A novel mutation in MPL (Y252H) results in increased thrombopoietin sensitivity in essential thrombocythemia

Abstract

Essential thrombocythemia (ET) is a rare type of myeloproliferative neoplasm characterized by clonal expansion of the megakaryocyte and platelet lineage. Here, we describe a novel mutation (Y252H) in the thrombopoietin (TPO) receptor, or MPL, in a JAK2 mutation-negative ET patient. The bone marrow examination revealed increased numbers of dysmorphic megakaryocytes with focal clustering. The x-inactivation pattern suggested clonal expansion of hematopoietic cells in the bone marrow. Furthermore, we found that the patient's bone marrow cells were hypersensitive to TPO in generating megakaryocyte colonies in vitro. More importantly, we demonstrated that this MPL Y252H mutant confers increased TPO/MPL-mediated cell growth and increased cell survival upon cytokine withdrawal in BaF3 cells, indicating it is a disease-driving mutation and may contribute to the development of ET in vivo. In summary, this is the first report describing a mutation in the extracellular domain of MPL underlying ET.

Figure 1. The MPL Y252H mutation enhances TPO-mediated megakaryocyte colony formation as well as TPO-dependent cell growth

(A) Hematoxylin & Eosin staining from the bone marrow biopsy of the patient is shown. Arrow indicates abnormal megakaryocyte clusters, while arrowheads indicate dysmorphic megakaryocytes with atypical nuclear chromatin patterns varying from widely dispersed nuclei to markedly hyperchromatic. (B) Bone marrow cells from the patient and a healthy control were subjected to Megacult colony assay with varying concentrations of TPO according to manufacturer’s instructions. Megakaryocyte colonies were enumerated from 4 replicates per group. p<0.001 by ANOVA. (C) Ba/F3 cells stably expressing vector alone, WT or the Y252H mutant MPL were generated by retroviral infection and live cell numbers were determined using an MTT assay. Results were normalized to the growth in IL-3. (D) Ba/F3 cells expressing vector alone, WT or Y252H MPL, were washed extensively and cultured in the absence of cytokines. Cell numbers were determined by Trypan blue staining using a hemacytometer. For all graphs in panels B-D, *: p<0.05; **: p<0.005.