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JMD 2007, Vol. 9, No. 1
Copyright © 2007 American Society for Investigative Pathology & Association for Molecular Pathology

Development of a Quantitative Real-Time Polymerase Chain Reaction Assay for the Detection of the JAK2 V617F Mutation

Elizabeth C. Wolstencroft*, Katy Hanlon*{dagger}, Lorna W. Harries*{dagger}, Graham R. Standen{ddagger}, Alexander Sternberg§ and Sian Ellard*{dagger}

From the Molecular Genetics Department, * Royal Devon and Exeter National Health Service (NHS) Foundation Trust, Exeter; Institute of Biomedical and Clinical Science, {dagger} Peninsula Medical School, Exeter; Department of Haematology, {ddagger} Bristol Royal Infirmary, Bristol; and Department of Haematology, § Royal Devon and Exeter NHS Foundation Trust, Exeter, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Achieving a specific diagnosis of polycythemia vera (PV) and other myeloproliferative disorders (MPDs) is often costly and complex. However, the recent identification of a V617F mutation in the JH2 domain of the JAK2 gene in a high proportion of patients suffering from MPDs may provide confirmation of a diagnosis. This is an acquired mutation and, as such, may only be present in a small number of cells within a sample. There is therefore a clinical need for highly sensitive detection techniques. We have developed a sensitive real-time polymerase chain reaction (PCR)-based approach for both detection and quantification of the JAK2 V671F mutation load, which allows determination of mutation status without the need for prior purification of granulocytes. We have performed a comparison of this assay with two previously published detection methods. Although an amplification refractory mutation system (ARMS) was shown to be slightly superior in terms of sensitivity, our real-time PCR method provides the potential for quantification of the JAK2 V617F mutation, having potential future applications in the monitoring of minimal residual disease or predicting outcome of disease severity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Myeloproliferative disorders (MPDs) are diseases characterized by proliferation of one or more myeloid cell lineages in the bone marrow and increased numbers of mature and immature cells in the peripheral blood. Examples include polycythemia vera (PV), essential thrombocythemia (ET), and idiopathic myelofibrosis (IMF). These conditions are reported to have an annual incidence of 2 to 3, 1.5 to 2, and 0.5 to 1.5 per 100,000 of the population, respectively.

The diagnosis of MPDs can be complex, expensive, and, in the case of ET, based solely on exclusion criteria. There is therefore a real clinical need for a faster, more specific, and more efficient test. The recent discovery of an acquired point mutation (c.1849G>T, p.V617F) within the Janus kinase 2 (JAK2) gene in a percentage of these patients has allowed a definitive diagnosis1, 2, 3, 4, 5 by providing a specific target for genetic testing. The mutation causes a valine to phenylalanine substitution (V617F) in the JH2 domain of the protein. This region has been suggested to play a direct role in the negative regulation of JAK2 signaling.6 The substitution of a valine residue by the large aromatic amino acid phenylalanine is likely to disrupt this regulation. The JAK2 V617F mutation has been shown to be present in the majority of patients with polycythemia vera (65 to 97%) and approximately half of patients with essential thrombocythemia (23 to 57%) or idiopathic myelofibrosis (43 to 50%).1, 3, 5

The identification of this acquired mutation establishes the presence of a clonal disorder and has allowed new approaches to the diagnosis and treatment of these diseases.7 Because this acquired mutation may be present only in a small proportion of cells, sensitive detection methods are required. Previously published detection techniques include an allele-specific PCR that uses a single common reverse primer and two forward primers, one specific to the mutant allele and one that serves to generate an internal control product1 ; an amplification refractory mutation system (ARMS), which uses two primer pairs to amplify specifically the normal and mutant sequences plus a positive control band in a single reaction5 ; direct fluorescent dye chemistry sequencing4 ; pyrosequencing5 ; restriction length polymorphism1 ; and real-time assays based on melt-curve analysis.8, 9 These approaches, although sensitive, are not quantitative. A recent publication demonstrated a correlation between hematological improvement and a reduction in the proportion of the JAK2 V617F mutant alleles.10 An approach that permits both detection and quantitation may therefore be of use in the future.

