A Sensitive Detection Method for MPLW515L or MPLW515K Mutation in Chronic Myeloproliferative Disorders with Locked Nucleic Acid-Modified Probes and Real-Time Polymerase Chain Reaction
2008; Elsevier BV; Volume: 10; Issue: 5 Linguagem: Inglês
10.2353/jmoldx.2008.080015
ISSN1943-7811
AutoresAlessandro Pancrazzi, Paola Guglielmelli, Vanessa Ponziani, Gaetano Bergamaschi, Alberto Bosi, Giovanni Barosi, Alessandro M. Vannucchi,
Tópico(s)Eosinophilic Disorders and Syndromes
ResumoAcquired mutations in the juxtamembrane region of MPL (W515K or W515L), the receptor for thrombopoietin, have been described in patients with primary myelofibrosis or essential thrombocythemia, which are chronic myeloproliferative disorders. We have developed a real-time polymerase chain reaction assay for the detection and quantification of MPL mutations that is based on locked nucleic acid fluorescent probes. Mutational analysis was performed using DNA from granulocytes. Reference curves were obtained using cloned fragments of MPL containing either the wild-type or mutated sequence; the predicted sensitivity level was at least 0.1% mutant allele in a wild-type background. None of the 60 control subjects presented with a MPLW515L/K mutation. Of 217 patients with myelofibrosis, 19 (8.7%) harbored the MPLW515 mutation, 10 (52.6%) with the W515L allele. In one case, both the W515L and W515K alleles were detected by real-time polymerase chain reaction. By comparing results obtained with conventional sequencing, no erroneous genotype attribution using real-time polymerase chain reaction was found, whereas one patient considered wild type according to sequence analysis actually harbored a low W515L allele burden. This is a simple, sensitive, and cost-effective procedure for large-scale screening of the MPLW515L/K mutation in patients suspected to have a myeloproliferative disorder. It can also provide a quantitative estimate of mutant allele burden that might be useful for both patient prognosis and monitoring response to therapy. Acquired mutations in the juxtamembrane region of MPL (W515K or W515L), the receptor for thrombopoietin, have been described in patients with primary myelofibrosis or essential thrombocythemia, which are chronic myeloproliferative disorders. We have developed a real-time polymerase chain reaction assay for the detection and quantification of MPL mutations that is based on locked nucleic acid fluorescent probes. Mutational analysis was performed using DNA from granulocytes. Reference curves were obtained using cloned fragments of MPL containing either the wild-type or mutated sequence; the predicted sensitivity level was at least 0.1% mutant allele in a wild-type background. None of the 60 control subjects presented with a MPLW515L/K mutation. Of 217 patients with myelofibrosis, 19 (8.7%) harbored the MPLW515 mutation, 10 (52.6%) with the W515L allele. In one case, both the W515L and W515K alleles were detected by real-time polymerase chain reaction. By comparing results obtained with conventional sequencing, no erroneous genotype attribution using real-time polymerase chain reaction was found, whereas one patient considered wild type according to sequence analysis actually harbored a low W515L allele burden. This is a simple, sensitive, and cost-effective procedure for large-scale screening of the MPLW515L/K mutation in patients suspected to have a myeloproliferative disorder. It can also provide a quantitative estimate of mutant allele burden that might be useful for both patient prognosis and monitoring response to therapy. The Philadelphia chromosome-negative chronic myeloproliferative disorders include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF).1Vardiman JW Harris NL Brunning RD The World Health Organization (WHO) classification of the myeloid neoplasms.Blood. 