4-methylumbelliferyl-glucoside and Glycogen-Storage-Disease-Type-II

Pompe disease, a rare, autosomal, recessive, inherited, lysosomal storage disorder, is caused by mutations in the acid α-glucosidase (GAA) gene leading to a deficiency of the lysosomal GAA enzyme. Some GAA mutations eliminate all enzymatic activities, causing severe infantile Pompe disease; others allow residual GAA activity and lead to middle adulthood forms. Here, we report a cohort of 12 patients, belonging to 11 unrelated families, with infantile Pompe disease. The mutational analysis of GAA gene revealed a novel c.1494G > A (p.Trp498X) mutation in one patient and three known mutatio,ns including the c.1497G > A (p.Trp499X) mutation, in two patients, the c.1927G > A (p.Gly643Arg) mutation in one patient and the common c.236_246del (p.Pro79ArgfsX13) mutation in eight patients. The high prevalence of c.236_246del mutation in our cohort (58%) was supported by the existence of a common founder ancestor that was confirmed by its segregation of similar SNPs haplotype, including four intragenic SNPs of GAA gene. In addition, a 3D structure model and a docking were generated for the mutant p.Gly643Arg using the crystal structure of human GAA as template and the 4-methylumbelliferyl-α-D-glucopyranoside as substrate. The results showed that the arginine at position 643 caused electrostatic changes in neighboring regions, leading to the repulsion between the amino acids located in the catalytic cavity of the GAA enzyme, thus restricting access to its substrate. These structural defects could cause the impairment of the transport and maturation previously reported for p.Gly643Arg mutation.

Topics: alpha-Glucosidases; Catalytic Domain; Female; Glucosides; Glycogen Storage Disease Type II; Humans; Hymecromone; Infant; Male; Molecular Docking Simulation; Mutation; Protein Binding

Pompe disease is caused by a deficiency in acid α-glucosidase (GAA) and results in progressive, debilitating, and often life-threatening symptoms. Newborn screening has led to the early diagnosis of Pompe disease, but the best algorithm for screening has not yet been established.. GAA and neutral α-glucosidase (NAG) activities in dried blood spots (DBSs) were assayed using 4-methylumbelliferyl-β-d-glucopyranoside as the substrate. We also measure α-galactosidase A (GLA) activity in DBSs for comparison. A total of 473,738 newborns were screened for Pompe disease, and the data were analyzed retrospectively to determine the best screening algorithm.. The fluorescence assay used in the screening possessed good reproducibility, but the NAG/GAA ratio was superior in separating the true-positive from the false-positive cases. An NAG/GAA cutoff ratio≥60 produces a positive predictive value (PPV) of 63.4%, and in our sample, only two cases of later-onset Pompe disease would have been missed. The GLA/GAA ratio is not as effective as the NAG/GAA ratio.. A suitable control enzyme can improve the performance of newborn screening. Newborn screening for Pompe disease can be performed using the NAG/GAA ratio as a cutoff even in the presence of GAA partial deficiency.

Topics: Algorithms; alpha-Galactosidase; alpha-Glucosidases; Base Sequence; Glucosides; Glycogen Storage Disease Type II; Humans; Hymecromone; Infant, Newborn; Molecular Sequence Data; Neonatal Screening; Reproducibility of Results; Taiwan

Glycogenosis type II (GSD II, Pompe disease) is an autosomal recessive lysosomal storage disease that results from a deficiency of acid alpha-glucosidase (GAA). Patients with this disorder are unable to break down lysosomal glycogen, which consequently accumulates in the lysosome. To evaluate enzyme replacement therapy for GSD II patients, we have expressed human GAA cDNA in Chinese hamster ovary-K1 cells utilising a vector that places the cDNA under the transcriptional control of the human polypeptide chain elongation factor 1 alpha gene promoter. A clonal cell line that secreted precursor recombinant GAA at approximately 18 mg.l-1.day-1 was identified. The precursor recombinant GAA was purified to homogeneity, had a molecular mass of 110 kDa as measured by SDS/PAGE, and was shown to have pH optima and kinetic parameters similar to those of GAA purified from human tissues. The partial N-terminal amino acid sequence of recombinant GAA conformed to that derived from the nucleotide sequence of the cloned cDNA. The recombinant enzyme was taken up by cultured fibroblasts and skeletal muscle cells from GSD II patients, and was shown to correct the storage phenotype. Endocytosed GAA was localised to the lysosome and showed evidence of intracellular processing to a more mature form. Activity levels increased up to twice the normal value and uptake was prevented if cells were cultured in the presence of mannose 6-phosphate.

