Analyse de type multiplex de sept nouveaux biomarqueurs urinaires de la maladie de Fabry par spectrométrie de masse en tandem

Les maladies lysosomales (aussi appelées maladies de surcharge) sont causées par l'activité déficitaire d'une ou de plusieurs hydrolases lysosomales perturbant ainsi le catabolisme de macromolécules. Ce déficit enzymatique engendre leur 'accumulation dans les tissus, les organes et les liquides biologiques de la personne atteinte. Plus de 70 maladies lysosomales ont été décrites jusqu'à maintenant et, bien que rares individuellement, elles ont une prévalence combinée d'environ 1:7000. Il est primordial que des biomarqueurs (ou indicateurs de changement) reflétant la progression et la sévérité de la maladie, la réponse à une intervention thérapeutique donnée et le mécanisme de la pathogénèse soient disponibles pour les patients. La maladie de Fabry est une maladie liée à l'X et causée par l'activité déficitaire de l'enzyme a-galactosidase A. Jusqu'à maintenant, deux sphingolipides ont été étudiés comme biomarqueurs dans l'urine et le plasma des patients Fabry : le globotriaosylcéramide (Gb 3 ) et le globotriaosylsphingosine (lyso-Gb3 ). Notre équipe a récemment découvert sept nouveaux biomarqueurs de la maladie de Fabry par des études en métabolomique utilisant la spectrométrie de masse en temps de vol. Ces nouveaux biomarqueurs présentaient des rapports masse sur charge ( m/z ) de 758, 774, 784, 800, 802, 820 et 836. Des études de fragmentation ont démontré qu'ils étaient tous des molécules analogues au lyso-Gb 3 , présentant des modifications au niveau de la partie sphingosine de la molécule. Les objectifs de ce projet de recherche étaient : 1) d' élaborer et de valider une méthode de spectrométrie de masse en tandem pour la quantification relative de ces sept analogues du lyso-Gb 3 ; 2) d'évaluer leurs niveaux d'excrétion dans des échantillons d'urine de 164 patients Fabry et de 94 individus contrôles sains de référence; 3) d'établir des corrélations entre l'excrétion urinaire des différents analogues et le sexe du patient et la thérapie enzymatique de remplacement offerte comme traitement aux patients. Une méthodologie d'analyse des biomarqueurs par spectrométrie de masse en tandem a été développée et validée. Les contrôles de référence sains analysés selon cette méthodologie ne présentaient pas de trace de biomarqueurs, à l'exception de l'analogue m/z 836, ce qui est un atout au niveau du diagnostic. Des corrélations significatives ont été établies entre les niveaux d'excrétion du lyso-Gb3 dans l'urine et le sexe du patient. Les hommes atteints de la maladie de Fabry, qui sont habituellement affectés plus sévèrement que les femmes, avaient des niveaux d'excrétion urinaires supérieurs, suggérant que les biomarqueurs découverts pourraient être reliés à la sévérité de la maladie. L'excrétion urinaire des analogues diminue significativement après l'administration de la thérapie enzymatique de remplacement chez les hommes atteints de la maladie de Fabry, ce qui démontre leur efficacité au niveau du suivi du traitement chez ces derniers. Les analogues du lyso-Gb 3 semblent donc être des biomarqueurs prometteurs pour la maladie de Fabry. [symboles non conformes

Neonatal Urine Screening Program in the Province of Quebec: Technological Upgrade from Thin Layer Chromatography to Tandem Mass Spectrometry

The Quebec Neonatal Urine Screening Program was initiated in 1971 with overall screening inception of newborns in 1973. Forty-seven years later, over 3.5 million babies have been screened for up to 25 inborn errors of metabolism divided into two groups: (1) urea cycle disorders and organic acidurias; and (2) disorders of amino acid metabolism and transport. The main goal of this preventive genetic medicine program is the detection of treatable diseases before the onset of clinical symptoms. Urine specimens from 21-day-old babies are collected and dried on filter paper by parents at home. The participation is voluntary with a high compliance rate over the years (~90%). Specimens are analyzed by thin layer chromatography (TLC). The main objective of this evaluative research project was to assess the feasibility of a technological upgrade towards mass spectrometry. A 2.85-min flow injection method was devised, normal values established, and abnormal profiles confirmed using second-tier tests. The validated assays are sensitive, specific, and suitable for populational screening, as well as for high-risk screening laboratories. Triple H syndrome, which would not be detected in newborns by blood screening at two days of age was found to be positive in the urine of an affected patient

Multiplex Analysis of Novel Urinary Lyso-Gb₃‑Related Biomarkers for Fabry Disease by Tandem Mass Spectrometry

