High Resolution Melting Analysis for JAK2 Exon 14 and Exon 12 Mutations
2009; Elsevier BV; Volume: 11; Issue: 2 Linguagem: Inglês
10.2353/jmoldx.2009.080110
ISSN1943-7811
AutoresInmaculada Rapado, Silvia Grande, Enriqueta Albízua, Rosa Ayala, José‐Ángel Hernández‐Rivas, Miguel Gallardo, Florinda Gilsanz, Joaquín Martínez‐López,
Tópico(s)Acute Myeloid Leukemia Research
ResumoJAK2 mutations are important criteria for the diagnosis of Philadelphia chromosome-negative myeloproliferative neoplasms. We aimed to assess JAK2 exon 14 and exon 12 mutations by high-resolution melting (HRM) analysis, which allows variation screening. The exon 14 analysis included 163 patients with polycythemia vera, secondary erythrocytoses, essential thrombocythemia, or secondary thrombocytoses, and 126 healthy subjects. The study of exon 12 included 40 JAK2 V617F-negative patients (nine of which had polycythemia vera, and 31 with splanchnic vein thrombosis) and 30 healthy subjects. HRM analyses of JAK2 exons 14 and 12 gave analytical sensitivities near 1% and both intra- and interday coefficients of variation of less than 1%. For HRM analysis of JAK2 exon 14 in polycythemia vera and essential thrombocythemia, clinical sensitivities were 93.5% and 67.9%, clinical specificities were 98.8% and 97.0%, positive predictive values were 93.5% and 79.2%, and negative predictive values were 98.8% and 94.6, respectively. Correlations were observed between the results from HRM and three commonly used analytical methods. The JAK2 exon 12 HRM results agreed completely with those from sequencing analysis, and the three mutations in exon 12 were detected by both methods. Hence, HRM analysis of exons 14 and 12 in JAK2 shows better diagnostic values than three other routinely used methods against which it was compared. In addition, HRM analysis has the advantage of detecting unknown mutations. JAK2 mutations are important criteria for the diagnosis of Philadelphia chromosome-negative myeloproliferative neoplasms. We aimed to assess JAK2 exon 14 and exon 12 mutations by high-resolution melting (HRM) analysis, which allows variation screening. The exon 14 analysis included 163 patients with polycythemia vera, secondary erythrocytoses, essential thrombocythemia, or secondary thrombocytoses, and 126 healthy subjects. The study of exon 12 included 40 JAK2 V617F-negative patients (nine of which had polycythemia vera, and 31 with splanchnic vein thrombosis) and 30 healthy subjects. HRM analyses of JAK2 exons 14 and 12 gave analytical sensitivities near 1% and both intra- and interday coefficients of variation of less than 1%. For HRM analysis of JAK2 exon 14 in polycythemia vera and essential thrombocythemia, clinical sensitivities were 93.5% and 67.9%, clinical specificities were 98.8% and 97.0%, positive predictive values were 93.5% and 79.2%, and negative predictive values were 98.8% and 94.6, respectively. Correlations were observed between the results from HRM and three commonly used analytical methods. The JAK2 exon 12 HRM results agreed completely with those from sequencing analysis, and the three mutations in exon 12 were detected by both methods. Hence, HRM analysis of exons 14 and 12 in JAK2 shows better diagnostic values than three other routinely used methods against which it was compared. In addition, HRM analysis has the advantage of detecting unknown mutations. High resolution melting (HRM) analysis represents a new generation of mutation scanning technology. HRM molecular methods based on real-time PCR have recently been developed and allow screening of known and unknown mutations, including 1-bp substitutions. Furthermore, melting curve analysis with a high-resolution melting instrument is a simple, high performance, time saving, and low labor-intensive technique that is a sensitive and specific tool for detecting DNA variations.1Krypuy M Ashour Ahmed A Etemadmoghadam D Hyland SJ High resolution melting for mutation scanning of TP53 exons 5–8.BMC Cancer. 