by increasing glycine synthesis through increased expression of serine hydroxymethyltransferase 2., Conclusions: Using an in vivo RNA interference screen, we discovered that AGL, a glycogen debranching enzyme, has a biologically and statistically significant role in suppressing human cancer growth., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Glycogen storage diseases are important causes of myopathy and cardiomyopathy. We describe 10 patients from 8 families with childhood or juvenile onset of myopathy, 8 of whom also had rapidly progressive cardiomyopathy, requiring heart transplant in 4. The patients were homozygous or compound heterozygous for missense or truncating mutations in RBCK1, which encodes for a ubiquitin ligase, and had extensive polyglucosan accumulation in skeletal muscle and in the heart in cases of cardiomyopathy. We conclude that RBCK1 deficiency is a frequent cause of polyglucosan storage myopathy associated with progressive muscle weakness and cardiomyopathy., (© 2013 American Neurological Association.)

Background: Due partly to physicians' unawareness, many adults with Pompe disease are diagnosed with great delay. Besides, it is not well known which factors influence the rate of disease progression, and thus disease outcome. We delineated the specific clinical features of Pompe disease in adults, and mapped out the distribution and severity of muscle weakness, and the sequence of involvement of the individual muscle groups. Furthermore, we defined the natural disease course and identified prognostic factors for disease progression., Methods: We conducted a single-center, prospective, observational study. Muscle strength (manual muscle testing, and hand-held dynamometry), muscle function (quick motor function test), and pulmonary function (forced vital capacity in sitting and supine positions) were assessed every 3-6 months and analyzed using repeated-measures ANOVA., Results: Between October 2004 and August 2009, 94 patients aged between 25 and 75 years were included in the study. Although skeletal muscle weakness was typically distributed in a limb-girdle pattern, many patients had unfamiliar features such as ptosis (23%), bulbar weakness (28%), and scapular winging (33%). During follow-up (average 1.6 years, range 0.5-4.2 years), skeletal muscle strength deteriorated significantly (mean declines of -1.3% point/year for manual muscle testing and of -2.6% points/year for hand-held dynamometry; both p<0.001). Longer disease duration (>15 years) and pulmonary involvement (forced vital capacity in sitting position <80%) at study entry predicted faster decline. On average, forced vital capacity in supine position deteriorated by 1.3% points per year (p=0.02). Decline in pulmonary function was consistent across subgroups. Ten percent of patients declined unexpectedly fast., Conclusions: Recognizing patterns of common and less familiar characteristics in adults with Pompe disease facilitates timely diagnosis. Longer disease duration and reduced pulmonary function stand out as predictors of rapid disease progression, and aid in deciding whether to initiate enzyme replacement therapy, or when.

A high performance liquid chromatography method, adapted from an established urinary sugars method, has been developed for the analysis of a tetraglucose oligomer (Glc(4)) in urine. Pompe disease results from defects in the activity of lysosomal acid α-glucosidase (GAA) with patients typically excreting increased amounts of Glc(4). Rapid determination of GAA in dried blood spots is now possible. However, enzymatic analysis is unable to discriminate between patients with Pompe disease and those individuals harbouring pseudo deficiency mutations. This method was able to quantify Glc(4) levels in all patients analysed with an established diagnosis of Pompe disease, and all controls analysed had Glc(4) levels below the limit of detection for this method. Importantly the method was able to discriminate between an individual known to harbour a pseudo Pompe mutation and patients with Pompe disease, providing a useful supporting test to enzymatic analysis. Sequential measurement of urinary Glc(4) has been proposed to monitor the effects of enzyme replacement therapy (ERT). We observed a clear decrease in Glc(4) levels following commencement of treatment in three patients studied. Additionally, raised levels of Glc(4) were observed in patients with glycogen storage disease (GSD) type Ia and type III suggesting that this method may have applications in other GSDs.

