The genotype-phenotype correlation in Pompe disease

Pompe disease is an autosomal recessive lysosomal glycogen storage disorder that is caused by acid α-glucosidase (GAA) deficiency and is due to pathogenic sequence variations in the corresponding GAA gene. The correlation between genotypes and phenotypes is strict, in that patients with the most severe phenotype, classic infantile Pompe disease, have two pathogenic mutations, one in each GAA allele, that prevent the formation of GAA or totally obliterates its function. All patients with less progressive phenotypes have at least one sequence variation that allows normal or low level synthesis of GAA leading to the formation of analytically measurable, low level GAA activity in most cases. There is an overall trend of finding higher GAA enzyme levels in patients with onset of symptoms in adulthood when compared to patients who show clinical manifestations in early childhood, aged 0-5 years, with a rapidly progressive course, but who lack the severe characteristics of classic infantile Pompe disease. However, several cases have been reported of adult-onset disease with very low GAA activity, which in all those cases corresponds with the GAA genotype. The clinical diversity observed within a large group of patients with functionally the same GAA genotype and the same c.-32-13C?>?T haplotype demonstrates that modifying factors can have a substantial effect on the clinical course of Pompe disease, disturbing the GAA genotype-phenotype correlation. The present day challenge is to identify these factors and explore them as therapeutic targets.     

Branching enzyme activity of cultured amniocytes and chorionic villi: prenatal testing for type IV glycogen storage disease.

Although type IV glycogen storage disease (Andersen disease; McKusick 23250) is considered to be a rare, autosomally recessive disorder, of the more than 600 patients with glycogenosis identified in our laboratory by enzymatic assays, 6% have been shown to be deficient in the glycogen branching enzyme. Most of the 38 patients with type IV glycogen storage disease who are known to us have succumbed at a very early age, with the exception of one male teenager, an apparently healthy 7-year-old male, and several 5-year-old patients. Fourteen pregnancies at risk for branching enzyme deficiency have been monitored using cultured amniotic fluid cells, and four additional pregnancies have been screened using cultured chorionic villi. Essentially no branching enzyme activity was detectable in eight samples (amniocytes); activities within the control range were found in five samples (three amniocyte and two chorionic villi samples); and five samples appeared to have been derived from carriers. In two of the cases lacking branching enzyme activity, in which the pregnancies were terminated and fibroblasts were successfully cultured from the aborted fetuses, no branching enzyme activity was found. Another fetus, which was predicted by antenatal assay to be affected, was carried to term. Skin fibroblasts from this baby were deficient in branching enzyme. Pregnancies at risk for glycogen storage disease due to the deficiency of branching enzyme can be successfully monitored using either cultured chorionic villi or amniocytes.     

Genetic mapping of GBE1 and its association with glycogen storage disease IV in American Quarter horses

Comparative biochemical and histopathological data suggest that a deficiency in the glycogen branching enzyme (GBE) is responsible for a fatal neonatal disease in Quarter Horse foals that closely resembles human glycogen storage disease type IV (GSD IV). Identification of DNA markers closely linked to the equine GBE1 gene would assist us in determining whether a mutation in this gene leads to the GSD IV-like condition. FISH using BAC clones as probes assigned the equine GBE1 gene to a marker deficient region of ECA26q12-->q13. Four other genes, ROBO2, ROBO1, POU1F1, and HTR1F, that flank GBE1 within a 10-Mb segment of HSA3p12-->p11, were tightly linked to equine GBE1 when analyzed on the Texas A&M University 5000 rad equine radiation hybrid panel, while the GLB1, MITF, RYBP, and PROS1 genes that flank this 10-Mb interval were not linked with markers in the GBE1 group. A polymorphic microsatellite (GBEms1) in a GBE1 BAC clone was then identified and genetically mapped to ECA26 on the Animal Health Trust full-sibling equine reference family. All Quarter Horse foals affected with GSD IV were homozygous for an allele of GBEms1, as well as an allele of the most closely linked microsatellite marker, while a control horse population showed significant allelic variation with these markers. This data provides strong molecular genetic support for the candidacy of the GBE1 locus in equine GSD IV.     