2007; 7: 168Crossref PubMed Scopus (115) Google Scholar2Laurie AD Smith MP George PM Detection of factor VIII gene mutations by high-resolution melting analysis.Clin Chem. 2007; 53: 2211-2214Crossref PubMed Scopus (36) Google Scholar3Liew M Pryor R Palais R Meadows C Erali M Lyon E Wittwer C Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons.Clin Chem. 2004; 50: 1156-1164Crossref PubMed Scopus (502) Google Scholar The JAK2 V617F mutation has become an important diagnostic criterion for Philadelphia chromosome-negative myeloproliferative neoplasms (Ph-neg MPN), especially in polycythemia vera (PV).4Baxter 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: 1054-1061Abstract Full Text Full Text PDF PubMed Scopus (2300) Google Scholar5James 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 signalling causes polycythaemia vera.Nature. 2005; 434: 1144-1148Crossref PubMed Scopus (2846) Google Scholar6Kralovics 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: 1779-1790Crossref PubMed Scopus (2897) Google Scholar7Levine RL Wadleigh M Cools J Ebert BL Wernig G Huntly BJ Boggon TJ Wlodarska I Clark JJ Moore S Adelsperger J Koo S Lee JC Gabriel S Mercher T D'Andrea A Frohling S Dohner K Marynen P Vandenberghe P Mesa RA Tefferi A Griffin JD Eck MJ Sellers WR Meyerson M Golub TR Lee SJ Gilliland DG Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis.Cancer Cell. 2005; 7: 387-397Abstract Full Text Full Text PDF PubMed Scopus (2409) Google Scholar8Tefferi A Thiele J Orazi A Kvasnicka HM Barbui T Hanson CA Barosi G Verstovsek S Birgegard G Mesa R Reilly JT Gisslinger H Vannucchi AM Cervantes F Finazzi G Hoffman R Gilliland DG Bloomfield CD Vardiman JW Proposals and rationale for revision of the World Health Organization diagnostic criteria for polycythemia vera, essential thrombocythemia, and primary myelofibrosis: recommendations from an ad hoc international expert panel.Blood. 2007; 110: 1092-1097Crossref PubMed Scopus (755) Google Scholar9Zhao 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: 22788-22792Crossref PubMed Scopus (546) Google Scholar Accordingly, it has been included in the World Health Organization diagnostic criteria for Ph-neg MPN.8Tefferi A Thiele J Orazi A Kvasnicka HM Barbui T Hanson CA Barosi G Verstovsek S Birgegard G Mesa R Reilly JT Gisslinger H Vannucchi AM Cervantes F Finazzi G Hoffman R Gilliland DG Bloomfield CD Vardiman JW Proposals and rationale for revision of the World Health Organization diagnostic criteria for polycythemia vera, essential thrombocythemia, and primary myelofibrosis: recommendations from an ad hoc international expert panel.Blood. 2007; 110: 1092-1097Crossref PubMed Scopus (755) Google Scholar Since 2007, new exon 12 mutations involved in the pathogenesis of PV and idiopathic erythrocytosis have been described. These mutations are also important for the diagnosis of PV, as approximately 3% of PV cases have mutations in this exon.10Pietra D Li S Brisci A Passamonti F Rumi E Theocharides A Ferrari M Gisslinger H Kralovics R Cremonesi L Skoda R Cazzola M Somatic mutations of JAK2 exon 12 in patients with JAK2 (V617F)-negative myeloproliferative disorders.Blood. 2008; 111: 1686-1689Crossref PubMed Scopus (212) Google Scholar,11Scott LM Tong W Levine RL Scott MA Beer PA Stratton MR Futreal PA Erber WN McMullin MF Harrison CN Warren AJ Gilliland DG Lodish HF Green AR JAK2 exon 12 mutations in polycythemia vera and idiopathic erythrocytosis.N Engl J Med. 2007; 356: 459-468Crossref PubMed Scopus (987) Google Scholar In addition, other JAK2 exon 14 mutations that are distinct from JAK2 V617F, such as C616Y, D620E, and C618R, have been detected in patients with myoproliferative neoplasms (MPN).12Grunebach F Bross-Bach U Kanz L Brossart P Detection of a new JAK2 D620E mutation in addition to V617F in a patient with polycythemia vera.Leukemia. 2006; 20: 2210-2211Crossref PubMed Scopus (30) Google Scholar,13Schnittger S Bacher U Kern W Schroder M Haferlach T Schoch C Report on two novel nucleotide exchanges in the JAK2 pseudokinase domain: d620E and E627E.Leukemia. 