Abstract: Fabry disease is an X-linked, multisystemic lysosomal storage disorder characterized by the accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3) in biological fluids, vascular endothelium, heart, and kidneys. Treatment by enzyme replacement therapy is provided to both males and females depending on the severity of symptoms. We devised rapid and efficient tandem mass spectrometry methodologies using a Waters Quattro micro-HPLC Alliance system to quantify two urinary Fabry disease biomarkers: Gb3 (4 min-assay) and globotriaosylsphingosine (lyso-Gb3) (6 min-assay) analyses normalized to creatinine (creat). We characterized urinary lyso-Gb3 by time-of-flight mass spectrometry on a Waters Synapt UPLC-QTOF MS (Ultra-Performance Liquid Chromatography-Time-of-flight mass spectrometry) system, since it was previously only detected in blood. Validation of both methodologies for clinical use gave good coefficients of variation for intra-day and inter-day assays (<13%). Normal values were established for adult Gb3 (<25 ug/mmol creat) and lyso-Gb3 (none found in the control cohort). Other objectives of this study were to identify and characterize novel disease-specific biomarkers in patients affected with Fabry disease that reliably reflect disease progression and severity, and thereby facilitate the evaluation of new therapies by improved monitoring of the response to treatment. We employed a time-of-flight mass spectrometry metabolomic approach. Urine and plasma samples from untreated and treated Fabry patients were evaluated and compared to age-matched controls. All samples were analyzed on a UPLC-QTOF MS Synapt system. Results processed with MarkerLynx (Waters) were downloaded in EZInfo (Umetrics) for multivariate data analysis such as principal component analysis (PCA) and orthogonal partial least square-discriminant analysis (OPLS-DA). We detected specific analogs in both biological fluids under study: in plasma, we found three novel lyso-Gb3 analogs at m/z 802; m/z 804; and m/z 820 and four other urinary analogs of lyso-Gb3 at m/z 758; m/z 774; m/z 800; and m/z 836. A m/z 784 analog was found in both biological fluids. Area counts for Fabry analogs were compared to controls. We found that some urinary analogs presented higher area counts than lyso-Gb3. We confirmed that all analogs are lyso-Gb3 sphingosine moiety modifications. Correlations between the presence and amounts of various disease-specific analogs and specific indices of clinical severity are in progress. To our knowledge, this metabolomic study using time-of-flight mass spectrometry is the first to demonstrate the presence of analogs of lyso-Gb3 potentially quantifiable by tandem mass spectrometry. The next step will be to synthesize efficient standards to accurately measure the amounts of these biomarkers in biological fluids. This will lead to the evaluation of correlations to determine disease-severity and progression in Fabry disease patients.

Brief Biography of the Speaker:
Dr. Auray-Blais is the Director of the Quebec Provincial Mass Urinary Screening Program for hereditary metabolic disorders since its inception more than 35 years ago. She is a pioneer in mass urine screening having developed the infrastructure, techniques and methodology permitting the urinary screening of 2 800 000 newborn babies in the province of Quebec for 25 disorders of amino acids and organic acids. She holds a Ph.D. in radiobiology from the Faculty of Medicine and Health Sciences at the Universite de Sherbrooke and postdoctoral studies from Duke University Medical Center in North Carolina, US. She has a masters degree in Health Law from the Faculty of Law at the University de Sherbrooke and a bachelors degree in biochemistry. She is the author of 150 publications, abstracts and articles. She is a professor in the Service of genetics in the Department of Pediatrics at the Faculty of Medicine and Health Sciences at the Universite de Sherbrooke and a researcher at the Clinical Research Centre Etienne-Le Bel in the Mother-Child Axis. She is the Scientific Director for the Waters-CHUS Expertise Centre in Clinical Mass Spectrometry. She is the principal investigator and co-investigator in numerous research grants. She has received awards for her involvement and expertise in screening inborn errors of metabolism in newborns in the Province of Quebec.