Objective: To examine metabolism during exercise in 2 patients with muscle phosphorylase kinase (PHK) deficiency and to further define the phenotype of this rare glycogen storage disease (GSD)., Methods: Patient 1 (39 years old) had mild exercise-induced forearm pain, and EMG showed a myopathic pattern. Patient 2 (69 years old) had raised levels of creatine kinase (CK) for more than 6 months after statin treatment. Both patients had increased glycogen levels in muscle and PHK activity <11% of normal. Two novel pathogenic nonsense mutations were found in the PHKA1 gene. The metabolic response to anaerobic forearm exercise and aerobic cycle exercise was studied in the patients and 5 healthy subjects., Results: Ischemic exercise showed a normal 5-fold increase in plasma lactate (peak 5.7 and 6.9 mmol/L) but an exaggerated 5-fold increase in ammonia (peak 197 and 171 μmol/L; control peak range 60-113 μmol/L). An incremental exercise test to exhaustion revealed a blunted lactate response (5.4 and 4.8 mmol/L) vs that for control subjects (9.6 mmol/L; range 7.1-14.3 mmol/L). Fat and carbohydrate oxidation rates at 70% of peak oxygen consumption were normal. None of the patients developed a second wind phenomenon or improved their work capacity with an IV glucose infusion., Conclusion: Our findings demonstrate that muscle PHK deficiency may present as an almost asymptomatic condition, despite a mild impairment of muscle glycogenolysis, raised CK levels, and glycogen accumulation in muscle. The relative preservation of glycogenolysis is probably explained by an alternative activation of myophosphorylase by AMP and P(i) at high exercise intensities.

Glycogen storage diseases (GSD) affect primarily the liver, skeletal muscle, heart, and sometimes the central nervous system and the kidneys. These unique diseases are quite varied in age of onset of symptoms, morbidity, and mortality. Glycogen storage diseases are classified according to their individual enzyme deficiency. Each of these enzymes regulates synthesis or degradation of glycogen. Interestingly, there is great phenotypic variation and variable clinical courses even when a specific enzyme is altered by mutation. Depending on the specific mutation in an enzyme, a GSD patient may have a favorable or unfavorable prognosis. With neonatal or infantile forms, some GSDs lead to death within the first year of life, whereas other glycogen storage diseases are relatively asymptomatic or may cause only exercise intolerance. The paper provides a brief review and update of glycogen storage diseases, with respect to clinical features, genetic abnormalities, pathologic features, and treatment.

Glycogen storage disease (GSD) due to a deficient hepatic phosphorylase system defines a genetically heterogeneous group of disorders that mainly manifests in children. We investigated 45 unrelated children in whom a liver GSD VI or IX was suspected on the basis of clinical symptoms including hepatomegaly, increased serum transaminases, postprandial lactatemia and/or mild fasting hypoglycemia. Liver phosphorylase and phosphorylase b kinase activities studied in peripheral blood cells allowed to suspect diagnosis in 37 cases but was uninformative in 5. Sequencing of liver phosphorylase genes was useful to establish an accurate diagnosis. Causative mutations were found either in the PYGL (11 patients), PHKA2 (26 patients), PHKG2 (three patients) or in the PHKB (three patients) genes. Eleven novel disease causative mutations, five missense (p.N188K, p.D228Y, p.P382L, p.R491H, p.L500R) and six truncating mutations (c.501&#95;502ins361pb, c.528+2T>C, c.856-29&#95;c.1518+614del, c.1620+1G>C, p.E703del and c.2313-1G>T) were identified in the PYGL gene. Seventeen novel disease causative mutations, ten missense (p.A42P, p.Q95R, p.G131D, p.G131V, p.Q134R, p.G187R, p.G300V, p.G300A, p.C326Y, p.W820G) and seven truncating (c.537+5G>A, p.G396DfsX28, p.Q404X, p.N653X, p.L855PfsX87, and two large deletions) were identified in the PHKA2 gene. Four novel truncating mutations (p.R168X, p.Q287X, p.I268PfsX12 and c.272-1G>C) were identified in the PHKG2 gene and three (c.573&#95;577del, p.R364X, c.2427+3A>G) in the PHKB gene. Patients with PHKG2 mutations evolved towards cirrhosis. Molecular analysis of GSD VI or IX genes allows to confirm diagnosis suspected on the basis of enzymatic analysis and to establish diagnosis and avoid liver biopsy when enzymatic studies are not informative in blood cells., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The diagnosis of glycogen storage disease (GSD) type IX is often complicated by the complexity of the phosphorylase kinase enzyme (PHK), and molecular analysis is the preferred way to provide definitive diagnosis. Here we reported two novel mutations found in two GSD type IX patients with different residual enzyme activities from Hong Kong, China using genetic analysis and, provided the molecular interpretation of the deficient PHK activity. These two newly described mutations would be useful for the study of future GSD patients., (Copyright © 2010 Elsevier Inc. All rights reserved.)