In this study, we have developed and validated a novel real-time, quantitative allele-specific PCR assay using the TaqMan 7000 platform to detect the JAK2 V617F mutation in a cohort of 200 patients suffering form a variety of MPDs. We have validated our approach by comparing these results with those achieved using two previously published methods in terms of sensitivity and specificity. The development of an approach that is able to quantitate JAK2 mutation load as well as detect the presence of the mutation may prove to have clinical use in the future.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients
Samples were received from 200 consecutive patients initially referred for investigation of one or more of high hemoglobin, platelet count, neutrophilia, or blood film suggestive of an MPD. Patients were classified by their MPD diagnosis into five categories: idiopathic myelofibrosis (n = 9), polycythemia vera (n = 74), essential thrombocythemia (n = 51), secondary polycythemia (n = 49), apparent polycythemia (n = 7), or other (for example, unclassifiable myeloproliferative disease or CMML) (n = 10). The diagnostic criteria for polycythemia vera, apparent polycythemia, and secondary polycythemia were derived from the British Committee for Standards in Hematology Guidelines (2004) (http://www.bcshguidelines.com/pdf/polycythaemia.pdf). Diagnosis of essential thrombocythemia and idiopathic myelofibrosis was based on World Health Organization criteria.11 All cases of idiopathic myelofibrosis presented de novo. There was nothing histologically to imply a fibrotic transformation of polycythemia vera; however, this cannot be excluded. Those cases of myelofibrosis with know antecedent PV were classified as polycythaemia vera. This study was conducted in accordance with the Declaration of Helsinki as revised in 2000.

Sample Preparation
Total DNA was extracted from peripheral blood using the QIAamp DNA mini kit (Qiagen, Paisley, UK). DNA concentration and sample absorbance at 260 and 280 nm were measured using a ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). Samples with a 260:280 ratio less than 1.8 were rejected as unsuitable for analysis.

Real-Time Assay Development
Allele-specific real-time quantitative PCR was performed using the ABI Prism 7000 platform as previously described.12 An assay specific for the JAK2 V617F mutation was designed using the Assays-by-Design service from Applied Biosystems (Foster City, CA). The assay contains probes specific to the wild-type (G) and mutant (T) alleles labeled 5' with VIC and 6-fluorescein (6-FAM), respectively. Our assay uses a single set of primers, so mutation discrimination is achieved by the use of probes that differ only at the position of the V617F mutation (probe and primer sequences are given in Table 1Go ). This approach has the advantage that detection of the JAK2 mutation is independent of amplification efficiency. The labeling of the probes with different fluorophores allows detection of the two alleles in a single-tube analysis. The presence of a probe against the wild-type sequence acts as an internal control to assess quality of the DNA and also allows quantification of mutation load by calculation of a wild-type to mutant allele ratio.


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  		Table 1. Sequence of Primers and Probes Used in Quantitative Real-Time PCR

 
PCR reactions mixture contained 10 µl of TaqMan Universal PCR MasterMix (Applied Biosystems), 0.08 µmol/L of each probe, 0.36 µmol/L of each primer and 40 ng of DNA, in a total volume of 20 µl. PCR conditions were 2 minutes at 50°C and 10 minutes at 95°C, followed by 50 cycles of denaturation for 15 seconds at 95°C and annealing/extension for 1 minute at 60°C. The fluorescent signal intensities were recorded and analyzed during PCR in an ABI Prism 7000 sequence detector system (Applied Biosystems) using the SDS (version 1.0) software (Applied Biosystems).

The assay was validated for accuracy by the construction of standard curves using a 1:2 serial dilution of DNA containing both mutant and normal alleles. Each dilution was set up in triplicate to allow calculation of SE. The sensitivity of our real-time assay was determined by mixing experiments involving the dilution of pure V617F mutant DNA in normal DNA. Concentrations of mutant DNA ranged from 100 to 0.5%. The pure mutant JAK2 template was generated as previously described.1

Comparison of Detection Methods
Patient samples were simultaneously analyzed for the JAK2 V617F mutation by two previously described techniques1, 5 The sensitivity of the assays was compared using the {chi}2 test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Validation of the Assay
Standard curves were constructed for both wild-type and mutant probes (Figure 1)Go . The gradients of the standard curves for the two alleles were within 0.03, indicating that both alleles were detected with equal efficiency. Because primers are common, efficiency of amplification was unaffected by genotype. There was a high correlation between input DNA and crossing point [r2 values of 0.975 (wild-type) and 0.950 (mutant)].


Figure 1
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  		Figure 1. Standard curves for the mutant and wild-type JAK2 probes generated by 1:2 serial dilution of DNA.

