International consensus guidelines for phosphoglucomutase 1 deficiency (PGM1-CDG): Diagnosis, follow-up, and management.

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.

Enzyme dysfunction at atomic resolution: Disease-associated variants of human phosphoglucomutase-1.

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.

Phosphoglucomutase-1 deficiency: Early presentation, metabolic management and detection in neonatal blood spots.

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.

Preclinical Research in Glycogen Storage Diseases: A Comprehensive Review of Current Animal Models.

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.

From the seminal discovery of proteoglycogen and glycogenin to emerging knowledge and research on glycogen biology.

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.

Clinical and molecular genetic characterization of two patients with mutations in the phosphoglucomutase 1 (PGM1) gene.

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.

PHKG2 mutation spectrum in glycogen storage disease type IXc: a case report and review of the literature.

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.

A novel image-based high-throughput screening assay discovers therapeutic candidates for adult polyglucosan body disease.

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.

The variable clinical phenotype of three patients with hepatic glycogen synthase deficiency.

BACKGROUND: Glycogen synthase deficiency, also known as glycogenosis (GSD) type 0 is an inborn error of glycogen metabolism caused by mutations in the GYS2 gene, which is transmitted in an autosomal recessive trait. It is a rare form of hepatic glycogen storage disease with less than 30 cases reported in the literature so far. The disorder is characterized by fasting hyperketotic hypoglycemia without hyperalaninemia or hyperlactacidemia. It is a glycogenosis with lack of liver glycogen synthesis, therefore hepatomegaly is not observed in patients with glycogen synthase deficiency. Symptoms of fasting hypoglycemia in patients with glycogen storage disease type 0 (GSD0) usually appear for the first time in late infancy when weaning from overnight feeds. Seizures associated with low blood glucose may also occur, but they are rare. Clinical management is therefore based on frequent meals composed of high protein intake during the day and addition of uncooked cornstarch in the evening. CASE PRESENTATION: Herein we report three new cases of liver glycogen synthase deficiency (GSD0). The first patient presented at the 4 years of age with recurrent hypoglycemic seizures. The second patient who is the brother of the first patient presented at 15 months with asymptomatic incidental hypoglycemia. Glucose monitoring in both patients revealed daily fluctuations from fasting hypoglycemia to postprandial hyperglycemia and lactic acidemia. A third patient was consulted for ketotic hypoglycemia and postprandial hyperglycemia at the 5 years of age. CONCLUSIONS: Genetic analyses of the siblings revealed homozygosity for mutation c.736C>T on the GYS2 gene confirming the diagnosis. The third patient was found to be homozygous for c.1145G>A. GSD0 is more common than previously assumed. Recognition of the variable phenotypic spectrum of GSD0 and routine analysis of GYS2 are essential for the correct diagnosis.

The structure of brain glycogen phosphorylase-from allosteric regulation mechanisms to clinical perspectives.

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.

A highly prevalent equine glycogen storage disease is explained by constitutive activation of a mutant glycogen synthase.

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 Km 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.

PGM1 deficiency diagnosed during an endocrine work-up of low IGF-1 mediated growth failure.

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.

Defining the Phenotype and Assessing Severity in Phosphoglucomutase-1 Deficiency.

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.

Skeletal muscle disorders of glycogenolysis and glycolysis.

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.

Structural basis of glycogen branching enzyme deficiency and pharmacologic rescue by rational peptide design.

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.

Spectrum of metabolic myopathies.

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.

Mutations in hereditary phosphoglucomutase 1 deficiency map to key regions of enzyme structure and function.

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.

Compromised catalysis and potential folding defects in in vitro studies of missense mutants associated with hereditary phosphoglucomutase 1 deficiency.

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.

Phylogenomic analysis of glycogen branching and debranching enzymatic duo.

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.