Jak2 Exon 12 Mutational Status in a Cohort of Idiopathic Erithrocytosis V617F Negative Patients.
Authors: Bernardi Martina , Ruggeri Marco , Albiero Elena , Madeo Domenico , Finotto Silvia , Rodeghiero Francesco 
Published: November 2019

Abstract
Background: V617F gain-of-function mutation in exon 14 of Janus Kinase 2 gene (JAK2) has been recognized as a major pathogenetic event of many cases of chronic myeloproliferative disorders, in particular in Polycythemia Vera (PV). The molecular basis of myeloproliferative disorders in patients without the V617F is still unclear but, recently, four new heterozygous allelic variants affecting JAK2 exon 12 have been described in V617F negative patients receiving a diagnosis of PV or Idiopathic Erythrocytosis (IE) (Scott et al, NEJM 2007). Revised WHO criteria for PV have been proposed considering these mutations as indicative for PV diagnosis (Tefferi et al, 2007).
Aim: To determine the JAK2 exon 12 mutational status in patients with IE in a cohort of 40 consecutive IE V617F negative patients; 2 PV patients, V617F negative, were also included. 36 males and 6 females with a median age of 63 years (range: 25–85), median follow-up 109 months (range: 7–205) were studied. IE was diagnosed in presence of increased haemoglobin (>186 gr/L) and hematocrit levels (>51%) and normal white blood cells and platelet counts. In addition, no splenomegaly, and a normal or low serum erythropoietin (Epo) levels were present along with a bone marrow biopsy showing erythroid hyperplasia with normal megakaryopoiesis and granulopoiesis. They were treated only with phlebotomy therapy. PV was diagnosed according to the WHO criteria.
Methods: Granulocytes were isolated from the lower interface of a density gradient separation of peripheral blood samples; the granulocytes were then submitted to erythrocytes lysis (mean purity of the granulocytes 97%) and DNA was extracted. Exon 12 was amplified using the couple of JAK2exon12F (5′-ctcctctttggagcaattca-3′) and JAK2exon12R (5′-caatgtcacatgaatgtaaatcaa-3′) primers. The amplicons were submitted to denaturing high performance liquid chromatography (dHPLC) analysis and runs were performed at 53° C. The amplicons showing a heteroduplex profile were sequenced directly in both strands.
Results: 4 of the 40 (10%) IE patients were heterozygous positive for N542-E543del JAK2 exon 12 mutation; both PV patients were negative for JAK2 exon 12 mutations (figure 1).
Conclusion: N542-E543del JAK2 was found only in 10% of patients with a diagnosis of IE, a frequency lower than that previously reported (Scott et al, NEJM 2007). This result could be due to the low levels of mutation prevalence in peripheral blood granulocytes. The sensitivity could be increase using erythroid colonies grown on colture media. In addition, different patients selection criteria could explain our results. Accordingly with the proposed revised WHO criteria for Polycythemia, a diagnosis of PV instead of IE should be made in these four patients.
figure 1: Clinical features of patients with JAK2 exon 12 mutations and Idiopathic Erythrocytosis. figure 1:. Clinical features of patients with JAK2 exon 12 mutations and Idiopathic Erythrocytosis.