Analysis of urinary oligosaccharide excretion patterns by UHPLC/HRAM mass spectrometry for screening of lysosomal storage disorders

Abstract

Oligosaccharidoses, sphingolipidoses and mucolipidoses are lysosomal storage disorders (LSDs) in which defective breakdown of glycan-side chains of glycosylated proteins and glycolipids leads to the accumulation of incompletely degraded oligosaccharides within lysosomes. In metabolic laboratories, these disorders are commonly diagnosed by thin-layer chromatography (TLC) but more recently also mass spectrometry-based approaches have been published.To expand the possibilities to screen for these diseases, we developed an ultrahigh-performance liquid chromatography (UHPLC) with a high-resolution accurate mass (HRAM) mass spectrometry (MS) screening platform, together with an open-source iterative bioinformatics pipeline. This pipeline generates comprehensive biomarker profiles and allows for extensive quality control (QC) monitoring. Using this platform, we were able to identify α-mannosidosis, β-mannosidosis, α-N-acetylgalactosaminidase deficiency, sialidosis, galactosialidosis, fucosidosis, aspartylglucosaminuria, GM1 gangliosidosis, GM2 gangliosidosis (M. Sandhoff) and mucolipidosis II/III in patient samples. Aberrant urinary oligosaccharide excretions were also detected for other disorders, including NGLY1 congenital disorder of deglycosylation, sialic acid storage disease, MPS type IV B and GSD II (Pompe disease). For the latter disorder, we identified heptahexose (Hex7), as a potential urinary biomarker, in addition to glucose tetrasaccharide (Glc4), for the diagnosis and monitoring of young onset cases of Pompe disease. Occasionally, so-called "neonate" biomarker profiles were observed in young patients, which were probably due to nutrition.Our UHPLC/HRAM-MS screening platform can easily be adopted in biochemical laboratories and allows for simple and robust screening and straightforward interpretation of the screening results to detect disorders in which aberrant oligosaccharides accumulate.

Validation of Aspartylglucosaminidase Activity Assay for Human Serum Samples: Establishment of a Biomarker for Diagnostics and Clinical Studies

“…The development of an optimized method for the determination of AGA activity in AGU patient sera was based on previously published methods [ 6 , 7 , 8 , 11 , 15 ]. The measurement of AGA enzyme activity in human fibroblasts and leucocytes using a fluorometric substrate Asp-AMC (L-Aspartic acid β-(7-amido-4-methylcoumarin)) was originally proposed by Mononen et al and Voznyi et al [ 13 , 14 , 15 , 19 ].…”

“…In Finland, AGU is usually diagnosed after analysis of urine oligosaccharides, especially N-acetylglucosamine-asparagine (GlcNAc-Asn), and the diagnosis is verified by means of gene sequencing [ 10 , 11 ]. The patients outside of Finland, who often exhibit their own, family-specific pathogenic variants, are identified either by gene/exome sequencing, or urine analysis.…”

“…Nevertheless, findings of a genetic analysis or urine glycoasparagine measurement should always be verified by a second method, especially if a novel, potentially pathogenic variant of the AGA gene is identified [ 10 ]. Furthermore, glycoasparagines can also be present in the urine of patients with NGLY1 deficiency, which is a related neurodevelopmental disorder [ 11 , 16 , 17 , 18 ].…”

Validation of Aspartylglucosaminidase Activity Assay for Human Serum Samples: Establishment of a Biomarker for Diagnostics and Clinical Studies

“…The development of an optimized method for the determination of AGA activity in AGU patient sera was based on previously published methods [6,7,8,11,15]. The measurement of AGA enzyme activity in human fibroblasts and leucocytes using a fluorometric substrate Asp-AMC (L-Aspartic acid β-(7-amido-4-methylcoumarin) was originally proposed by Mononen et al and Voznyi et al [13,14,15,19].…”

“…2 of 11 ings of a genetic analysis or urine glycoasparagine measurement should always be verified by a second method, especially if a novel, potentially pathogenic variant of the AGA gene is identified [10]. Furthermore, glycoasparagines can also be present in the urine of patients with NGLY1 deficiency, which is a related neurodevelopmental disorder [11,16,17,18].…”