Absence of mutations of the EPO‐receptor gene in Greek patients with familiar polycythemia

To the Editor: Primary familiar and congenital polycythemia (PFCP) is a rare myeloproliferative, congenital and dominant disorder with an autosomal mode of inheritance (1). It is caused as a result from inherited defects in hypoxia sensing or from inherited vital defects in red blood cell precursors that cause augmented responsiveness to erythropoietin (EPO) (2, 3). These facts result in isolated proliferation of the erythroid progenitor cells and in erythrocytosis. Clinically, the patients suffer from headaches, hypertension, dizziness, nose bleeding and pruritus after bathing (2, 4). Moreover, there may be expected an increased risk of both haemorrhagic and thrombotic episodes (2), as well as an elevated incidence of cardiovascular diseases (4). In laboratory examination the findings are: increased red blood mass without raised white blood cell and platelet count, normal or low values of EPO levels and normal values of arterial Po2 and P50 (1, 2). There is also hypersensitivity of erythroid progenitors to EPO in serum containing clonogenic cultures (5). The EPO-receptor (EPO-R) is situated on the surface of erythroid cells (3). The EPO-R consists of 508 amino acids with three different domains: one extracellular, one single hydrophobic transmembrane area and a cytoplasmic domain (3, 5). The EPO-R gene is situated on chromosome 19 and contains eight exons: exons I–V code for the extracellular domain, exon VI for the transmembrane and exons VII and VIII the cytoplasmic domain (3). The cytoplasmic domain contains the COOH-terminal of the receptor, which also contains a negative regulatory domain (5). There is widespread suspicion that abnormalities of the EPO-R gene are responsible in the pathogenesis of the disorder. Moreover, 12 mutations in patients with familiar polycythemia have been recognised. Ten of these mutations have a result in truncated EPO-Rs lacking the cytoplasmic COOH-terminal of the receptor (4). The aim of this study was to investigate the DNA taken from peripheral blood of patients with PFCP and detect potential mutations on the EPO-receptor gene. We studied eight families with familiar polycythemia of Greek origin. Peripheral blood from 15 healthy individuals, who were used as controls, was drawn. The data concerning the patients are shown in Table 1. Lymphocyte genomic DNA was extracted from peripheral blood and polymerase chain reaction (PCR) was employed using primers as described before (5). Single-strand conformational polymorphism (SSCP) analysis and sequencing was performed to detect mutations in the exon VIII, which is the specific area of the EPO-R gene responsible for the cytoplasmic domain of the EPOR, using the same protocol (5). The SSCP screening of the PCR products showed identical pattern in the patients and the controls, in native polyacrylamide gel under strict conditions. The sequence analysis of the PCR products revealed the wild-type nucleotide sequence in all patients. In the light of these results we conclude that in the exon VIII of the EPO-R gene, which was investigated, there is no detectable point mutation, which could underlie the molecular pathogenesis of the disease. Our results can lead to the conclusion that the molecular cause of polycythemias in Greek patients cannot be attributed to exon VIII alterations. It must be mentioned that in the literature, EPO-R mutations can be detected in only 10–20% of all