2002; 100: 2292-2302Crossref PubMed Scopus (1786) Google Scholar,2Tefferi 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 (759) Google Scholar Their molecular pathogeneses remained poorly characterized until the description of an acquired point mutation in exon 14 of JAK2 (V617F), which involves the substitution of a key valine for a phenylalanine residue in the JH2 pseudokinase domain.3James 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 (2864) Google Scholar4Baxter 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 (2299) Google Scholar5Levine 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 (2426) 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 (2918) Google Scholar The frequency of the JAK2V617F mutation among patients with PV is more than 95%, whereas approximately half of those who are V617F-negative harbor different abnormalities located in JAK2 exon 12.7Scott 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 (994) Google Scholar On the other hand, approximately 60% of patients with ET or PMF possess the JAK2V617F mutant. In 25 to 30% of patients with either PV or PMF, the JAK2V617F mutation is harbored in a homozygous state due to mitotic recombination involving the short arm of chromosome 9.3James 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 (2864) Google Scholar4Baxter 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 (2299) Google Scholar5Levine 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 (2426) 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 (2918) Google Scholar,8Kralovics R Guan Y Prchal JT Acquired uniparental disomy of chromosome 9p is a frequent stem cell defect in polycythemia vera.Exp Hematol. 2002; 30: 229-236Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar Homozygous patients are rare in ET and account for only 2 to 4%.9Vannucchi AM Antonioli E Guglielmelli P Rambaldi A Barosi G Marchioli R Marfisi RM Finazzi G Guerini V Fabris F Randi ML De Stefano V Caberlon S Tafuri A Ruggeri M Specchia G Liso V Rossi E Pogliani E Gugliotta L Bosi A Barbui T Clinical profile of homozygous JAK2V617F mutation in patients with polycythemia vera or essential thrombocythemia.Blood. 2007; 110: 840-846Crossref PubMed Scopus (367) Google Scholar The burden of the V617F allele influences disease phenotype9Vannucchi AM Antonioli E Guglielmelli P Rambaldi A Barosi G Marchioli R Marfisi RM Finazzi G Guerini V Fabris F Randi ML De Stefano V Caberlon S Tafuri A Ruggeri M Specchia G Liso V Rossi E Pogliani E Gugliotta L Bosi A Barbui T Clinical profile of homozygous JAK2V617F mutation in patients with polycythemia vera or essential thrombocythemia.Blood. 2007; 110: 840-846Crossref PubMed Scopus (367) Google Scholar,10Tefferi A Lasho TL Schwager SM Strand JS Elliott M Mesa R Li CY Wadleigh M Lee SJ Gilliland DG The clinical phenotype of wild-type, heterozygous, and homozygous JAK2V617F in polycythemia vera.Cancer. 2006; 106: 631-635Crossref PubMed Scopus (193) Google Scholar and is associated with a greater risk of thrombosis in both ET9Vannucchi AM Antonioli E Guglielmelli P Rambaldi A Barosi G Marchioli R Marfisi RM Finazzi G Guerini V Fabris F Randi ML De Stefano V Caberlon S Tafuri A Ruggeri M Specchia G Liso V Rossi E Pogliani E Gugliotta L Bosi A Barbui T Clinical profile of homozygous JAK2V617F mutation in patients with polycythemia vera or essential thrombocythemia.Blood. 2007; 110: 840-846Crossref PubMed Scopus (367) Google Scholar and PV11Vannucchi AM Antonioli E Guglielmelli P Longo G Pancrazzi A Ponziani V Bogani C Ferrini PR Rambaldi A Guerini V Bosi A Barbui T Prospective identification of high-risk polycythemia vera patients based on JAK2(V617F) allele burden.Leukemia. 