Phosphofructokinase deficiency; past, present and future

Phosphofructokinase deficiency (Tarui disease, glycogen storage disease VII, GSD VII) stands out among all the GSDs. PFK deficiency was the first recognized disorder that directly affects glycolysis. Ever since the discovery of the disease in 1965, a wide range of biochemical, physiological and molecular studies of the disorder have greatly expanded our understanding of the function of normal muscle, general control of glycolysis and glycogen metabolism. The studies of PFK deficiency vastly enriched the field of glycogen storage diseases, as well as the field of metabolic and neuromuscular disorders. This article cites a historical overview of this clinical entity and the progress that has been made in molecular genetic area. We will also present the results of a search in-silico, which allowed us to identify a previously unknown sequence of the human platelet PFK gene (PFK-P). In addition, we will describe phylogenetic analysis of evolution of PFK genes.     

Glycogen branching enzyme (<Emphasis Type="Italic">GBE1</Emphasis>) mutation causing equine glycogen storage disease IV

Comparative biochemical and histopathological evidence suggests that a deficiency in the glycogen branching enzyme, encoded by the GBE1 gene, is responsible for a recently identified recessive fatal fetal and neonatal glycogen storage disease (GSD) in American Quarter Horses termed GSD IV. We have now derived the complete GBE1 cDNA sequences for control horses and affected foals, and identified a C to A substitution at base 102 that results in a tyrosine (Y) to stop (X) mutation in codon 34 of exon 1. All 11 affected foals were homozygous for the X34 allele, their 11 available dams and sires were heterozygous, and all 16 control horses were homozygous for the Y34 allele. The previous findings of poorly branched glycogen, abnormal polysaccharide accumulation, lack of measurable GBE1 enzyme activity and immunodetectable GBE1 protein, coupled with the present observation of abundant GBE1 mRNA in affected foals, are all consistent with the nonsense mutation in the 699 amino acid GBE1 protein. The affected foal pedigrees have a common ancestor and contain prolific stallions that are likely carriers of the recessive X34 allele. Defining the molecular basis of equine GSD IV will allow for accurate DNA testing and the ability to prevent occurrence of this devastating disease affecting American Quarter Horses and related breeds.The nucleotide sequence data reported in this article have been submitted to GenBank and have been assigned the accession numbers AY505107–AY505110.     

Glycogen debranching enzyme: purification, antibody characterization, and immunoblot analyses of type III glycogen storage disease.

Type III glycogen storage disease is caused by a deficiency of glycogen debranching-enzyme activity. Many patients with this disease have both liver and muscle involvement, whereas others have only liver involvement without clinical or laboratory evidence of myopathy. To improve our understanding of the molecular basis of the disease, debranching enzyme was purified 238-fold from porcine skeletal muscle. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified enzyme gave a single band with a relative molecular weight of 160,000 that migrated to the same position as purified rabbit-muscle debranching enzyme. Antiserum against porcine debranching enzyme was prepared in rabbit. The antiserum reacted against porcine debranching enzyme with a single precipitin line and demonstrated a reaction having complete identity to those of both the enzyme present in crude muscle and the enzyme present in liver extracts. Incubation of antiserum with purified porcine debranching enzyme inhibited almost all enzyme activity, whereas such treatment with preimmune serum had little effect. The antiserum also inhibited debranching-enzyme activity in crude liver extracts from both pigs and humans to the same extent as was observed in muscle. Immunoblot analysis probed with anti-porcine-muscle debranching-enzyme antiserum showed that the antiserum can detect debranching enzyme in both human muscle and human liver. The bands detected in human samples by the antiserum were the same size as the one detected in porcine muscle. Five patients with Type III and six patients with other types of glycogen storage disease were subjected to immunoblot analysis. Although anti-porcine antiserum detected specific bands in all liver and muscle samples from patients with other types of glycogen storage disease (Types I, II, and IX), the antiserum detected no cross-reactive material in any of the liver or muscle samples from patients with Type III glycogen storage disease. These data indicate (1) immunochemical similarity of debranching enzyme in liver and muscle and (2) that deficiency of debranching-enzyme activity in Type III glycogen storage disease is due to absence of debrancher protein in the patients that we studied.     

Identification of the first deletion–insertion involving the complete structure of GAA gene and part of CCDC40 gene mediated by an Alu element

Pompe disease is an uncommon autosomal recessive glycogen storage disorder caused by deficiency of acid α-glucosidase. Classic infantile form triggers severe cardiomyopathy, hypotonia, and respiratory failure, leading to death within the first two years of life. The majority of patients with Pompe disease have been reported to have point mutations in the GAA gene. We report the first complex deletion–insertion encompassing the complete structure of GAA gene and a large fragment of the gene CCDC40 in a patient with very severe form of Pompe disease. Sequencing analysis of breakpoints allowed us to determine the potential implication of an Alu repeat in the pathogenic mechanism. We suggest that molecular strategy of Pompe disease should include systematic analysis of large rearrangements.     

