Background: Gaucher disease (GD) is a rare, inherited, and autosomal recessive disorder caused by mutations in the GBA1 gene leading to a deficiency of a lysosomal enzyme, acid β-glucocerebrosidase, and glucosylsphingosine (Lyso-Gb1) toxic overload. GD diagnosis and its subsequent management are very challenging due to the wide spectrum of symptoms and complications. The introduction of enzyme replacement therapy (ERT) 30 years ago was considered a great revolution in the treatment of GD patients. However, the exact cause remains unknown, thus a deeper comprehension of the underlying pathogenetic mechanisms is required to provide valuable alternatives for its management. In this context, a "multi-omics" approach could be useful to enable novel biomarker discovery directly on dried blood spot (DBS) by using untargeted metabolomics and proteomics combination that can profile the phenotypical status produced by the genetic background, metabolic and protein characterizations.

Methods: Metabolites and proteins from DBS specimens of a homozygous GD patient, before and after ERT, and a healthy control at pediatric age were analysed with an orbitrap platform (Orbitrap Exploris^TM 120 and Orbitrap Fusion^TM Tribrid for metabolomics and proteomics strategies, respectively). For both "omics" analyses, the molecular features have been investigated first for statistics through Thermo Scientific^TM Compound Discoverer^TM and Proteome Discoverer^TM, then for further combined functional analysis using Ingenuity Pathway Analysis (IPA^® tool, QIAGEN).

Results: Overall, more than 500 metabolites and 200 proteins were robustly quantified. Among the known GD biomarkers, routinely used in the clinical practice, LysoGb1 and chitotriosidase-1 were confirmed as overexpressed at baseline before ERT, while they decreased after ERT, demonstrating the robustness of our method as proof of concept. Simultaneously, among the other features with the same trend, a new putative biomarker emerged from the metabolomics approach, that is likely compatible with 2-deoxy-2,3-dihydro-N-acetylneuraminic acid, a ganglioside derivative. According to proteomics analysis, galectin-3 binding protein was quantified in GD DBS with the same trend of the metabolites explained above suggesting it as a new putative marker since galectin-3, together with chitotriosidase-1, is already described as sign of macrophage activation. Moreover, functional analysis revealed the modulation of inflammatory pathways involving several classes of sphingolipids, which are reverted by ERT. In conclusion, the DBS "multi-omics" strategy, can help unravel the pathogenesis of GD, the most common sphingolipidosis in the Caucasian population, by identifying new biomarkers and therapeutical targets, and supporting the disease management over time.