Functional and genetic characterization of the non-lysosomal glucosylceramidase 2 as a modifier for Gaucher disease

Abstract

Background: Gaucher disease (GD) is the most common inherited lysosomal storage disorder in humans, caused by mutations in the gene encoding the lysosomal enzyme glucocerebrosidase (GBA1). GD is clinically heterogeneous and although the type of GBA1 mutation plays a role in determining the type of GD, it does not explain the clinical variability seen among patients. Cumulative evidence from recent studies suggests that GBA2 could play a role in the pathogenesis of GD and potentially interacts with GBA1.

Methods: We used a framework of functional and genetic approaches in order to further characterize a potential role of GBA2 in GD. Glucosylceramide (GlcCer) levels in spleen, liver and brain of GBA2-deficient mice and mRNA and protein expression of GBA2 in GBA1-deficient murine fibroblasts were analyzed. Furthermore we crossed GBA2-deficient mice with conditional Gba1 knockout mice in order to quantify the interaction between GBA1 and GBA2. Finally, a genetic approach was used to test whether genetic variation in GBA2 is associated with GD and/ or acts as a modifier in Gaucher patients. We tested 22 SNPs in the GBA2 and GBA1 genes in 98 type 1 and 60 type 2/3 Gaucher patients for single- and multi-marker association with GD.

Results: We found a significant accumulation of GlcCer compared to wild-type controls in all three organs studied. In addition, a significant increase of Gba2-protein and Gba2-mRNA levels in GBA1-deficient murine fibroblasts was observed. GlcCer levels in the spleen from Gba1/Gba2 knockout mice were much higher than the sum of the single knockouts, indicating a cross-talk between the two glucosylceramidases and suggesting a partially compensation of the loss of one enzyme by the other. In the genetic approach, no significant association with severity of GD was found for SNPs at the GBA2 locus. However, in the multi-marker analyses a significant result was detected for p.L444P (GBA1) and rs4878628 (GBA2), using a model that does not take marginal effects into account.

Conclusions: All together our observations make GBA2 a likely candidate to be involved in GD etiology. Furthermore, they point to GBA2 as a plausible modifier for GBA1 in patients with GD.

Figure 1

Quantitative mass spectrometric analysis of HexCer (sum of GlcCer and GalCer). (A) Total HexCer amount in liver, spleen, and brain of 6 -month-old GBA2-deficient (KO) and wild-type (WT) mice. (B) Amount of the HexCer species carrying the typical neuronal ceramide anchor with a stearic acyl residue. *: P < 0.05, n = 4 animals per group. (C) TLC of a representative brain lipid sample in which GlcCer and GalCer are separated. Note the increased GlcCer in the KO. The double band reflects heterogeneity of its ceramide anchor composition.

Figure 2

mRNA expression of GBA2 and GBA1 in GBA1-deficient embryonic mice fibroblasts [24]expressed as fold change GBA2 versus GBA1. 18S-RNA was used as internal control. Western blot analysis for GBA2 in GBA1-deficient embryonic mice fibroblasts, beta-actin was used as loading control.

Figure 3

GlcCer levels in GBA-deficient mice. Thin layer chromatography (TLC) analysing glycosphingolipids from spleen of 12-month-old GBA1-deficient, GBA2-deficient, and GBA1/GBA2-deficient mice. Representative TLC analysis shown neutral sphingolipids of 5 mg (wet weight). WT: wild-type, KO: knockout mice, GlcCer: glucosylceramide, LacCer: lactosylceramide, SM: sphingomyelin.