Enzyme analysis for Pompe disease in leukocytes; superior results with natural substrate compared with artificial substrates.

Enzyme analysis for Pompe disease in leukocytes has been greatly improved by the introduction of acarbose, a powerful inhibitor of interfering alpha-glucosidases, which are present in granulocytes but not in lymphocytes. Here we show that the application of acarbose in the enzymatic assay employing the artificial substrate 4-methylumbelliferyl-alpha-D: -glucoside (MU-alphaGlc) is insufficient to clearly distinguish patients from healthy individuals in all cases. Also, the ratios of the activities without/with acarbose only marginally discriminated Pompe patients and healthy individuals. By contrast, when the natural substrate glycogen is used, the activity in leukocytes from patients (n = 82) with Pompe disease is at most 17% of the lowest control value. The use of artificial substrate in an assay with isolated lymphocytes instead of total leukocytes is a poor alternative as blood samples older than one day invariably yield lymphocyte preparations that are contaminated with granulocytes. To diagnose Pompe disease in leukocytes we recommend the use of glycogen as substrate in the presence of acarbose. This assay unequivocally excludes Pompe disease. To also exclude pseudo-deficiency of acid alpha-glucosidase caused by the sequence change c.271G>A (p.D91N or GAA2; homozygosity in approximately 1:1000 caucasians), a second assay employing MU-alphaGlc substrate plus acarbose or DNA analysis is required.

Molecular and functional characterization of eight novel GAA mutations in Italian infants with Pompe disease.

We characterized 29 unrelated patients presenting with the severe form of Pompe disease (Glycogen Storage Disease Type II, acid maltase deficiency) and identified 26 pathogenic mutations divided over 28 different genotypes. Among the eight new mutations, five were exonic point mutations (c.572A>G, c.1124G>T, c.1202A>G, c.1564C>G and c.1796C>A) leading to codon changes (p.Y191C, p.R375L, p.Q401R, p.P522A and p.S599Y); two were intronic point mutations (c.-32-3C>A and c.1636+5G>C) affecting mRNA processing; one was a single base deletion (c.742delC) generating a truncated protein (p.L248PfsX20). A comprehensive evaluation, based on different methodological approaches, confirmed the detrimental effect of the eight mutations on the protein and its function. Structural alterations potentially induced by the five missense mutations were also predicted through visual inspection of the atomic model of the GAA protein, in terms of both function and spatial orientation of specific residues as well as disturbance generated by amino acid substitutions. Although the remarkable heterogeneity of the mutational spectrum in Pompe disease was already known, our data demonstrate and confirm the power of molecular and functional analysis in predicting the natural course of Pompe disease.

Cardiac evaluation in children and adults with Pompe disease sharing the common c.-32-13T>G genotype rarely reveals abnormalities.

BACKGROUND AND OBJECTIVE: Pompe disease is an inherited metabolic disorder caused by deficiency of acid alpha-glucosidase. All affected neonates have a severe hypertrophic cardiomyopathy, leading to cardiac failure and death within the first year of life. We investigated the presence and extent of cardiac involvement in children and adults with Pompe disease with the common c.-32-13T>G genotype to determine the usefulness of cardiac screening in these patients with relatively 'milder' phenotypes. METHODS: Cardiac dimensions and function were evaluated through echocardiography, electrocardiography and Holter monitoring. The total group comprised 68 patients with Pompe disease, of whom 22 patients had disease onset before the age of 18. RESULTS: Two patients (3%) had cardiac abnormalities possibly related to Pompe disease: Electrocardiography showed a Wolff-Parkinson-White pattern in an 8-year-old girl, and one severely affected adult patient had a mild hypertrophic cardiomyopathy. This hypertrophy did not change during treatment with recombinant human alpha-glucosidase. In addition, four adult patients showed minor cardiac abnormalities which did not exceed the prevalence in the general population and were attributed to advanced age, hypertension or pre-existing cardiac pathology unrelated to Pompe disease. CONCLUSIONS: Cardiac involvement is rare in Pompe patients with the common c.-32-13T>G genotype. The younger patients were not more frequently affected than the adults. Electrocardiographic evaluation appears to be appropriate as initial screening tool. Extensive cardiac screening seems indicated only if the electrocardiogram is abnormal or the patient has a history of cardiac disease.

