A new allelic discrimination assay using locked nucleic acid-modified nucleotides (LNA) probes for detection of JAK2 V617F mutation

Discovery of an acquired mutation in the Janus kinase 2 (JAK2 V617F) in patients with Philadelphia chromosome negative (Ph-) myeloproliferative diseases (MPD) was reported by multiple groups of authors in 2005 [1]. The presence of the mutation confirms clonality in MPD, and according to some authors, it may be relevant even prognostically [2]. The reported proportions of patients carrying this mutation are highly variable, depending on the respective MPD entity and on the sensitivity of the method used for its detection [3]. The newly developed allelic discrimination assay that we wish to report on is based on real-time RTPCR [4,5]. It is very simple, high throughput and enables detection of 2% of the mutated allele. It uses two dual labelled TaqMan probes with locked nucleic acid (LNA)-modified nucleotides. LNA is a nucleic acid analog that contains a 20-O, 40-C methylene bridge. This bicyclic structure locks the ribose group into a C30-endo conformation. Introduction of LNA monomers into oligonucleotides increases thermal stability of heteroduplexes within þ3/þ88C per modification. This translates in increased specificity of the allelic discrimination [6]. The probes differ at the polymorphic site; one of them is complementary to the wild-type JAK2 allele and the other to the mutated one. Each probe is labelled with a different reporter dye on its 50-end (6-carboxyfluorescein (FAM) for the probe complementary to the wild type allele, 2,7-dimethoxy-4,5dichloro-6-carboxyfluorescein (JOE) for the mutant one in our hands) and a black hole quencher dye (BHQ1) is attached to the 30-end of both the probes. During PCR cycling, the probes specifically hybridize to the targeted sequences (mutated and wild type, respectively). Then the Taq polymerase cleaves the probe with its 50-30 nuclease activity, so that the reporter dye and quencher dye become separated [Figure 1(A)]. This leads to an increase of fluorescence intensity. Patients homozygous for the JAK2 mutation show only JOE signal, whereas increase of only the FAM signal indicates wild type homozygous genotype. Heterozygous patients show various ratios of both signals. To verify the results obtained by our new method, all samples were tested by the common allelespecific (AS)-PCR developed by Baxter et al. [7]. Peripheral blood samples from 261 patients with already diagnosed or suspected Ph-MPD were obtained. Patients with WHO-defined polycythaemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF) were included in the study, as well as patients with undifferentiated MPD (MPD-U), secondary polyglobulia (SP) and secondary thrombocytosis (ST). Mononuclear cells from peripheral blood were removed using Histopaque1077 (Sigma – Aldrich, St. Louis, MO) density gradient, and erythrocytes were lysed using red cell lysis solution (155 mmol NH4Cl, 10 mmol NH4HCO3, 0.1 mmol EDTA) in order to obtain relatively pure granulocytes. The contamination by other cells was estimated in smears of the separated cells and routinely did not exceed 10%. This granulocyte-rich cell fraction was washed twice with phosphate-buffered saline (137 mmol NaCl, 2.7 mmol KCl, 4.3 mmol Na2HPO4 7H2O, 1.4 mmol KH2PO4), and aliquots of 56 10 6 cells