2007; 21: 1952-1959Crossref PubMed Scopus (259) Google Scholar as well as with disease progression in PMF.12Barosi G Bergamaschi G Marchetti M Vannucchi AM Guglielmelli P Antonioli E Massa M Rosti V Campanelli R Villani L Viarengo G Gattoni E Gerli G Specchia G Tinelli C Rambaldi A Barbui T JAK2 V617F mutational status predicts progression to large splenomegaly and leukemic transformation in primary myelofibrosis.Blood. 2007; 110: 4030-4036Crossref PubMed Scopus (190) Google Scholar This variable mutation frequency of patients with different clinical phenotypes,13Nussenzveig RH Swierczek SI Jelinek J Gaikwad A Liu E Verstovsek S Prchal JF Prchal JT Polycythemia vera is not initiated by JAK2V617F mutation.Exp Hematol. 2007; 35: 32-38Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar together with the demonstration that at least certain wild-type JAK2 patients with ET14Antonioli E Guglielmelli P Pancrazzi A Bogani C Verrucci M Ponziani V Longo G Bosi A Vannucchi AM Clinical implications of the JAK2 V617F mutation in essential thrombocythemia.Leukemia. 2005; 19: 1847-1849Crossref PubMed Scopus (214) Google Scholar,15Gale RE Allen AJ Nash MJ Linch DC Long-term serial analysis of X-chromosome inactivation patterns and JAK2 V617F mutant levels in patients with essential thrombocythemia show that minor mutant-positive clones can remain stable for many years.Blood. 2007; 109: 1241-1243Crossref PubMed Scopus (46) Google Scholar or PMF (unpublished data) present clonal hematopoiesis using assays for the X chromosome inactivation pattern (X-CIP),16Chen GL Prchal JT X-linked clonality testing: interpretation and limitations.Blood. 2007; 110: 1411-1419Crossref PubMed Scopus (50) Google Scholar prompted a search for additional mutations in other genes involved in the JAK2/STAT pathway. Recently, two novel, recurrent, molecular abnormalities (W515L and W515K) were identified in MPL, the gene encoding the receptor for thrombopoietin.17Pikman Y Lee BH Mercher T McDowell E Ebert BL Gozo M Cuker A Wernig G Moore S Galinsky I DeAngelo DJ Clark JJ Lee SJ Golub TR Wadleigh M Gilliland DG Levine RL MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia.PLoS Med. 2006; 3: e270Crossref PubMed Scopus (1091) Google Scholar Thrombopoietin, the main humoral regulator of thrombopoiesis in humans,18Kaushansky K The molecular mechanisms that control thrombopoiesis.J Clin Invest. 2005; 115: 3339-3347Crossref PubMed Scopus (408) Google Scholar has been implicated in the pathogenesis of myelofibrosis in murine models.19Villeval JL Cohen-Solal K Tulliez M Giraudier S Guichard J Burstein SA Cramer EM Vainchenker W Wendling F High thrombopoietin production by hematopoietic cells induces a fatal myeloproliferative syndrome in mice.Blood. 1997; 90: 4369-4383Crossref PubMed Google Scholar,20Yan XQ Lacey D Hill D Chen Y Fletcher F Hawley RG McNiece IK A model of myelofibrosis and osteosclerosis in mice induced by overexpressing thrombopoietin (mpl ligand): reversal of disease by bone marrow transplantation.Blood. 1996; 88: 402-409PubMed Google Scholar The 515 tryptophan residue is located in a unique amphipathic domain of MPL in the transmembrane-cytoplasmic hinge region that prevents spontaneous activation of the receptor.21Staerk J Lacout C Sato T Smith SO Vainchenker W Constantinescu SN An amphipathic motif at the transmembrane-cytoplasmic junction prevents autonomous activation of the thrombopoietin receptor.Blood. 