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00019606-201103000-00005ArticleDiagnostic Molecular PathologyDiagnostic Molecular Pathology© 2011 by Lippincott Williams & Wilkins.20March 2011 p 34–39MPL Mutation Profile in JAK2 Mutation-negative Patients With Myeloproliferative DisordersOriginal ArticlesMa, Wanlong BSc; Zhang, Xi BSc; Wang, Xiuqiang BSc; Zhang, Zhong MD; Yeh, Chen-Hsiung PhD; Uyeji, Jennifer BSc; Albitar, Maher MDDepartment of Hematology/Oncology, Quest Diagnostics Nichols Institute, San Juan Capistrano, CAReprints: Maher Albitar, MD, Department of Hematology/Oncology, Quest Diagnostics Nichols Institute, San Juan Capistrano, CA 92675 (e-mail: [email protected]).AbstractMutations in the thrombopoietin receptor gene (myeloproliferative leukemia, MPL) have been reported in patients with JAK2 V617F-negative chronic myeloproliferative disorders (MPDs). We evaluated the prevalence of MPL mutations relative to JAK2 mutations in patients with suspected MPDs. A total of 2790 patient samples submitted for JAK2 mutation analysis were tested using real-time polymerase chain reaction and bidirectional sequencing of plasma RNA. JAK2 V617F-negative samples were tested for JAK2 exons 12 to 14 mutations, and those with negative results were then tested for mutations in MPL exons 10 and 11. Of the 2790 patients, 529 (18.96%) had V617F, 12 (0.43%) had small insertions or deletions in exon 12, and 7 (0.25%) had other JAK2 mutations in exons 12 to 14. Of the 2242 JAK2 mutation-negative patients, 68 (3.03%) had MPL mutations. W515L was the predominant MPL mutation (n=46; 68%), and 10 (15%) patients had other W515 variants. The remaining MPL mutations (n=12, 17%) were detected at other locations in exons 10 and 11 and included 3 insertion/deletion mutations. The S505N mutation, associated with familial MPD, was detected in 3 patients. Overall, for every 100 V617F mutations in patients with suspected MPDs, there were 12.9 MPL mutations, 2.3 JAK2 exon 12 mutations, and 1.3 JAK2 exons 13 to 14 mutations. These findings suggest that MPL mutation screening should be performed before JAK2 exons 12 to 14 testing in JAK2 V617F-negative patients with suspected MPDs.Myeloproliferative disorders (MPDs) are multipotent hematopoietic stem cell disorders characterized by the excess production of maturing blood cells. The 3 most common MPDs, after chronic myeloid leukemia (CML), are polycythemia vera (PV), essential thrombocythemia (ET), and idiopathic myelofibrosis (IMF). Although CML is characterized by the presence of the Philadelphia chromosome, these non-CML MPDs seem to lack any consistent, specific karyotypic abnormalities, although 9p deletion has been reported in approximately 30% of the patients with PV.1 The JAK2 V617F mutation has recently emerged as a specific molecular abnormality in non-CML MPDs, found in about 95% of PV patients, 35% to 70% of ET patients, and 50% of IMF patients.2–5 Mutations in JAK2 exon 12 and, to a lesser extent, exons 13 and 14, have been found in rare cases of V617F-negative non-CML MPDs.6–9Mutations in the thrombopoietin receptor myeloproliferative leukemia (MPL) gene have also been reported in certain patients with familial MPD.10–14 The reporting of the MPL S505A mutation in a Japanese family prompted the investigation of acquired MPL mutations in patients with MPDs. This led to the discovery of 2 activating MPL mutations, W515L and W515K, in approximately 5% of IMF and ET patients.12,13 These mutations are in the juxtamembrane region of the MPL protein and are believed to lead to constitutive activation of the receptor proliferation of the myeloid elements. Significantly higher platelet counts have been reported in patients with MPL W515 mutations.12,14 Rare additional mutations in the MPL gene, outside the W515 codon, have been reported in individual patients, but at very low frequencies.14–16Current guidelines require testing for the JAK2 V617F mutation when establishing diagnosis in patients with suspected MPD, who are negative for the Philadelphia chromosome.1,2,17 The results of our earlier study of the JAK2 mutation profiles indicated that testing should be expanded to the entire pseudokinase domain coding region of JAK2.8 In this study, we expanded our analysis to encompass mutations in exons 10 and 11 of the MPL gene and exons 12 to 14 of JAK2. In addition