DETECTION OF JAK2 EXON 12 MUTATIONS BY HETERODUPLEX ANALYSIS AND PYROSEQUENCING

Abstract

Background: Somatic mutations in codons 533-547 of JAK2 exon 12 are highly specific to confirm the diagnosis of polycythemia vera (PV). We have previously proposed techniques for the detection and quantification of JAK2 exon 12 allele burden using a pyrosequencing method (Subbotina T et al, Haematologica 2014). However, due to the high cost of sequencing, developing a two-stage algorithm for detect mutations in JAK2 exon 12 using inexpensive screening is of immediate practically necessity.

Aims: The aim of this study was to demonstrate the feasibility of using heteroduplex analysis with separation of the PCR product by electrophoresis on non-denaturing PAGE as the preliminary screening test for detection of JAK2 exon 12 mutations.

Methods: 274 patients with PV or unclear erythrocytosis and with a low JAK2V617F allele burden or unmutated JAK2V617 (51 women, mean age 52.2±15.7 years and 223 men, mean age 43.6±15.6 years) were included in this study. The informed consents from these patients were obtained. The PCR with the additional stage of formation heteroduplexes was performed using the Real-time PCR kit (Syntol, Russia) and CFX 96 Real Time System (Biorad, USA). PCR products were analyzed by electrophoresis in 8% PAGE. The presence of the mutations was identified and confirmed by pyrosequencing method with PyroMark Q24 (Qiagen, Germany). To verify the presence of mutations, the DNA sequences extracted from the clinical samples were cloned into pGem-T vector using standard protocol («Promega», USA), and obtained clones were sequenced using reagents and equipment of the «Applied Biosystems» (USA). JAK2 exon 12 varianceMUT was calculated as a measure of relative changes in allele burden between the baseline and follow-up sample (Theocharides A et al, Haematologica, 2008).

Results: We detected JAK2 exon 12 mutation in five out 274 patients. The results of electrophoresis on non-denaturing PAGE are reported in Figure 1. The type of №1-5 patient mutations was determined by pyrosequencing: N542-E543del (c.1624_1629delAATGAA); I540-E543delinsKK (с.1619_1627 TCAgAAATg<AAA); N542_E543del (c.1623_1628delAAATGA) and R541_E543>K (c.1622_1627delGAAATG) and p.H538_K539>L (c.1612_1616CACAA>TT). These mutations have been already described. Main characteristics of 5 patients with JAK2 exon 12-mutated PV are reported in Table 1. The PV diagnosis of №1, 2, 3 and 5 patients was confirmed by bone marrow trephine biopsies histological examination. All five patients with JAK2 exon 12-mutated PV have an increased number of red blood cells, along with an accompanying increase in the concentration of hemoglobin and hematocrit level in the peripheral blood. Some of them had increase number of leukocytes and platelets in the disease dynamics. №1-4 patients was treated phlebotomy only and did not received any cytoreductive treatment to date. Patient №5 receives hydroxyurea (HU). Importantly, two out five patients with JAK2 exon 12-mutated PV also have a mutation JAK2V617 (<1%). JAK2 exon 12 allele burden in sample from №1 patient is significantly increased in the disease dynamics.

Summary/Conclusion: The proposed variant of the heteroduplex analysis with separation of the PCR product by electrophoresis on non-denaturing PAGE can be recommended for use as the preliminary screening test which is carried out before the confirming pyrosequencing. The two-stage approach allows to optimize the algorithm of the JAK2 exon 12 mutation detection and to improve the efficiency of testing for patients suspected of having PV in whom a JAK2V617F mutation is not detected or detected in a low allele burden. In five out 274 patients we detected JAK2 exon 12 mutation and confirmed the diagnosis of PV.