JAK2 V617F Mutation Detection: Laboratory Comparison of Two Kits Using RFLP and qPCR

Abstract

Aim: The JAK2 V617F mutation has been implicated in a variety of diseases mainly related to myeloproliferative disorders including polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis with an increased demand for testing using molecular techniques. The latter are diversified and all aim to simplify the methods employed for detection. Materials and Methods: In this study, two detection kits were compared: one using polymerase chain reaction (PCR)-restriction fragment length polymorphisms (RFLP) (JAK2 Activating Mutation assay; InVivoScribe Technologies, San Diego, CA) and the other using real-time quantitative PCR (JAK2 MutaScreen Kit assay; Ipsogen, Marseilles, France). Results: A total of 80 reactions were compared using the two techniques and the results showed a perfect concordance between the two methods. Conclusion: We conclude that both PCR-RFLP and quantitative PCR are extremely useful and sensitive techniques for the detection of the JAK2 V617F mutation with quantitative PCR being more time effective and less expensive.

Introduction

JAK2 is an intracytoplasmic tyrosine kinase that has an essential role in signal transduction from several hemopoietic growth factor receptors. The international published literature so far points to the extreme importance of this gene and its acquired point mutation, V617F (Saharinen et al., 2000; Komura et al., 2003; Ugo et al., 2004). The latter substitutes a phenylalanine for a valine at position 617 of the JAK2 protein and occurs in the JH2 pseudokinase domain that negatively regulates the kinase domain (Saharinen et al., 2000). The JAK2 V617F mutation appears to trigger cytokine-independent activation of JAK-STAT, phosphoinositide 3-kinase, AKT kinase, mitogen-activated protein kinase, and extracellular signal-regulated protein kinase pathways (Kralovics et al., 2005; Zhao et al., 2005), all of which are involved in the regulation of erythropoietin receptor signaling (Witthuhn et al., 1993; Socolovsky et al., 1999; Myklebust et al., 2002; Ugo et al., 2004).

JAK2 V617F has been implicated in a variety of clinical conditions mainly associated with the group of myeloproliferative disorders. This mutation has been observed in around 95% of the polycythemia vera patients, 50-60% of the essential thrombocythemia patients or idiopathic myelofibrosis patients (Baxter et al., 2005; Jones et al., 2005; Campbell et al., 2006; Levine et al., 2006) and in a small number of patients with chronic myelomonocytic leukemia, chronic neutrophilic leukemia, hypereosinophilic syndrome, myelodysplasia, and acute myeloid leukemia (Jelinek et al., 2005; Jones et al., 2005; Levine et al., 2005; Scott et al., 2005; Steensma et al., 2005; Campbell et al., 2006; Fröhling et al., 2006). On the other hand, JAK2 V617F has not been reported in lymphoid cancers or in nonhematopoietic disorders (Levine et al., 2005; Scott et al., 2005).

Currently, several useful and sensitive assays are available to test for the V617F JAK2 mutation including restriction enzyme digestion (Baxter et al., 2005), real-time polymerase chain reaction (PCR) (Levine, 2005), allele-specific PCR (Campbell et al., 2006), and pyrosequencing (Jelinek et al., 2005), which are all important methods that serve to provide the most accurate detection results and simplify the diagnostic workup. However, techniques differ in their result turnaround time, cost, as well as ease of use. In this study, two detection kits were compared: one using PCR-restriction fragment length polymorphisms (RFLP) (JAK2 Activating Mutation assay; InVivoScribe Technologies, CA) and the other using real-time quantitative PCR (JAK2 MutaScreen Kit assay; Ipsogen, Marseille, France). This descriptive report is the experience of a tertiary care center with a bone marrow transplantation center and a high referral rate and admissions of oncology patients.

Materials and Methods

Samples and DNA extraction

This study was conducted at the American University of Beirut Medical Center, which is a tertiary care Lebanese center. The extraction of the DNA material was done using the PEL-FREEZ extraction kit (PEL-FREEZ; DYNAL, Oslo, Norway) and genomic material stored at −80°C. This DNA material originated from samples belonging to patients referred for JAK2 testing in our center. A total of 40 samples were run in duplicate; thus, 80 reactions were compared between the two techniques utilized for the V617F mutation detection.

