A Genetic Mutation on Murine Chromosome 7 Causes Thrombocytosis Associated with Thrombopoietin Excess - A Model of Jak2V617F Negative Idiopathic Thrombocytosis.

A substantial proportion of patients with Essential Thrombocythemia (ET) have evidence of an acquired Jak2V617F mutation that causes the myeloproliferative phenotype. Jak2V617F negative ET patients presumably have mutations in other related pathways or alternatively may have chronic thrombocytosis secondary to factors extrinsic to the hemopoietic system. This second hypothesis is supported by clonality studies that demonstrate at least 30% of women with ET have evidence of polyclonal granulocytes. As part of an ENU mutation screen to identify novel genes associated with thrombocytosis, we have studied a mutant mouse strain, called plt2, with autosomal recessive thrombocytosis associated with increased thrombopoietin production. This mutation is linked to mouse chromosome 7, and therefore is unrelated to mutations in Jak2, thrombopoietin or the thrombopoietin receptor, c-Mpl. plt2/plt2 mice have platelet counts of 2038±347x10⁹/L (n=107) increased by 47% compared to wild-type mice (1386±223; n=107, p<0.001). This thrombocytosis was associated with increased numbers of megakaryocyte progenitor cells (plt2/plt2 28.8±4.6; n=4, Meg-CFC/5x105 cells to maximal cytokine stimulation compared to wild-type 18.3±5.5; n=4, p<0.05) and megakaryocytes (108±15; n=7 megakaryocytes per hpf compared to 70±13; n=7, p<0.01) that demonstrated a normal maturation profile based on ploidy measurement. plt2/plt2 mutant mice exhibited mild hepatomegaly (liver weight 1.6±0.2g; n=34 compared to 1.3±0.2g; n=23, p<0.001) and serum thrombopoietin levels were elevated by 76% (4930±1309pg/mL; n=18 compared to 2802±1031; n=15, p<0.001). There was no difference in relative TPO transcription between plt2/plt2 mice and wild-type mice in the liver (p=0.23), however, the TPO content of whole liver lysates was increased by 50% in the mutant mice (150%±50; n=8 per 1g liver weight compared to 100±32; n=8, p=0.03). To examine how the plt2 mutation interacts with other components of the TPO pathway, plt2/plt2 mice were intercrossed with mice lacking the TPO receptor (Mpl^−/−). Mpl^−/−plt2/plt2 mice have thrombocytopenia with platelet counts that are indistinguishable from Mpl^−/−/ mice confirming that the plt2 mutation causes thrombocytosis by acting through the Mpl receptor. Surprisingly, Mpl^+/−plt2/plt2 mice, with only one copy of the Mpl gene, had marked thrombocytosis with platelet counts of 2656±459x10⁹/L (n=25) increased by 92% over wild-type mice suggesting an additive effect between excess thrombopoietin and Mpl heterozygosity on platelet count. We have utilized ENU mutagenesis to identify a novel mouse pedigree with heritable thrombocytosis linked to chromosome 7 that demonstrates altered cytokine production can mimic the chronic thrombocytosis that is the cardinal diagnostic feature of ET.