 
The sensitivity of the assay was determined by generating serial dilutions of the 100% mutant DNA with 100% wild-type DNA to generate samples with the JAK2 V617F mutant allele present at levels of 100, 10, 7.5, 5, 2.5, 1, and 0.5%. This method showed the assay to be sensitive enough to detect 2.5% mutant DNA (Figure 2A)Go . A small nonspecific signal from the mutant probe binding to wild-type sequences was noted. This is due to cross-hybridization, which is a common observation where sequences differ by a single base and is easily distinguished from genuine low positives (Figure 2B)Go . Such signals are inherent to exonuclease assays where discrimination is at the single bp level.13


Figure 2
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  		Figure 2. A: Mixing curve showing limit of detection of the real-time PCR assay. 1, 100% mutant; 2, 10% mutant; 3, 7.5% mutant; 4, 5% mutant; 5, 2.5% mutant; 6, 1% mutant; 7, 0.5% mutant; and 8, 100% wild type. B: The nonspecific hybridization of the mutant probe to wild-type sequence can easily be distinguished from genuine low positives.

 
Comparison of JAK2 V617F Detection Methods
Samples from 200 patients with MPD were tested for the presence of the JAK2 V617F mutation by three methods (Table 2)Go . Using the real-time approach, 76/196 (39%) of the samples gave a positive result. Two tests were inconclusive, and two samples failed to give a result. Of the 69 patients who tested positive by the Baxter et al1 allele-specific PCR method, 68 samples were also positive by the real-time PCR technique (one was inconclusive). All samples found to be positive by the real-time and allele-specific PCR techniques were also detected by the Jones et al5 ARMS technique. The ARMS assay detected the V617F mutation in a further five samples where the real-time PCR test was negative and 12 where the allele-specific PCR test was negative. We have shown the ARMS assay as described by Jones et al5 to be more sensitive than either the real-time technique ({chi}2, P = 0.0125) or the allele-specific PCR method described by Baxter et al1 ({chi}2, P = 0.0001). These results are consistent with the sensitivity of these assays in other laboratories, which have been reported as 1 to 2% and 3% for the ARMS and allele-specific PCR methods, respectively.1, 5


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  		Table 2. Number of Patients in Each of the Six MPD Categories Tested As Positive for the JAK2 V617F Mutation by the Three Different Mutation Detection Techniques

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previously published techniques for detection of the JAK2 V617F mutation include allele-specific PCR,1 an ARMS,5 direct fluorescent dye chemistry sequencing,4 pyrosequencing,5 restriction length polymorphism,1 and real-time assays based on melt-curve analysis.8, 9 We have developed and validated a quantitative real-time PCR assay to detect the JAK2 V617F mutation and have shown this assay to be able to sensitive enough to detect 2.5% mutant DNA in an artificial mixture of pure mutant and wild-type DNA. However, it should be noted that in a more heterogeneous mixed population without prior purification of granulocytes, this sensitivity may be compromised.

Each of these techniques provides a faster, cheaper, and less invasive diagnostic tool than previously available techniques for the diagnosis of myeloproliferative disorders such as the red cell mass test. Of these tests, the allele-specific PCR,1 the ARMS method,5 and real-time assays based on either melt-curve analysis8, 9 or TaqMan technology have proved to be the most sensitive. However, because both allele-specific PCR and ARMS techniques rely on the discrimination of genotypes at the level of amplification, differences in amplification efficiency between allele-specific primers may be a complicating factor. This is not an issue with the real-time approach because mutation detection uses a common set of primers and is thus independent of amplification. As previously stated, although each of these assays provide a faster diagnostic tool for myeloproliferative disorders than those previously available, recent advances in TaqMan technology allow up to 96 samples to be analyzed in 35 minutes.

A comparison of the real-time PCR technique with the allele-specific PCR1 and ARMS5 methods revealed that this assay was able to detect the V617F mutation in 76 (39%) of all samples tested, including seven samples that were negative by the allele-specific PCR technique.1 The ARMS assay5 detected the V617F mutation in a further five samples where the real-time PCR test was negative. In keeping with other studies, we were able to detect the V617F mutation in the majority of patients with PV by all three methods (60 to 70%; Table 2Go ) and nearly half of the patients with ET (33 to 43%; Table 2Go ). It was noted that in our study, all three methods detected a higher than expected number of V617F-positive results for patients with myelofibrosis (56 to 67%; Table 2Go ). This may be due to the relatively small sample number within this category for this study and differences in classification criteria.

In conclusion, we have developed a novel quantitative real-time PCR approach to the detection of the V617F mutation of the JAK2 gene associated with polycythemia vera and other MPDs. We have also performed a comprehensive validation of our approach compared with two previously published methods.1, 5 An ARMS detection method5 proved to be slightly more sensitive than the real-time PCR approach (1 to 2% versus 2.5%), and we now routinely use this technique in our laboratory. However, unlike this ARMS detection method and other previously published techniques,1, 4, 5, 8, 9 our technique has the potential for quantitative analysis. Recent reports indicate a possible correlation of hematological improvement with reduced V617F mutation load.10 Our method may therefore have future applications for monitoring minimal residual disease, predicting disease progression or thrombotic events.


