Plasma neurofilament light, glial fibrillary acidic protein and lysosphingolipid biomarkers for pharmacodynamics and disease monitoring of GM2 and GM1 gangliosidoses patients

Abstract

GM2 and GM1 gangliosidoses are genetic, neurodegenerative lysosomal sphingolipid storage disorders. The earlier the age of onset, the more severe the clinical presentation and progression, with infantile, juvenile and late-onset presentations broadly delineated into separate phenotypic subtypes. Gene and substrate reduction therapies, both of which act directly on sphingolipidosis are entering clinical trials for treatment of these disorders. Simple to use biomarkers for disease monitoring are urgently required to support and expedite these clinical trials. Here, lysosphingolipid and protein biomarkers of sphingolipidosis and neuropathology respectively, were assessed in plasma samples from 33 GM2 gangliosidosis patients, 13 GM1 gangliosidosis patients, and compared to 66 controls. LysoGM2 and lysoGM1 were detectable in 31/33 GM2 gangliosidosis and 12/13 GM1 gangliosidosis patient samples respectively, but not in any controls. Levels of the axonal damage marker Neurofilament light (NF-L) were highly elevated in both GM2 and GM1 gangliosidosis patient plasma samples, with no overlap with controls. Levels of the astrocytosis biomarker Glial fibrillary acidic protein (GFAP) were also elevated in samples from both patient populations, albeit with some overlap with controls. In GM2 gangliosidosis patient plasma NF-L, Tau, GFAP and lysoGM2 were all most highly elevated in infantile onset patients, indicating a relationship to severity and phenotype. Plasma NF-L and liver lysoGM2 were also elevated in a GM2 gangliosidosis mouse model, and were lowered by treatment with a drug that slowed disease progression. These results indicate that lysosphingolipids and NF-L/GFAP have potential to monitor pharmacodynamics and pathogenic processes respectively in GM2 and GM1 gangliosidoses patients.

Keywords: Biomarker; CLN2, neuronal ceroid lipofuscinosis type 2; CNS, central nervous system; CSF, cerebrospinal fluid; EDTA, Ethylenediaminetetraacetic acid; GBA2, non-lysosomal glucocerebrosidase; GCS, glucosylceramide synthase; GD3, Gaucher disease type 3; GFAP, Glial fibrillary acidic protein; Gangliosidosis; GlcSph, glucosylsphingosine; LC-MS/MS, liquid chromatography coupled with tandem mass spectrometry; LLOQ, lower limit of quantification; LSD, lysosomal storage disorders; Lysosome; Lysosphingolipid; MRI, magnetic resonance imaging; MS, multiple sclerosis; NF-L, Neurofilament light chain; NPC, Niemann Pick disease type C; Neurofilament; QC, quality control; lysoGb3, globotriaosylsphingosine.

Fig. 1

Plasma levels of lysosphingolipid biomarkers, lysoGM2 and lysoGM1. (A) and (D): Boxplot showing respective biomarker levels per disease. LysoGM2 and lysoGM1 values which were below the respective LLOQ, were replaced with the LLOQ (10 and 0.5 nM respectively). (B), (E) and (F): scatter plots for biomarker levels vs age at sampling for GM2 and GM1 gangliosidoses. The Spearman correlations are given in the scatter plots. When longitudinal values were measured in samples from a patient, the points for that patient are connected by a line in the scatter plot. (C): Biomarker levels by age at onset, with points colored by age at sampling, only the first sample from each patient is included. The same color scheme for disease is used in A,B,D,E and F. Points are shaped by the different sites the samples came from (3 shapes for control, and 2 for the patient samples). Fig. 1B is redrawn with coloring by age at onset in Supplemental fig. 3. Summary statistics of the biomarker per group and per age of onset grouping are provided in the supplement (Supplemental table 1 and Supplemental table 2).

Fig. 2

Plasma levels of the neuronal damage markers NF-L and Tau. (A) and (E) boxplots of each biomarker per disease group. Each group was compared to the control group using a non-parametric Wilcoxon test and p values corrected using the method of Bonferroni. P < 0.0001, ****; p < 0.001, ***; p < 0.01, **; p < 0.05, *; ns, not significant. (B,C,F,G) Scatter plots for biomarker levels vs age at sampling for GM2 and GM1 gangliosidoses, with the control group included in both cases. The Spearman correlations are given in the scatter plots. When longitudinal values were measured in samples from a patient, the points for that patient are connected by a line in the scatter plot. (D) and (H) Biomarker levels by age at onset, with points colored by age at sampling, only the first sample from each patient is included. The same color scheme for disease is used in A-F. Points are shaped by the different sites the samples came from (3 shapes for control, and 2 for the patient samples). Figs. B and F are redrawn with coloring by age at onset in Supplemental fig. 3.

Fig. 3

Plasma levels of the astrocytosis marker GFAP. (A) boxplot of GFAP levels per disease group. Each group was compared to the control group using a non-parametric Wilcoxon test and p values corrected using the method of Bonferonni. P < 0.0001, ****; p < 0.001,***; p < 0.01, **; p < 0.05, *; ns, not significant. (B) and (C): scatter plots for biomarker levels vs age at sampling for GM2 and GM1 gangliosidoses, with the control group included in both cases. The Spearman correlations are given in the scatter plots. When longitudinal values were measured in samples from a patient, the values for that patient are connected by a line in the scatter plot. (D): Biomarker levels by age at onset, with points colored by age at sampling only the first sample from each patient is included. The same color scheme for disease is used in A,B and C. Points are shaped by the different sites the samples came from (3 shapes for control, and 2 for the patient samples). Fig. 3B is redrawn with coloring by age at onset in Supplemental fig. 3.

Fig. 4

Limited correlation of plasma biomarkers to one another in GM2 and GM1 gangliosidoses patients. (A) and (B) heatmaps with hierarchical clustering and (C) and (D) correlation matrices were generated using GM2 and GM1 gangliosidoses patient samples when age at sampling was <7 years, with only the first sample from each patient included. For the heatmaps clustering is based on Pearson correlation, with patients on the x-axis and biomarkers on the y-axis. For correlation matrices correlations are Spearman, and r (filled circle) is displayed only if p < 0.05.

Fig. 5

Treatment effect of sinbaglustat on plasma NF-L and liver lysoGM2 in Hexb^−/− mice at 105 days of age. Data was analyzed using Dunnett's test to compare each group to Hexb^−/−, vehicle. P < 0.001,***; p < 0.01 **; p < 0.05, *; ns, not significant. As no lysoGM2 standard was available at time of study the units for lysoGM2 are peak area/ unit wet weight. LysoGM2 was measured in liver rather than plasma, due to the limited quantity of available plasma being used for other measurements.