2006; 107: 1864-1871Crossref PubMed Scopus (112) Google Scholar In fact, the tryptophan to leucine (W>L) substitution confers factor-independent growth to Ba/F3 cells that is associated with constitutive activation of downstream signaling pathways.17Pikman Y Lee BH Mercher T McDowell E Ebert BL Gozo M Cuker A Wernig G Moore S Galinsky I DeAngelo DJ Clark JJ Lee SJ Golub TR Wadleigh M Gilliland DG Levine RL MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia.PLoS Med. 2006; 3: e270Crossref PubMed Scopus (1091) Google Scholar Expression of MPLW515L in a murine bone marrow transplantation model resulted in an acute myeloproliferative disorder phenotype that recapitulated several aspects of human myelofibrosis.17Pikman Y Lee BH Mercher T McDowell E Ebert BL Gozo M Cuker A Wernig G Moore S Galinsky I DeAngelo DJ Clark JJ Lee SJ Golub TR Wadleigh M Gilliland DG Levine RL MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia.PLoS Med. 2006; 3: e270Crossref PubMed Scopus (1091) Google Scholar In humans, the MPL mutation occurs in multipotent hematopoietic stem cells, myeloid cells, purified B and T lymphocytes, and natural killer cells.22Hu WY Zhao Y Ishii T Sozer S Shi J Zhang W Bruno E Hoffman R Xu M Haematopoietic cell lineage distribution of MPLW515L/K mutations in patients with idiopathic myelofibrosis.Br J Haematol. 2007; 137: 378-379Crossref PubMed Scopus (11) Google Scholar23Pardanani A Lasho TL Finke C Markovic SN Tefferi A Demonstration of MPLW515K, but not JAK2V617F, in in vitro expanded CD4+ T lymphocytes.Leukemia. 2007; 21: 2206-2207Crossref PubMed Scopus (20) Google Scholar24Chaligne R James C Tonetti C Besancenot R Le Couedic JP Fava F Mazurier F Godin I Maloum K Larbret F Lecluse Y Vainchenker W Giraudier S Evidence for MPL W515L/K mutations in hematopoietic stem cells in primitive myelofibrosis.Blood. 2007; 110: 3735-3743Crossref PubMed Scopus (72) Google ScholarThe MPLW515L/K mutation has been detected in 5 to 7% of patients with PMF and in 1% of those with ET17Pikman Y Lee BH Mercher T McDowell E Ebert BL Gozo M Cuker A Wernig G Moore S Galinsky I DeAngelo DJ Clark JJ Lee SJ Golub TR Wadleigh M Gilliland DG Levine RL MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia.PLoS Med. 2006; 3: e270Crossref PubMed Scopus (1091) Google Scholar,25Pardanani AD Levine RL Lasho T Pikman Y Mesa RA Wadleigh M Steensma DP Elliott MA Wolanskyj AP Hogan WJ McClure RF Litzow MR Gilliland DG Tefferi A MPL515 mutations in myeloproliferative and other myeloid disorders: a study of 1182 patients.Blood. 2006; 108: 3472-3476Crossref PubMed Scopus (832) Google Scholar,26Guglielmelli P Pancrazzi A Bergamaschi G Rosti V Villani L Antonioli E Bosi A Barosi G Vannucchi AM Anaemia characterises patients with myelofibrosis harbouring Mpl mutation.Br J Haematol. 2007; 137: 244-247Crossref PubMed Scopus (121) Google Scholar; it has not been described in PV or myelodysplastic syndromes, including refractory anemia with ringed sideroblasts and thrombocytosis,27Steensma DP Caudill JS Pardanani A McClure RF Lasho TL Tefferi A MPL W515 and JAK2 V617 mutation analysis in patients with refractory anemia with ringed sideroblasts and an elevated platelet count.Haematologica. 2006; 91: ECR57PubMed Google Scholar or in acute myeloid leukemia.25Pardanani AD Levine RL Lasho T Pikman Y Mesa RA Wadleigh M Steensma DP Elliott MA Wolanskyj AP Hogan WJ McClure RF Litzow MR Gilliland DG Tefferi A MPL515 mutations in myeloproliferative and other myeloid disorders: a study of 1182 patients.Blood. 2006; 108: 3472-3476Crossref PubMed Scopus (832) Google Scholar The coexistence of the MPLW515L/K and JAK2V617F mutations in myelofibrosis has also been reported.25Pardanani AD Levine RL Lasho T Pikman Y Mesa RA Wadleigh M Steensma DP Elliott MA Wolanskyj AP Hogan WJ McClure RF Litzow MR Gilliland DG Tefferi A MPL515 mutations in myeloproliferative and other myeloid disorders: a study of 1182 patients.Blood. 2006; 108: 3472-3476Crossref PubMed Scopus (832) Google Scholar,26Guglielmelli P Pancrazzi A Bergamaschi G Rosti V Villani L Antonioli E Bosi A Barosi G Vannucchi AM Anaemia characterises patients with myelofibrosis harbouring Mpl mutation.Br J Haematol. 