2006; 20: 2195-2197Crossref PubMed Scopus (62) Google Scholar Despite this, the described JAK2 mutations are neither specific for this group of diseases, nor are they present in all patients with classical Ph-neg MPN (especially in essential thrombocythemia [ET] and primary myelofibrosis). In addition, infrequent occurrence of the JAK2 V617F mutation has been reported in chronic myelomonocytic leukemia, atypical MPN, myelodysplastic syndromes, systemic mastocytosis, and acute myeloid leukemia.14Steensma DP Dewald GW Lasho TL Powell HL McClure RF Levine RL Gilliland DG Tefferi A The JAK2 V617F activating tyrosine kinase mutation is an infrequent event in both "atypical" myeloproliferative disorders and myelodysplastic syndromes.Blood. 2005; 106: 1207-1209Crossref PubMed Scopus (404) Google Scholar Assay sensitivity should be carefully considered when interpreting test results. If low-sensitivity techniques are used, some positive cases will not be detected, whereas if the technique is highly sensitive, false positive results may arise. Highly sensitive methods used to determine the presence of the JAK2 V617F mutation (such as denaturing high-performance liquid chromatography, pyrosequencing, and allele-specific oligonucleotide [ASO]-PCR) are replacing direct sequencing and are able to determine more precisely the frequency of this mutation in Ph-neg MPN.9Zhao 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: 22788-22792Crossref PubMed Scopus (546) Google Scholar,15Levine RL Belisle C Wadleigh M Zahrieh D Lee S Chagnon P Gilliland DG Busque L X-inactivation-based clonality analysis and quantitative JAK2V617F assessment reveal a strong association between clonality and JAK2V617F in PV but not ET/MMM, and identifies a subset of JAK2V617F-negative ET and MMM patients with clonal hematopoiesis.Blood. 2006; 107: 4139-4141Crossref PubMed Scopus (202) Google Scholar16Poodt J Fijnheer R Walsh I Hermans M A sensitive and reliable semi-quantitative real-time PCR assay to detect JAK2 V617F in blood.Hematol Oncol. 2006; 24: 227-233Crossref PubMed Scopus (32) Google Scholar17Rapado I Albizua E Ayala R Hernández J Garcia-Alonso L Grande S Gallardo M Gilsanz F Martinez-Lopez J Validity test study of JAK2 V617F and allele burden quantification in the diagnosis of myeloproliferative diseases.Ann Hematol. 2008; 87: 741-749Crossref PubMed Scopus (31) Google Scholar18Sidon P El Housni H Dessars B Heimann P The JAK2V617F mutation is detectable at very low level in peripheral blood of healthy donors.Leukemia. 2006; 20: 1622Crossref PubMed Scopus (97) Google Scholar19Sidon P Heimann P Lambert F Dessars B Robin V El Housni H Combined locked nucleic acid and molecular beacon technologies for sensitive detection of the JAK2V617F somatic single-base sequence variant.Clin Chem. 2006; 52: 1436-1438Crossref PubMed Scopus (23) Google Scholar20Jones 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: 2162-2168Crossref PubMed Scopus (727) Google Scholar We aimed to compare the diagnostic values of the HRM method with a number of other described techniques for detecting mutations in exon 14 and exon 12 of the JAK2 gene to diagnose MPN. For exon 14 analysis, HRM diagnostic values were compared with three methods: two based on hybridization probes, one of which included a peptide nucleic acid probe (PNA) specific for the wild-type allele, and the third using ASO primers for the JAK2 V617F mutant allele and a minor groove binder TaqMan probe. In the case of exon 12, dideoxynucleotide sequencing was used to confirm HRM results. For the study of JAK2 exon 14 mutations, 289 subjects were enrolled and peripheral blood samples were collected between January 2002 and February 2007. The samples came from three centers in Madrid (Spain) and included 31 cases of PV, 35 cases of secondary erythrocytoses or primary non-myeloproliferative erythrocytoses, 60 cases of ET, 37 cases of secondary thrombocytoses, and 126 healthy subjects with normal hemoglobin and platelet levels as controls. In the case of exon 12, 40 JAK2 V617F-negative