Reed, M; (2017) Characterisation of pathogenic pathways within the bone microenvironment of Gaucher Disease which contribute to bone pathology and haematological malignancy. Doctoral thesis , UCL (University College London).

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Abstract

Introduction: Gaucher disease is a recessive disorder, mutation of the GBA1 gene leads to a reduction in β-glucocerebrosidase (GBA) activity, accumulation of glucosylceramide and abnormal levels of other sphingolipids. Features include hepatosplenomegaly, cytopenia and bone disease including osteopenia and osteonecrosis. Enzyme replacement is the most common therapy however bone manifestations can be slow to respond, some patients show no improvement or continue to suffer bone events. Gaucher patients are reported to have a higher incidence of multiple myeloma with an estimated risk ratio up to 51.1. Aim: To investigate the bone marrow microenvironment in GD by partial recreation of this environment using co-culture systems. Methods: Co-culture of up to 3 cell types, histochemical staining, immunofluorescence imaging, functional assay, enzyme activity, therapy and lipid assays, cytotoxicity assay, flow cytometry. Results: Gaucher patient (GD) peripheral blood mononuclear cell derived osteoclast cultures (OC) generated more osteoclasts and at earlier time points than control cultures. GD osteoclasts were larger, had more nuclei and resorbed more bone. Addition of GBA inhibitor CBE to control OC increased osteoclast generation, size and nuclei number. Addition of glucosylceramide to OC increased osteoclast numbers in both control and GD cultures. In vitro GD osteoclast numbers correlated with active bone disease, bone pain and anaemia. Addition of GD-specific therapies to GD OC decreased osteoclast generation, also observed in a selection of patients receiving GD-specific therapies. Uncoupling between GD osteoblast precursors and osteoclasts was observed. Culture of control OC with plasma cell line NCI-H929 increased osteoclast generation. CBE inhibition of human osteoblast cell line SaOS-2 reduced calcium deposition. Glucosylceramide increased NCI-H929 cell number after 7 days. NCI-H929 viability increased when in contact with osteoclasts in SaOS-2 co-cultures. Conclusion: Osteoblast and osteoclast dysregulation may contribute to bone disease in GD. The GD bone microenvironment may provide a pro-survival environment for plasma cells.

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