Quantification of Specific Urinary Oligosaccharide Biomarkers for Diagnosis and Treatment Monitoring of...

Alpha-mannosidosis (AM, OMIM# 248500), an ultra-rare lysosomal storage disorder (LSD), is caused by insufficient activity of alpha-mannosidase, an enzyme involved in degradation of N-glycan oligosaccharides. Hence, specific oligosaccharides accumulate in tissues, resulting in a progressive multi-organ disease. Therapeutic options exist but treatment outcome depends on early diagnosis. Thus, multiple methods have been developed for analysis of these oligosaccharides in urine that are qualitative or semi-quantitative, limiting their use for treatment monitoring. A rapid quantitative method without derivatization was developed for AM biomarkers GlcNAc(Man)₂, GlcNAc(Man)₃, and GlcNAc(Man)₄ in spot urine samples using ultra-performance liquid chromatography coupled to tandem-mass spectrometry. Urine samples of 208 controls, 20 AM patients, and 26 patients with other LSDs were analyzed. Method validation proved high recoveries (88%-108%) and precisions (standard deviation < 8%), low limits of quantification (0.12 μg GlcNAc(Man)₂/mL), and high sample stability. We observed a clear separation between controls and AM patients (0.0-9.0 vs 39.4-99.3 μmol GlcNAc(Man)₂/mmol creatinine, p < 0.0001). Furthermore, GlcNAc(Man)₂ concentration showed significant differences between untreated patients and those treated with enzyme replacement therapy (ERT; n = 13, p = 0.0006) or hematopoietic stem cell transplantation (HSCT; n = 3, p = 0.0143), and between both treatments (p = 0.0143). Thus, the developed method is well-suited for selective screening for AM and offers a simple way to monitor treatment efficacy.