Topics: alpha-Glucosidases; Amino Acid Sequence; Animals; Cells, Cultured; CHO Cells; Cricetinae; Endocytosis; Enzyme Precursors; Glucan 1,4-alpha-Glucosidase; Glucosides; Glycogen; Glycogen Storage Disease Type II; Humans; Hydrogen-Ion Concentration; Hymecromone; Lysosomes; Mannosephosphates; Molecular Sequence Data; Muscle, Skeletal; Peptide Elongation Factor 1; Peptide Elongation Factors; Protein Processing, Post-Translational; Recombinant Proteins; Sequence Analysis

We have defined one type of acid alpha-glucosidase and two types of neutral alpha-glucosidases from quail skeletal muscle on the basis of differences in the elution patterns on a DEAE-cellulose column. The appearance of the two neutral alpha-glucosidase isoenzymes was age-dependent. A decrease in acid alpha-glucosidase activity was demonstrated in Japanese quails with glycogenosis type II. The characteristics of these three alpha-glucosidase isoenzymes are described.

Topics: Age Factors; alpha-Glucosidases; Animals; Chromatography, Affinity; Chromatography, DEAE-Cellulose; Coturnix; Glucosidases; Glucosides; Glycogen Storage Disease Type II; Glycoside Hydrolase Inhibitors; Hydrogen-Ion Concentration; Hymecromone; Molecular Weight; Muscles; Substrate Specificity

We report on a 2-yr-old boy with progressive muscular weakness and respiratory failure. There was no clinical evidence of heart muscle involvement. Autopsy showed marked intralysosomal glycogen deposition in skeletal muscle and liver with no histological evidence of glycogen deposition in cardiac muscle. The activity of the lysosomal enzyme alpha-1,4-glucosidase was deficient in skin fibroblasts, skeletal muscle, cardiac muscle, and liver; however, the enzymatic activity in peripheral blood leukocytes was in the low normal range. The child's mother had normal enzymatic activity in leukocytes but heterozygote levels in skin fibroblasts.

Topics: alpha-Glucosidases; Child, Preschool; Fibroblasts; Genes, Recessive; Genetic Variation; Glucosides; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Hymecromone; Leukocytes; Male; Muscles; Muscular Diseases; Respiratory Insufficiency

The diagnosis of Pompe's disease by the assay of acid alpha-glucosidase in kidney and leucocytes was not previously possible because of the presence of another component which had activity at pH 4.0, but was not deficient in the disease. This problem was resolved either by the use of the inhibitors, turanose, maltose and citrate, or by isoelectric precipitation at pH 5.0, which enabled the estimation of acid alpha glucosidase in kidney and leucocytes.

Topics: alpha-Glucosidases; Glucosides; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Hymecromone; Kidney; Leukocytes; Umbelliferones

The possible interference of neutral alpha-D-glucosidase in the diagnosis of Pompe's disease using 4-methylumbelliferyl-alpha-D-glucopyranoside as substrate for the assay of acid alpha-D-glucosidase was investigated. The pH profile of alpha-D-glucosidase in control skin fibroblasts and amniotic fluid cells showed two peaks of activity. The shape of the pH profile depended upon whether or not the extract was added to the buffer before the substrate. If extract was added to the buffer before the substrate, a greater separation was obtained between the two peaks of activity. The neutral alpha-D-glucosidase activity could be totally removed by preliminary precipitation at pH 5.0. Following acid region whilst Pompe's cells had no activity enabling a clear distinction to be made between carriers and the disease state.

Topics: Amniotic Fluid; Cells, Cultured; Drug Stability; Female; Fibroblasts; Glucosidases; Glucosides; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Hydrogen-Ion Concentration; Hymecromone; Kinetics; Pregnancy; Prenatal Diagnosis; Skin