The overall objective of this proposal is to identify defects of the erythropoietin receptor (EPO-R - a newly distinguished proto-oncogene) in patients with primary familial and congenital polycythemias and polycythemia vera and to study the role of EPO-R defects in the pathogenesis of these diseases. In those individuals in whom no mutation of EPO-R mutation is found the possibility of persistence, or delayed turn-off, of the autocrine stimulation of erythropoiesis by EPO will be examined. The cause of primary polycythemias at a molecular level is not understood. Based on the in vitro behavior of erythroid progenitors, we hypothesize that the mechanism is an increased sensitivity of erythroid progenitors to erythropoietin (EPO) in patients with familial and congenital polycythemias and polycythemia vera and this hypersensitivity may be related to defects of the EPO-R. We have identified various mutations of the EPO-R in some patients with primary familial and congenital polycythemias. The proposed work will search for defects in the EPO-R by sequencing all of the important regulatory regions of EPO-R cDNA (Specific Aim 1). In order to determine the relevance of these defects to disease phenotype, mutated EPO-R cDNA will be transfected into a suitable cell line, and the EPO sensitivity of these transfectants will be measured (Specific Aim 2). Finally, we will correlate clinical features of these patients with the in vitro behavior of their erythroid progenitors (Specific Aim 3). We will develop the animal model of the polycythemia by expressing the wild and mutated EPO- R genomic DNA and cDNA in transgenic mice and confirm the veracity of our causative role of EPO-R mutation in etiology of polycythemia (Specific aim 4). Finally, in those individuals in whom no mutation of EPO-R mutation is found the possibility of persistence, or delayed turn- off, of the autocrine stimulation of erythropoiesis by EPO will be examined (Specific Aim 5). These studies should provide important insights into the pathogenesis of primary polycythemias as well as erythroleukemias. Such information should contribute to better understanding of, and more effectively targeted therapeutic interventions in, myeloproliferative and leukemia disorders.
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