Abstract(1) Objective-The chronic myeloproliferative disorders (MPD), polycythemia vera (PV), essential thrombocytosis (ET) and idiopathic myelofibrosis (IMF), are characterized by a spectrum of clinical features and linked by common genetic lesions in JAK2 and MPL. However, the clinical phenotypes in genetically undefined MPD patients are similar to those patients with JAK2 and MPL lesions. We, therefore, sought to determine whether there were JAK2 or MPL lesions in a welldefined, JAK2 V617F-negative MPD cohort, and to determine if clinical associations could be identified based on variations identified in these genes.(2) Methods-We examined the JAK2 and MPL genes in JAK2 V617F-negative PV, IMF and idiopathic erythrocytosis (ERT) patients for sequence variations.(3) Results-We identified two previously unrecognized JAK2 mutations and three previously unrecognized MPL mutations in JAK2 V617F-negative PV, erythrocytosis and IMF patients. We identified JAK2 exon 12 lesions in 30% of JAK2 V617F-negative PV patients, and either JAK2 V617F or JAK2 exon 12 lesions in 9% of ERT patients In IMF, in addition to the MPL gene mutation, W515K, we identified three additional mutations: 204P and 2 intervening sequence transitions: IVS 11/12 and 10/11.(4) Conclusions-While the clinical phenotype of JAK2 exon 12 lesions in the MPD was predominantly erythroid, there was significant disease spectrum overlap between JAK2 V617F and JAK2 exon 12 mutations. By contrast, MPL gene mutations were not associated with erythrocytosis, but segregated primarily with the phenotypes of thrombocytosis, extramedullary disease, myelofibrosis and osteosclerosis.

The chronic myeloproliferative disorders (MPD), polycythemia vera (PV), essential thrombocytosis (ET) and idiopathic myelofibrosis (IMF), are linked by a common genetic lesion, JAK2V617F, and abnormalities of the thrombopoietin receptor, Mpl. JAK2 is an obligate Mpl chaperone and is responsible for its cell surface expression. We observed a reciprocal relationship between neutrophil JAK2V617F allele percentage and platelet Mpl expression in JAK2V617F-positive PV, IMF and ET patients. However, severely impaired platelet Mpl expression was also present in the majority of JAK2V617F-negative PV and IMF patients, with the most severe reductions in IMF. We previously identified a constitutional variation in the Mpl gene in the distal N-terminal domain (K39N) associated with thrombocytosis, and an Mpl splice variant involving the same region that was increased in PV and IMF patients compared to controls. Given the association of Mpl genetic lesions and protein expression abnormalities in the MPD, we hypothesized that other lesions in Mpl may be present in the MPD. To test this, we sequenced all of the exons, the flanking intervening sequences and the 5′-proximal untranslated region of the Mpl gene in 1 ET, 3 PV and 4 IMF patients, all of whom were JAK2V617F-negative and all of whom had markedly reduced (<5%) platelet Mpl protein. In one IMF patient, we identified a two base substitution at nucleotide 1543 of the Mpl gene which results in a tryptophan to lysine substitution at amino acid 515 (W515K); the wild-type sequence was not identified from either peripheral blood CD34+ cells or neutrophils isolated from this patient. The patient developed IMF at the age of 26, and had the disease 23 years before succumbing to complications of extensive extramedullary hematopoiesis and anemia. In a second male patient, we identified a heterozygous nucleotide transition in the intervening sequence distal to exon 10 close to the splice donor site; the mutation was also detected in buccal cell DNA. This patient developed IMF at age 35 and has had the disease for 31 years characterized by anemia and extramedullary hematopoiesis. To test whether this or other lesions in exon 10 of the Mpl gene were present, we directly sequenced exon 10 in 9 patients with JAK2V617F-negative PV, 21 with JAK2V617F-negative IMF, and 13 with JAK2V617F-positive IMF. Neither these nor other lesions in exon 10 were identified in these 43 MPD patients; no other unreported SNPs were identified either. We did not identify the recently described MplW515L reported as present in 4/44 JAK2V617F-negative IMF patients (PLoS Med.2006;3:e270) in our 21 JAK2V617F-negative IMF patients. These data indicate that, in contrast to other cytokine receptors, genetic and epigenetic variations of Mpl have unique roles in the pathogenesis of the chronic MPD. We conclude that significant disruptions of Mpl, whether acquired or constitutional, at the genomic level as described above or at the proteomic level as we have previously described, associate with a chronic MPD characterized by significant extramedullary hematopoiesis.