Truncated Human Erythropoietin Receptor Causes Fetal Polycythemia and Is Associated with a Prolonged Primitive Erythropoiesis and Hyperactive Definitive Erythropoiesis.
Authors: Yoon Donghoon , Prchal Josef 
Published: April 2020

Abstract
Gain-of-function mutations that cause truncations of the cytoplasmic domain of the human erythropoietin receptor (EpoR) result in the dominantly inherited disorder, primary familial congenital polycythemia. The EpoR truncation causes polycythemia, which is not always seen at birth, and in vitro hypersensitivity of erythroid precursors to erythropoietin (Epo). We have replaced the murine EpoR (MEpoR) gene with either a wild-type human EPOR (wthEPOR) gene or a mutant human EPOR gene (mthEPOR). This mutation, initially identified in a family with polycythemia, produces a truncated EpoR with a deletion of the cytoplasmic C-terminal domain after the first tyrosine residue. To investigate the effects of wthEPOR and mthEPOR genes on fetal erythropoiesis, we examined mouse fetuses homozygous for either wthEPOR or mthEPOR from E12.5 up to 5 weeks postnatally (PN). The mthEPOR fetuses were polycythemic and the wthEPOR fetuses were anemic during the early embryonic stage (E12.5∼E16.5) and at the adult stage (3 weeks and older). However, during the perinatal period (E18.5∼PN2 weeks), both of the phenotypes temporarily disappeared. To study the pathophysiology of the phenotypes, we analyzed erythropoiesis using differential expression of CD71 and TER119 surface antigens by FACS in the peripheral blood (PB) and fetal liver (FL). PB from wthEPOR and mthEPOR had a significantly higher population of earlier progenitors (proerythroblasts and early basophilic erythroblasts) from E12.5 to E16.5 compared to MEpoR. From E18.5 on, however, all genotypes showed a similar pattern. At E14.5, FL of wthEPOR and mthEPOR homozygotes were similar to MEpoR FLs at E12.5. Since a dynamic switch of primitive to definitive erythropoiesis occurs around E14.5, we evaluated nucleated erythroid cell populations in PB from E12.5∼E15.5 using the DNA dye DRAQ5 followed by FACS analysis. From E13.5 to E14.5, mthEPOR fetuses contained more nucleated cells than MEpoR fetuses (43 to 55% vs 37 to 29%; mthEPOR vs MEpoR), whereas wthEPOR fetuses had comparable numbers to MEpoR fetuses (36 to 25% vs 37 to 29%; wthEPOR vs MEpoR). Interestingly, all genotypes from E15.5 and thereafter contained over 90% enucleated cells with no significant difference in their PB. To understand the molecular mechanism of these features, we have analyzed Stat5 phosphorylation after Epo stimulation in FL from E14.5 and E18.5. At both stages, the mthEPOR FL had a markedly increased and sustained Epo-sensitive Stat5 phosphorylation compared to MEpoR FL. In contrast, in wthEPOR FL Stat5 phosphorylation was significantly lower even at the highest concentration of Epo. The membrane-localized EpoR levels were measured by fluorescence labeled anti-EpoR antibody. All genotypes showed comparable levels of EpoR on the membrane. We demonstrate here that with the exception of the perinatal period, mthEPOR causes polycythemia whereas wthEPOR causes anemia, even in fetal life. We propose that the polycythemia of mthEPOR during early embryogenesis (E12.5∼E14.5) is due to a delayed switch from primitive to fetal erythropoiesis, followed by hyperactive definitive erythropoiesis. These characteristics (the prolonged primitive erythropoiesis and hyperactive definitive erythropoiesis) could be explained, at least in part, by hyperactive and sustained Stat5 activation.