Haploinsufficiency in RP genes is coupled to an attenuated hypusination in erythroid progenitors. (A) CD34⁺ BM progenitors from a healthy control and a patient with del(5q)-MDS were expanded ex vivo. Immunoblots showing hypusinated and total eIF5A levels at days 0 and 3 of EPO-induced differentiation are presented (left). Hypusination in CD34⁺ BM progenitors from a second patient with del(5q)-MDS was compared with progenitors from a patient with refractory anemia, after a 3-day differentiation in the absence (−) or presence (+) of EPO and blots are shown (middle). Quantification of the ratios of eIF5A/actin and eIF5A^H/eIF5A for progenitors from 3 patients with del(5q)-MDS are presented (n = 3). (B) CD34⁺ BM progenitors from a RPL11-haploinsufficient patient with DBA [c.27_28 delGA (p.N10FS)] were evaluated for hypusinated and total eIF5A levels at isolation (day 0) and then expanded for 4 days followed by 3 days of EPO-induced differentiation (left). The change in eIF5A^H/eIF5A after differentiation is shown. Peripheral blood (PB) CD34⁺ progenitors from a second RPL11-haploinsufficient patient (c.164dup; p.Tyr55Ter) were evaluated after ex vivo differentiation with a 4-day expansion followed by a 3 day EPO stimulation. eIF5A^H immunoblots are shown (middle). Quantifications of the ratios of eIF5A/actin and eIF5A^H/eIF5A for the 2 patients with DBA and 2 controls at day 3 of EPO-induced differentiation are presented (right). (C) The differentiation of shCTRL- and shRPL11-transduced progenitors to a vs n erythroid (GlyA⁺) vs myeloid (CD11b⁺) fate was evaluated by flow cytometry at day 3 of rEPO-induced differentiation. Representative CD11b/GlyA dot plots (left) and quantifications are presented (right, n = 10). (D) Hypusination was evaluated in human progenitors transduced with shCTRL and shRPL11 vectors at day 3 of differentiation. Representative immunoblots of eIF5A^H and total eIF5A levels in shCTRL- and shRPL11-transduced progenitors (left) and quantifications ± SEM of eIF5A^H/eIF5A and eIF5A/actin are presented (right, n = 6). Levels in control cells are set at “1.” (E) OCRs of shCTRL- and shRPL11-transduced progenitors were evaluated at day 1 of differentiation and representative data (left, n = 3-6 technical replicates) and mean basal OCR levels ± SEM are presented (right, n = 3 independent experiments). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗P < .0001.