Molecular characterization of Philadelphia-negative myeloproliferative neoplasms

A somatic gain-of-function mutation in exon 14 of the Janus kinase 2 (JAK2) gene is found in Philadelphia-negative myeloproliferative neoplasms, which include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Hematopoietic cells can be either heterozygous or homozygous for JAK2 (V617F) mutation, producing variable proportions of mutant alleles and a relation was observed between mutation status and clinical phenotype. Moreover, while the vast majority of patients with PV carry the V617F mutation in JAK2 exon 14, mutations in exon 12 have been recently reported in V617F-negative patients with PV. For the evaluation of granulocyte JAK2 (V617F) mutation status, we developed both a quantitative real-time polymerase chain reaction (PCR)-based and a microelectronic chip assay as a model of microarray-based approach. A significant correlation was found between the percentages of JAK2 (V617F) mutant alleles estimated by the two methods. However, the real-time PCR approach proved to be more sensitive in the detection of the minority mutant allele than the microelectronic chip. To further characterize JAK2 exon 12 mutations, we performed analysis in 128 patients with JAK2 (V617F)-negative myeloproliferative neoplasms. Direct sequencing analysis of JAK2 exon 12 led to the identification of five different mutations in cases where the levels of the mutated alleles were higher than 10%

Somatic mutations of JAK2 exon 12 in patients with JAK2 (V617F)-negative myeloproliferative disorders

We searched for JAK2 exon 12 mutations in patients with JAK2 (V617F)-negative myeloproliferative disorders. Seventeen patients with polycythemia vera (PV), including 15 sporadic cases and 2 familial cases, carried deletions or duplications of exon 12 in circulating granulocytes but not in T lymphocytes. Two of the 8 mutations detected were novel, and the most frequent ones were N542-E543del and E543-D544del. Most patients with PV carrying an exon 12 mutation had isolated erythrocytosis at clinical onset, unlike patients with JAK2 (V617F)-positive PV, most of whom had also elevations in white blood cell and/or platelet counts. Both patients with familial PV carrying an exon 12 mutation had an affected sibling with JAK2 (V617F)-positive PV. Thus, several somatic mutations of JAK2 exon 12 can be found in a myeloproliferative disorder that is mainly characterized by erythrocytosis. Moreover, a genetic predisposition to acquisition of different JAK2 mutations is inherited in families with myeloproliferative disorders

Deep sequencing reveals double mutations in cis of MPL exon 10 in myeloproliferative neoplasms

Somatic mutations of MPL exon 10, mainly involving a W515 substitution, have been described in JAK2 (V617F)-negative patients with essential thrombocythemia and primary myelofibrosis. We used direct sequencing and high-resolution melt analysis to identify mutations of MPL exon 10 in 570 patients with myeloproliferative neoplasms, and allele specific PCR and deep sequencing to further characterize a subset of mutated patients. Somatic mutations were detected in 33 of 221 patients (15%) with JAK2 (V617F)-negative essential thrombocythemia or primary myelofibrosis. Only one patient with essential thrombocythemia carried both JAK2 (V617F) and MPL (W515L). High-resolution melt analysis identified abnormal patterns in all the MPL mutated cases, while direct sequencing did not detect the mutant MPL in one fifth of them. In 3 cases carrying double MPL mutations, deep sequencing analysis showed identical load and location in cis of the paired lesions, indicating their simultaneous occurrence on the same chromosome