A real-time polymerase chain reaction assay for rapid, sensitive, and specific quantification of the JAK2V617F mutation using a locked nucleic acid-modified oligonucleotide

Abstract

The JAK2V617F mutation has emerged as an essential molecular determinant of myeloproliferative neoplasms (MPNs). The aim of this study was to evaluate the analytical and clinical performances of a real-time PCR (qPCR) assay using a combination of hydrolysis probes and a wild-type blocking oligonucleotide, all containing locked nucleic acid (LNA) bases. Moreover, we validated a procedure for precise quantification of the JAK2V617F allele burden. We used DNA samples from patients suspected to suffer from MPN and dilutions of HEL cells, carrying the mutation, to compare the LNA-qPCR assay to two previously published methods. All assays detected the same 36 JAK2V617F positive patients of 116 suspected MPN diagnostic samples. No amplification of normal donor DNA was observed in the LNA-qPCR, and the assay was able to detect and reproducibly quantify as few as 0.4% of the JAK2V617F allele in wild-type alleles. Quantification of the JAK2V617F allele burden showed similar proportion levels among the different MPN entities as described by other groups. In conclusion, the LNA-qPCR is a rapid, robust, sensitive, and highly specific assay for quantitative JAK2V617F determination that can be easily implemented in clinical molecular diagnostic laboratories. Moreover, precise quantification allows determination of JAK2V617F burden at diagnosis as well as the evaluation of response to JAK2 inhibitors.

Figure 1

Schematic overview of the mutation-specific LNA-based qPCR reaction. At the left part of the figure, the perfect matching between the hydrolysis probe and the mutated DNA sequence allows annealing of the probe to the target DNA and consequently the Taq polymerase can hydrolyze the probe. In contrast, the single bp mismatch between the LNA oligonucleotide sequence and the mutated DNA sequence prevents specifically its annealing. At the right part of the figure, the LNA oligonucleotide anneals to wild-type target DNA, thereby preventing the detection of wild-type JAK2 sequences. The LNA oligonucleotide thus allows preferential amplification of the mutated DNA if present. Moreover, the mutated specific hydrolysis probe cannot efficiently hybridize to wild-type target because of a single bp mismatch between both. LNA indicates locked nucleic acid.

Figure 2

Proportion of JAK2V617F allele burden of the 36 JAK2 mutant patients included in this study. Box-and-Whisker presentation of the results from patients with PV (n = 11), ET (n = 14), PMF (n = 8), and post-PV MF (n = 3). The central box represents 25th to 75th percentile, and the middle line the median. The black vertical line extends from the minimum to the maximum value, excluding “outside” and “far out” values. P values are represented for comparisons between all disease entities. PV indicates polycythemia vera; ET, essential thrombocythemia; PMF, primary myelofibrosis; post-PV MF, post-polycythemic myelofibrosis.