Clinical diversity in glycogenosis type II. Biosynthesis and in situ localization of acid alpha-glucosidase in mutant fibroblasts.

The molecular basis of clinical diversity in glycogenosis type II (Pompe's disease) was investigated by comparing the nature of acid alpha-glucosidase deficiency in cultured fibroblasts from 30 patients. Biosynthetic forms of acid alpha-glucosidase with different molecular mass were separated electrophoretically and identified by immunoblotting. Immuno-electron microscopy was employed to determine the intracellular localization of mutant enzyme. Our studies illustrate that maturation of acid alpha-glucosidase is associated with transport to the lysosomes. Deficiency of catalytically active mature enzyme in lysosomes is common to all clinical phenotypes but, in the majority of cases, is more profound in early onset than in late onset forms of the disease. Thus, the results suggest that the clinical course of glycogenosis type II is primarily determined by the amount of functional acid alpha-glucosidase. The role of secondary factors can, however, not be excluded because three adult patients were identified with very low activity and little enzyme in the lysosomes.

Intravenous administration of phosphorylated acid alpha-glucosidase leads to uptake of enzyme in heart and skeletal muscle of mice.

The lysosomal storage disorder glycogenosis type II is caused by acid alpha-glucosidase deficiency. In this study we have investigated the possible applicability of mannose 6-phosphate receptor-mediated enzyme replacement therapy to correct the enzyme deficiency in the most affected tissues. Bovine testes acid alpha-glucosidase containing phosphorylated mannose residues was intravenously administered to mice and found to be taken up by heart (70% increase of activity) and skeletal muscle (43% increase); the major target organs. The uptake of nonphosphorylated human placenta acid alpha-glucosidase by heart and skeletal muscle appeared to be significantly less efficient, whereas uptake of dephosphorylated bovine testes enzyme was not detectable. The phosphorylated bovine testes acid alpha-glucosidase remained present in mouse skeletal muscle up to 9-15 d after administration, with a half-life of 2-4 d. Besides being measured in skeletal muscle and heart, uptake of phosphorylated bovine testes and nonphosphorylated human placenta acid alpha-glucosidase was measured in several other organs, but not in brain. The increase of acid alpha-glucosidase activity was highest in liver and spleen. We concluded that application of mannose 6-phosphate receptor-mediated enzyme replacement therapy may offer new perspectives for treatment of glycogenesis type II.

Seven cases of Pompe disease from Greece.

We present seven cases of Pompe disease (McKusick 232300; glycogen storage disease type II; acid maltase deficiency) from Greece. The onset of symptoms varied from early childhood to late adulthood, and the patients had quite variable duration of disease. All but one of them had muscle weakness and all had mildly to highly elevated serum creatine kinase. The diagnosis in all cases was confirmed by the finding of acid alpha-glucosidase (EC 3.2.1.3/20) deficiency in cultured skin fibroblasts. Thirteen mutant alleles were identified and nine different pathogenic mutations were encountered. Four were new: c.2071_2072insAGCCG leads to frameshift and total loss of function; c.1856G > A (p.Ser619Asn) leads to 90-95% loss of function; and the splice-site mutations c.1552-3C > G and c.2331+4A > G reduce the number of correct splicing events by more than 90%. The splice-site mutation c.-32-13T > G (IVS1-13T > G) was encountered four times and seems equally common among Greek and other caucasians. The other mutations: c.925G > A (p.Gly309Arg), c.[307T > G; 271G > A] (p.Cys103Gly; Asp91Asn), c.271del and c.1655T > C (p.Leu552Pro) have been reported earlier. Our study highlights the heterogeneity of Pompe disease in Greece and provides tools for diagnosis and carrier detection.

Childhood Pompe disease: clinical spectrum and genotype in 31 patients.