GSD-1 (glycogen storage disease type 1) is caused by an inherited defect in glucose-6-phosphatase activity, resulting in a massive accumulation of hepatic glycogen content and an induction of de novo lipogenesis. The chlorogenic acid derivative S4048 is a pharmacological inhibitor of the glucose 6-phosphate transporter, which is part of glucose-6-phosphatase, and allows for mechanistic studies concerning metabolic defects in GSD-1. Treatment of mice with S4048 resulted in an ~60% reduction in blood glucose, increased hepatic glycogen and triacylglycerol (triglyceride) content, and a markedly enhanced hepatic lipogenic gene expression. In mammals, hepatic expression of lipogenic genes is regulated by the co-ordinated action of the transcription factors SREBP (sterol-regulatory-element-binding protein)-1c, LXRα (liver X receptor α) and ChREBP (carbohydrate-response-element-binding protein). Treatment of Lxra-/- mice and Chrebp-/- mice with S4048 demonstrated that ChREBP, but not LXRα, mediates the induction of hepatic lipogenic gene expression in this murine model of GSD-1. Thus ChREBP is an attractive target to alleviate derangements in lipid metabolism observed in patients with GSD-1.

Repeated evaluation of biotinidase (BTD) activity was carried out for a long-term follow-up in patients with hepatic glycogen storage diseases (GSDs). The results indicated inter-intra variability among the GSD-Ia, GSD-III and GSD-IX patients. In addition, a c.1330G>C transversion in the BTD gene, resulting in a p.Asp444His substitution was detected in one allele of a GSD-Ia patient with sustained normal enzyme activity. Thus far, it is necessary to be cautious in the interpretation of the results of BTD activity as a presumptive GSD diagnostic element. It is not known why plasma BTD activity increases in GSDs patients, or the clinical importance of the increment. When viewed from a global perspective, there are some lines of biotin biology that could indicate a relationship between BTD´s behavior and GSDs.

Conversion to glycogen is a major fate of ingested glucose in the body. A rate-limiting enzyme in the synthesis of glycogen is glycogen synthase encoded by two genes, GYS1, expressed in muscle and other tissues, and GYS2, primarily expressed in liver (liver glycogen synthase). Defects in GYS2 cause the inherited monogenic disease glycogen storage disease 0. We have generated mice with a liver-specific disruption of the Gys2 gene (liver glycogen synthase knock-out (LGSKO) mice), using Lox-P/Cre technology. Conditional mice carrying floxed Gys2 were crossed with mice expressing Cre recombinase under the albumin promoter. The resulting LGSKO mice are viable, develop liver glycogen synthase deficiency, and have a 95% reduction in fed liver glycogen content. They have mild hypoglycemia but dispose glucose less well in a glucose tolerance test. Fed, LGSKO mice also have a reduced capacity for exhaustive exercise compared with mice carrying floxed alleles, but the difference disappears after an overnight fast. Upon fasting, LGSKO mice reach within 4 h decreased blood glucose levels attained by control floxed mice only after 24 h of food deprivation. The LGSKO mice maintain this low blood glucose for at least 24 h. Basal gluconeogenesis is increased in LGSKO mice, and insulin suppression of endogenous glucose production is impaired as assessed by euglycemic-hyperinsulinemic clamp. This observation correlates with an increase in the liver gluconeogenic enzyme phosphoenolpyruvate carboxykinase expression and activity. This mouse model mimics the pathophysiology of glycogen storage disease 0 patients and highlights the importance of liver glycogen stores in whole body glucose homeostasis.

