Diagnosis of Lysosomal Storage Disorders: Gaucher Disease

Britt A. Johnson1, Angela Dajnoki1, Olaf Bodamer1

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 17.15
DOI:  10.1002/0471142905.hg1715s82
Online Posting Date:  July, 2014
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Gaucher Disease (GD) is a progressive lysosomal storage disorder caused by deficiency of glucocerebrosidase (GBA). The clinical phenotype follows a spectrum ranging from severe early‐onset to milder late‐onset disease. The absence of neurological involvement defines GD type I, whereas neuronopathic features define GD type II and III. Early diagnosis may be important for timely initiation of enzyme replacement therapy to prevent disease complications, although the enzyme does not cross the blood brain barrier. Diagnosis of GD can be readily achieved by analysis of GBA in leukocytes, fibroblasts, and/or dried blood spots using fluorometric, microfluidic or mass spectrometry‐based assays. Low GBA activities are typically confirmed through molecular analysis of the GBA gene. GBA analysis in dried blood spots may be attractive for high‐throughput screening of at‐risk individuals and/or newborn infants. The method detailed in this unit is based on GBA analysis by tandem mass spectrometry following incubation of dried blood spots with the GBA‐specific substrate D‐glucosyl‐β1‐1′‐N‐dodecanoyl‐D‐erythro‐sphingosine [C12‐glucocerebroside (C36H69NO8)] and internal standard N‐myristoyl‐D‐erythro‐sphingosine [C14‐ceramide (C32H63NO3)]. GBA activities in more than 2,000 newborn infants showed a mean of 22.0 ± 13.8 μmol/hr/liter (median: 19.9 μmol/hr/liter; 95% CI: 21.41‐22.59 μmol/hr/liter). GBA activities in an adult population (n >1,200) showed generally lower enzyme activities than newborns, with a mean of 9.87 ± 9.35 μmol/hr/liter (median: 8.06 μmol/hr/liter). GBA activities in ten adult patients with confirmed GD were less than 4.2 μmol/hr/liter and in seven infants and children with GD less than 1.24 μmol/hr/liter. This method is robust, sensitive, and suitable for high‐throughput analysis of hundreds of samples. Curr. Protoc. Hum. Genet. 82:17.15.1‐17.15.6. © 2014 by John Wiley & Sons, Inc.

Keywords: dried blood spot; tandem mass spectrometry; Gaucher Disease; lysosomal storage disorder; glucocerebrosidase; GBA

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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
  • Extraction buffer (see recipe)
  • GBA assay cocktail (see recipe)
  • Ethyl acetate (B&J Honeywell)
  • Methanol (B&J Honeywell)
  • Distilled water (Braun)
  • Acetonitrile (B&J Honeywell)
  • Silica gel (Sigma‐Aldrich)
  • 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 (BD Falcon, Fisher Scientific)
  • 96‐well plate vacuum manifold (Porvair Sciences)
  • Tandem mass spectrometer (API 3000) and autosampler
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Literature Cited

Literature Cited
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  Chamoles, N.A., Blanco, M., and Gaggioli, D. 2001a. Fabry disease: enzymatic diagnosis in dried blood spots on filter paper. Clin. Chim. Acta 308:195‐196.
  Chamoles, N.A., Blanco, M., and Gaggioli, D. 2001b. Diagnosis of alpha‐l‐iduronidase deficiency in dried blood spots on filter paper: the possibility of newborn diagnosis. Clin. Chem. 47:780‐781.
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  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 Jesús, 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. Lancet 379:335‐341.
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