BACKGROUND: As little information is available on children with non-classic presentations of Pompe disease, we wished to gain knowledge of specific clinical characteristics and genotypes. We included all patients younger than 18 years, who had been evaluated at the Pompe Center in Rotterdam, the Netherlands, between 1975 and 2012, excluding those with the classic-infantile form. None were treated with enzyme replacement therapy at the time of evaluation. We collected information on first symptoms, diagnosis, use of a wheelchair and/or respirator, and enzyme and mutation analysis and assessed muscle strength, pulmonary function, and cardiac parameters. RESULTS: Thirty-one patients participated. Median age at symptom onset was 2.6 years (range 0.5-13y) and at diagnosis 4.0 years. Most first problems were delayed motor development and problems related to limb-girdle weakness. Fatigue, persistent diarrhea and problems in raising the head in supine position were other first complaints. Ten patients were asymptomatic at time of diagnosis. Five of them developed symptoms before inclusion in this study. Over 50 % of all patients had low or absent reflexes, a myopathic face, and scoliosis; 29 % were underweight. Muscle strength of the neck flexors, hip extensors, hip flexors, and shoulder abductors were most frequently reduced. Pulmonary function was decreased in over 48 % of the patients; 2 patients had cardiac hypertrophy. Patients with mutations other than the c.-32-13T > G were overall more severely affected, while 18 out of the 21 patients (86 %) with the c.-32-13T > G/'null' genotype were male. CONCLUSIONS: Our study shows that Pompe disease can present with severe mobility and respiratory problems during childhood. Pompe disease should be considered in the differential diagnosis of children with less familiar signs such as disproportional weakness of the neck flexors, unexplained fatigue, persistent diarrhea and unexplained high CK/ASAT/ALAT. Disease presentation appears to be different from adult patients. The majority of affected children with GAA genotype c.-32-13T > G/'null' appeared to be male.

Binding sites of iron transferrin on rat reticulocytes. Inhibition by specific antibodies.

1. In the process of iron uptake by precursors of the erythrocytes probably more than one membrane component is involved; besides the specific transferrin receptor, another membrane component with a high iron activity after incubation with 59Fe can be isolated. 2. A striking resemblance exists between rat and human reticulocyte components which are involved in the process of iron uptake. 3. Incubation of reticulocytes with Fab fragments of an antibody against the membrane receptor for transferrin causes a concentration-dependent decrease in transferrin binding and iron uptake. 4. The membrane receptor complex isolated is still heterogeneous; analytical ultracentrifugation studies suggest a molecular weight lower than 230,000. 5. Intact immature red cells are necessary for specific binding of transferrin with the receptor followed by iron uptake. This is the only mechanism for iron uptake. 6. Immunofluorescence studies showed that the receptor for transferrin is localized at the outside of the cell membrane.

Cell-free translation of human lysosomal alpha-glucosidase: evidence for reduced precursor synthesis in an adult patient with glycogenosis type II.

Early events in the biosynthesis of alpha-glucosidase (EC 3.2.1.20) were studied in a wheat-germ cell-free translation system, using control and mutant RNA. In vitro, the primary translation product of the alpha-glucosidase mRNA is a 100 kDa protein. When canine microsomal membranes are added to the translation system, the nascent alpha-glucosidase precursor is cotranslationally transported across the microsomal membranes, yielding a 110 kDa glycosylated form. This protein has the same electrophoretic characteristics as the alpha-glucosidase precursor observed after in vivo labeling of control fibroblasts. Inhibition of glycosylation in vivo by tunicamycin or deglycosylation of the in vivo synthesized alpha-glucosidase precursor by glycopeptidase F reveals a core protein similar in molecular mass to the primary translation product. Total RNA from a patient with the adult form of glycogenosis type II is not able to direct the synthesis of normal amounts of alpha-glucosidase in vitro. Northern blot analysis of the RNA, using cloned alpha-glucosidase cDNA sequences as a probe, demonstrates that in this patient the amount of the 3.4 kb alpha-glucosidase mRNA is highly reduced. The results indicate that the synthesis or stability of the mRNA is affected.