Molecular characterization of glycogen storage disease type III

Deficiency of the glycogen debranching enzyme (gene, AGL) causes glycogen storage disease type III (GSD-III), an autosomal recessive disease affecting glycogen metabolism. Most GSD-III patients have AGL deficiency in both the liver and muscle (type IIIa), but some have it in the liver but not muscle (type IIIb). Cloning of human AGL cDNAs and determination of the genomic structure and mRNA isoforms of AGL have allowed for the study of GSD-III at the molecular level. In turn, the resulting information has greatly facilitated our understanding of the molecular basis of this storage disease with remarkable clinical and enzymatic variability. In this review, we summarize all 31 GSD-III mutations in the literature and discuss their clinical and laboratory implications. Most of the mutations are nonsense mutations caused by a nucleotide substitution or small insertion or deletion; only one is caused by a missense amino acid change. Some important genotype-phenotype correlation have emerged, in particular, that exon 3 mutations (17delAG and Q6X) are specifically associated with GSD-IIIb. Three other mutations have appeared to have some phenotype correlation. Specifically, the splice mutation IVS32-12A>G was found in GSD-III patients having mild clinical symptoms, while the mutations 3965delT and 4529insA are associated with a severe phenotype and early onset of clinical manifestations. A molecular diagnostic scheme has been proposed to diagnose GSD-III noninvasively. The characterization of AGL mutations in GSD-III patients has also helped the structure-function analysis of this bifunctional enzyme important for glycogen metabolism.     

Variability of biochemical and clinical phenotype in X-linked liver glycogenosis with mutations in the phosphorylase kinase PHKA2 gene

X-linked liver glycogenosis (XLG) resulting from phosphorylase kinase (Phk) deficiency is one of the most common forms of glycogen storage disease. It is caused by mutations in the gene encoding the liver isoform of the Phk α subunit (PHKA2). In the present study, we address the issue of phenotypic and allelic heterogeneity in XLG. We have identified mutations in seven male patients. One of these patients represents the variant biochemical phenotype, XLG subtype 2 (XLG2), where Phk activity is low in liver but normal or even elevated in erythrocytes. He carries a K189E missense mutation, which adds to the emerging evidence that XLG2 is associated with missense mutations clustering at a few sites. Two patients display clinical phenotypes unusual for liver Phk deficiency, with dysfunction of the kidneys (proximal renal tubular acidosis) or of the nervous system (seizures, delayed cognitive and speech abilities, peripheral sensory neuropathy), respectively, in addition to liver glycogenosis. In the patient with kidney involvement, we have identified a missense mutation (P399S) and a trinucleotide deletion (2858del3) leading to the replacement of two amino acids by one new residue (N953/L954I), and a missense mutation has also been found in the patient with neurological symptoms (G1207W). These two cases demonstrate that PHKA2 mutations can also be associated with uncommon clinical phenotypes. Finally, in four typical XLG cases, we have identified three truncating mutations (70insT, R352X, 567del22) and an in-frame deletion of eight well-conserved amino acids (2452del24). Together, this study adds eight new mutations to the previously known complement of sixteen PHKA2 mutations. All known PHKA2 mutations but one are distinct, indicating pronounced allelic heterogeneity of X-linked liver glycogenosis with mutations in the PHKA2 gene. Received: 17 October 1997 / Accepted: 23 December 1997     

The role of immune tolerance induction in restoration of the efficacy of ERT in Pompe disease

Pompe disease is a lysosomal storage disorder caused by deficiency in the enzyme acid α-glucosidase (GAA). Pompe disease is characterized by the accumulation of glycogen, predominantly in muscle tissue, leading to progressive muscle weakness, loss of motor, respiratory, and, in the infantile-onset form, cardiac function. Disease progression is highly variable depending on phenotype, but premature death due to respiratory complications occurs in most patients. Beginning in 2006, approved alglucosidase alfa enzyme replacement therapies [recombinant human (rh) GAA] have been available to treat Pompe patients. Treatment of classic infantile-onset patients, who manifest the severest form of the disease, with alglucosidase alfa (Myozyme?) has led to extended survival and an evolving understanding of the pathophysiology and course of the disease. Moreover, such treatment has brought to light the role of the immune response in abrogating the efficacy of rhGAA in classic infantile-onset patients with severe genetic mutations. Thus, optimization of treatment for such patients includes development and utilization of strategies to prevent or eliminate immune responses, including modulating the immune system (prophylactic and therapeutic immune tolerance induction regimens) and engineering the enzyme to be less immunogenic and more effective. Future research is also critical for evaluating and mitigating novel disease-associated pathologies uncovered by prolonged survival of infantile-onset patients including development of novel therapeutics, and for protein design strategies to increase delivery of enzyme replacement therapy to critical target tissues. Such efforts would be greatly bolstered by further development of predictive animal models and biomarkers to facilitate clinical trials and patient management. Published 2012. This article is a U.S. Government work and is in the public domain in the USA.     

