Fluorescent In Situ Staining and Flow Cytometric Procedures as New Pre-Diagnostic Tests for Sialidosis, GM1 Gangliosidosis and Niemann–Pick Type C

Abstract

Background: Early diagnosis is essential in the field of lysosomal storage disorders for the proper management of patients and for starting therapies before irreversible damage occurs, particularly in neurodegenerative conditions. Currently, specific biomarkers for the diagnosis of lysosomal storage disorders are lacking in routine laboratory practice, except for enzymatic tests, which are available only in specialized metabolic centers. Recently, we established a method for measuring and verifying changes in GM1 ganglioside levels in peripheral blood lymphocytes in patients with GM1 gangliosidosis. However, fresh blood is not always available, and using frozen/thawed lymphocytes can lead to inaccurate results. Methods: We used frozen/thawed fibroblasts obtained from stored biopsies to explore the feasibility of fluorescent imaging and flow-cytometric methods to track changes in storage materials in fibroblasts from patients with three lysosomal neurodegenerative conditions: GM1 gangliosidosis, Sialidosis, and Niemann–Pick type C. We used specific markers for each pathology. Results and Conclusions: We demonstrated that with our methods, it is possible to clearly distinguish the levels of accumulated metabolites in fibroblasts from affected and unaffected patients for all the three pathologies considered. Our methods proved to be rapid, sensitive, unbiased, and potentially applicable to other LSDs.

Identification of GM1-Ganglioside Secondary Accumulation in Fibroblasts from Neuropathic Gaucher Patients and Effect of a Trivalent Trihydroxypiperidine Iminosugar Compound on Its Storage Reduction

“…These results were further corroborated by flow-cytometry analysis. In fibroblasts of patients carrying the L444P mutation, a ~50% increase in GlcCer accumulation ( Figure 2 A) was observed, accompanied by a ~60% increase in GM1 storage ( Figure 2 B), comparable to the one observed in patients affected by GM1 gangliosidosis [ 20 , 21 ]. On the other hand, the contents of GlcCer and GM1 in fibroblasts of patients carrying the N370S mutation did not vary significantly compared to the control ( Figure 2 A,B).…”

“…The R131C mutant allele was also identified in type 2 (acute neuronopathic) GD [ 23 , 28 ]. Surprisingly, the levels of GM1 also appeared to be dramatically increased in fibroblasts bearing these mutations, up to the levels observed for infantile patients affected by GM1 gangliosidosis [ 20 , 21 ]. Little or no changes in the GlcCer and GM1 amounts were found for the fibroblasts with the N370S mutation, which is associated with GD type 1.…”

“…In vitro enzymatic-activity tests use soluble synthetic fluorogenic substrates in cell lysates, which may not take into account potential enzyme activators and may deliver inaccurate results that may not match the clinical course of the disease. We have recently developed an approach based on specific fluorescence labelling coupled to microscopy and flow cytometry capable of detecting higher levels of accumulated metabolites (e.g., sialic acid, GM1 ganglioside, and cholesterol) in fibroblasts from patients affected by several LSDs, such as GM1 gangliosidosis, Niemann–Pick type C, and sialidosis [ 20 , 21 ]. These methods allow one to directly correlate the amount of accumulated material with the severity of the disease.…”