Diagnosing Lysosomal Storage Disorders: Pompe Disease

Olaf A. Bodamer1, Angela Dajnoki1

1 Division of Clinical and Translational Genetics, Dr. John T. MacDonald Foundation, Department of Human Genetics, University of Miami Miller School of Medicine, Miami, Florida
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 17.11
DOI:  10.1002/0471142905.hg1711s75
Online Posting Date:  October, 2012
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Pompe disease is a lysosomal storage disorder caused by a deficiency of acid alpha glucosidase (GAA). Diagnosis of Pompe disease is typically based on an enzyme analysis of blood or tissues, such as fibroblasts, followed by confirmation through molecular testing. The advent of fluorometric and mass spectrometry methods for enzyme analysis in dried blood spots (DBS) has simplified the diagnostic approach for Pompe disease, facilitating high‐throughput screening of at‐risk populations and newborn infants. The following unit will provide the detailed analytical protocol for measurement of GAA activity in DBS using tandem mass spectrometry. Curr. Protoc. Hum. Genet. 75:17.11.1‐17.11.6. © 2012 by John Wiley & Sons, Inc.

Keywords: dried blood spot; acid maltase; alpha glucosidase; tandem mass spectrometry; Pompe disease; glycogen

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:

  • Filter card (Whatman multipart 903 neonatal screening paper) containing dried blood or used blank without blood
  • Extraction buffer (see recipe)
  • GAA assay cocktail (see recipe) Ethyl acetate (Merck)
  • Methanol (Merck)
  • Distilled water (Braun)
  • Nitrogen
  • Silica gel (Sigma)
  • Acetonitrile (Fischer Scientific)
  • Hand‐held puncher for 3‐mm punches or DBS puncher (automated system; Perkin Elmer)
  • 96‐well flat‐bottom plate (Greiner Bio‐One)
  • Silicone plate sealer (Pall)
  • NCS incubator (Perkin Elmer) with shaker
  • 96‐well deep‐well plate (Brand)
  • Centrifuge (Beckmann Coulter)
  • Minivap (Porvair Sciences) or custom‐made system
  • 96‐well deep 0.45‐µm polypropylene filter plate (Pall)
  • 96‐well conical‐bottom plate (Fisher Scientific BD Falcon)
  • 96‐well plate vacuum manifold (Porvair Sciences)
  • Tandem mass spectrometer (API 2000) and autosampler
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Literature Cited

Literature Cited
   Chamoles, N.A., Niizawa, G., Blanco, M., Gaggioli, D., and Casentini, C. 2004. Glycogen storage disease type II: Enzymatic screening in dried blood spots on filter paper. Clin. Chim. Acta 347:97‐102.
   Dajnoki, A., Muhl, A., Fekete, G., Keutzer, J., Orsini, J., Dejesus, V., Zhang, X.K., and Bodamer, O.A. 2008. Newborn screening for Pompe Disease by measuring acid alpha‐glucosidase activity using tandem mass spectrometry. Clin. Chem. 54:1624‐1629.
   Desnuelle, C. and Salviati, L. 2011. Challenges in diagnosis and treatment of late‐onset Pompe disease. Curr. Opin. Neurol. 24:443‐448.
   Duffner, P.K., Caggana, M., Orsini, J.J., Wenger, D.A., Patterson, M.C., Crosley, C.J., Kurtzberg, J., Arnold, G.L., Escolar, M.L., Adams, D.J., Andriola, M.R., Aron, A.M., Ciafaloni, E., Djukic, A., Erbe, R.W., Galvin‐Parton, P., Helton, L.E., Kolodny, E.H., Kosofsky, B.E., Kronn, D.F., Kwon, J.M., Levy, P.A., Miller‐Horn, J., Naidich, T.P., Pellegrino, J.E., Provenzale, J.M., Rothman, S.J., and Wasserstein, M.P. 2009. Newborn screening for Krabbe disease: The New York State model. Pediatr. Neurol. 40:245‐255.
   Gelb, M.H., Turecek, F., Scott, C.R., and Chamoles, N.A. 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.
   Hirschhorn, R. and Reuser, A.J. 2001. Glycogen storage disease type II: Acid α‐glucosidase (acid maltase) deficiency. In The Metabolic and Molecular Basis of Inherited Disease, 8th ed. (C.R. Scriver, A.L. Beaudet, C. Valle, and W.S. Sly, eds.) pp. 3389‐3420. McGraw‐Hill, New York.
   Li, Y., Scott, C.R., Chamoles, N.A., Ghavami, A., Pinto, B.M., Turecek, F., and Gelb, M.H. 2004. Direct multiplex assay of lysosomal enzymes in dried blood spots for newborn screening. Clin. Chem. 50:1785‐1796.
   Mechtler, T.P., Stary, S., Metz, T.F., De Jesus, V.R., Greber‐Platzer, S., Pollak, A., Herkner, K.R., Streubel, B., and Kasper, D.C. 2012. Neonatal screening for lysosomal storage disorders: Feasibility and incidence from a nationwide study in Austria. Lancet 379:335‐341.
   Müller, K.B., Rodrigues, M.D., Pereira, V.G., Martins, A.M., and D'Almeida, V. 2010. References values for lysosomal enzymes activities using dried blood spots samples‐a Brazilian experience. Diagn. Pathol. 5:65‐68.
   Sista, R.S., Eckhardt, A.E., Wang, T., Graham, C., Rouse, J.L., Norton, S.M., Srinivasan, V., Pollack, M.G., Tolun, A.A., Bali, D., Millington, D.S., and Pamula, V.K. 2011. Digital microfluidic platform for multiplexing enzyme assays: implications for lysosomal storage disease screening in newborns. Clin. Chem. 57:1444‐1451.
   Van der Ploeg, A.T. and Reuser, A.J. 2008. Pompe's disease. Lancet 372:1342‐1353.
   Wittmann, J., Karg, E., Turi, S. et al. 2012. Newborn screening for lysosomal storage disorders in Hungary. J. Inherit Metab. Dis. In press.
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