Fabry disease is a lysosomal storage disorder caused by the absence or reduction of α-galactosidase A enzyme activity. The enzymatic deficiency results in the impaired catabolism of neutral sphingolipids with terminal α-galactosyl residues and subsequent accumulation in several tissues. Biomarkers reflecting disease severity and progression, the response to therapeutic intervention, and details of molecular pathogenesis are needed. Until now, two sphingolipids were targeted as biomarkers in urine and plasma of Fabry patients: globotriaosylceramide (Gb₃) and globotriaosylsphingosine (lyso-Gb₃). Using metabolomic approaches, our group recently discovered seven novel urinary lyso-Gb₃-related Fabry disease biomarkers with mass-to-charge ratios (<i>m</i>/<i>z</i>) of 758, 774, 784, 800, 802, 820, and 836. All these biomarkers exhibited modifications of the lyso-Gb₃ sphingosine moiety. The aims of the present study were to devise and validate a specific tandem mass spectrometry multiplex methodology for the relative quantification of these seven analogues and to evaluate their urinary excretion levels in samples from 164 Fabry patients and 94 healthy controls. We found no detectable analogues in healthy controls, except for trace amounts of the analogue with <i>m</i>/<i>z</i> 836. Significant correlations were established between lyso-Gb₃ analogue levels in urine and gender (<i>p</i> < 0.001). Fabry males had higher excretion levels compared to females with the disease. Lyso-Gb₃ analogue levels correlated well with enzyme replacement therapy (ERT) status in males (<i>p</i> < 0.05). The urinary analogue distributions varied among Fabry patients. However, the analogues with <i>m</i>/<i>z</i> 802, 820, and 836 were generally more abundant in the majority of patients. Lyso-Gb₃ analogues are promising urinary biomarkers for Fabry disease

Mass Spectrometry Analysis of Globotriaosylsphingosine and Its Analogues in Dried Blood Spots

Fabry disease (FD) is an X-linked lysosomal storage disorder where impaired α-galactosidase A enzyme activity leads to the intracellular accumulation of undegraded glycosphingolipids, including globotriaosylsphingosine (lyso-Gb3) and related analogues. Lyso-Gb3 and related analogues are useful biomarkers for screening and should be routinely monitored for longitudinal patient evaluation. In recent years, a growing interest has emerged in the analysis of FD biomarkers in dried blood spots (DBSs), considering the several advantages compared to venipuncture as a technique for collecting whole-blood specimens. The focus of this study was to devise and validate a UHPLC-MS/MS method for the analysis of lyso-Gb3 and related analogues in DBSs to facilitate sample collection and shipment to reference laboratories. The assay was devised in conventional DBS collection cards and in Capitainer®B blood collection devices using both capillary and venous blood specimens from 12 healthy controls and 20 patients affected with FD. The measured biomarker concentrations were similar in capillary and venous blood specimens. The hematocrit (Hct) did not affect the correlation between plasma and DBS measurements in our cohort (Hct range: 34.3–52.2%). This UHPLC-MS/MS method using DBS would facilitate high-risk screening and the follow-up and monitoring of patients affected with FD

Mass Spectrometry Analysis of Globotriaosylsphingosine and Its Analogues in Dried Blood Spots

Fabry disease (FD) is an X-linked lysosomal storage disorder where impaired &alpha;-galactosidase A enzyme activity leads to the intracellular accumulation of undegraded glycosphingolipids, including globotriaosylsphingosine (lyso-Gb3) and related analogues. Lyso-Gb3 and related analogues are useful biomarkers for screening and should be routinely monitored for longitudinal patient evaluation. In recent years, a growing interest has emerged in the analysis of FD biomarkers in dried blood spots (DBSs), considering the several advantages compared to venipuncture as a technique for collecting whole-blood specimens. The focus of this study was to devise and validate a UHPLC-MS/MS method for the analysis of lyso-Gb3 and related analogues in DBSs to facilitate sample collection and shipment to reference laboratories. The assay was devised in conventional DBS collection cards and in Capitainer&reg;B blood collection devices using both capillary and venous blood specimens from 12 healthy controls and 20 patients affected with FD. The measured biomarker concentrations were similar in capillary and venous blood specimens. The hematocrit (Hct) did not affect the correlation between plasma and DBS measurements in our cohort (Hct range: 34.3&ndash;52.2%). This UHPLC-MS/MS method using DBS would facilitate high-risk screening and the follow-up and monitoring of patients affected with FD

Contribution of tandem mass spectrometry to the diagnosis of lysosomal storage disorders

International audienceTandem mass spectrometry (MS/MS) is a highly sensitive and specific technique. Thanks to the development of triple quadrupole analyzers, it is becoming more widely used in laboratories working in the field of inborn errors of metabolism. We review here the state of the art of this technique applied to the diagnosis of lysosomal storage disorders (LSDs) and how MS/MS has changed the diagnostic rationale in recent years. This fine technology brings more sensitive, specific, and reliable methods than the previous biochemical ones for the analysis of urinary glycosaminoglycans, oligosarcharides, and sialic acid. In sphingolipidoses, the quantification of urinary sphingolipids (globotriaosylecramide, sulfatides) is possible. The measurement of new plasmatic biomarkers such as oxysterols, bile acids, and lysosphingolipids allows the screening of many sphingolipidoses and related disorders (Niemann-Pick type C), replacing tedious biochemical techniques. Applied to amniotic fluid, a more reliable prenatal diagnosis or screening of LSDs is now available for fetuses presenting with antenatal manifestations. Applied to enzyme measurements, it allows high throughput assays for the screening of large populations, even newborn screening. The advent of this new method can modify the diagnostic rationale behind LSDs