2007; 137: 244-247Crossref PubMed Scopus (121) Google Scholar,28Lasho TL Pardanani A McClure RF Mesa RA Levine RL Gilliland DG Tefferi A Concurrent MPL515 and JAK2V617F mutations in myelofibrosis: chronology of clonal emergence and changes in mutant allele burden over time.Br J Haematol. 2006; 135: 683-687Crossref PubMed Scopus (83) Google Scholar In a study of 217 patients with PMF, we found that this mutation was associated with more severe anemia and greater transfusion support.26Guglielmelli P Pancrazzi A Bergamaschi G Rosti V Villani L Antonioli E Bosi A Barosi G Vannucchi AM Anaemia characterises patients with myelofibrosis harbouring Mpl mutation.Br J Haematol. 2007; 137: 244-247Crossref PubMed Scopus (121) Google Scholar Therefore, a search for MPLW515 mutations in either PMF or ET may have both diagnostic and prognostic implications; indeed, the discovery of any of these molecular abnormalities establishes the presence of a clonal myeloproliferation, representing a major diagnostic criterion in the recent proposal for revision to the World Health Organization classification of myeloid neoplasms.2Tefferi 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 (759) Google ScholarPreviously published assay methods for MPLW515L/K mutations have been represented by direct sequencing and melting curve analysis; these methods have a low sensitivity, unable to detect fewer than 10% and 3% mutant cells in a wild-type background, respectively, and are not suitable for high-throughput screening. Therefore, the aim of this study was to develop a novel real-time polymerase chain reaction (PCR) assay that could be used in a large series of patients and would be sensitive enough to detect a low MPL mutant allele burden. The latter point might be relevant to novel drugs that target members of the constitutively activated JAK/STAT pathway, as they are expected to enter the therapeutic scenario soon.29Pardanani A JAK2 inhibitor therapy in myeloproliferative disorders: rationale, preclinical studies and ongoing clinical trials.Leukemia. 2008; 22: 23-30Crossref PubMed Scopus (183) Google Scholar This novel real-time PCR assay was validated by retrospectively analyzing a population of 217 patients with myelofibrosis who had been previously genotyped using direct sequencing.26Guglielmelli P Pancrazzi A Bergamaschi G Rosti V Villani L Antonioli E Bosi A Barosi G Vannucchi AM Anaemia characterises patients with myelofibrosis harbouring Mpl mutation.Br J Haematol. 2007; 137: 244-247Crossref PubMed Scopus (121) Google ScholarMaterials and MethodsPatientsThe characteristics of 217 patients with myelofibrosis analyzed in this study were previously described in detail26Guglielmelli P Pancrazzi A Bergamaschi G Rosti V Villani L Antonioli E Bosi A Barosi G Vannucchi AM Anaemia characterises patients with myelofibrosis harbouring Mpl mutation.Br J Haematol. 2007; 137: 244-247Crossref PubMed Scopus (121) Google Scholar; 150 patients had PMF, 30 had prefibrotic myelofibrosis,1Vardiman JW Harris NL Brunning RD The World Health Organization (WHO) classification of the myeloid neoplasms.Blood. 2002; 100: 2292-2302Crossref PubMed Scopus (1786) Google Scholar and 37 had postpolycythemic/post-thrombocythemic myelofibrosis. Sixty healthy blood donors were used as controls. Furthermore, we evaluated 50 patients with PV who harbor the JAK2V617F mutation and were diagnosed according to the proposal for revision to the World Health Organization criteria.2Tefferi 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 (759) Google Scholar All patients and controls gave their consent for sample donation and informed consent for the study.Sample PreparationPeripheral blood granulocytes were separated by differential centrifugation over a Ficoll-Ipaque gradient. Briefly, 20 ml of whole blood were collected into