The uptake of iron by reticulocytes. The influence of purification of the ghosts on iron-containing components in the ghost suspension.

1. Rat reticulocytes previously incubated with ((59)Fe, (125)I)-labelled transferrin were hemolysed to yield labelled ghosts. 2. The solubilized ghosts can be fractionated, by gel filtration on Sepharose 2B and 6B, into several (59)Fe- and (125)I-containing compounds, classified as A, B(1) and B(2). 3. These fractions were prepared from ghosts which were obtained at different centrifugation rates and further purified by sucrose density gradient centrifugation in order to obtain membrane compounds purified from mitochondrial and lysosomal impurities. The influence of these purification steps on the appearance and the (59)Fe/(125)I activity of the three components was investigated. 4. The first Sepharose 2B fraction with high molecular mass, greater than 10(6) daltons, is an intracellular product of mitochondrial and lysosomal origin which precipitates with the membrane fractions during the preparation of the ghost. The first Sepharose 6B fraction, B(1), with M(r) approximately 10(6) seems to be a real membrane component. The second Sepharose 6B fraction, B(2), with M(r) approximately 230 000 represents a real membrane receptor transferrin.

Determination of the structure of the carbohydrate chains of acid alpha-glucosidase from human placenta.

Acid alpha-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) from human placenta (70 and 76 kDa) was found to contain 4 N-glycosidic carbohydrate chains per molecule. Sugar analysis of purified enzyme revealed the presence of mannose, N-acetylglucosamine and fucose at a molar ratio of 5.0:2.0:0.6. In addition, trace amounts of galactose and N-acetylneuraminic acid were detected. The sugar chains were liberated from the polypeptides by the hydrazinolysis procedure and subsequently fractionated by gel filtration and HPLC. Purified compounds were investigated by 500-MHz 1H-NMR spectroscopy. Oligomannoside-type chains of intermediate size, e.g., Man5GlcNAcGlcNAc-ol and Man7GlcNAcGlcNAc-ol, and N-type chains of smaller size e.g., Man2-3GlcNAc[Fuc]0-1GlcNAc-ol, were demonstrated to be present at a ratio of 2:3. In addition, a small amount of sialylated N-acetyllactosamine-type chains has been found. The possible biosynthetic route of the fucose-containing small-size chains is discussed.

Expression of cDNA-encoded human acid alpha-glucosidase in milk of transgenic mice.

Enzyme replacement therapy is at present the option of choice for treatment of lysosomal storage diseases. To explore the feasibility of lysosomal enzyme production in milk of transgenic animals, the human acid alpha-glucosidase cDNA was placed under control of the alpha S1-casein promoter and expressed in mice. The milk contained recombinant enzyme at a concentration up to 1.5 micrograms/ml. Enzyme purified from milk of transgenic mice was internalized via the mannose 6-phosphate receptor and corrected enzyme deficiency in fibroblasts from patients. We conclude that transgenically produced human acid alpha-glucosidase meets the criteria for therapeutic application.

Frequency of glycogen storage disease type II in The Netherlands: implications for diagnosis and genetic counselling.

Glycogen storage disease type II (GSD H) is an autosomal recessive myopathy. Early and late-onset phenotypes are distinguished - infantile, juvenile and adult. Three mutations in the acid alpha-glucosidase gene are common in the Dutch patient population: IVS1(-13T-->G), 525delT and delexon18. 63% of Dutch GSD II patients carry one or two of these mutations, and the genotype-phenotype correlation is known. To determine the frequency of GSD II, we have screened an unselected sample of neonates for the occurrence of these three mutations. Based on the calculated carrier frequencies, the predicted frequency of the disease is 1 in 40000 divided by 1 in 138 000 for infantile GSD II and 1 in 57 000 for adult GSD II. This is about two to four times higher than previously suggested, which is a reason to become more familiar with the presentation of GSD II in its different clinical forms and to adjust the risk assessment for genetic counselling.

Adult forms of glycogenosis type II. A defect in an early stage of acid alpha-glucosidase realization.