Assignment of the human glycogen debrancher gene to chromosome 1p21

Glycogen debranching enzyme is a monomeric protein containing two independent catalytic activities of glycantransferase and glucosidase that are both required for glycogen degradation. Its deficiency causes type III glycogen storage disease. A majority of the patients with this disease have deficient enzyme activity in both liver and muscle (type IIIa) but approximately 15% of them lack enzyme activity only in the liver (type IIIb); however, the enzyme is a monomer and appears to be identical in all the tissues. The cDNA coding for the complete human muscle debranching enzyme has recently been isolated. Using the cDNA clones, the debrancher gene was localized to human chromosome 1 by somatic cell hybrid analysis. Regional assignment to chromosome band 1p21 was determined by in situ hybridization. Mapping of the debrancher gene to a single chromosome site is consistent with our hypotheses that a single gene encodes both liver and muscle debrancher protein.     

A newly identified c.1824_1828dupATACG mutation in exon 13 of the GAA gene in infantile-onset glycogen storage disease type II (Pompe disease)

Pompe disease or glycogen storage disease type II is a glycogen storage disorder associated with malfunction of the acid α-glucosidase enzyme (GAA; EC.3.2.1.3) leading to intracellular aggregations of glycogenin muscles. The infantile-onset type is the most life-threatening form of this disease, in which most of patients suffer from cardiomyopathy and hypotonia in early infancy. In this study, a typical case of Pompe disease was reported in an Iranian patient using molecular analysis of the GAA gene. Our results revealed a new c.1824_1828dupATACG mutation in exon 13 of the GAA gene. In conclusion, with the finding of this novel mutation, the genotypic spectrum of Iranian patients with Pompe disease has been extended, facilitating the definition of disease-related mutations.     

Regional mapping of a liver alpha-subunit gene of phosphorylase kinase (PHKA) to the distal region of human chromosome Xp.

X-linked liver glycogenosis (XLG) is a glycogen storage disorder resulting from deficient activity of phosphorylase kinase (PHK). PHK consists of four different subunits: alpha, beta, gamma, and delta. Several genes encoding PHK subunits have been cloned and localized, but only the muscle alpha-subunit (PHKA) gene has been assigned to the X chromosome, in the region Xq12----q13. However, we have previously excluded the muscle PHKA gene as a candidate gene for the XLG mutation, as linkage analysis indicated that the mutation responsible for XLG is located in Xp22 and not in Xq12----q13. We report here the chromosomal localization by in situ hybridization of a liver PHKA gene to the distal region of chromosome Xp. Strong hybridization signals were observed on the distal part of the short arm of a chromosome identified as the X chromosome by cohybridization with an X chromosome-specific centromeric probe. The localization of this gene in the same chromosomal region as the disease gene responsible for XLG suggests that the liver PHKA gene is a highly likely candidate gene for the XLG mutation.     

The molecular basis of the type 1 glycogen storage diseases.

Microsomal glucose-6-phosphatase catalyses the last step in liver glucose production. Glucose-6-phosphatase deficiency, now termed type 1 glycogen storage disease, was first described almost 40 years ago but until recently very little was known about the molecular basis of the various type 1 glycogen storage diseases. Recently we have shown that at least six different proteins are needed for normal glucose-6-phosphatase activity in liver. Four of the proteins have been purified and three cloned. Study of the type 1 glycogen storage diseases has stimulated investigations of the mechanisms of small molecule transport across the endoplasmic reticulum membrane and demonstrated the existence of novel endoplasmic reticulum transport proteins for glucose and phosphate.     

