Molecular and cellular pathogenesis of the erythroid defect and the therapeutic effect of glucocorticoids in Diamond-Blackfan anaemia
File(s)
Author(s)
Iskander, Deena Mohsen
Type
Thesis
Abstract
Diamond-Blackfan Anaemia (DBA) is a rare inherited bone marrow (BM) failure syndrome characterised by selective anaemia, congenital anomalies and predisposition to malignancy. DBA is caused by monoallelic, loss-of-function mutations in ribosomal protein (RP) genes. Delineation of the precise erythroid defect underpinning anaemia in DBA has been hampered by a lack of markers that define cells giving rise to burst- and colony-forming unit-erythroid (BFU-E and CFU-E) colonies, i.e., the clonogenic assays that quantify early and late erythroid progenitor (EP) potential respectively. By combining flow-cytometry, cell-sorting and single cell clonogenic assays, I identify Lin-CD34+CD38+CD45RA-CD123-CD71+CD41a-CD105-CD36- BM cells as early EP (EEP) and Lin-CD34+/-CD38+CD45RA-CD123-CD71+CD41a-CD105+CD36+ cells as late EP (LEP) giving rise to BFU-E and CFU-E respectively. By applying these definitions to DBA, I reveal, for the first time, that both quantitative and qualitative defects in EEP and LEP contribute to defective erythropoiesis in DBA and its restoration by corticosteroids (CS). I also demonstrate that, while an EP defect associated with profound loss of erythroblasts (EB) is present in transfusion-dependent (TD) patients with RPS gene-associated DBA, EP/EB are relatively preserved in TD patients with RPL gene variants. Transcriptome profiling by RNA-sequencing of FACS-purified EB from RPL genotype patients compared with age-matched controls, provides a unique dataset showing a distinct DBA molecular signature, characterised by p53 and inflammatory signalling pathways. Remarkably, the cellular differences between RPL and RPS gene-associated DBA correlate with divergent clinical phenotypes: patients with RPL rather than RPS gene pathogenic variants are more likely to present with anaemia at an older age, and to respond to CS. Finally, although DBA is rare, I demonstrate its wider utility as a paradigm of disordered erythroid commitment, providing insight into normal haematopoiesis. The strategies described for prospective isolation of EP/EB constitute an original contribution to knowledge that will facilitate more incisive study of normal and aberrant erythropoiesis.
Version
Open Access
Date Issued
2017-11
Date Awarded
2018-03
Copyright Statement
Creative Commons Attribution Non-Commercial No Derivatives license
Advisor
Karadimitris, Anastasios
Roberts, Irene
Sponsor
Bloodwise
Imperial College Healthcare NHS Trust
Grant Number
P43385
P45704
Publisher Department
Centre for Haematology
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)