patients (nine of them with PV and 31 with splanchnic vein thrombosis) and 30 healthy subjects were assessed. JAK2 mutational analysis was performed on DNA from whole peripheral blood. All samples were obtained with informed consent, and all procedures were approved by the ethics committee from our institution. Genomic DNA was isolated from whole venous peripheral blood by using the large volume MP DNA isolation kit (Roche Applied Sciences, Manheim, Ge). Primer sequences and product sizes are shown in Table 1. The primers used for HRM analysis of exon 14 were the same as those used with the hybridization probes in the LightCycler 2.0 (Roche Applied Sciences, Manheim, Ge). The forward primer used to sequence exon 12 was the same as that used for the HRM analysis of exon 12 mutations. Amplification conditions were identical for the two exons. PCR reactions were performed in a 10-μl volume and included 3 mmol/L MgCl2, 5 μl High Resolution Master Mix (Roche Applied Sciences, Manheim, Ge), 0.3 μmol/L primers, and 10 to 20 ng of DNA. Cycling and melting conditions were as follows: one cycle at 95°C for 10 minutes; 40 cycles at 95°C for 10 seconds, 55°C for 15 seconds, 72°C for 15 seconds; and a melt from 60°C to 95°C at 0.25°C per second.Table 1Oligonucletotides Sequences for JAK2 Exons 14 and 12 MethodsProduct size (bp)Exon 14 HP, HP with PNA and HRM Forward5′-TTCTTTGAAGCAGCAAGTATGATGA-3′180 Reverse5′-CTGACACCTAGCTGTGATCC-3′ Probes5′-LC Red640-AAAACCAAATGCTTGTGAGAAAGCT-PH-3′5′-TCGTCTCCACAGACACATACTCCATAA-FL-3′ PNA5′-TCCACAGACATACT-3′ ASO qPCR Forward5′-GGTTTTAAATTATGGAGTATGTT-3′60 Reverse5′-AGAAAGCCTGTAGTTTTACTTACTCTCG-3′ Probe5′-6-FAM-TGTGGAGACGAGAGTAA-MGB-3′Exon 12 Sequencing PCR Forward5′-CTCCTCTTTGGAGCAATTCA-3′500 Reverse5′-GAGAACTTGGGAGTTGCGATA-3′ HRM Forward5′-CTCCTCTTTGGAGCAATTCA-3′280 Reverse5′-GATTTACATTCATGTGACATTGG-3′HP, hybridization probes; PH, phosphate; FL, fluorescein; FAM, 6-carboxyfluorescein; MGB, minor groove binder. Open table in a new tab HP, hybridization probes; PH, phosphate; FL, fluorescein; FAM, 6-carboxyfluorescein; MGB, minor groove binder. HRM analysis was performed with the LightCycler 480 Gene Scanning Software (Roche Applied Sciences, Manheim, Ge). The normalized graph and the normalized temperature-shifted difference graph (difference graph) from the gene scanning analysis were used to analyze the data (Figure 1). The normalized graph was generated by monitoring the dissociation of fluorescent dye from double-stranded DNA with an increase in temperature. The High-Resolution Melting Dye (Roche Applied Sciences, Manheim, Ge) used in this study can only fluoresce when it is intercalated into double-stranded DNA. The normalized graph shows the degree of reduction in fluorescence over a temperature range (typically 70°C to 95°C). All samples, including the wild-type, were plotted according to their melting profiles. In the difference graph, the melting profiles of each sample were compared with those of the wild-type (which were converted to a horizontal line). Significant deviations from the horizontal line were indicative of sequence changes within the amplicon. Samples with aberrant melting curves were recorded as HRM mutation positive. Analysis of the JAK2 V617F gene mutation was performed by real-time PCR using a melting curve-based, LightCycler assay with hybridization probes. Sequences of the primers and probes are shown in Table 1. Reactions were performed with 0.5 μmol/L of both forward and reverse primers, 0.2 μmol/L of both sense and anchor hybridization probes, 4.5 mmol/L of MgCl2, 1 μl of Fast Start DNA Master Hybridization Probe (Roche Applied Sciences, Manheim, Ge), and 50 to 100 ng of DNA, all in a final volume of 10 μl. PCR conditions were as follows: an initial hold at 95°C for 10 minutes; 35 cycles of 95°C for 0 seconds, 58°C for 10 seconds, and 72°C for 10 seconds; and a melt from 50°C to 80°C at 0.2°C per second. Fluorescence was detected continuously. Data were analyzed using the LightCycler software v3.5 (Roche Applied