Abstract
Diamond Blackfan anaemia (DBA) is a rare congenital red cell aplasia, characterised by a steroid responsive intrinsic erythropoietic failure. Mutations affecting RPS19 are found in up to 25% of DBA, but the pathophysiological link between ribosomal gene haploinsufficiency and the specific erythroid phenotype of DBA remains elusive. One potential mechanism is that ribosomal insufficiency might affect the level of expression of critical erythroid proteins, resulting in a block in terminal erythroid differentiation and expansion. The aim of this study was to elucidate the cellular and molecular events occurring in response to erythropoietin (EPO) in DBA. We applied a 2-phase erythroid culture system in which we have previously demonstrated a consistent and profound failure of erythroid expansion in DBA cultures, localised to early in the second EPO-dependent phase of culture. The timing of the onset of proliferation in response to EPO was studied by tetrazolium dye assay of enriched erythroid progenitor cells in phase II of culture. An increase in cell number was detectable following 72hrs of EPO exposure in DBA (n=5) as well as control cultures, but with a subsequently slower proliferation rate in DBA. The proliferation rate of both DBA and control cells was enhanced by the addition of dexamethasone. Transcriptional profiles of control and DBA erythroid progenitors were then compared by microarray analysis after 24hrs exposure to EPO. Cy3/Cy5 labelled aRNA was competitively hybridised on an in-house array containing 395 transcript exon identifiers in ENSEMBL. The results showed little difference in expression between control and DBA after 24hrs of EPO exposure, the only significant changes being in the expression of just 4 of the 251 genes expressed above background (CDKN2D, TNFRSF6, MAPKK and CXCR), which were upregulated in DBA (p<0.0001 on t-test analysis). The gene expression profile from 0–96 hrs following EPO exposure was then studied in control and DBA (n=3) erythroid progenitors by real time PCR. There was little change in the first 48hr of exposure to EPO. At 72hrs the expression of both EPOR and α-GLOBIN was increased 3–6 fold in both DBA and controls in comparison with pre-EPO, confirming the occurrence of an erythroid-specific transcriptional response to EPO in DBA erythroid progenitors. These results are consistent with a qualitatively normal onset of terminal erythroid maturation in DBA, rather than a block in differentiation, and tend to favour an alternative hypothesis that the erythroid failure in DBA is the result of ribosomal insufficiency at the simultaneous onset of both rapid cell proliferation and a high rate of globin synthesis. In keeping with this, the expression of RPS19 showed 1.5–2 fold increase at 72hrs after EPO exposure, with 3.5 fold increase in the presence of dexamethasone, with equivalent results in both DBA and controls. We now aim to study translational and cell cycle events during this important wave of erythroid proliferation to further elucidate the molecular basis for the specific erythroid defect in DBA, despite the ubiquitous cellular requirement for ribosomal biogenesis.
A constitutively activated erythropoietin receptor stimulates proliferation and contributes to transformation of multipotent, committed nonerythroid and erythroid progenitor cells.
