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

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.