Mutations in the liver isoform of the Phosphorylase Kinase (PhK) alpha subunit (PHKA2 gene) cause X-linked liver glycogenosis (XLG), the most frequent type of PhK deficiency (glycogen-storage disease type IX). XLG patients can be divided in two subgroups, with similar clinical features but different activity of PhK (decreased in liver and blood cells for XLG-I and low in liver but normal or enhanced in blood cells for XLG-II). Here, we show that the PHKA2 missense mutations and small in-frame deletions/insertions are concentrated into two domains of the protein, which were recently described. In the N-terminal glucoamylase domain, mutations (principally leading to XLG-II) are clustered within the predicted glycoside-binding site, suggesting that they may have a direct impact on a possible hydrolytic activity of the PhK alpha subunit, which remains to be demonstrated. In the C-terminal calcineurin B-like domain (domain D), mutations (principally leading to XLG-I) are clustered in a region predicted to interact with the regulatory region of the PhK catalytic subunit and in a region covering this interaction site. Altogether, these results show that PHKA2 missense mutations or small in-frame deletions/insertions may have a direct impact on the PhK alpha functions and provide a framework for further experimental investigation.

Objective: To determine insulin sensitivity, proportions of muscle fiber types, and activities of glycogenolytic and glycolytic enzymes in Belgians with and without polysaccharide storage myopathy (PSSM)., Animals: 10 Quarter Horses (QHs) and 103 Belgians in which PSSM status had been determined., Procedures: To determine insulin sensitivity, a hyperinsulinemic euglycemic clamp (HEC) technique was used in 5 Belgians with PSSM and 5 Belgians without PSSM. Insulin was infused i.v. at 3 mU/min/kg for 3 hours, and concentrations of blood glucose and plasma insulin were determined throughout. An i.v. infusion of glucose was administered to maintain blood glucose concentration at 100 mg/dL. Activities of glycogenolytic and glycolytic enzymes were assessed in snap-frozen biopsy specimens of gluteus medius muscle obtained from 4 Belgians with PSSM and 5 Belgians without PSSM. Percentages of type 1, 2a, and 2b muscle fibers were determined via evaluation of >or= 250 muscle fibers in biopsy specimens obtained from each Belgian used in the aforementioned studies and from 10 QHs (5 with PSSM and 5 without PSSM)., Results: Belgians with and without PSSM were not significantly different with respect to whole-body insulin sensitivity, muscle activities of glycogenolytic and glycolytic enzymes, or proportions of muscle fiber types. However, Belgians had an increased proportion of type 2a and decreased proportion of type 2b muscle fibers, compared with proportions in QHs, regardless of PSSM status., Conclusions and Clinical Relevance: PSSM in Belgians may be attributable to excessive glycogen synthesis rather than decreased glycogen utilization or enhanced glucose uptake into muscle cells.

Polysaccharide storage myopathy (PSSM) is a novel glycogenosis in horses characterized by abnormal glycogen accumulation in skeletal muscle and muscle damage with exertion. It is unlike glycogen storage diseases resulting from known defects in glycogenolysis, glycolysis, and glycogen synthesis that have been described in humans and domestic animals. A genome-wide association identified GYS1, encoding skeletal muscle glycogen synthase (GS), as a candidate gene for PSSM. DNA sequence analysis revealed a mutation resulting in an arginine-to-histidine substitution in a highly conserved region of GS. Functional analysis demonstrated an elevated GS activity in PSSM horses, and haplotype analysis and allele age estimation demonstrated that this mutation is identical by descent among horse breeds. This is the first report of a gain-of-function mutation in GYS1 resulting in a glycogenosis.

We report two new cases of liver glycogen synthase deficiency (GSD0). The first patient presented at the age of 8 months with recurrent hypoglycemic seizures. The second patient presented at 14 months with asymptomatic incidental hyperglycemia. Glucose monitoring in both patients revealed daily fluctuations from fasting hypoglycemia to postprandial hyperglycemia. Genetic analysis of the GYS2 gene confirmed the diagnosis. GSD0 is more common than previously assumed. Recognition of the variable phenotype spectrum of GSD0 and routine analysis of GYS2 are essential for the correct diagnosis.