The activity of acid alpha-glucosidase in cultured fibroblasts from adult patients with the lysosomal storage disease glycogenosis type II is only 10% of normal. A normal activity per molecule is found for the mature as well as for the precursor form of acid alpha-glucosidase in adult mutant fibroblasts. Excessive lysosomal breakdown of mature enzyme purified from mutant fibroblasts and taken up by acceptor cells does not occur. However, the NH4Cl-stimulated secretion of a precursor form of acid alpha-glucosidase by adult mutant fibroblasts is markedly reduced. The results are indicative of a defect during the production of acid alpha-glucosidase.

The involvement of iron and lipid peroxidation in the pathogenesis of HCB induced porphyria.

Hexachlorobenzene (HCB) induces a porphyria characterized by a diminished activity of the enzyme uroporphyrinogen decarboxylase (URO-D), presumably due to inactivation by reactive metabolites of HCB. We studied the effect of iron on HCB porphyria in female rats, to determine whether the iron dependent process of lipid peroxidation was involved in the pathogenesis of porphyria. We showed that malondialdehyde formation is increased in rat liver tissue of porphyric rats and that high molecular weight proteins due to cross-linking are formed. We also showed that the induction of porphyria by HCB is dependent on the presence of iron. Our findings suggest that lipid peroxidation is involved in the toxicity of HCB and that the aggravating effects of iron on HCB are mediated by lipid peroxidation.

Binding of human isotransferrin variants to microvillous and basal membrane vesicles from human term placenta.

Transferrin (Tf)-dependent iron transfer from mother to fetus is mediated by Tf receptors (TfRs) which are present on both microvillous and basal membranes of human placental syncytiotrophoblast. We used microvillous and basal membrane vesicles, both isolated from the same human term placenta, to investigate the binding of [125I]-labelled diferric bi-bi antennary tetra-sialo Tf (bb Tf), bi-tri-antennary penta-sialo Tf (bt Tf) and tri-tri-antennary hexa-sialo Tf (tt Tf). To diminish the effect of endogenous Tf, membrane vesicles were washed before binding of [125I]-Tf. The number of TfRs on microvillous membranes was 6.1 +/- 2.4 (mean +/- s.d., n = 15) times higher than that on basal membranes, whereas the affinity of TfRs on basal membranes was 3.9 +/- 0.4 (mean +/- s.d., n = 15) times higher than that of TfRs on microvillous membranes, irrespective the isoTf used. The affinity constants of TfRs on both microvillous and basal membranes were higher for bb Tf than for bt Tf and higher for bt Tf than for tt Tf. However, these latter differences were rather small and probably not of physiological importance.

Ferritin in cultured human cytotrophoblasts: synthesis and subunit distribution.

The present study aims at the role of ferritin in the regulation of syncytiotrophoblast free iron levels. The differentiated cytotrophoblast cell in culture is used as a model for this maternal-fetal interface. Cytotrophoblast cells isolated from term placentae are cultured in iron-poor (Medium 199), iron-depleted [desferrioxamine(DFO)] and iron-supplemented [diferric transferrin (hTF-2Fe), ferric ammonium citrate (FAC)] medium. Distribution and de novo synthesis of isoferritins is studied, together with the cellular iron concentration and the ferritin iron saturation. Compared to ferritin isolated from total placenta, ferritin obtained from villous tissue is enriched with acidic isoforms. This observation is in agreement with measured light (L) to heavy (H) subunit ratios < 1 of de novo synthesized ferritin in cultured cytotrophoblast cells. Neither iron-poor culture medium, nor hTf-2Fe supplemented medium affects the cellular iron or ferritin concentration. FAC increased the cellular ferritin iron saturation and (by synthesis) the acidic isoferritin concentrations. The results strongly suggest, that the term syncytiotrophoblast is able to balance transferrin-mediated iron uptake and iron release. In case of FAC supplementation, the syncytiotrophoblast is unable to keep intracellular iron low, and ferritin synthesis is stimulated. The predominance of acidic ferritins and the preferential synthesis of H subunits can be functionally explained by the established fact that iron incorporation in acidic ferritins is faster due to the presence of ferroxidase centres. Damage by free iron catalysed hydroxyl radical formation is therefore minimized.

