Two-phase culture in Diamond Blackfan anemia: localization of erythroid defect

Abstract

The erythroid defect in Diamond Blackfan anemia (DBA) is known to be intrinsic to the stem cell, but its molecular pathophysiology remains obscure. Using a 2-phase liquid erythroid culture system, we have demonstrated a consistent defect in DBA, regardless of clinical severity, including 3 first-degree relatives with normal hemoglobin levels but increased erythrocyte adenosine deaminase activity. DBA cultures were indistinguishable from controls until the end of erythropoietin (Epo)-free phase 1, but failed to demonstrate the normal synchronized wave of erythroid expansion and terminal differentiation on exposure to Epo. Dexamethasone increased Epo sensitivity of erythroid progenitor cells, and enhanced erythroid expansion in phase 2 in both normal and DBA cultures. In DBA cultures treated with dexamethasone, Epo sensitivity was comparable to normal, but erythroid expansion remained subnormal. In clonogenic phase 2 cultures, the number of colonies did not significantly differ between normal cultures and DBA, in the presence or absence of dexamethasone, and at both low and high Epo concentrations. However, colonies were markedly smaller in DBA under all conditions. This suggests that the Epo-triggered onset of terminal maturation is intact in DBA IntroductionDiamond Blackfan anemia (DBA) is a rare congenital red cell aplasia, which classically presents with profound aregenerative anemia in early infancy, often in association with physical anomalies and growth retardation. [1][2][3] The anemia responds to steroid therapy in up to 70% of patients, but eventually around 40% of affected individuals are dependent on long-term transfusion programs. [1][2][3][4] Spontaneous remissions may occur, even in patients who have never previously achieved transfusion independence. [1][2][3][4][5] Sustained remissions have occasionally been reported in response to interleukin-3 (IL-3). 6,7 Recently, clinical responses to prolactin or metoclopramide have been described, but only after prolonged administration. 8 Remissions, whether spontaneous or treatment induced, are usually associated with a residual erythropoietic defect shown by persistent mild anemia and macrocytosis, 1-3 with increased erythrocyte adenosine deaminase (eADA) activity. [9][10][11] The pathophysiologic basis for this characteristic pattern of erythroid failure and remission in DBA remains elusive, even after the identification of the gene encoding ribosomal protein S19 (RPS19) as the first DBA gene, 12 mutated in up to 25% of affected individuals. 13 RPS19 was not an obvious candidate gene for this disorder, being ubiquitously expressed in its normal ribosomal role. Gene transfection studies support the association between RPS19 mutations and impaired erythroid maturation, 14 but the specific contribution of RPS19 to normal and abnormal erythropoiesis has yet to be defined.The demonstration of impaired erythroid differentiation in vitro of enriched erythroid progenitor cells 15 and of purified CD34 ϩ cells 16 from patients with DBA provides strong evidence fo...

Ribosomal Protein S19 Deficiency Leads to Reduced Proliferation and Increased Apoptosis but Does Not Affect Terminal Erythroid Differentiation in a Cell Line Model of Diamond-Blackfan Anemia

“…Hence, abnormalities in EPO-R expression, EPO binding, or EPO signal transduction may be present. Ohene-Abuakwa et al speculate that the defect lies downstream of the EPO-R, influencing survival and/or proliferation of erythroid progenitors in DBA cells [26]. Our findings presented here demonstrate that the level of EPO-R expression and EPO signal transduction is normal in an RPS19-deficient DBA model cell line.…”

Ribosomopathies: human disorders of ribosome dysfunction

“…81,82 Similarly, primary CD34 ϩ cells from DBA patients have a higher number of apoptotic cells compared with normal CD34 ϩ cells, 83 and mononuclear cells derived from patients with DBA fail to proliferate in response to erythropoietin and form smaller erythroid colonies in methylcellulose compared with normal patients. 84 The hematopoietic defect caused by RPS19 deficiency can be rescued by forced overexpression of RPS19. Expression of a RPS19 transgene by retroviral vectors in CD34 ϩ bone marrow cells from DBA patients significantly improved erythropoiesis.…”

Diamond-Blackfan anemia: erythropoiesis lost in translation

“…In their study peripheral blood mononuclear cells from healthy donors and patients with DBA were compared in a 2-phase culture system. 13 During the initial pre-Epo-dependant phase both normal and DBA-cell numbers declined 2-fold. When Epo was added for the second phase of culture, a wave of erythroid proliferation was seen in normal cells, whereas DBA cells failed to respond to Epo.…”