Summary
Enzyme analysis for Pompe disease in leukocytes has been greatly improved by the introduction of acarbose, a powerful inhibitor of interfering α-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-α-d-glucoside (MU-αGlc) 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 α-glucosidase caused by the sequence change c.271G>A (p.D91N or GAA2; homozygosity in approximately 1:1000 caucasians), a second assay employing MU-αGlc substrate plus acarbose or DNA analysis is required.
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Abbreviations
- DBS:
-
dried blood spot on filter paper
- GSD II:
-
glycogen storage disease type II, Pompe disease, acid maltase deficiency
- Glcn :
-
Glycogen
- MU:
-
4-methylumbelliferone
- MU-αGlc:
-
4-methylumbelliferyl-α-d-glucoside
- NEM:
-
N-ethylmaleimide
References
Bembi B, Cerini E, Danesino C, et al (2008) Diagnosis of glycogenosis type II. Neurology 71: S4–11. doi:10.1212/WNL.0b013e31818da91e.
Chien YH, Chiang SC, Zhang XK, et al (2008) Early detection of Pompe disease by newborn screening is feasible: results from the Taiwan screening program. Pediatrics 122: e39–45. doi:10.1542/peds.2007-2222.
Dajnoki A, Muhl A, Fekete G, et al (2008) Newborn screening for Pompe disease by measuring acid alpha-glucosidase activity using tandem mass spectrometry. Clin Chem 54: 1624–1629. doi:10.1373/clinchem.2008.107722.
De Jesus VR, Zhang XK, Keutzer J, et al (2009) Development and evaluation of quality control dried blood spot materials in newborn screening for lysosomal storage disorders. Clin Chem 55: 158–164. doi:10.1373/clinchem.2008.111864.
Gasparotto N, Tomanin R, Frigo AC, et al (2008) Rapid diagnostic testing procedures for lysosomal storage disorders: alpha-glucosidase and beta-galactosidase assays on dried blood spots. Clin Chim Acta 402(1–2): 38–41. doi:10.1016/j.cca.2008.12.006.
Gelb MH, Turecek F, Scott CR, Chamoles NA (2006) Direct multiplex assay of enzymes in dried blood spots by tandem mass spectrometry for the newborn screening of lysosomal storage disorders. J Inherit Metab Dis 29: 397–404. doi:10.1007/s10545-006-0265-4.
Hirschhorn R, Reuser AJJ (2001) Glycogen storage disease type II (GSDII). In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds; Childs B, Kinzler KW, Vogelstein B, assoc. eds. The Metabolic and Molecular Bases of Inherited Disease, 8th edn. New York: McGraw-Hill, 3389–3420.
Jack RM, Gordon C, Scott CR, Kishnani PS, Bali D (2006) The use of acarbose inhibition in the measurement of acid alpha-glucosidase activity in blood lymphocytes for the diagnosis of Pompe disease. Genet Med 8: 307–312. doi:10.1097/01.gim.0000217785.19262.9e.
Kallwass H, Carr C, Gerrein J, (2007) Rapid diagnosis of late-onset Pompe disease by fluorometric assay of alpha-glucosidase activities in dried blood spots. Mol Genet Metab 90: 449–452. doi:10.1016/j.ymgme.2006.12.006.
Li Y, Scott CR, Chamoles NA, et al (2004) Direct multiplex assay of lysosomal enzymes in dried blood spots for newborn screening. Clin Chem 50: 1785–1795. doi:10.1373/clinchem.2004.035907.
Martiniuk F, Bodkin M, Tzall S, Hirschhorn R (1990) Identification of the base-pair substitution responsible for a human acid α-glucosidase allele with lower “affinity” for glycogen (GAA 2) and transient gene expression in deficient cells. Am J Hum Genet 47: 440–445.
Okumiya T, Keulemans JL, Kroos MA, et al (2006) A new diagnostic assay for glycogen storage disease type II in mixed leukocytes. Mol Genet Metab 88: 22–28. doi:10.1016/j.ymgme.2005.10.016.
Swallow DM, Corney G, Harris H, Hirschhorn R (1975) Acid α-glucosidase: a new polymorphism in man demonstrable by ‘affinity’ electrophoresis. Ann Hum Genet 38: 391–406. doi:10.1111/j.1469-1809.1975.tb00629.x.
Swallow DM, Kroos M, Van der Ploeg AT, et al (1989) An investigation of the properties and possible clinical significance of the lysosomal α-glucosidase GAA*2 allele. Ann Hum Genet 53: 177–184. doi:10.1111/j.1469-1809.1989.tb01782.x.
van der Ploeg AT, Reuser AJ (2008) Pompe’s disease. Lancet 372: 1342–1353. doi:10.1016/S0140-6736(08)61555-X.
van Diggelen OP, Zhao H, Kleijer WJ, et al (1990) A fluorimetric enzyme assay for the diagnosis of Morquio disease type A (MPS IV A). Clin Chim Acta 187: 131–139. doi:10.1016/0009-8981(90)90339-T.
Winchester B, Bali D, Bodamer OA, et al. (2008) Methods for a prompt and reliable laboratory diagnosis of Pompe disease: report from an international consensus meeting. Mol Genet Metab 93: 275–281. doi:10.1016/j.ymgme.2007.09.006.
Zhang H, Kallwass H, Young SP, et al (2006) Comparison of maltose and acarbose as inhibitors of maltase-glucoamylase activity in assaying acid alpha-glucosidase activity in dried blood spots for the diagnosis of infantile Pompe disease. Genet Med 8: 302–306. doi:10.1097/01.gim.0000217781.66786.9b.
Zhang XK, Elbin CS, Chuang WL, et al (2008) Multiplex enzyme assay screening of dried blood spots for lysosomal storage disorders by using tandem mass spectrometry. Clin Chem 54: 1725–1728. doi:10.1373/clinchem.2008.104711.
Acknowledgements
We thank Ton de Wit for communicating the cytometry experiments.
This study was supported by a grant from Genzyme Corporation, Cambridge MA, USA and was part of the Dutch TI Pharma initiative to commence a project on Sustainable Orphan Drug Development through Registries and Monitoring (T6–208).
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Communicating editor: Guy Besley
Competing interests: None declared
References to electronic databases: Glycogen storage disease II (Pompe disease): #232300. Acid α-glucosidase: EC 3.2.1.3/20.
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van Diggelen, O.P., Oemardien, L.F., van der Beek, N.A.M.E. et al. Enzyme analysis for Pompe disease in leukocytes; superior results with natural substrate compared with artificial substrates. J Inherit Metab Dis 32, 416–423 (2009). https://doi.org/10.1007/s10545-009-1082-3
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DOI: https://doi.org/10.1007/s10545-009-1082-3