Effect of velaglucerase alfa enzyme replacement therapy on red blood cell properties in Gaucher disease
Authors: Melanie Franco, Nelly Reihani, Mickael Marin, Marine De Person, Thierry Billette de Villemeur, Christian Rose, Yves Colin, Fathi Moussa, Nadia Belmatoug, Caroline Le Van Kim
Journal: American Journal of Hematology (2017)

Gaucher disease (GD) is a rare autosomal recessive disease,1 caused by a deficiency in the lysosomal enzyme glucocerebrosidase, leading to the accumulation of glucosylceramide (GL1) and its deacylated form glucosylsphingosine (lysoGL1) in cells of the monocyte/macrophage lineage. Lipid-laden macrophages that transform into Gaucher cells are thought to be responsible for GD symptoms but some studies have suggested that other cell types may contribute to GD pathophysiology.1 GD is classified into three types depending on the absence (type 1) or presence (types 2 and 3) of neurological features. In GD 1 and 3, visceral enlargement (splenomegaly and hepatomegaly), hematological manifestations (anemia, thrombocytopenia) and bone involvement (bone pain, osteonecrosis, and avascular necrosis) are common symptoms. Enzyme replacement therapy (ERT) with a recombinant form of human glucocerebrosidase has been the standard of care for GD, improving clinical parameters such as hemoglobin (Hb) concentration, platelet counts, spleen and liver volumes and bone damages. Velaglucerase alfa (VPRIV; Shire Human Genetic Therapies, Cambridge, MA) is one of the molecules currently available to treat GD by ERT.2 We have previously shown that red blood cells (RBCs) from GD patients exhibit membrane defects. Notably, GD RBCs have abnormal morphologies, deformability, aggregation and adhesion properties to laminin, owing to overexpression and activation of the adhesion molecule Lu/BCAM, the unique erythroid laminin receptor.3 We have also demonstrated an unexpected dyserythropoiesis responsible for central anemia in GD.4 These findings have revealed that abnormal erythroid properties may contribute to the occurrence of ischemic events and anemia in GD. However, no studies investigated the relationship between the degree of impairment in RBC properties and the severity of the disease. To further elucidate the role of RBCs in the pathophysiology of GD we investigated whether ERT normalizes RBC parameters. The effects of velaglucerase alfa treatment on membrane RBC properties in 15 ERT-treated GD patients (GD ERT) were compared with those in 16 untreated patients (GD UT) (comparative study). Seven out of 16 GD UT patients were also followed for at least 1 year after starting the treatment (longitudinal study). As expected, the response to ERT decreased the sizes of the spleen and the liver and increased Hb and Hematocrit (Hct) values as well as platelet counts.5 GD ERT patients also exhibited lower chitotriosidase activity and CC chemokine ligand 18 (CCL18) levels as compared with GD UT patients. Surprisingly, ERT neither reduced the RBC adhesiveness to laminin nor the expression of Lu/BCAM (see Table 1). Although we have no clear explanations for these results, they are most probably due in part to the short duration of the treatment. ERT did not seem to prevent bone events (see Table 1), but the total number of GD patients exhibiting bone defects at the time of blood sampling (seven patients) is too small to conclude. For the same reason, correlation studies between RBC adhesion and bone defects could not be done. Because bones defects could occur many years after the onset of ERT treatment, it will be of interest to investigate in the future the effect of ERT and the potential relationship between RBC abnormalities and bone infarct occurrence in a larger cohort. We showed previously that untreated GD patients exhibit a lower elongation index (EI) at shear stresses below 5.33 Pa, as well as higher percentage of abnormal morphologies as compared to that observed in healthy controls.3 ERT normalized the deformability parameter as well as the percentage of RBCs with abnormal morphologies (see Table 1). The low EI in RBCs from GD patients correlated well with the markers of disease activity, i.e., the low Hb levels (P = 0.008 and ρ = 0.44), high CCL 18 and chitotriosidase levels (P = 0.009 and ρ = −0.57; P = 0.02 and ρ = −0.57, respectively). Similarly, the percentage of RBCs exhibiting abnormal morphology in GD patients correlated with low Hb, high CCL18 levels (P = 0.0007 and ρ = −0.58; P = 0.002 and ρ = 0.60, respectively) and high chitotriosidase activity (P = 0.01 and ρ = −0.54). Interestingly, we observed a negative correlation between the EI and the percentage of RBCs exhibiting abnormal morphologies (P = 0.004 and ρ = −0.54). We also investigated the effects of ERT on RBC properties in the longitudinal study. Although the number of investigated patients is too low to conclude, our results showed that ERT treatment tends to improve the deformability of RBCs and ameliorates significantly RBC morphology (P = 0.0625, n = 5 and P = 0.0156, n = 7 respectively, not shown). The accumulation of LysoGL1 in the plasma of patients has recently been described as the most relevant biomarker of GD.6 To investigate whether lipid overload might be an important factor in membrane abnormalities of GD RBCs, we measured lysoGL1 levels in RBC membranes by LC/MS and compared these levels with those from healthy controls. We observed an increase in the RBC membrane lysoGL1 level of GD UT patients compared with healthy controls (median of 0.15 for the control group vs. 0.69 pmol mg−1 of protein for the GD UT group, not shown). Interestingly, ERT significantly normalized the lysoGL1 content in RBCs (see Table 1). Again, we observed that the lysoGL1 content in GD RBCs correlated well with low Hb levels (P = 0.0002 and ρ = −0.90) and high CCL18 levels (P = 0.01 and ρ = 0.86). Finally, significant correlations were found between lysoGL1 overload in GD RBC membranes and abnormal deformability and morphology (P = 0.013 and ρ = −0.76; P = 0.0002 and ρ = 0.89, respectively), suggesting that this lipid may be a critical mediator of RBC defects. The present study indicates that abnormal RBC morphology and deformability were normalized by ERT in a cohort of GD patients. Furthermore, patients with the highest degree of RBC abnormalities (deformability, morphology, lysoGL1 overload) showed elevated markers of activity in GD (CCL18 levels and chitotriosidasis activity). In conclusion, we showed that ERT is not only efficient in correcting hematological parameters, but also in normalizing RBC properties. This study is the first to describe an overload of lysoGL1 in RBC membranes of untreated GD patients which clearly correlate with other biomarkers of GD. Although the precise mechanism leading to the erythroid defects remains to be resolved, this study highlights the RBCs as important contributors to the pathophysiology of GD. Additional Supporting Information may be found in the online version of this article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.