to identifying novel MPL mutations, our results may help define the most appropriate molecular testing strategy for patients with non-CML MPDs who lack the V617F mutation.PATIENTS AND METHODSPatient SamplesStudy samples comprised a total of 2790 consecutive ethylene diaminetetra-acetic acid peripheral blood samples from patients with suspected Philadelphia chromosome-negative MPDs submitted to Quest Diagnostics for JAK2 mutation analysis. Samples testing negative for JAK2 V617F were screened for mutations in JAK2 exons 12 to 14; samples negative for JAK2 V617F and exons 12 to 14 mutations were then tested for MPL mutations in exons 10 and 11. All mutation testing was performed on RNA isolated from plasma. In addition, MPL mutation testing was performed on paired plasma and cell samples in 21 of these patients. The study was approved by an independent review board.RNA Extraction From PlasmaPatient whole blood samples were shipped at room temperature and processed within 24 hours to separate plasma. Total nucleic acids were isolated from the resulting ethylene diaminetetra-acetic acid plasma samples with the EasyMag extraction kit (BioMerieux Inc, Durham, NC) according to the manufacturer's instructions, and the same extract was used for JAK2 and MPL testing.Sequence AnalysisJAK2 V617 and Exons 12 to 14 MutationsA primer pair with 18-nucleotide M13 tags (lower case) was designed to yield a 491-bp product encompassing the JAK 2 V617F and exons 12 to 14 mutations: 5′-tgt aaa acg acg gcc agt CTA AAT GCT GTC CCC CAA AG-3′ (forward) and 5′-cag gaa aca gct atg acc CCA TGC CAA CTG TTT AGC AA-3′ (reverse). The real-time polymerase chain reaction (RT-PCR) amplification from patient RNA was performed using the Superscript III one-step RT-PCR systems with Platinum Taq (Invitrogen, Carlsbad, CA), under the following thermocycler conditions: an initial step at 55°C for 30 minutes and 94°C for 2 minutes, followed by 40 cycles of 94°C for 15 seconds, 60°C for 30 seconds, and 68°C for 1 minute, with a final extension step at 68°C for 7 minutes. The PCR product was then purified and sequenced in both forward and reverse directions using an ABI PRISM 3730XL genetic analyzer. Sequencing data were base-called using a sequencing analysis software, and assembled and analyzed by the SeqScape software using the GenBank accession number NM 004972 as reference. The overall repeat rate in plasma testing because of poor RNA or poor sequencing was 1% to 2% in JAK2 testing and 3% to 5% in MPL testing. A case was considered homozygous/hemizygous when the wild-type peak constituted less than 30% of the mutant peak.MPL Exons 10 to 11 MutationsA primer pair with 18-nucleotide M13 tags (lower case) was designed to yield a 366-bp PCR product encompassing exons 10 and 11 of the MPL gene: 5′-tgt aaa acg acg gcc agt GCG ATC TCG CTA CCG TTT AC-3′ (forward) and 5′-cag gaa aca gct atg acc GAG GAC TTG GGG AGG ATT TC-3′ (reverse). Amplification from patient RNA was performed using Superscript III one-step RT-PCR (Invitrogen). The PCR product was then purified and sequenced in both forward and reverse directions using an ABI PRISM 3730XL genetic analyzer. Sequencing data were base-called using a sequencing analysis software, and assembled and analyzed by the SeqScape software using the GenBank accession number NM 005373 as reference.RESULTSJAK2 MutationsThe frequencies of JAK2 mutations in this group of patients were similar to those in our earlier report.8 The JAK2 V617F mutation was detected in 529 of the 2790 patients (18.96%) (Table 1). Twelve patients had exon 12 mutations (0.43% of all patients and 2% of JAK2 mutations), most of which were deletions or insertions. Few mutations were detected in exons 13, 14, and 15 (0.025% of all patients and 1% of all JAK2 mutations; Table 1). Overall, 19.6% of patients had mutations in the JAK2 gene. For every 100 JAK2 V617F-positive patients, 2.3 had mutations in JAK2 exon 12, and 1.3 had mutations in exons 13 or 14.JOURNAL/dimp/04.03/00019606-201103000-00005/table1-5/v/2021-02-17T200029Z/r/image-tiffSummary of JAK2 Mutations 2790 Patients With Suspected Non-CML MPDsMPL MutationsThe remaining samples (2242; 80.4%) had no JAK2 mutations and were tested for MPL mutations in exons 10 and 11. No discrepancy was noted between the results from cell and plasma analysis in any of the 21 paired