Sequence Analysis of MPL Exon 10 of ET and MF JAK2 V617F Negative Patients Reveals a New Mutation

Background. Diagnosis and classification of myeloproliferative disorders was greatly enhanced by reports describing JAK2V617F mutation in Polycythemia Rubra Vera (PRV) patients and approximately 50% of Essential Thrombocythemia (ET) and Myelofibrosis (MF) patients. More recently mutations mapping to the juxtamembrane region of the thrombopoietin receptor MPL were described in 5–10% of the patients with MF and 1% of the patients with ET. The reported data imply the Exon 10 MPL mutations lead to receptor activation in the absence of thrombopoietin binding and constitutive phosphorylation of JAK2, STAT3, STAT5, AKT and ERK. However, thus far only mutations mapping to codons 515 and 505 have been published. The aim of this study was to determine the frequency of JAK2V617F and MPL exon 10 mutations in MF and ET patients seen at the Hematology Department, Centro Hospitalar de Coimbra.

Methods. Sixty three patients with ET and 12 with MF, diagnosed according to the Polycythemia Vera Study Group (PVSG) criteria, were studied. Genomic DNA was extracted from peripheral blood samples collected into EDTA. Samples were tested for JAK2 V617F mutation by allele specific polymerase chain reaction (ASO-PCR). Subsequently these samples were screened for MPL exon 10 mutations by direct sequencing.

Results. 45 (71.4%) of the 63 patients with ET and 6 (50%) of the 12 MF patients had JAK2V617F mutation. Of the remaining 6 MF patients 3 had MPL mutations; two with W515L and one with W515K amino acid substitutions. Direct sequencing failed to detect exon 10 MPL mutations in 17 of the 18 ET JAK2 V617F negative patients. The remaining patient had a previously unreported MPL mutation mapping to codon 524 where C was substituted by T, predicting a replacement of arginine residue by cysteine (R524C).

Conclusions. JAK2 V617F mutation was detected in 71% of the ET patients and in 50% of the MF. MPL mutations were found in 2 out of 12 MF patients (17%) and in one of the 63 ET patients (1.6%). The MPL codon 515 mutations are reported to activate mpl protein independent of ligand binding. The prevalence of MPL mutations among MF patients is higher than the described in previous reports, however this maybe due to the small number of patients studied. Whilst there is genotype and phenotype correlation between MPD and MPL mutations described the precise functional role in the development of MPD remain unclear. Similarly, whether the previously unreported MPL R524C mutant allele presented here leads to activation of mpl protein like codon 515 mutations is unclear. But nonetheless such mutations assist in accurate diagnosis and classification of Philadelphia negative MPD patients with the view to optimization of clinical management of these patients.