Sciences, Manheim, Ge). Mutated alleles, with melting peaks at 59°C, were distinguished from wild-type alleles, whose melting peaks occurred at 63°C. Amplification conditions were identical to those described above for JAK2 analysis with hybridization probes. A specific PNA for the wild-type allele was designed and synthesized following specific rules and using Applied Biosystem's reagents (Palo Alto, CA) (Table 1). The specific PNA for the JAK2 V617 wild-type allele was added to the reaction at a concentration of 1.25 μmol/L. Mutated and wild-type alleles were identified, as described above for PCR detection of JAK2 V617F. JAK2 V617F ASO quantitative PCR (ASO qPCR) was performed with the ABI PRISM 7900 (Applied Biosystems, Palo Alto, CA), and a forward ASO primer spanning the JAK2 V617F mutation region. A reverse primer and a minor groove binder TaqMan probe were also used (sequences are indicated in Table 1). The PCR was performed in a 10-μl reaction volume with 0.5 μmol/L of both forward and reverse primers, 0.12 μmol/L of the minor groove binder probe, 5 μl of TaqMan Fast Start Universal PCR Master Mix (Applied Biosystems, Palo, CA), and 10 to 20 ng of DNA. The first cycle was a 10 minutes denaturation at 95°C. The following 40 cycles included denaturation at 95°C for 1 second, and annealing at 55°C for 25 seconds. We performed bidirectional sequencing analysis to detect JAK2 mutations in exon 12. The reaction mixture of 10 μl contained 1.25 mmol/L MgCl2, 1 μmol/L of each primer (Table 1), 50 ng of genomic DNA, and 1.5 μl of Fast Start DNA Master Hybridization Probe. PCR cycling conditions were as follows: an initial denaturation at 95°C for 10 minutes; 35 cycles of 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 30 seconds; and one cycle of 72°C for 10 minutes. Amplification products were purified with ExoSapIT (GE Health care, Little Chalfont, UK), followed by sequencing with Big Dye Terminator v3.1 (Applied Biosystems, Foster City, CA), according to the manufacturer's protocol. The sequencing products were run on a 3100 Genetic Analyser (Applied Biosystems, Palo Alto, CA). The sequencing data were visualized using Sequencing Analysis 5.1.1 (Applied Biosystems, Palo Alto, CA). A six-point standard curve was generated for each exon as a measure of the sensitivity of all of the assays. The standard curves arose from serial dilutions of positive control DNA in negative control DNA, to obtain proportions of 1 to 10−4 in a final, constant amount of 10 ng of genomic DNA. For the assessment of HRM analysis of exon 14, JAK2 V617F-positive DNA from homozygous human erythroleukemia cells (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Ge) was diluted into samples from healthy subjects, without exon 14 mutations. For the assessment of HRM analysis of exon 12, mutated DNA from a patient was used as a positive control. Each point in the dilution curve was measured in duplicate. Intra- and interday coefficients of variation were calculated in three ways for both exons 14 and 12, ie, the crossing point (CP) cycle, and the number of DNA molecules added to reaction (N0), and amplified at this cycle (NCP). Intraday variation of the HRM analysis of exon 14 was determined from five values obtained in the same run, in samples prepared with five different reaction mixes, and at four levels of JAK2 V617F allele burden (100%, 22.4%, 1.2%, 0%) (Table 2). The interday precision corresponded to runs performed over five days, with a 100% allele burden. Intraday variation of the HRM analysis of exon 12 was determined from five values obtained in the same run of mutated and non-mutated DNA samples prepared with five different reaction mixes (Table 2). The interday variation was performed over five days using a mutated patient sample.Table 2Intra- and Inter-Day Coefficients of Variation (n = 5) of Crossing Point Cycle Values, the Number of DNA Molecules at CP, and the Number of DNA Molecules Added to the Reaction for JAK2 Exons 14 and 12 in HRM MethodsIntra-day CV (%)Inter-day CV (%)Sample*JAK2 V617F allele burden in samples 1–4: 100%, 22.4%, 1.2%, and 0%. Samples 5 and 6 corresponded to JAK2 exon 12 mutated and not mutated, respectively.CPNCPN0CPNCPN0Exon 14 10.618.99.60.669.910.2 20.7712.413.0 30.549.69.5 40.417.47.7Exon 12 50.9213.313.50.9313.714.1 60.517.47.0CP, crossing point cycle values; NCP, the number of DNA molecules at CP; N0, number of DNA molecules added to the reaction.