EDTA-containing polypropylene tubes and processed within 4 hours. Blood was diluted 1:1 with Ca2+/Mg2+-free phosphate-buffered saline, carefully layered over 15 ml of Ficoll-Ipaque in a 50-ml tube, and centrifuged at 800 × g at room temperature for 20 minutes. Both the upper fraction and the Ficoll layer were carefully removed, while the lower fraction was collected and transferred to a fresh tube. Lysis of red blood cells was performed by the addition of a 10X volume of 1X BD PharmLyse solution (Becton Dickinson BD, San Jose, CA), centrifugation of the tube after a 15-minute incubation at room temperature, and removal of the supernatant. This step was repeated twice. After two washes in Ca2+/Mg2+-free phosphate-buffered saline, the dry granulocyte pellet was stored at −20°C until processed. Cytosmears of these cellular preparations routinely showed a granulocyte content greater than 95%. DNA was extracted using the QIAmp DNA blood kit (Qiagen, GmbH, Hilden, Germany) and quantified using the NanoDrop technology (ND-1000 spectrophotometer; NanoDrop Technology, Wilmington, DE). All samples used for real-time PCR had 260:280 ratios greater than 1.8.Real-Time PCR Assay DevelopmentMPL maps to human chromosome 1p33–34.2. GenBank sequence AL139289 was used for the design of primers and probes according to the Primer 3 software at http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi; accessed January 2007 and the MELT-CALC (version 2.0) software at http://www.meltcalc.de; accessed January 2007. The Tm of the locked nucleic acid (LNA) probes was verified using Exiqon Tm prediction (http://lna-tm.com; accessed March 2007). Sequences of primers and probes are provided in Table 1. This assay makes use of a single set of primers, with the advantage that amplification of mutant and wild-type alleles has the same efficiencies, while specificities are obtained with different probes that were modified according to the LNA chemistry (Sigma-Proligo, Paris, France).Table 1Sequences of Primers and Probes Used for Real-Time PCR Assay of the MPLW515L or W515K MutationReal-time PCR primers/probesSequenceMPL forward primer5′-agcctggatctccttggtgac-3′MPL reverse primer5′-accgccagtctcctgcct-3′MPL wild-type probe5′-ctgctg+Aggt+Ggc+Agtttc-3′MPL 515W>L probe5′-ctgc+Tgagg+T+Tgcag+T+Ttc-3′MPL 515W>K probe5′-tgc+Tgctgagg+A+Ag cagtttcc-3′LNA bases are indicated by a capital letter with a plus sign before it. Probes were labeled with 5-carboxyfluorescein at their 5′ termini while Black Hole Quencher-1 was attached to the 3′ termini. The underlined nucleotides correspond to the 515 codon; the wild-type sequence is TGG, the W515L sequence is TTG, and the W515K sequence is AAG. Open table in a new tab Real-time PCR was performed using both the ABI Prism 7000 platform and the StepOne real-time PCR system (Applied Biosystems, Foster City, CA) with similar results. Forty nanograms of granulocyte DNA were used in each real-time PCR assay. Three different real-time reactions were set up in triplicate (one each for W515L, W515K, and wild-type control) for each DNA sample. A 20-μl reaction contained 1X TaqMan universal PCR Master Mix (Applied Biosystems), 300 nmol/L each primer, and 200 nmol/L each LNA-modified probe. Control wells without template (NTC) were included in each assay. Amplification and detection were performed under the following conditions: initial hold at 50°C for 2 minutes, hold at 95°C for 10 minutes followed by 55 cycles at 95°C for 15 seconds and 66°C or 62°C for 1 minute for the case of MPL unmutated and W515L probe or W515K probe, respectively. The fluorescent signal intensities were recorded and analyzed during PCR amplification using the SDS software (Applied Biosystems). The mean ΔCT of triplicate determinations (CTMPLW515L/K − CTMPLwild-type) was