Objective: To evaluate whether biochemical or genetic alterations in AMP-activated protein kinase (AMPK) play a role in the development of polysaccharide storage myopathy (PSSM) in Quarter Horses., Animals: 30 PSSM-affected and 30 unaffected (control) Quarter Horses., Procedures: By use of an established peptide phosphotransfer assay, basal and maximal AMPK activities were measured in muscle biopsy samples obtained from 6 PSSM-affected and 6 control horses. In 24 PSSM-affected and 24 control horses, microsatellite markers identified from the chromosomal locations of all 7 AMPK subunit genes were genotyped with a fluorescent DNA fragment analyzer. Alleles of 2 of the AMPK gamma subunit genes were genotyped via DNA sequencing. Allele frequencies of DNA markers in or near the AMPK subunit genes were measured in isolated genomic DNA., Results: No differences in basal or maximal muscle AMPK enzyme activities between PSSM-affected and control horses were detected. There were also no differences in allele frequencies for microsatellite markers near any of the 7 AMPK subunit genes between the 2 groups. Furthermore, previously known and newly identified alleles of 2 equine AMPK gamma subunit genes were also not associated with PSSM., Conclusions and Clinical Relevance: These results have provided no evidence to indicate that AMPK plays a causative role in PSSM in American Quarter Horses.

Mutation in PRKAG2 encoding the gamma2 subunit of the AMP activated protein kinase (AMPK) cause human cardiomyopathy characterized by hypertrophy, Wolff-Parkinson-White syndrome, conduction system disease and glycogen storage in the myocardium. AMPK is a master metabolic regulator activated by hormones and energy deficient states. A heterotrimer enzyme comprising the catalytic alpha- and regulatory beta-and gamma-subunits was preserved through evolution and is ubiquitously expressed among mammalian tissues. AMPK is activated by AMP and inhibited by ATP that competes for binding to the regulatory sites on the gamma-subunit. Upstream kinases which phosphorylate Thr172 on the catalytic subunit activate the enzyme during exercise, ischemia, in response to sympathetic stimulation and hormones such as leptin and adiponectin. AMPK operates by phosphorylating its target proteins such as Acetyl CoA Carboxylase. Its classic functions include decreased fat synthesis in liver and adipose tissues, increased fatty acid oxidation, stimulating muscle glucose uptake and glycolysis. Altogether, these activities serve to restore the cellular and whole body energy balance. Human mutations which disrupt the nucleotide-binding affinity of the gamma2 subunit lead to loss of inhibition by ATP and inappropriate activate AMPK under resting conditions. As a result, myocytes recruit energy metabolites in excess of demand, causing storage of glycogen. Will AMPK ever emerge as a therapeutic target? Bench experiments suggest its potential in treating diabetes, ischemia and cell cycle regulation but much work is needed until these developments reach the bedside.

Phosphofructokinase deficiency (Tarui disease) was the first disorder recognized to directly affect glycolysis. Since the discovery of the disease, in 1965, a wide range of biochemical, physiological and molecular studies have greatly contributed to our knowledge concerning not only phosphofructokinase function in normal muscle but also on the general control of glycolysis and glycogen metabolism. Studies on phosphofructokinase deficiency vastly enriched the field of glycogen storage diseases, making a relevant improvement also in the molecular genetic area. So far, more than one hundred patients have been described with prominent clinical symptoms characterized by muscle cramps, exercise intolerance, rhabdomyolysis and myoglobinuria, often associated with haemolytic anaemia and hyperuricaemia. The muscle phosphofructokinase gene is located on chromosome 12 and about 20 mutations have been described. Other glycogenoses have been recognised in the distal part of the glycolytic pathway: these are infrequent but some may induce muscle cramps, exercise intolerance and rhabdomyolysis. Phosphoglycerate Kinase, Phosphoglycerate Mutase, Lactate Dehydrogenase, beta-Enolase and Aldolase A deficiencies have been described as distal glycogenoses. From the molecular point of view, the majority of these enzyme deficiencies are sustained by "private" mutations.

A 10-wk-old infant girl with severe hypertrophy of the septal and atrial walls by cardiac ultrasound, developed progressive ventricular wall thickening and died of aspiration pneumonia at 5 mo of age. Postmortem examination revealed ventricular hypertrophy and massive atrial wall thickening due to glycogen accumulation. A skeletal muscle biopsy showed increased free glycogen and decreased activity of phosphorylase b kinase (PHK). The report of a pathogenic mutation (R531Q) in the gene (PRKAG2) encoding the gamma2 subunit of AMP-activated protein kinase (AMPK) in three infants with congenital hypertrophic cardiomyopathy, glycogen storage, and "pseudo PHK deficiency" prompted us to screen this gene in our patient. We found a novel (R384T) heterozygous mutation in PRKAG2, affecting an arginine residue in the N-terminal AMP-binding domain. Like R531Q, this mutation reduces the binding of AMP and ATP to the isolated nucleotide-binding domains, and prevents activation of the heterotrimer by metabolic stress in intact cells. The mutation was not found in DNA from the patient's father, the only available parent, and is likely to have arisen de novo. Our studies confirm that mutations in PRKAG2 can cause fatal infantile cardiomyopathy, often associated with apparent PHK deficiency.