* JAK2 V617F allele burden in samples 1–4: 100%, 22.4%, 1.2%, and 0%. Samples 5 and 6 corresponded to JAK2 exon 12 mutated and not mutated, respectively. Open table in a new tab CP, crossing point cycle values; NCP, the number of DNA molecules at CP; N0, number of DNA molecules added to the reaction. All data were included in a relational database, Access 2003, and analyzed by SPSS 13.0 (SPSS Inc, Chicago, Illinois). For the three methods, clinical sensitivities, clinical specificities, positive predictive values and negative predictive values were determined by contingency tables. Receiving operating characteristic plots were performed to establish the best sensitivity and specificity levels for a quantitative diagnostic test. The best cut-off specificity level was established at over 97%, and sensitivity cut-off was assessed according to the referred specificity level. Comparisons between the diagnostic tests were performed using the kappa coefficient. PCR efficiencies were calculated from the linear regression slopes of each method. In the case of HRM, this was 1.89, with hybridization probes it was 1.93, for hybridization probes with PNA it was 1.90, and for ASO qPCR it was 1.96. Linearity in the investigated range was high for all of the methods, where R2 > 0.98. For HRM analysis, analytical sensitivity obtained from the dilution curves was 1−0.5%. In the case of hybridization probes alone, analytical sensitivity was 10%, while hybridization probes with PNA gave an analytical sensitivity around 1−0.5%, and in ASO qPCR, analytical sensitivity was 0.01%. The coefficients of variation were calculated according to CP values. In this study the intra- and interday coefficients of variation were always below 1%. When N0 or NCP coefficients of variation were determined, results were between 7.4 and 13.0% (Table 2). We also compared the diagnostic values of the four methods for the JAK2 V617F mutation in the most frequent MPN (PV and ET) (Table 3). In the case of HRM analysis of JAK2 V617F for a PV diagnosis, sensitivity was 93.5%, specificity was 98.8%, positive predictive value was 93.5%, and negative predictive value was 98.8%. In the case of an ET diagnosis, sensitivity was 67.9%, specificity was 97.0%, positive predictive value was 79.2%, and negative predictive value was 94.6%.Table 3Comparison of Diagnostic Validity in PV and ET for JAK2 V617F Mutation with PCR MethodsHRMHPHP with PNAASO qPCR*Receiving operating characteristic curve results, discriminating selected value 1.0%.Polycythemia vera Sensitivity93.5 (85.2–102.6)87.1 (75.0–101.1)93.5 (85.2–102.6)93.5 (85.2–102.6) Specificity98.8 (97.1–100.5)100 (100)98.8 (97.1–100.5)99.4 (98.2–100.6) PPV93.5 (85.2–102.6)100 (100)93.5 (85.2–102.6)96.7 (90.5–103.3) NPV98.8 (97.1–100.5)97.6 (95.3–100.0)98.8 (97.1–100.5)98.8 (97.1–100.5)Essential thrombocythemia Sensitivity67.9 (52.6–87.6)57.1 (41.4–78.7)67.9 (52.6–87.6)78.6 (64.8–95.4) Specificity97.0 (94.4–99.7)100 (100)97.0 (94.4–99.7)96.3 (93.5–99.2) PPV79.2 (64.5–97.2)100 (100)79.2 (64.5–97.2)78.6 (64.8–95.4) NPV94.6 (91.3–98.1)93.2 (89.5–97.0)94.6 (91.3–98.1)96.3 (93.5–99.2)HP, hybridization probes.