   Acknowledgments
 
We thank Dr. M.V. Joyner, Dr. R. Lee, Dr. M. Pocock, Dr. C. Rudin, and Dr. P. Vyas for providing samples for this study.


   Footnotes
 
Address reprint requests to Sian Ellard, Institute of Biomedical and Clinical Science, Peninsula Medical School, Molecular Genetics Department, Royal Devon and Exeter NHS Foundation Trust, Barrack Rd., Exeter, UK EX2 5DW. E-mail: Sian.Ellard{at}rdehc-tr.swest.nhs.uk

Accepted for publication August 16, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, Swanton S, Vassiliou GS, Bench AJ, Boyd EM, Curtin N, Scott MA, Erber WN, Green AR: Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 2005, 365:1054-1061[Medline]
  2. James C, Ugo V, Le Couedic JP, Staerk J, Delhommeau F, Lacout C, Garcon L, Raslova H, Berger R, Bennaceur-Griscelli A, Villeval JL, Constantinescu SN, Casadevall N, Vainchenker W: A unique clonal JAK2 mutation leading to constitutive signaling causes polycythaemia vera. Nature 2005, 434:1144-1148[CrossRef][Medline]
  3. Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR, Tichelli A, Cazzola M, Skoda RC: A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 2005, 352:1779-1790[Abstract/Free Full Text]
  4. Zhao R, Xing S, Li Z, Fu X, Li Q, Krantz SB, Zhao ZJ: Identification of an acquired JAK2 mutation in polycythemia vera. J Biol Chem 2005, 280:22788-22792[Abstract/Free Full Text]
  5. Jones AV, Kreil S, Zoi K, Waghorn K, Curtis C, Zhang L, Score J, Seear R, Chase AJ, Grand FH, White H, Zoi C, Loukopoulos D, Terpos E, Vervessou EC, Schultheis B, Emig M, Ernst T, Lengfelder E, Hehlmann R, Hochhaus A, Oscier D, Silver RT, Reiter A, Cross NC: Widespread occurrence of the JAK2 V617F mutation in chronic myeloproliferative disorders. Blood 2005, 106:2162-2168[Abstract/Free Full Text]
  6. Lindauer K, Loerting T, Liedl KR, Kroemer RT: Prediction of the structure of human Janus kinase 2 (JAK2) comprising the two carboxy-terminal domains reveals a mechanism for autoregulation. Protein Eng 2001, 14:27-37[Abstract/Free Full Text]
  7. Campbell PJ, Scott LM, Buck G, Wheatley K, East CL, Marsden JT, Duffy A, Boyd EM, Bench AJ, Scott MA, Vassiliou GS, Milligan DW, Smith SR, Erber WN, Bareford D, Wilkins BS, Reilly JT, Harrison CN, Green AR: Definition of subtypes of essential thrombocythaemia and relation to polycythaemia vera based on JAK2 V617F mutation status: a prospective study. Lancet 2005, 366:1945-1953[CrossRef][Medline]
  8. Murugesan G, Aboudola S, Szpurka H, Verbic MA, Maciejewski JP, Tubbs RR, Hsi ED: Identification of the JAK2 V617F mutation in chronic myeloproliferative disorders using FRET probes and melting curve analysis. Am J Clin Pathol 2006, 125:625-633[CrossRef][Medline]
  9. McClure R, Mai M, Lasho T: Validation of two clinically useful assays for evaluation of JAK2 V617F mutation in chronic myeloproliferative disorders. Leukemia 2006, 20:168-171[Medline]
  10. Jones AV, Silver RT, Waghorn K, Curtis C, Kreil S, Zoi K, Hochhaus A, Oscier D, Metzgeroth G, Lengfelder E, Reiter A, Chase AJ, Cross NC: Minimal molecular response in polycythemia vera patients treated with imatinib or interferon alpha. Blood 2006, 107:3339-3341[Abstract/Free Full Text]
  11. Jaffe ES Harris NL Stein H Vardiman JW eds. WHO Classification of Tumours. Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. 2001 IARC Press Lyon, France
  12. Harries LW, Ellard S, Jones RW, Hattersley AT, Bingham C: Abnormal splicing of hepatocyte nuclear factor-1 beta in the renal cysts and diabetes syndrome. Diabetologia 2004, 47:937-942[CrossRef][Medline]
  13. Singh R, Ellard S, Hattersley A, Harries LW: Rapid and sensitive real-time PCR method for detection and quantification of 3243A>G mitochondrial point mutation. J Mol Diagn 2006, 8:225-230[Abstract/Free Full Text]




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