title: Tandem mass spectrometry multiplex analysis of methylated and non-methylated urinary Gb3 isoforms in Fabry disease patients.
abstract: BACKGROUND
  Fabry disease is a lysosomal storage disorder leading to the accumulation of glycosphingolipids in biological fluids and tissues. Globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3) are currently used for Fabry screening and diagnosis. However, these biomarkers are not always increased in Fabry patients with residual enzyme activity. We recently identified 7 urinary methylated Gb3-related isoforms. The aims of this study were (1) to develop and validate a novel LC-MS/MS method for the relative quantification of methylated and non-methylated Gb3 isoforms normalized to creatinine, (2) to evaluate these biomarkers in Fabry patients and healthy controls, and (3) to assess correlations between biomarker urinary excretion with age, gender, treatment and genotype of patients.
  
  
  METHODS
  Urine samples from 150 Fabry patients and 95 healthy controls were analyzed. Samples were purified and injected in the tandem mass spectrometer working in positive electrospray ionization. Relative quantification was performed for 15 methylated and non-methylated Gb3 isoforms.
  
  
  RESULTS
  Significant correlations (p<0.001) were established between Gb3 isoform concentrations, gender and treatment. Five patients with the late-onset cardiac mutation p.N215S showed abnormal concentrations of methylated Gb3 isoforms compared to their non-methylated homologues.
  
  
  CONCLUSIONS
  Methylated Gb3 isoforms might be helpful urinary biomarkers for Fabry patients with late-onset cardiac variant mutations.
authors.affiliation: Division of Medical Genetics, Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001, 12th Avenue North, Sherbrooke, Quebec J1H 5N4, Canada.
authors.forename: Mona
authors.initials: M
authors.lastname: Abaoui
authors.affiliation: Division of Medical Genetics, Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001, 12th Avenue North, Sherbrooke, Quebec J1H 5N4, Canada.
authors.forename: Michel
authors.initials: M
authors.lastname: Boutin
authors.affiliation: Division of Medical Genetics, Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001, 12th Avenue North, Sherbrooke, Quebec J1H 5N4, Canada.
authors.forename: Pamela
authors.initials: P
authors.lastname: Lavoie
authors.affiliation: Division of Medical Genetics, Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001, 12th Avenue North, Sherbrooke, Quebec J1H 5N4, Canada. Electronic address: Christiane.auray-blais@usherbrooke.ca.
authors.forename: Christiane
authors.initials: C
authors.lastname: Auray-Blais
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D002138:Calibration; D002648:Child; D002675:Child, Preschool; D000795:Fabry Disease / Q000378:metabolism / Q000652:urine*; D005260:Female; D006017:Glycolipids / Q000737:chemistry / Q000378:metabolism / Q000652:urine*; D006801:Humans; D007223:Infant; D008297:Male; D008745:Methylation; D008875:Middle Aged; D015394:Molecular Structure; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism / Q000652:urine; D013107:Sphingolipids / Q000737:chemistry / Q000378:metabolism / Q000652:urine*; D053719:Tandem Mass Spectrometry; D014281:Trihexosylceramides / Q000737:chemistry / Q000378:metabolism / Q000652:urine*; D055815:Young Adult
chemical_list: D006017:Glycolipids; D020033:Protein Isoforms; D013107:Sphingolipids; D014281:Trihexosylceramides; C063288:globotriaosyl lysosphingolipid; C018549:globotriaosylceramide
keywords: Fabry disease; Globotriaosylceramide; Methylated Gb(3) isoforms; Non-methylated Gb(3) isoforms; Tandem mass spectrometry; UPLC–MS/MS