Cori's disease is a glycogen storage disorder characterized by a deficiency in the glycogen debranching enzyme, amylo-1,6-glucosidase,4-alpha-glucanotransferase (AGL). Here, we demonstrate that the G1448R genetic variant of AGL is unable to bind to glycogen and displays decreased stability that is rescued by proteasomal inhibition. AGL G1448R is more highly ubiquitinated than its wild-type counterpart and forms aggresomes upon proteasome impairment. Furthermore, the E3 ubiquitin ligase Malin interacts with and promotes the ubiquitination of AGL. Malin is known to be mutated in Lafora disease, an autosomal recessive disorder clinically characterized by the accumulation of polyglucosan bodies resembling poorly branched glycogen. Transfection studies in HepG2 cells demonstrate that AGL is cytoplasmic whereas Malin is predominately nuclear. However, after depletion of glycogen stores for 4 h, approximately 90% of transfected cells exhibit partial nuclear staining for AGL. Furthermore, stimulation of cells with agents that elevate cAMP increases Malin levels and Malin/AGL complex formation. Refeeding mice for 2 h after an overnight fast causes a reduction in hepatic AGL levels by 48%. Taken together, these results indicate that binding to glycogen crucially regulates the stability of AGL and, further, that its ubiquitination may play an important role in the pathophysiology of both Lafora and Cori's disease.

Glycogen storage disease type IX (GSD type IX) results from a deficiency of hepatic phosphorylase kinase activity. The phosphorylase kinase holoenzyme is made up of four copies of each of four subunits (alpha, beta, gamma and delta). The liver isoforms of the alpha-, beta- and gamma-subunits are encoded by PHKA2, PHKB and PHKG2, respectively. Mutation within these genes has been shown to result in GSD type IX. The diagnosis of GSD type IX is complicated by the spectrum of clinical symptoms, variation in tissue specificity and severity, and its inheritance, either X-linked or autosomal recessive. We investigated 15 patients from 12 families with suspected GSD type IX. Accurate diagnosis had been hampered by enzymology not being diagnostic in five cases. Clinical symptoms included combinations of hypoglycaemia, hepatosplenomegaly, short stature, hepatopathy, weakness, fatigue and motor delay. Biochemical findings included elevated lactate, urate and lipids. We characterised causative mutations in the PHKA2 gene in ten patients from eight families, in PHKG2 in two unrelated patients and in the PHKB gene in three patients from two families. Seven novel mutations were identified in PHKA2 (p.I337X, p.P498L, p.P869R, p.Y116&#95;T120dup, p.R1070del, p.R916W and p.M113I), two in PHKG2 (p.L144P and p.H48QfsX5) and two in PHKB (p.Y419X and c.2336+965A>C). There was a severe phenotype in patients with PHKG2 mutations, a mild phenotype with patients PHKB mutations and a broad spectrum associated with PHKA2 mutations. Molecular analysis allows accurate diagnosis where enzymology is uninformative and identifies the pattern of inheritance permitting counselling and family studies.

AMP-activated protein kinase (AMPK) responds to impaired cellular energy status by stimulating substrate metabolism for ATP generation. Mutation of the gamma2 regulatory subunit of AMPK in humans renders the kinase insensitive to energy status and causes glycogen storage cardiomyopathy via unknown mechanisms. Using transgenic mice expressing one of the mutant gamma2 subunits (N488I) in the heart, we found that aberrant high activity of AMPK in the absence of energy deficit caused extensive remodeling of the substrate metabolism pathways to accommodate increases in both glucose uptake and fatty acid oxidation in the hearts of gamma2 mutant mice via distinct, yet synergistic mechanisms resulting in selective fuel storage as glycogen. Increased glucose entry in the gamma2 mutant mouse hearts was directed through the remodeled metabolic network toward glycogen synthesis and, at a substantially higher glycogen level, recycled through the glycogen pool to enter glycolysis. Thus, the metabolic consequences of chronic activation of AMPK in the absence of energy deficiency is distinct from those previously reported during stress conditions. These findings are of particular importance in considering AMPK as a target for the treatment of metabolic diseases.

