The A3669G Polymorphism of GR Is a Host Genetic Modifier Associated with Polycythemia Vera and Primary Myelofibrosis

Abstract 3067

The Philadelphia chromosome negative myeloproliferative neoplasms (MPN) include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) which are all associated with a gain-of-function mutation (JAK2V617F) of the JAK2 gene. There is ongoing discussion on the mechanism by which this single mutation may cause diseases with three different phenotypes. The consensus is that while the JAK2V617F may increase proliferation of hematopoietic cells in general, the lineage affected is determined by host genetic modifiers still to be identified. The glucocorticoid receptor (GR) is an important regulator of erythropoiesis in vivo and in vitro. It exerts its activity by synergistic transcriptional and membrane-associated mechanisms. Upon activation, GR forms a transcriptional complex with phosphorylated STAT-5 (P-STAT-5) which binds and alters the expression of a subset of the erythropoietin (EPO)- and stem cell factor (SCF)-target genes. Physical interaction between GR and the EPO receptor (EPO-R) on the cell membrane suppresses the ability of both receptors to phosphorylate STAT-5 when activated together. Insufficient GR/P-STAT-5 transcriptional activity has been suggested to result in delayed erythroid maturation and to induce eythrocytosis in patients exposed to excessive glucocorticoids. We had previously determined that erythroid cells (EBs) generated ex vivo from peripheral blood mononuclear cells of patients with PV and those obtained from normal donors (ND) stimulated with dexamethasone have similar biological (increased proliferation with delayed maturation) and biochemical (great levels of NFE-2, WT-1 and GATA2 and low levels of GATA1 and β-globin expression) properties (Varricchio et al,Blood 2009;114:3899a). This similarity is counterintuitive because STAT-5 phosphorylation was reduced in normal EBs exposed to dexamethasone and EPO while in EBs from PV patients it was constitutive due to the presence of the JAK2V617F mutation. In addition to the canonical GRα signalling isoform, cells may express the dominant negative GRβ isoform which binds poorly to glucocorticoids because its unique structure impairs ligand binding and determines nuclear retention. Expression of GRβ is regulated by the presence of the A3669G SNP in exon 9 which specifically stabilizes GRβ mRNA. The presence of this polymorphism has been associated with glucocorticoid resistance but its effects on erythropoiesis are unknown. To clarify the similarities between the biological properties of EBs expanded ex vivo from ND in the presence of dexamethasone and those obtained from PV patients, we determined the levels of expression (at the mRNA and protein level) of GRα and GRβ in EBs expanded ex vivo from 16 ND and 16 PV patients. While EBs from ND expressed GRα only, those expanded from PV patients expressed both GRα and GRβ. Therefore, in spite of constitutive STAT-5 phosphorylation, in EBs from PV patients formation of transcriptionally active GR/P-STAT-5 complexes is reduced by expression of GRβ. To clarify the mechanism underlying expression of GRβ in EBs obtained from PV patients, the frequency of the A3669G polymorphism was determined by specific PCR followed by sequencing of the amplified products using DNA obtained from the mononuclear cells of ND (7) and patients with PV (10). DNA from patients with PMF (10) and ET (14) was analysed for comparison. The polymorphism was detected in 60% of patients with PV but not in ND, indicating that GRβ mRNA is particularly stable in EBs from PV patients. In addition, the polymorphism was observed in 50% of patients with PMF but was not detected in patients with ET. A trend toward higher JAK2V617F allele burden was observed in A3669G positive PV patients (69.8±23.0) compared to A3669G negative PV patients (37.3±22.6) and ET patients (28.13±27.9) while the JAK2V617F allele burden in patients with PMF with or without the A3669G SNP was the same (51.2±37.5 vs 42.8±17.9). These data suggest that in addition to mutations of EPO-R and Von Hippel-Lindau gene, erythrocytosis may be induced by excess GR activation by glucocorticoids or, in association with the JAK2V617F mutation, by the presence of the A3669G SNP which favours GRβ expression. Moreover, the A3669G SNP may represent a host genetic modifier and a potential diagnostic tool to predict development of erythrocytosis in patients with MPN and GRβ may represent a potential therapeutic target for PV.