Molecular analysis of the erythropoietin receptor system in patients with polycythaemia vera

Erythropoietin (EPO) is a potent regulator of the viability, proliferation and differentiation of erythroid progenitor cells. Its effect is mediated by binding to the erythropoietin receptor (EPO-R), a member of a new cytokine receptor family. Alterations of the EPO/EPO-R system have recently been shown to be involved in the pathogenesis of familial erythrocytosis and polycythaemia vera (PV). In order to define whether genetic changes in the EPO-R gene and its ligand play a role in the development of PV, the structure and expression levels of the EPO-R and EPO genes were examined in samples from bone marrow and/or peripheral blood mononuclear cells of 24 patients with PV. As expected, EPO serum levels were low and no detectable level of EPO mRNA was found by reverse polymerase chain reaction (RT-PCR) in the peripheral blood mononuclear cells of our PV patients. To search for structural alterations of the EPO-R, cDNA samples were subjected to PCR and SSCP analysis as well as sequencing. Heterogenous expression of EPO-R mRNA was observed without any structural changes, as revealed by RT-SSCP analysis using overlapping primers spanning the whole coding region of the EPO-R gene. Structural integrity of the EPO-R was further confirmed by sequencing of cloned PCR products. These data suggest that the mechanisms for the development of PV do not involve structural changes of the EPO-R gene.