Truncated Human EpoR Causes Polycythemia in Fetal Erythropoiesis through Stat5 Hyperactivation.

Gain-of-function mutations causing truncations of the cytoplasmic domain of the human erythropoietin receptor (EPOR) result in a dominantly inherited disorder-primary familiar congenital polycythemia. The consequence of EpoR truncation is polycythemia and in vitro hypersensitivity of erythroid precursors to erythropoietin (Epo). We have replaced the murine EpoR gene with a wild-type human EPOR (whEPOR) gene or a mutant human EPOR gene (mthEPOR) that we initially identified in a family with polycythemia. This mutation produces a truncated EpoR mutant with deleted cytoplasmic C-terminal domain after the first tyrosine residue of the intracellular domain. We reported that the mice containing mthEPOR allele have polycythemia and the adult whEPOR mice have anemia. To study the effects of these genotypes on fetal erythropoiesis, we examined fetuses E14.5 homozygotes wild type mouse EpoR, and human EPOR alleles. Hematocrit results show: MthEPOR fetuses were polycythemic compared to mouse EpoR (mthEPOR vs mouse EpoR; 34.58 ± 1.24 % vs 27.49 ± 0.90 %, P<0.001 by t-test), and whEPOR were anemic (whEPOR vs mouse EpoR; 18.61 ± 0.79 % vs 27.49 ± 0.89 %, P<0.001 by t-test). To study the pathophysiology of these phenotypes, we analyzed the erythroid differentiation using differential expression of CD71 and TER119 surface antigens by FACS in the fetal liver cells (FLs). FLs from mthEPOR homozygote had increased population on early and late basophilic erythroblast stage compared to mouse EpoR, but the populations of differentiated erythroid progenitors from mthEPOR homozygote were significantly lower than mouse EpoR and whEPOR homozygotes. We proposed that the erythroid progenitors from mthEPOR exit fetal liver to fetal blood circulation prematurely. The peripheral blood from mthEPOR and mouse EpoR s was analyzed by morphology and FACS. We show that the majority of population in peripheral blood from mthEPOR homozygote were earlier erythroid progenitors (high CD71/low TER119 population in mthEPOR 47.53 %, vs 13.14 % mouse EpoR; high CD71/high TER119 population from mthEPOR 11.49 % vs 65.00 % mouse EpoR). To evaluate the Epo signal transduction we tested the kinetics and dose responses of Epo at Stat5 phosphorylation level. The mthEPOR expressing FLs showed a markedly increased and sustained Epo sensitivity compared to mouse EpoR expressing FLs. In contrast, in whEPOR expressing FLs, Stat5 phosphorylation was significantly lower even at the highest concentration of Epo. We conclude that mthEPOR causes fetal polycythemia and is associated with premature exit of early erythroid progenitors from fetal liver to fetal circulation. In contrast whEPOR is associated with more severe anemia than we observed in adult erythropoiesis. The degree of Epo induced Stat5 activation plays an important role in generation of anemic and polycythemic phenotypes.