A new UHPLC-MS/MS method for the screening of urinary oligosaccharides expands the detection of storage disorders

Background Oligosaccharidoses are storage disorders due to enzymatic defects involved in the breakdown of the oligosaccharidic component of glycosylated proteins. The defect cause the accumulation of oligosaccharides (OS) and, depending on the lacking enzyme, results in characteristic profiles which are helpful for the diagnosis. We developed a new tandem mass spectrometry method for the screening of urinary OS which was applied to identify a large panel of storage disorders. Methods The method was set-up in urine and dried urine spots (DUS). Samples were analysed, without derivatization and using maltoheptaose as internal standard, by UHPLC-MS/MS with MRM acquisition of target OS transitions, including Glc4, the biomarker of Pompe disease. The chromatographic run was < 30 min. Samples from patients with known storage disorders were used for clinical validation. Results The method allowed to confirm the diagnosis of oligosaccharidoses (sialidosis, α-/β-mannosidosis, fucosidosis, aspartylglucosaminuria) and of GM1 and GM2 (Sandhoff type) gangliosidosis, by detecting specific OS profiles. In other storage disorders (mucolipidosis II and III, mucopolysaccharidosis type IVB) the analyisis revealed abnormal OS excretion with non-specific profiles. Besides Pompe disease, the tetrasaccharide Glc4 was increased also in disorders of autophagy (Vici syndrome, Yunis-Varon syndrome, and Danon disease) presenting cardiomuscular involvement with glycogen storage. Overall, results showed a clear separation between patients and controls, both in urine and in DUS. Conclusion This new UHPLC/MS-MS method, which is suitable for rapid and easy screening of OS in urine and DUS, expands the detection of storage disorders from oligosaccharidoses to other diseases, including the novel category of inherited disorders of autophagy.

Urine oligosaccharide tests for the diagnosis of oligosaccharidoses

This review discusses the development of capillary electrophoresis with laser-induced detection and mass spectrometry techniques for the analysis of urinary oligosaccharides to screen for human oligosaccharidoses and related disorders. Capillary electrophoresis is suitable for the analysis of urinary oligosaccharides. It has a high resolution efficiency, and when it is coupled to a laser-induced fluorescence detector system, it offers an optimal analytical sensitivity. Mass spectrometry techniques have evolved as powerful tools for glycan analyses, are important tools for the analysis of oligosaccharide structures, and offer precise results, analytical versatility, very high sensitivity, high precision and high speed. Mass spectrometry is tolerant of mixtures and is a natural choice for the analysis of this class of molecules. The urine oligosaccharide profiles from healthy controls and patients diagnosed with different lysosomal storage diseases (fucosidosis, α-mannosidosis, GM1 gangliosidosis, GM2 gangliosidosis type Sandhoff, glycogen storage disease type 2 and 3, aspartylglucosaminuria, Schindler disease and galactosialydosis) and related disorders (glycogen storage diseases) are presented. In summary, these procedures are powerful tools for the rapid identification and characterization of these disorders. In all cases, the oligosaccharide profiles are strongly informative, identified the abnormal compounds, and are good alternatives to traditional screening tests that use thin layer chromatography. These tests have great sensibility and resolution and are automatable, enabling extended screening of these diseases, both as a first step in diagnosis or for confirming the pathogenicity of mutations that may be detected with next generation sequencing techniques.

Protein Glycosylation and Diseases: Blood and Urinary Oligosaccharides as Markers for Diagnosis and Therapeutic Monitoring

Background: N- and O-oligosaccharide variants on glycoproteins (glycoforms) can lead to alterations in protein activity or function that may manifest themselves as overt disease. Approach: This review summarizes those diseases that are known to be the result of an inherited or acquired glycoprotein oligosaccharide structural alteration and that are diagnosed in blood or urine by chemical characterization of that oligosaccharide alteration. Content: The biochemical synthesis steps and catabolic pathways important in determining glycoprotein function are outlined with emphasis on alterations that lead to modified function. Clinical and biochemical aspects of the diagnosis are described for inherited diseases such as I-cell disease, congenital disorders of glycosylation, leukocyte adhesion deficiency type II, hereditary erythroblastic multinuclearity with a positive acidified serum test, and Wiskott-Aldrich syndrome. We also review the laboratory use of measurements of glycoforms related to acquired diseases such as alcoholism and cancer. Conclusions: Identification of glycoprotein glycoforms is becoming an increasingly important laboratory contribution to the diagnosis and management of human diseases as more diseases are found to result from glycan structural alterations.

Early infantile form of galactosialidosis in a female baby with a prenatal diagnosis of fetal ascites: First case in Brazil

We present the first case of an early infantile form of galactosialidosis among Brazilians. This very rare and severe lysosomal storage disease has only a dozen patients clearly diagnosed worldwide. Clinical, pathological and biochemical features were consistent with previously published findings. We detected the disorder in a 7-month-old female baby with prenatal diagnosis of ascites. Evolution of the storage disease was monitored through routine thin-layer chromatography (TLC) for urinary oligosaccharides as part of a screening program for inborn errors of metabolism (IEM) in high-risk children, carried out in Rio de Janeiro.

Identification of urinary oligosaccharides by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

A new method of urinary oligosaccharides identification by matrix-assisted laser desorption time-of-flight mass spectrometry is presented. The method involves three steps: coupling of the urinary oligosaccharides with 8-aminonaphthalene-1,3,6-trisulfonic acid; fast purification over a porous graphite carbon extraction column; and mass spectrometric analysis. Identification of urinary oligosaccharides is based on the patterns and values of the pseudomolecular ions observed. We report here the patterns in urines from patients with Pompe disease, alpha and beta mannosidoses, galacto-sialidosis, and GM1 gangliosidosis. The protocols described here allowed facile and sensitive identification of the pathognomonic oligosacchariduria present in lysosomal diseases and can be extended to any pathological oligosacchariduria.