JAK2 exon 12 mutations in polycythemia vera and idiopathic erythrocytosis

Abstract

Background: The V617F mutation, which causes the substitution of phenylalanine for valine at position 617 of the Janus kinase (JAK) 2 gene (JAK2), is often present in patients with polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. However, the molecular basis of these myeloproliferative disorders in patients without the V617F mutation is unclear.

Methods: We searched for new mutations in members of the JAK and signal transducer and activator of transcription (STAT) gene families in patients with V617F-negative polycythemia vera or idiopathic erythrocytosis. The mutations were characterized biochemically and in a murine model of bone marrow transplantation.

Results: We identified four somatic gain-of-function mutations affecting JAK2 exon 12 in 10 V617F-negative patients. Those with a JAK2 exon 12 mutation presented with an isolated erythrocytosis and distinctive bone marrow morphology, and several also had reduced serum erythropoietin levels. Erythroid colonies could be grown from their blood samples in the absence of exogenous erythropoietin. All such erythroid colonies were heterozygous for the mutation, whereas colonies homozygous for the mutation occur in most patients with V617F-positive polycythemia vera. BaF3 cells expressing the murine erythropoietin receptor and also carrying exon 12 mutations could proliferate without added interleukin-3. They also exhibited increased phosphorylation of JAK2 and extracellular regulated kinase 1 and 2, as compared with cells transduced by wild-type JAK2 or V617F JAK2. Three of the exon 12 mutations included a substitution of leucine for lysine at position 539 of JAK2. This mutation resulted in a myeloproliferative phenotype, including erythrocytosis, in a murine model of retroviral bone marrow transplantation.

Conclusions: JAK2 exon 12 mutations define a distinctive myeloproliferative syndrome that affects patients who currently receive a diagnosis of polycythemia vera or idiopathic erythrocytosis.

Figure 1. Somatic Mutations of JAK2 Exon 12 in Patients with Polycythemia Vera or Idiopathic Erythrocytosis

Panel A shows DNA-sequence traces from peripheral-blood granulocytes and T lymphocytes and from erythropoietin-independent erythroid colonies. Nucleotides are indicated by capital letters, with N representing sites at which wild-type and mutant nucleotides are apparent at the same position. The traces reveal four acquired mutations within JAK2 exon 12 (indicated by arrowheads), often with low-level involvement in granulocytes. Panel B (top) shows the alignment of wild-type and mutant exon 12 JAK2 alleles (shown in red) (nucleotides are indicated by capital letters and amino acids by bold capital letters; dashes indicate the positions of deleted nucleotides). The amino acid alignment across multiple species (Panel B, bottom) shows conservation of the mutated amino acids, indicated in red.

Figure 2. Erythroid Hyperplasia with Normal Granulopoiesis and Megakaryopoiesis in Patients with JAK2 Exon 12 Mutations

Bone marrow trephine sections, stained with hematoxylin and eosin, from a control patient (Panel A) and from one patient with V617F-positive polycythemia vera (PV) (Panel B; shown at twice the magnification in Panel C) show the marked hypercellularity with trilineage hyperplasia that is characteristic of patients with polycythemia vera. There are increased numbers of megakaryocytes, some of which are morphologically abnormal and present in clusters (arrowheads). In contrast, trephine sections from Patient 5, with a K539L JAK2 mutation, were only mildly hypercellular and showed isolated erythroid hyperplasia (Panel D; shown at twice the magnification in Panel E; hematoxylin and eosin). Megakaryocytes appear to be morphologically normal and are not clustered. In Panel F (shown at the same magnification as that in Panel E), Wright–Giemsa staining highlights cells of the erythroid lineage. B denotes bone, and F fat.

Figure 3. Proliferation and Increased Signaling in the Absence of Exogenous Cytokine from Jak2 Exon 12 Mutations

BaF3/EpoR cells (10⁵ per cubic millimeter) — transduced with an empty retroviral vector or stably expressing wild-type murine Jak2 or Jak2 with V617F, F537-K539delinsL, H538QK539L, K539L, or N542-E543del mutations — were cultured in the absence of interleukin-3 for 4 days (Panel A). On days 2 and 4, we assessed cell numbers and viability in quadruplicate using trypan-blue exclusion. Results reflect four independent experiments; mean (±SD) counts for each cell line at both time points are shown. BaF3/EpoR cells transduced with an empty MSCViresGFP retroviral vector (Panel B), or BaF3/ EpoR cells containing wild-type Jak2 or Jak2 with V617F, F537-K539delinsL, H538QK539L, N542-E543del, or K539L mutations were depleted of cytokines for 4 hours. Cells were lysed and underwent immunoprecipitation (IP) with antibody specific for Jak2 or Stat5; Western blot (WB) was then performed with antibodies against phosphotyrosine (4G10), total Jak2, phosphotyrosine-694 Stat5, or total Stat5 (Panel B). BaF3/EpoR cells expressing the Jak2 alleles were analyzed by Western blot with antibodies specific for phosphorylated or total extracellular regulated kinase 1 (Erk1) and 2 (Erk2) (Panel C). BaF3/EpoR cells stimulated with 10 U per milliliter of erythropoietin for 10 minutes were used as positive controls in Panels B and C. Plus signs indicate presence and minus signs absence of exogenous Jak2 or erythropoietin.

Figure 4. A Myeloproliferative Phenotype, Resulting from Retroviral Expression of K539L Jak2, in a Murine Model of Bone Marrow Transplantation

Panel A shows the mean (±SD) hematocrit, white-cell count, and platelet count in the peripheral blood of BALB/c mouse recipients of bone marrow expressing wild-type, V617F, or K539L Jak2. Mice (five in each group) were evaluated 38 days after transplantation. P values are shown for the comparison with recipients of wild-type Jak2. Panels 2nd 3rd B, C, and D (hematoxylin and eosin) show representative peripheral-blood smears from mice 38 days after transplanation.