Diagnosing Lysosomal Storage Disorders: Mucopolysaccharidosis Type I

Britt A. Johnson1, Angela Dajnoki1, Olaf A. 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 17.17
DOI:  10.1002/0471142905.hg1717s84
Online Posting Date:  January, 2015
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Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder due to deficiency of alpha iduronidase (IDUA). Progressive storage of dermatan and heparan sulfate throughout the body lead to a multiorgan presentation including short stature, dysostosis multiplex, corneal clouding, hearing loss, coarse facies, hepatosplenomegaly, and intellectual disability. Diagnosis of MPS I is based on IDUA enzyme analysis in leukocytes or dried blood spots (DBS) followed by molecular confirmation of the IDUA gene mutations in individuals with low enzyme activity. The advent of mass spectrometry methods for enzyme analysis in DBS has enabled high‐throughput screening for MPS I in symptomatic individuals and newborn infants. The following unit provides the detailed analytical protocol for measurement of IDUA activity in DBS using tandem mass spectrometry. © 2015 by John Wiley & Sons, Inc.

Keywords: dried blood spot; alpha‐iduronidase; tandem mass spectrometry; MPS I; Mucopolysaccharidosis type I; Hurler Syndrome; Scheie Syndrome

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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)
  • IDUA assay cocktail (see recipe)
  • HPLC‐grade water (J.T. Baker)
  • Ethyl acetate (Merck)
  • Methanol (Merck)
  • Nitrogen
  • Silica gel (Sigma, 230‐400 mesh)
  • Acetonitrile (Fisher Scientific)
  • Formic acid
  • 96‐well deep‐well plate (VWR)
  • Hand‐held puncher for 3‐mm punches or DBS puncher (automated system; Perkin Elmer)
  • Multichannel pipets
  • Silicone plate sealer (Pall)
  • iEMS Incubator/Shaker for 96 well plates (Thermo scientific)
  • Sonicator
  • 96‐well round‐bottom solvent resistant plate (Corning)
  • Centrifuge (Beckmann Coulter)
  • Vortex mixer, optional
  • MiniVap (Perkin Elmer) or custom‐made system
  • 96‐well 0.45‐μm polypropylene filter plate (Pall)
  • 96‐well plate vacuum manifold (Porvair Sciences)
  • Tandem mass spectrometer (eg, API 4000) and autosampler
  • 96‐well plate conical‐bottom solvent‐resistant plate (NUNC)
  • NOTE: A multichannel pipet is used when multiple samples are analyzed.
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Literature Cited

Literature Cited
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