calculated, and the percentage of mutant alleles in the sample was obtained by comparison with a reference curve of serial dilutions of mutant plasmid mixtures in wild-type plasmid DNA. Both positive and negative controls were included in each assay.Preparation of Cloned MPL FragmentsTo prepare a reference curve for the quantification of the mutant alleles, a 277-bp fragment of MPL from healthy subjects (wild-type sequence) or from patients harboring either the W515L or W515K mutation was subcloned into the pCR2.1 TOPO vector using the TOPO TA cloning kit (Invitrogen, Groningen, The Netherlands). The sequences of the forward and reverse primers for PCR were 5′-TGGGCCGAAGTCTGACCCTTT-3′ and 5′-AGAGGTGACGTGCAGGAAGTGGCGAAGC-3′, respectively. Recombinant plasmids were isolated and bi-directional sequencing was performed to validate the sequences before plasmid amplification. A dilution of the stock plasmid solution was prepared and preliminarily tested according to the standardization principles described in the Final Report on Plasmid Standards for Real Time PCR and GM Enforcement Testing developed by Department for Environment, Food, and Rural Affairs of the government of the United Kingdom at http://www.g-inspectorate.gov.uk/documents/PLASMID.pdf; accessed July 2007.Comparison of Conventional Sequencing and Real-Time PCR AssayDNA samples from 217 patients with myelofibrosis were genotyped for the MPLW5151L/K mutation using conventional sequencing. MPL mutation sequencing was performed by amplifying a 248-bp region of MPL exon 10 using the following primers: forward, 5′-TAGCCTGGATCTCCTTGGTG-3′ and reverse, 5′-AGAGGTGACGTGCAGGAAGT-3′. PCR products were subjected to bidirectional sequencing analysis using an ABI PRISM 3730DNA Analyzer (Applied Biosystems, Foster City, CA) as described.26Guglielmelli P Pancrazzi A Bergamaschi G Rosti V Villani L Antonioli E Bosi A Barosi G Vannucchi AM Anaemia characterises patients with myelofibrosis harbouring Mpl mutation.Br J Haematol. 2007; 137: 244-247Crossref PubMed Scopus (121) Google ScholarResultsIn preliminary experiments we attempted to conduct a real-time PCR assay for the MPLW515L/K allele using conventional 5′-5-carboxyfluorescein- and 3′-Black Hole Quencher-1-labeled probes, with lengths varying from 16 to 28 bp; these probes were differentially spaced along the mutation site. However, we failed to obtain satisfactory results due to a high background generated by nonspecific binding of the probes for mutant alleles to the wild-type sequence (not shown in detail). Therefore, we subsequently explored the use of LNA-modified probes because of their anticipated greater specificity of hybridization and thermal stability. Both the primers and LNA probes, listed in Table 1, were chosen for their optimal performance among other sequences and were used in all of the experiments described herein. These probes were derived from a 28-bp probe originally designed for conventional PCR, progressively shortened by the introduction of LNA residues at various positions so that the final calculated Tms of the individual probes were similar.30Latorra D Arar K Hurley JM Design considerations and effects of LNA in PCR primers.Mol Cell Probes. 2003; 17: 253-259Crossref PubMed Scopus (88) Google Scholar The Tms of the probes selected from a panel of five different sets that were experimentally tested in real-time PCR assays were 73°C, 74°C, and 72°C for the wild-type, W515L, and W515K probes, respectively. The probe corresponding to codon 515 had two LNA nucleotides for the mutant sequences and one LNA base in the wild-type sequence (underlined in Table 1). In our original assay design, we performed single real-time PCRs for each of these probes, all labeled with 5-carbox
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