Glycogen storage diseases (GSDs) are characterized by abnormal inherited glycogen metabolism in the liver, muscle, and brain and divided into types 0 to X. GSD type I, glucose 6-phosphatase system, has types Ia, Ib, Ic, and Id, glucose 6-phosphatase, glucose 6-phosphate translocase, pyrophosphate translocase, and glucose translocase deficiencies, respectively. GSD type II is caused by defective lysosomal alpha-glucosidase (GAA), subdivided into 4 onset forms. GSD type III, amylo-1,6-glucosidase deficiency, is subdivided into 6 forms. GSD type IV, Andersen disease or amylopectinosis, is caused by deficiency of the glycogen-branching enzyme in numerous forms. GSD type V, McArdle disease or muscle phosphorylase deficiency, is divided into 2 forms. GSD type VI is characterized by liver phosphorylase deficiency. GSD type VII, phosphofructokinase deficiency, has 2 subtypes. GSD types VIa, VIII, IX, or X are supposedly caused by tissue-specific phosphorylase kinase deficiency. GSD type 0, glycogen synthase deficiency, is divided into 2 subtypes.

Background: The widespread use of routine biochemical assays has led to increased incidental findings of hypertransaminasemia. We aimed to evaluate the prevalence of different causes of raised aminotransferase levels in children referred to a university department of pediatrics., Methods: We investigated 425 consecutive children (age range, 1-18 years) with isolated hypertransaminasemia. All patients had raised aminotransferase levels on at least two occasions in the last month before observation. Cases due to major hepatotropic viruses were excluded., Results: During the first 6 months of observation, 259 children showed normalized liver enzymes. Among the remaining 166 patients with hypertransaminasemia lasting for more than 6 months, 75 had obesity-related liver disease; 51, genetic disorders; 7, autoimmune hepatitis; 5, cholelithiasis; 3, choledochal cyst; and 3, celiac disease. Among the 51 children with genetic disorders, 18 had Wilson disease; 14, muscular dystrophy; 4, alpha-1-antitrypsin deficiency; 4, Alagille syndrome; 4, hereditary fructose intolerance; 3, glycogen storage disease (glycogenosis IX); 2, ornithine transcarbamylase deficiency; and 2, Shwachman's syndrome. In 22 children, the hypertransaminasemia persisted for more than 6 months in the absence of a known cause., Conclusions: Genetic disease accounted for 12% of cases of isolated hypertransaminasemia observed in a tertiary pediatric department. A high level of suspicion is desirable for an early diagnosis of these disorders, which may present with isolated hypertransaminasemia and absence of typical clinical signs.

Pompe's disease is a neuromuscular disorder caused by deficiency of lysosomal acid alpha-glucosidase. Recombinant human alpha- glucosidase is under evaluation as therapeutic drug. In light of this development we studied the natural course of cases not fitting the definition of classic infantile Pompe's disease. Our review of 109 reports including 225 cases shows a continuous spectrum of phenotypes. The onset of symptoms ranged from 0 to 71 years. Based on the available literature, no criteria to delineate clinical sub-types could be established.A common denominator of these cases is that first symptoms were related to or caused by muscle weakness. In general, patients with a later onset of symptoms seemed to have a better prognosis. Respiratory failure was the most frequent cause of death. CK, LDH, ASAT, ALAT and muscle glycogen levels were frequently but not always elevated. In most cases a muscle biopsy revealed lysosomal pathology, but normal muscle morphology does not exclude Pompe's disease. In 10% of the cases in which the enzyme assay on leukocytes was used, a normal alpha-glucosidase activity was reported. Data on skeletal muscle strength and function, pulmonary function, disability, handicap and quality of life were insufficiently reported in the literature. Studies of non-classic Pompe's disease should focus on these aspects, before enzyme replacement therapy becomes generally available.

