High‐Risk Screening for Fabry Disease: Analysis by Tandem Mass Spectrometry of Globotriaosylceramide (Gb3) in Urine Collected on Filter Paper

Christiane Auray‐Blais1, Pamela Lavoie1, Michel Boutin1, Mona Abaoui1

1 Division of Medical Genetics, Department of Pediatrics, Faculty of Medicine and Health Sciences, Centre de recherche du CHUS, Université de Sherbrooke, Sherbrooke, Quebec
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 17.26
DOI:  10.1002/cphg.34
Online Posting Date:  April, 2017
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Fabry disease is a complex, panethnic lysosomal storage disorder. It is characterized by the accumulation of glycosphingolipids in tissues, organs, the vascular endothelium, and biological fluids. The reported incidence in different populations is quite variable, ranging from 1:1400 to 1:117,000. Its complexity lies in the marked genotypic and phenotypic heterogeneity. Despite the fact that it is an X‐linked disease, more than 600 mutations affect both males and females. In fact, some females may be affected as severely as males. The purpose of this protocol is to focus on the high‐risk screening of patients who might have Fabry disease using a simple, rapid, non‐invasive high performance liquid chromatography‐tandem mass spectrometry (HPLC‐MS/MS) method for urinary globotriaosylceramide (Gb3) analysis. Urine filter paper samples are easily collected at home by patients and sent by regular mail. This method has been successfully used for high‐risk screening of patients with ophthalmologic manifestations and in an on‐going study for high‐risk screening of Fabry disease in patients with chronic kidney diseases. © 2017 by John Wiley & Sons, Inc.

Keywords: Fabry disease; filter paper sample; globotriaosylceramide (Gb3); high‐risk screening; mass spectrometry; urine

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Table of Contents

  • Introduction
  • Basic Protocol 1: Analysis of Globotriaosylceramide in Urine Collected on Filter Paper by Tandem Mass Spectrometry
  • Support Protocol 1: Urine Collection on Filter Paper
  • Support Protocol 2: Preparation of Positive Quality Controls
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Analysis of Globotriaosylceramide in Urine Collected on Filter Paper by Tandem Mass Spectrometry

  • Healthy control urine sample (for the calibration curve)
  • Urine samples to be analyzed ( protocol 2)
  • Quality control samples ( protocol 3)
  • Gb 3 calibration curve working solutions:
    • STD I Gb 3 isoform working solution (see recipe)
    • STD II Gb 3 isoform working solution (see recipe)
  • C17‐Gb 3 internal standard working solution (1 µg/100 µl; see recipe)
  • Creatinine standard curve working solution (10 mM; see recipe)
  • Creatinine‐d 3 internal standard working solution (40 μg/100 μl; see recipe)
  • Methanol (MeOH), LC‐MS grade (EMD Millipore, cat. no. MX0486‐1)
  • Water (H 2O), LC‐MS grade (EMD Millipore, cat. no. WX0001‐1)
  • 99% formic acid (Thermo Fisher Scientific, cat. no. AC27048‐0010)
  • Ammonium acetate (Sigma‐Aldrich, cat. no. A1542)
  • Whatman‐GE 903 filter papers, 10 × 10 cm
  • Ultraviolet lamp
  • 5‐cm pneumatic punch (homemade or commercial equivalent)
  • Graphite pencil
  • Parafilm
  • 20‐ml clear borosilicate vials
  • Pasteur pipets (Ultident, cat. no. 170‐CTB700‐3)
  • 2‐ml vials, screw tread, 12 × 32 mm, clear glass (Chromspec, cat. no. C779100WM)
  • Screw cap 9 mm, preslit PTFE/silicone septum (Chromspec, cat. no. C779200XBBM)
  • Vortex
  • Quattro micro tandem quadrupole mass spectrometer equipped with an Alliance 2795 HPLC system with autosampler (Waters or equivalent system but the method must be optimized and validated on a case‐by‐case basis)
  • Zorbax Bonus‐RP Guard column cartridge (4.6 × 12.5 mm; Agilent Technologies)
  • MassLynx version 4.1 (Waters)
  • New Brunswick rotary shaker

Support Protocol 1: Urine Collection on Filter Paper

  • 100 ml urine from an untreated Fabry female presenting a low level of Gb 3 (low quality control [LQC])
  • 100 ml urine from an untreated Fabry male presenting a high level of Gb 3 (high quality control [HQC])
  • 200, 5‐cm diameter filter paper disks (Whatman‐GE 903)
  • Magnetic stirrer and bar
  • Graphite pencil
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Literature Cited

Literature Cited
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