Tandem mass spectrometry multiplex analysis of methylated and non-methylated urinary Gb3 isoforms in Fabry disease patients
Introduction
Fabry disease (OMIM 301500) is a multisystemic, X-linked lysosomal storage disorder caused by mutations in the GLA gene, leading to the deficiency of alpha-galactosidase A (α-GAL A, EC 3.2.1.22) and resulting in the accumulation of glycosphingolipids in organs, tissues, and biological fluids [1], [2]. Over 600 mutations in the GLA gene have been identified to date [3]. Fabry male patients are usually more clinically affected than women. However, some female carriers may develop the disease with symptoms as severe as those in affected men [4]. Enzyme replacement therapy (ERT) treatment (as well as other treatments still under study [5], [6], [7], [8]) is available for patients who present specific manifestations of the disease [9], [10]. In Canada, indications for treatment are summarized by the Canadian Fabry Disease Initiative (CFDI) guidelines [11], [12] (Supplementary Data section Table S-1). The incidence of Fabry disease is difficult to establish with estimations varying from 1:1250 to 1:117,000 [13], [14], [15], which is probably an underestimate of the true incidence considering milder variants which are difficult to diagnose [16], [17], [18].
Glycosphingolipid biomarkers, such as globotriaosylceramide (Gb3, or GL-3, also known as ceramide trihexoside (CTH)) [19], [20] and galabiosylceramide (Ga2 or ceramide dihexoside (CDH)) [19], [20], [21], [22], are found to be abnormally increased in Fabry patients. Moreover, a recent study showed that increased concentrations of urinary Gb3 in patients with heart disease who are not affected with Fabry disease are positively associated with near-term mortality [23]. The identification of a deacylated form of Gb3, globotriaosylsphingosine (lyso-Gb3), was also reported to be increased in plasma of Fabry patients [24], [25], [26], [27], as well as in urine [16], [28], [29]. Recent time of flight mass spectrometry metabolomic studies have led to the discovery of lyso-Gb3 related analogues with modified sphingosine moieties, thus emphasizing the variability of plasma [27] and urine [28] biomarkers in Fabry disease patients. In fact, most patients with the late-onset cardiac variant mutation p.N215S have normal concentrations of urinary Gb3 isoforms [30] and often normal concentrations of lyso-Gb3 itself, but abnormal concentrations of urinary analogues of lyso-Gb3 [31]. The recent development of a method for the relative quantitative analysis of seven analogues of lyso-Gb3 might be useful and reliable for the screening and diagnosis of patients with late-onset cardiac variants of the disease [31]. Considering the striking genotypic and phenotypic variability in Fabry disease patients, the search for novel Fabry biomarkers, which correlate with disease severity and progression, is still on-going. Recent metabolomic studies, using time of flight mass spectrometry, revealed 5 groups of Fabry disease Gb3-related biomarkers [32], [33].
More specifically, the group of methylated Gb3-related isoforms is comprised of seven isoforms with different fatty-acid chains and methylation of the nitrogen atom (Fig. 1). The position of the methyl group was determined precisely by tandem mass spectrometry experiments [33]. For example, the isoform Gb3(d18:1)(C22:0)Me had fragments at m/z 296.3 corresponding to the methylated sphingosine, and at m/z 354.4, corresponding to a methylated fatty acid, which confirmed the methylation on the nitrogen atom common to the sphingosine and fatty acid fragments [33]. Methyltransferases-catalyzed methylation has previously been reported in various biological processes [34], [35], [36], [37]. It is noteworthy to mention that “isoforms” is the term applied to modifications on the Gb3 fatty acid chain, whereas the term “analogues” is applied to modifications on the Gb3 sphingosine chain [33].
Section snippets
Ethics approval
This project was approved by the Research Ethics Board (REB) of the Faculty of Medicine and Health Sciences of the Centre hospitalier universitaire de Sherbrooke (CHUS).
Urine collection
Urine samples were collected from Fabry patients and healthy controls and stored at − 20 °C until analysis. Fabry disease diagnosis was confirmed by enzyme analysis in leucocytes and/or identification of a pathogenic mutation in the GLA gene. Urine samples from 150 Fabry patients and 95 healthy controls were analyzed. Table 1
Method validation
The precision of the method was evaluated with intraday (n = 5) and interday (n = 5) assays for methylated and non-methylated Gb3 isoforms and for urinary creatinine. Intraday CVs on the measured concentrations of all Gb3 isoforms were ≤ 18% for LQC and ≤ 12% for HQC. The creatinine intraday precision was good with CVs ≤ 5% for both LQC and HQC. For the interday, LQC and HQC CVs were ≤ 18% for all Gb3 isoforms and ≤ 10% for creatinine. All precision assay results are in the Supplementary Data section
Conclusions
A novel UPLC–MS/MS method was developed and validated for the relative quantification of methylated and non-methylated Gb3 isoforms. Urinary creatinine was simultaneously analyzed to allow direct sample normalization. A liquid–liquid extraction with methyl tert-butyl ether was devised, and optimized. The tandem mass spectrometry method was also validated prior to analysis of samples. Urinary biomarker concentrations were evaluated in 150 Fabry patients and 95 age- and gender-matched healthy
Acknowledgments
This research was funded by a Grant-in-Aid of research from the Canadian Institutes of Health Research (CIHR) (179883). We are grateful to Waters Corporation for their continued scientific support and partnership. We would like to thank Dr. Joe T. R. Clarke for his scientific expertise. We would like to acknowledge the dedicated collaboration of colleagues and coordinating-nurses at the Canadian Fabry Disease Initiative. We thank all Fabry patients and healthy reference controls who generously
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2017, Molecular Genetics and MetabolismCitation Excerpt :Evaluating plasma and urine samples of patients with FD, Auray-Blais et al. identified methylated isoforms of the globotriaosylceramide (Gb3) [157]. In another study, Abaoui et al. found seven methylated Gb3 isoforms in urine samples from 150 patients FD not seen in controls [158]. Additionally, Hübner et al. reported methylation at position 78,504 in the promoter region (CpG island) of calcitonin receptor in patients with FD on enzyme replacement therapy, suggesting that this modification could be used as a biomarker for either treatment or disease severity, as more critical patients tend to be on enzyme replacement therapy [159].