High‐Risk Screening of Fabry Disease: Analysis of Fifteen Urinary Methylated and Non‐Methylated Gb3 Isoforms Using Tandem Mass Spectrometry

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

1 Division of Medical Genetics, Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Centre de Recherche–CHUS, Sherbrooke, Quebec
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 17.24
DOI:  10.1002/cphg.24
Online Posting Date:  October, 2016
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Fabry disease is a multisystemic, X‐linked lysosomal storage disorder caused by mutations in the GLA gene, leading to α‐galactosidase A deficiency and resulting in the accumulation of glycosphingolipids in different tissues and biological fluids. Glycosphingolipid biomarkers, such as globotriaosylceramide (Gb3) isoforms, globotriaosylsphingosine (lyso‐Gb3) and related analogs, and galabiosylceramide (Ga2) isoforms and analogs, are found to be abnormally increased in urine and in plasma of Fabry patients and have the potential to be used as specific biomarkers of the disease. This unit presents a protocol for the relative quantification of fifteen urinary isoforms of Gb3 analyzed simultaneously with creatinine by ultra‐performance liquid chromatography (UPLC) coupled to tandem mass spectrometry (MS/MS). In order to purify urine samples, a liquid‐liquid extraction is performed and samples are analyzed by MS/MS in positive electrospray ionization mode. These biomarkers are useful for screening, diagnosis, and long‐term monitoring of Fabry disease patients. We have shown that the methylated Gb3 isoforms are particularly useful for screening Fabry patients who present with late‐onset cardiac variant mutations. © 2016 by John Wiley & Sons, Inc.

Keywords: Fabry disease; globotriaosylceramide; Gb3; methylated globotriaosylceramide isoforms; non‐methylated Gb3 isoforms; tandem mass spectrometry; UPLC–MS/MS; urine

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Table of Contents

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

  • Urine samples to be analyzed: patients and healthy controls
  • Heptadecanoyl ceramide trihexoside (Gb 3(d18:1)(C17:0)) standard curve working solution (see recipe)
  • Creatinine standard curve working solutions (see recipe)
  • 2 mM creatinine‐D3 internal standard working solution (see recipe)
  • 0.790 μM N‐octadecanoyl‐D3‐ceramide trihexoside (Gb 3(d18:1)(C18:0)D3) internal standard working solution (see recipe)
  • Positive quality control samples (see recipe)
  • Methanol (MeOH; LC‐MS grade; EMD Millipore, cat. no. MX0486‐1)
  • Methyl tert‐butyl ether (MTBE; LC‐MS grade; Sigma‐Aldrich, cat. no. 650560)
  • Dry ice
  • Acetone (EMD Millipore, cat. no. AX0120‐8)
  • Ammonium formate (Fisher, cat. no. A666‐500)
  • Formic acid (99%; Fisher, cat. no. AC27048‐0010)
  • Screw‐cap 13 × 100–mm glass culture tubes with PTFE‐lined phenolic caps (Fisher Scientific cat. no. 14‐933 A)
  • Nitrogen evaporator
  • 2‐ml vial, screw‐thread, 12 × 32 mm, clear glass (Chromspec, cat. no. C779100WM)
  • Glass insert, 250 µl, 6 × 29 mm (Chromspec, cat. no. C221020M)
  • Screw‐cap 9 mm, preslit PTFE/silicone septum (Chromspec, cat. no. C779200XBBM)
  • Centrifuge
  • Xevo TQ‐S mass spectrometer equipped with an Acquity I‐Class UPLC system with autosampler (Waters Corp.)
  • MassLynx version 4.1(Waters)
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Literature Cited

Literature Cited
  Abaoui, M., Boutin, M., Lavoie, P., and Auray‐Blais C. 2016. Tandem mass spectrometry multiplex analysis of methylated and non‐methylated urinary Gb3 isoforms in Fabry disease patients. Clin. Chim. Acta 452:191‐198. doi: 10.1016/j.cca.2015.11.018.
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