Toward deconstructing the phenotype of late-onset Pompe disease

Pompe disease (glycogen storage disease type 2 or acid maltase deficiency) is a rare autosomal recessive lysosomal storage disorder. Since the advent of ERT a lot has been learned about the phenotypic spectrum especially in the late onset patients. We describe in detail 44 patients diagnosed with late-onset Pompe disease (LOPD) at our neuromuscular department from 1985 to 2011 and compare them to patients with LOPD in the literature of the past 40 years. Study of the Munich LOPD group revealed varying musculoskeletal and cardio-cerebrovascular manifestation patterns. Several of these symptom patterns commonly appeared in conjunction with one another, highlighting the multisystem involvement of this condition. Common symptom patterns include: (i) Classic limb girdle and diaphragmatic weakness, (ii) rigid spine syndrome (RSS), scoliosis, and low body mass, and (iii) several cardio-cerebrovascular manifestation patterns. The most common presentation, limb girdle and diaphragmatic weakness, appeared in 78% (34/44) of our patients and over 80% of those in the literature. Sixteen percent (7/44) of our patients presented with rigid spine, scoliosis, and low body mass. Although scoliosis had a reported frequency of 33% in the general LOPD patient population, the literature only occasionally reported low body mass and RSS. Importantly, a multisystem extramuscular finding accompanied by cardio-cerebrovascular manifestations was found in 29% (13/44) of our LOPD patients; the literature showed an increasing prevalence of this latter finding. By examining the phenotype of patients with confirmed LOPD, we found a more subtle clinical multisystem involvement in LOPD. Whether patients presenting with the different symptom patterns respond differently to enzyme replacement therapy remains a key question for future research. ? 2012 Wiley Periodicals, Inc.     

Characterization of cyanobacterial glycogen isolated from the wild type and from a mutant lacking of branching enzyme

Cyanobacteria produce glycogen as their primary form of carbohydrate storage. The genomic DNA sequence of Synechocystis sp. PCC6803 indicates that this strain encodes one glycogen-branching enzyme (GBE) and two isoforms of glycogen synthase (GS). To confirm the putative GBE and to demonstrate the presence of only one GBE gene, we generated a mutant lacking the putative GBE gene, sll0158, by replacing it with a kanamycin resistance gene through homologous recombination. GBE in sll0158(-) mutant was eliminated; the mutant strain produced less glucan, equivalent to 48% of that produced by the wild type. In contrast to the wild-type strain that had 74% of the glucan being water-soluble, the mutant had only 14% of the glucan water-soluble. Molecular structures of glucans produced by the mutant and the wild type were characterized by using high-performance size-exclusion and anion-exchange chromatography. The glycogen produced by the wild type displayed a molecular mass of 6.6 x 10(7) daltons (degree of polymerization (DP) 40700) and 10% branch linkages, and the alpha-D-glucan produced by the mutant displayed a molecular mass of 4.7-5.6 x 10(3) daltons (DP 29-35) with slight branch linkages. The results indicated that sll0158 was the major functional GBE gene in Synechocystis sp. PCC6803.     

Genetic heterogeneity and the diagnosis of hepatic glycogenoses

Glycogen storage diseases constitute a highly heterogeneous group of disorders, because of the many complex enzyme systems involved in glycogen metabolism, and also because of the diversity of molecular defects connected with gene mutations. To illustrate these features, the authors studied four types of liver glycogen storage diseases, respectively caused by deficiencies of glucose-6-phosphatase, debranching enzyme, phosphorylase and phosphorylase kinase. In each case, the role and functional characteristics of the enzyme system are described, as well as the bioclinical aspects of the deficiency. The only reliable way of diagnosing glycogen storage disease is by assaying the activity of the enzyme concerned. Assay procedure must take account of various factors, especially the progress made in understanding the nature and mechanism of action of enzyme systems, the possible tissular heterogeneity of the deficiency and the functional characteristics of certain enzymes.     

Generalized accumulation of neutral glycosphingolipids with G M2 ganglioside accumulation in the brain

Analyses have been made of glycosphingolipids from visceral organs and brain of a patient with an unusual lipid storage disorder diagnosed initially as classical Tay-Sachs disease. Levels of the lipids from fresh-frozen sections of gray and white matter, kidney, spleen, liver, and heart from this patient were compared with those of normal juvenile controls, and the fatty acid composition of accumulated glycosphingolipids was compared with reference compounds. This patient was found to have abnormally high concentrations of a globoside in liver, kidney, and spleen, asialo G(M2) ganglioside in brain and liver, and G(M2) ganglioside in the brain. On the basis of these findings along with the clinical manifestations of Tay-Sachs disease with visceral involvement (hepatosplenomegaly) and demonstration of total deficiency of both A and B components of beta-N-acetylhexosaminidase activity, this glycosphingolipidosis is the same as two previously reported cases of G(M2) gangliosidosis with globoside accumulation and total beta-N-acetylhexosaminidase deficiency.