* Receiving operating characteristic curve results, discriminating selected value 1.0%. Open table in a new tab HP, hybridization probes. In the ASO quantitative analysis of the JAK2 V617F mutation, receiving operating characteristic curves were generated to select the adequate threshold level of the JAK2 V617F mutant allele burden. This was done to obtain the best relationship between sensitivity and specificity for the diagnosis of MPN. A 1% JAK2 V617F allele burden was found to be the best level with high specificity to distinguish PV or ET from secondary conditions. With this threshold value, the area under the receiving operating characteristic curve was 0.96 for PV, and 0.94 for ET (Table 3). We correlated the HRM results with the other three analytical methods,17Rapado I Albizua E Ayala R Hernández J Garcia-Alonso L Grande S Gallardo M Gilsanz F Martinez-Lopez J Validity test study of JAK2 V617F and allele burden quantification in the diagnosis of myeloproliferative diseases.Ann Hematol. 2008; 87: 741-749Crossref PubMed Scopus (31) Google Scholar and good kappa coefficients were reached. The kappa coefficient between HRM and the hybridization probe method was 0.76 (SD ± 0.05), between the HRM and the hybridization probe with PNA it was 1.00 (SD ± 0.00), and between the HRM and ASO quantitative method it was 0.91 (SD ± 0.03). The HRM PCR results matched with the results from the hybridization probe with PNA PCR, in all patients (Table 4). Two PV samples (2/31) were negative with all of the methods. In ET, 19 cases were negative with the HRM method, 13 of these were also negative with the ASO quantitative method, and the other six were slightly more than the 1% JAK2 V617F allele burden.Table 4JAK2 V617F Positive Results Obtained by the Four MethodsHRMHPHP with PNAASO qPCRNTN%N%N%N%Polycythemia vera312994278729942994Secondary erythrocytoses3500000000Essential thrombocythemia604168345741684778Secondary thrombocytoses3738003838Healthy subjects12622002211HP, hybridization probes. Open table in a new tab HP, hybridization probes. Three secondary thrombocytoses were positive with the HRM method, in contrast to no positive results in secondary erythrocytoses (Table 4). These secondary thrombocytoses were also positive with the hybridization probe with PNA method, and the ASO quantitative method (allele burden between 2.5% and 2.8%). Two secondary thrombocytoses were only positive by ASO qPCR (JAK2 V617F allele burden 0.99) was also observed. An analytical sensitivity of 1% was determined by diluting DNA from an exon 12-mutated sample into samples from healthy subjects with no mutation in this region. When the assay variations were calculated with CP values, the intra- and interday coefficients of variation were below 1%. The coefficients of variation of N0 or NCP values were between 7.0% and 14.1% (Table 2). The JAK2 exon 12 HRM results correlated 100% with sequencing results for detecting mutations in the 40 JAK2 V617F-negative patients with a clinical diagnosis of PV or splanchnic vein thrombosis, and the 30 healthy subjects. Only three JAK2 V617F-negative cases with a PV phenotype showed mutations in exon 12: two deletions of two and three amino acids and an 11-amino acid duplication. Using sequencing as a reference method, the clinical sensitivity, clinical specificity, positive predictive values, and negative predictive values of HRM for the detection of mutations within JAK2 exon 12 could be considered to be 100%. Figure 1B shows the difference plots of the high resolution melting curves of JAK2 exon 12 in four samples. The wild-type amplicon has been selected as the baseline curve. The other three curves correspond to the two mutations where curve A represents the duplication, and curves B and C, the two deletions. An assessment of JAK2 mutations has become an essential and relevant tool for the diagnosis of Ph-neg MPN. Improving the methods used for detecting JAK2 mutations will significantly benefit these patients. HRM methods, which represent a new generation of real-time PCR techniques, add some advantages over the previously used methods, and could improve diagnosis performance.1Krypuy M Ashour Ahmed A Etemadmoghadam D Hyland SJ High resolution melting for mutation scanning of TP53 exons 5–8.BMC Cancer. 2007; 7: 168Crossref PubMed Scopus (115) Google Scholar2Laurie AD Smith MP George PM Detection of factor VIII gene mutations by high-resolution melting analysis.Clin Chem. 2007; 53: 2211-2214Cr
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