Non-transferrin iron uptake by trophoblast cells in culture. Significance of a NADH-dependent ferrireductase.

This study shows that trophoblast cells in culture are able to take up 59Fe from both Fe(III)nitrilotriacetate (NTA) and Fe-ascorbate. Fe in the presence of ascorbate is assumed to be Fe(III) in equilibrium with Fe(II). Kinetic parameters for non-transferrin iron uptake are determined from initial rate experiments, yielding Vmax=366 pmol/mg protein/5 min and Km=0.96 microM for Fe(III)NTA and Vmax=4043 pmol/mg protein/5 min and Km= 1.3 microM for Fe-ascorbate. Since trophoblast cells in culture reduce extracellular Fe(III)CN, and uptake of 59Fe from Fe-ascorbate is higher than that from Fe(III)NTA, it is suggested that reduction of Fe(III) precedes uptake. Uptake of 59Fe from both Fe-ascorbate and Fe(III)NTA is inhibited by Fe(II)chelator ferrozine and membrane-impermeable Fe(III)CN, further supporting this hypothesis. Studies with microvillous membrane vesicles (MMV) and basal membrane vesicles (BMV) reveal the presence of a NADH-dependent ferrireductase. Reduction of Fe(III)CN follows Michaelis-Menten kinetics, both with respect to [NADH] and [Fe]. NADPH is ineffective as electron donor. The rate of Fe(III)CN reduction by BMV is 2.5 times higher compared to MMV, while Km values for Fe(III)CN and NADH are not significantly different. These results reveal that a transmembrane NADH-dependent ferrireductase plays a role in uptake of non-transferrin iron. The possibility that this enzyme system is involved in iron transfer across the basal membrane is discussed.

Breakdown of lysosomal glycogen in cultured fibroblasts from glycogenosis type II patients after uptake of acid alpha-glucosidase.

Fibroblast cultures from patients with different clinical subtypes of glycogenosis type II were compared with respect to residual acid alpha-glucosidase activity and lysosomal glycogen content. Lysosomal glycogen storage was most pronounced in fibroblasts from patients with the rapidly progressive infantile form of the disease, and the most severe enzyme deficiency. In fibroblasts from adult patients with more than 10% of the control activity storage did not occur, and 15% of the total cellular glycogen was found in the lysosomes as in control cells. The strict correlation between residual acid alpha-glucosidase activity and lysosomal glycogen accumulation was further illustrated in two adult Pompe patients with an unusually low enzyme activity. The mild clinical course is unexplained in these particular cases. The enzyme deficiency in all the different mutant cell lines was corrected by the uptake of bovine testis acid alpha-glucosidase from the culture medium. As a result of this, the lysosomal glycogen storage disappeared, and the balance between lysosomal and cytoplasmic glycogen was restored to normal. The implications of this study as a model for enzyme replacement therapy are discussed.

Regulation of transferrin receptor expression and distribution in in vitro cultured human cytotrophoblasts.

During gestation the transplacental iron transport is very important to the fetus. Iron uptake by the placenta can be studied in cultured cytotrophoblasts. The influence of culture time and human differic transferrin on the number and distribution of transferrin receptors (TfRs) was investigated in human cytotrophoblasts. Cytotrophoblasts cultured for 2.5 h had few TfRs (0.28 pmol/mg protein). With time, total TfR amounts increase (4.14 pmol/mg protein at 70 h). They increase to a higher level in cells cultured in iron-poor medium, indicating that iron has an effect on the TfR synthesis/breakdown ratio. TfRs were distributed between two 'active' (located at the cell surface and intracellularly) and one 'inactive' (located intracellularly) receptor pools. TfR distribution among these pools was modulated by culture time and iron. Trophoblasts regulated iron uptake by variation of number of surface TfRs via changes in total TfRs and redistribution of TfRs among the receptor pools.