JAK2 activating mutation assay and PCR

The protocol of The JAK-2 Activating Mutation (InVivoScribe Technologies) was followed exactly as stated by the manufacturer to screen for the V617F mutation. To start with, PCR was used to amplify the portion of the JAK-2 region that acquired this mutation. The thermocycler program consisted of an initial denaturation step of 95°C for 7 min, followed by 34 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 1 min, and final extension step of 72°C for 10 min. The JAK-2 amplicon products were then digested with BsaXI (New England BioLabs, Ipswich, MA) restriction enzyme and placed at 37°C overnight. Finally, the products were run on a 2% agarose/TBE gel and viewed under ultraviolet light. “Normal” samples following BsaXI digestion generated 140, 97, and 30 bp products. “Homozygous” samples having the V617F mutation no longer have the BsaXI restriction site and thus keep the 267 bp amplicon intact. “Heterozygous” samples generate the 267 amplicon in addition to the 140, 97 and 30 bp products.

JAK2 MutaScreen Kit and quantitative PCR

The JAK-2 MutaScreen Kit (Ipsogen Cancer Profiler) was used for the detection of JAK2 V617F mutation in genomic DNA. The assay followed exploits the quantitative PCR Double Dye Hydrolysis Oligonucleotide Probes principle. Quantitative PCR results were obtained promptly by real-time detection of fluorescent signals during and/or following the PCR cycling and without any post-PCR processing. The amplification mix included forward and reverse primers and double-dye probes that were labeled with a 5′ reporter and 3′quencher dyes. The assay followed used the 5′-3′ exonuclease activity of the Taq polymerase. PCR was used to amplify the portion of the JAK-2 region that had the mutation. The thermocycler program consisted of an initial step of 55°C for 2 min, then 95°C for 10 min, followed by 50 cycles of 92°C for 15 s, and 60°C for 1 min. At the end of the amplification run, the LightCycler 2.0 instrument (Roche Diagnostics, Indianapolis, IN) was used to obtain the FAM/VIC (530/560) ratio. The mean ratio of the samples tested was simply compared with the reference sample mean ratio value. If the mean ratio of sample was equal or superior to the mean ratio of the reference sample, then the sample was considered “mutant.” However, if the mean ratio of the sample was less than the mean ratio of the reference sample, the sample was considered as “Wild” type.

Results

All 80 reactions (from the 40 myeloproliferative samples run in duplicate) were 100% concordant between the two techniques for the JAK2 V617F mutation detection.

Discussion

The diagnosis of myeloproliferative disorders has been recently associated with increasing demand for testing and detection of the V617F mutation in the JAK2 gene. Identification of this mutation is vital, not only for diagnosis and classification of the myeloproliferative disorders but also for the development of novel targeted therapies similar to imatinib in the treatment of chronic myeloid leukemia. In addition, the detection of this mutation has been associated with response to treatment. For example, the presence of V617F mutation in patients with essential thrombocythemia is associated with a better response to hydroxyurea (Campbell et al., 2005).

Different laboratories use different techniques for detection of this mutation and the advanced technology in molecular diagnostics is getting reflected on all types of gene mutation detection whether in homebrew tests or ready-made kits marketed by different companies. In this study we evaluated the performance of two such new detection kits, each employing a different technique: the first employs PCR-RFLP and the second utilizes real-time quantitative PCR. The high concordance (100%) between the two techniques makes them highly useful for direct application in any laboratory; however, quantitative PCR has been standing out as a faster tool for generation of a result with a minimal turnaround time.

In professional hands, the PCR-RFLP assay needs at least 24 h to generate a result that is mainly related to the needed enzymatic digestion, usually overnight. On the other hand, a real-time quantitative technique for the detection of JAK2 V617F mutation can be performed within the same day and in less than 4 h, including the needed steps for DNA extraction and PCR setup and amplification reactions.

Another parameter to consider for those laboratories that can afford either one of these techniques is costlier. In our medical center and per single patient, the cost of a PCR-RFLP reaction is around 110 USD while that for a run using a quantitative PCR technique around 78 USD. Needless to mention here that quantitative PCR requires the presence of a real-time PCR instrument which is not needed for those labs employing PCR-RFLP.

In terms of sensitivity, both of these techniques are quite comparable with around 1 copy of mutant gene per 100 cells which is very acceptable in diagnostic terms since the clone of cells that harbors the JAK2 V617F mutation, and usually is pathognomonic of the symptomatology and presentation of the disease in question, is present in a sufficient detectable load to classify the patient as either “positive” or “negative” for the mutation.

In conclusion, the two commonly used methods for detection of JAK2 V617F mutation, PCR-RFLP and quantitative PCR, are acceptable diagnostic techniques for implementation in molecular diagnostic laboratories; however, the latter remains faster, saves more technical time, and is more cost effective for those laboratories that can afford a real-time PCR instrument and both can be considered in laboratories that lack a sequencer, knowing that the “gold standard” for point mutation testing remains at the level of direct sequencing for the highest sensitivity and accuracy of detection.

Footnotes

Disclosure Statement

No competing financial interests exist.

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