Absolute erythrocytosis are rare disorders characterized by a significant increase of the red blood cell mass which is reflected by an increased number of circulating erythrocytes, high hematocrit and hemoglobin. Primary familial and congenital polycythemia (PFCP, familial erythrocytosis type 1, OMIM #133100) is an inherited form of this disease, caused by erythropoietin (EPO) hypersensitivity of erythroid precursor cells. A number of mutations in the intracellular region of the EPO receptor (EPOR) have been found in recent years to be associated with manifestation of the disease.

The objective of the study was to explore the mechanisms by which these mutations in the cytosolic domain of EPOR may induce the erythrocytosis phenotype. The research strategy was to construct chimeric receptors that contain the extracellular and transmembrane regions of the human epidermal growth factor receptor (EGFR) fused to the cytosolic domain of the human EPOR. The moiety of the EPOR intracellular region was mutated according to 4 novel mutations discovered in the cytosolic domain of the EPOR in PFCP patients. This design enabled EPOR signaling to be triggered by EGF binding to the extracellular domain of the chimera. The working hypothesis was that since the cytosolic, EPOR region of the chimera was generated based on mutations discovered in PFCP patients, any differences in the chimera-related effects would be specifically attributed to the mutations. The experiments were performed in an in vitro system, in which the Wild-Type (WT) EGFR– EPOR and the EGFR– EPOR chimeras bearing the mutated EPOR cytosolic domain were stably transfected into the EPO-dependent human erythroleukemia cell-line UT7. As these cells lack the EGF receptor, the natural EPOR signaling can be avoided when the transfected cells are grown with, or activated by, EGF. Using this system we have analyzed 4 novel EPOR mutations discovered in PFCP patients: 2 deletions which lead to frameshifts, premature stop codons and truncations (Del1387-1390 and Del1378-1412), a nonsense mutation (C1371A) which results in truncation of the receptor, and a missense mutation (G1445A) which results in the substitution of arginine 437 to histidine.

A panel of experiments was performed on UT7 cells expressing the WT EGFR-EPOR chimera or EGFR– EPOR chimeras bearing the EPOR cytosolic PFCP-associated mutations. We addressed the degradation rates of the chimeric receptors, the kinetics of EGF induced STAT5 and MAPK/ERK signaling cascades and the levels of glycan-maturation and cell-surface expression of the chimeras.

Cell viability analysis revealed that UT7 cells expressing either one of the 4 mutated chimeric receptors proliferated even under very low (0.01 ng/ml) EGF concentrations, as opposed to cells expressing the WT chimera, which did not proliferate under these conditions. UT7 cells expressing the mutated chimeras also displayed enhanced growth, which may indicate that indeed the mutations are the underlying cause of PFCP. Notably, our findings are the first description of a missense EPOR mutation that is associated with over-activity of the EPOR and with development of a polycythemic phenotype.

Furthermore, we found that each of the 4 EPOR mutations differentially affected signaling and metabolism of the chimeric receptors. Hence, (i) Del1387-1390, showed higher levels of glycan-mature protein, as well as slower degradation rates, and conferred slower attenuation of EGF-induced pSTAT5. (ii) Del1378-1412 exhibited slower degradation rates and higher levels of glycan-mature protein. (iii) The nonsense mutation, C1371A, displayed higher cell surface levels, slower degradation rates and relayed constitutive activation of STAT5. (iv) The missense mutation, G1445A, mediated a slower attenuation of EGF induced MAPK/ERK signaling.

Our study supports the evidence regarding the role of the cytosolic region of EPOR in metabolism of the receptor and sheds new light on the mechanisms underlying erythrocytosis-inducing mutations in the EPOR.

HH, MFMcM, MJP, CB, HC and DN are members MPN&MPNr-EuroNet (COST Action BM0902)


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.

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