Diagnosing Lysosomal Storage Disorders: Mucopolysaccharidosis Type II

Britt A. Johnson1, Otto P. van Diggelen2, Angela Dajnoki1, Olaf A. Bodamer1

1 University of Miami Miller School of Medicine, Miami, Florida, 2 Erasmus University Medical College, Rotterdam, The Netherlands
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 17.14
DOI:  10.1002/0471142905.hg1714s79
Online Posting Date:  October, 2013
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Mucopolysaccharidosis type II (MPS II) is an X‐linked lysosomal storage disorder caused by a deficiency of iduronate 2‐sulfatase (IDS). Progressive, intralysosomal accumulation of the glycosaminoglycans (GAGs) dermatan and heparan sulfate in almost all tissues leads to multi‐organ involvement in affected males but to virtual absence of symptoms in heterozygote female carriers due to preferential inactivation of the mutant allele. Diagnosis of MPS II in males is based on IDS analysis in leukocytes, fibroblasts, plasma, or dried blood spots (DBS), whereas IDS activities may be within the normal range in heterozygote females. The advent of fluorometric and mass spectrometry methods for enzyme analysis in DBS has simplified the diagnostic approach for MPS II males. Molecular analysis of the IDS gene confirms the diagnosis of MPS II in males and is the only diagnostic test to confirm carrier status in females. This unit provides detailed analytical protocols for measurement of IDS activity in DBS and plasma using a fluorometric assay. Curr. Protoc. Hum. Genet. 79:17.14.1‐17.14.9. © 2013 by John Wiley & Sons, Inc.

Keywords: dried blood spot; alpha‐iduronate‐2‐sulfatase; fluorometry; mucopolysaccharidosis Type II; MPS II; Hunter Syndrome; mucopolysaccharide; glycosaminoglycans

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Table of Contents

  • Introduction
  • Basic Protocol 1: MPS II Dried Blood Spot Assay
  • Alternate Protocol 1: MPS II Plasma Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
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Basic Protocol 1: MPS II Dried Blood Spot Assay

  • Filter card (Whatman multipart 903 neonatal screening filter paper) containing dried blood from patient suspected to have MPS II
  • Filter card with normal and abnormal dried blood control samples
  • 0.2% bovine serum albumin (BSA; see recipe)
  • Substrate (4‐methylumbelliferyl‐iduronide‐2‐sulfate; Moscerdam Substrates #M2; http://moscerdam.com/)
  • Stop buffer (see recipe)
  • Pi/Ci buffer (see recipe)
  • Purified lysosomal enzymes, reconstituted (supplied with substrate; see recipe)
  • 4‐methylumbelliferyl standard (4 MU; see recipe)
  • Hand‐held puncher for 3‐mm punches or DBS puncher (automated system; Perkin Elmer)
  • 96‐well flat‐bottom plate (Greiner Bio‐One)
  • NCS 96‐well plate incubator (Perkin Elmer) with shaker
  • Multichannel pipettor and tips
  • Reagent reservoirs for use with multichannel pipettors (VWR)
  • 96‐well conical‐bottom plate (Fisher Scientific BD Falcon)
  • Silicone plate sealer (Pall)
  • Aluminum foil
  • White 96‐well flat‐bottom plate (Greiner Bio‐One)
  • 96‐well plate fluorometer (e.g., Bio‐Tek Synergy HT)

Alternate Protocol 1: MPS II Plasma Assay

  Additional Materials
  • Plasma from patient suspected to have MPS II
  • Plasma from normal and abnormal control samples
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Literature Cited

Literature Cited
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  Civallero, G., Michelin, K., de Mari, J., Viapiana, M., Burin, M., Coelho, J.C., and Giugliani, R. 2006. Twelve different enzyme assays on dried‐blood filter paper samples for detection of patients with selected inherited lysosomal storage diseases. Clin. Chim. Acta 372:98‐102.
  Nelson, J., Crowhurst, J., Carey, B., and Greed, L. 2003. Incidence of the mucopolysaccharidoses in Western Australia. Am. J. Med. Genet. A 123A:310‐313.
  Neufeld, E.F. and Muenzer, J. 2001. The mucopolysaccharidoses. In The Metabolic and Molecular Basis of Inherited Disease, 8th ed. (C.R Scriver, A.L. Beaudet, W.S. Sly, and D. Valle, eds.) pp. 3421‐3452. McGraw‐Hill, New York.
  Poorthuis, B.J., Wevers, R.A., Kleijer, W.J., Groener, J.E., de Jong, J.G., van Weely, S., Niezen‐Koning, K.E., and van Diggelen, O.P. 1999. The frequency of lysosomal storage diseases in The Netherlands. Hum. Genet. 105: 151‐156.
  Tuschl, K., Gal, A., Paschke, E., Kircher, S., and Bodamer, O.A. 2005. Mucopolysaccharidosis type II in females: Case report and review of literature. Pediatr. Neurol. 32:270‐272.
  Voznyi, Y.V., Keulemans, J.L., and van Diggelen, O.P. 2001. A fluorimetric enzyme assay for the diagnosis of MPS II (Hunter disease). J. Inherit. Metab. Dis. 24:675‐680.
  Wang, R.Y., Bodamer, O.A., Watson, M.S., and Wilcox, W.R. 2011. Lysosomal storage diseases: Diagnostic confirmation and management of presymptomatic individuals. Genet. Med. 13:457‐484.
  Wraith, J.E., Scarpa, M., Beck, M., Bodamer, O.A., De Meirleir, L., Guffon, N., Meldgaard Lund, A., Malm, G., Van der Ploeg, A.T., and Zeman, J. 2008. Mucopolysaccharidosis type II (Hunter syndrome): A clinical review and recommendations for treatment in the era of enzyme replacement therapy. Eur. J. Pediatr. 167:267‐277.
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