Novel approach for pre-diagnosis and follow-up of patients affected by Gaucher disease based on specific flow cytometry and fluorescent labeling

Gaucher disease (GD) belongs to the lysosomal storage disorders (LSDs) and results from biallelic mutations in the gene GBA1, disrupting the function of the lysosomal glycoside hydrolase glucocerebrosidase (Gcase) and causing an accumulation of glucosylceramide (Glc-cer), as well as secondary storage materials, including ganglioside GM1. GD can be divided into three types, based on the presence and progression of neurological manifestations: while the type 1 is not related with any neuronal damage, types 2 and 3 typically cause neuronal impairments. Moreover, in patients carrying GBA1 mutations it has also been observed an increased incidence of Parkinson’s disease. Up to date, the measurement of in vitro enzymatic activity of Gcase with fluorescent substrates represent one of the most used techniques to evaluate therapeutic effects of drugs, potentially useful for the treatment of GD. However, an in vitro increase of Gcase activity might not lead to a reduction of Glc-cer accumulation in cells. In this contest, by using frozen/thawed fibroblasts, we explored the possibility of using specific fluorescent markers together with flow-cytometry and confocal microscopy in order to monitor changes in storage materials directly in fibroblasts from patients with different GD phenotypes.

We demonstrated that with our methods it is possible to clearly distinguish the levels of accumulated Glc-cer and GM1 in fibroblasts from affected and unaffected patients, providing the basis for a rapid, sensitive and unbiased approach, applicable in routine laboratory practice, to monitor the efficacy of therapeutic strategies on reducing the storage materials directly in cells.

Thanks to the Union European – NextGenerationEU for financial support in research project FluoPCPark and Cassa di Risparmio di Pistoia e Pescia Foundation for financial support in research project IDANEUROPARK.