Diagnosing Lysosomal Storage Disorders: Fabry Disease

Olaf A. Bodamer1, Britt Johnson1, 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 17.13
DOI:  10.1002/0471142905.hg1713s77
Online Posting Date:  April, 2013
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Fabry disease (FD) is an X‐linked lysosomal storage disorder due to deficiency of alpha galactosidase A (GLA). Progressive, intralysosomal accumulation of neutral glycosphingolipids in endothelial cells and podocytes leads to multi‐organ involvement in affected males and to a lesser extent in affected females. Diagnosis of FD is based on GLA analysis in leukocytes or dried blood spots (DBS) in FD males while GLA activities may be within the normal range in FD females. The advent of fluorometric and mass spectrometry methods for enzyme analysis in DBS has simplified the diagnostic approach for FD males, facilitating high‐throughput screening of at risk populations and newborn infants. However, the diagnostic mainstay for FD females remains molecular analysis of the GLA gene. The following unit will provide the detailed analytical protocol for measurement of GLA activity in DBS using tandem mass spectrometry. Curr. Protoc. Hum. Genet. 77:17.13.1‐17.13.7. © 2013 by John Wiley & Sons, Inc.

Keywords: dried blood spot; galactosidase alpha; tandem mass spectrometry; Fabry disease; glycosphingolipid

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Table of Contents

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

  • Filter card (Whatman multipart 903 neonatal screening filter paper) containing dried blood
  • Extraction buffer (see recipe)
  • GLA assay cocktail [see recipe; internal standard and GLA substrate are provided free of charge through the Centers for Disease Control and Prevention (CDC; http://www.cdc.org)]
  • Ethyl acetate (Merck)
  • Methanol (Merck)
  • Distilled water (Braun)
  • Nitrogen
  • Silica gel (Sigma, 230‐400 mesh)
  • 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)
  • Blank filter paper
  • 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 conical‐bottom plate (Fisher Scientific BD Falcon)
  • 96‐well deep 0.45‐µm polypropylene filter plate (Pall)
  • 96‐well plate vacuum manifold (Porvair Sciences)
  • Tandem mass spectrometer (API 2000) and autosampler
NOTE: A multi‐channel pipettor is used when multiple samples are analyzed.
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Literature Cited

Literature Cited
   Dajnoki, A., Fekete, G., Keutzer, J., Orsini, J.J., De Jesus, V.R., Chien, Y.H., Hwu, W.L., Lukacs, Z., Muhl, A., Zhang, X.K., and Bodamer, O. 2010. Newborn screening for Fabry disease by measuring GLA activity using tandem mass spectrometry. Clin. Chim. Acta. 411:1428‐1431.
   Desnick, R.J., Ioannou, Y.A., and Eng, C.M. 2001. α‐Galactosidase A deficiency: Fabry Disease. In The Metabolic and Molecular Basis of Inherited Disease, 8th ed. (C.R. Scriver, W.S. Sly, B. Childs, A.L. Beaudet, D. Valle, K.W. Kinzler, and B. Vogelstein, eds.) pp. 3733‐3774. McGraw‐Hill, New York.
   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.
   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.
   MacDermot, K.D., Holmes, A., and Miners, A.H. 2001a. Anderson‐Fabry disease: Clinical manifestations and impact of disease in a cohort of 60 obligate carrier females. J. Med. Genet. 38:769‐775.
   MacDermot, K.D., Holmes, A., and Miners, A.H. 2001b. Anderson‐Fabry Disease: Clinical manifestations and impact of disease in a cohort of 98 hemizygous males. J. Med. Genet. 38:750‐760.
   Pisani, A., Visciano, B., Roux, G.D., Sabbatini, M., Porto, C., Parenti, G., and Imbriaco, M. 2012. Enzyme replacement therapy in patients with Fabry disease: State of the art and review of the literature. Mol. Genet. Metab. 107:267‐275.
   Schaefer, E., Mehta, A., and Gal, A. 2005. Genotype and phenotype in Fabry disease: Analysis of the Fabry Outcome Survey. Acta Paediatr. Suppl. 94:87‐92.
   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.
   Wittman, J., Karg, E., Turi, S., Legnini, E., Wittmann, G., Giese, A.K., Lukas, J., Gölnitz, U., Klingenhäger, M., Bodamer, O., Mühl, A., and Rolfs, A. 2012. Newborn screening for lysosomal storage disorders in Hungary. J. Inherit. Metab. Dis. 6:117‐125.
   Zarate, Y.A. and Hopkin, R.J. 2008. Fabry's Disease. Lancet 372:1427‐1435.
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