Objectives: To document 2 apparently incongruous clinical disorders occurring in the same infant: congenital myopathy with myophosphorylase deficiency (McArdle disease) and mitochondrial hepatopathy with liver failure and mitochondrial DNA depletion., Methods: An infant girl born to consanguineous Moroccan parents had severe congenital hypotonia and hepatomegaly, developed liver failure, and died at 5 months of age. We studied muscle and liver biopsy specimens histochemically and biochemically, and we sequenced the whole coding regions of the deoxyguanosine kinase (dGK) and myophosphorylase (PYGM) genes., Results: Muscle biopsy specimens showed subsarcolemmal glycogen accumulation and negative histochemical reaction for phosphorylase. Liver biopsy specimens showed micronodular cirrhosis and massive mitochondrial proliferation. Biochemical analysis showed phosphorylase deficiency in muscle and cytochrome c oxidase deficiency in liver. We identified a novel homozygous missense G-to-A mutation at codon 456 in exon 11 of PYGM, as well as a homozygous 4-base pair GATT duplication (nucleotides 763-766) in exon 6 of dGK, which produces a frame shift and a premature TGA stop codon at nucleotides 766 to 768, resulting in a truncated 255-amino acid protein. Both mutations were absent in 100 healthy individuals., Conclusions: Our data further expand the genetic heterogeneity in patients with McArdle disease; confirm the strong relationship between mitochondrial DNA depletion syndrome, liver involvement, and dGK mutations; and suggest that genetic "double trouble" should be considered in patients with unusual severe phenotypes.

Muscle-specific deficiency of phosphorylase kinase (Phk) causes glycogen storage disease, clinically manifesting in exercise intolerance with early fatiguability, pain, cramps and occasionally myoglobinuria. In two patients and in a mouse mutant with muscle Phk deficiency, mutations were previously found in the muscle isoform of the Phk alpha subunit, encoded by the X-chromosomal PHKA1 gene (MIM # 311870). No mutations have been identified in the muscle isoform of the Phk gamma subunit (PHKG1). In the present study, we determined Q1the structure of the PHKG1 gene and characterized its relationship to several pseudogenes. In six patients with adult- or juvenile-onset muscle glycogenosis and low Phk activity, we then searched for mutations in eight candidate genes. The coding sequences of all six genes that contribute to Phk in muscle were analysed: PHKA1, PHKB, PHKG1, CALM1, CALM2 and CALM3. We also analysed the genes of the muscle isoform of glycogen phosphorylase (PYGM), of a muscle-specific regulatory subunit of the AMP-dependent protein kinase (PRKAG3), and the promoter regions of PHKA1, PHKB and PHKG1. Only in one male patient did we find a PHKA1 missense mutation (D299V) that explains the enzyme deficiency. Two patients were heterozygous for single amino-acid replacements in PHKB that are of unclear significance (Q657K and Y770C). No sequence abnormalities were found in the other three patients. If these results can be generalized, only a fraction of cases with muscle glycogenosis and a biochemical diagnosis of low Phk activity are caused by coding, splice-site or promoter mutations in PHKA1, PHKG1 or other Phk subunit genes. Most patients with this diagnosis probably are affected either by elusive mutations of Phk subunit genes or by defects in other, unidentified genes.

Membrane bound glycogen storage in muscle is characteristic for the lysosomal storage disorder acid maltase (acid alpha-glucosidase) deficiency while in phosphofructokinase and phosphorylase deficiency, glycogen is stored free in the cytoplasm. Using immunohistochemistry, we examined whether acid maltase deficiency had an effect on early endosomes, recycling endosomes and trans-Golgi network, vesicle systems linked to lysosomes. Vacuolated glycogen containing fibres stained intensely for the lysosomal marker lysosomal-membrane-protein-1 within fibres and at the sarcolemma. There was a similar increase in immunoreactivity for markers of early endosomes (rab5), recycling endosomes (transferrin receptor) and the trans-Golgi network. In acid maltase deficiency, but not in normal muscle or other glycogenoses, staining for the insulin responsive glucose transporter 4 was markedly increased and partially co-localised with all vesicular markers. Our results suggest an effect of acid maltase deficiency extending to various vesicle systems linked to lysosomes. The enzyme defect may also affect the homoeostasis of receptors cycling through these organelles such as glucose transporter 4.