samples tested. These 21 samples included 3 containing an MPL mutation. In addition, more than 80 plasma samples from a normal control group were tested for MPL mutations, none of which showed any mutation. Although the sensitivity of detection in plasma was shown to be better than in cells for JAK2 as we have reported earlier,8 it is difficult to show the limit of detection for MPL without studying a large number of paired cell and plasma samples.Of these 2242 patients, 68 (3.03%) had MPL mutations (Table 2). That is, for every 100 patients with suspected MPDs who were positive for JAK2 V617F, 12.9 had mutations in MPL. As expected, most of the mutations (n=58; 86%) were in codon W515 (Fig. 1). However, a wide variety of codon W515 mutations were detected (Table 2). The most common substitution was W515L (TGG>TTG), comprising 67.7% of all the MPL mutations (Fig. 1); 3 patients showed homozygous W515L mutation. We also detected 5 other types of W515 mutations present in 10 patients (10/68, 15%). In these mutations, TGG was mutated to AGG, AAG, TCG, AAA, and GCG resulting 515K, 515R, 515S, 515G, and 515A. In the mutations to AAA and AAG, the possibility that these are bi-allelic mutations and each allele carries 1 nucleic acid change cannot be ruled out. One of these patients had a mutation in codon G540 in addition to W515L.JOURNAL/dimp/04.03/00019606-201103000-00005/table2-5/v/2021-02-17T200029Z/r/image-tiffMPL Mutation Profile in Patients With JAK2-negative Suspected Non-CML MPDs (n=2242)JOURNAL/dimp/04.03/00019606-201103000-00005/figure1-5/v/2021-02-17T200029Z/r/image-jpegSequence analysis of myeloproliferative leukemia gene (MPL) mutations at codon W515. For both panels (A and B), direct and indirect sequencing are shown for the patient and control; the mutated nucleotides are indicated with arrows. A, Examples of various mutations detected at W515. The expected amino acids are shown. B, Examples of homozygous/hemizygous mutations at codon W515.The other point mutations were in codons V501, S505, V507, R514, and A519 in exon 10, and codon D545 in exon 11, accounting for 12% (8/68) of all MPL mutations (Fig. 2). Only 3 patients showed mutations in codon 505, which has been reported to be associated with a familial form of MPD manifesting mainly as familial ET.11 One of these 3 patients had a second MPL mutation (V501L). The second mutation was most likely acquired, but the possibility of it being in the germline could not be ruled out. We also discovered 3 novel MPL insertion/deletion mutations in exons 10 and 11 (3/68, 4.4%) (Fig. 3). One mutation was a 12-bp insertion (GCT CTG GTG ATC) at C1533-G1534 of exon 10, adding 4 new amino acids (ALVI) in-frame at position T496-A497. Interestingly, this was in a homozygous or hemizygous state. The patient had a confirmed diagnosis of IMF. The second patient had a deletion of 4 amino acids with insertion of 2 new amino acids at codon W515 (W515-P518 del/insKT). This mutation was in-frame and in the heterozygous state, and the patient had MPD not otherwise specified. The third indel mutation was in exon 11 at codon R525, involving a deletion of 2 nucleotides (AG) and insertion of T, leading to a frameshift and a stop codon after 13 amino acids. The patient also had confirmed chronic MPD not otherwise specified.JOURNAL/dimp/04.03/00019606-201103000-00005/figure2-5/v/2021-02-17T200029Z/r/image-jpegExamples of novel mutations involving various codons in myeloproliferative leukemia gene (MPL). Both direct and indirect sequencing are shown for the patient and control. The mutated nucleotides are indicated with arrows.JOURNAL/dimp/04.03/00019606-201103000-00005/figure3-5/v/2021-02-17T200029Z/r/image-jpegExamples of insertion/deletion mutations. A, Deletion of ag and insertion of t at nucleotide position 1618 (arrow), leading to an R525C mutation with frameshift and termination at amino acid 14. B, Deletion of tggcagtttcct and insertion of aaaat at nucleotide 1588 (arrow), leading to a W515-p518 deletion and insertion of 2 amino acids (KT). The first trace is a control, the middle trace is the forward sequence, and the bottom trace is the reverse direction. C, Forward and reverse insertion of 12 nucleotides (gctctggtgatc) (arrow) leading to the insertion of 4 new amino acids (ALVI) at codon T496.Detection of PolymorphismsMultiple polymorphic sites in MPL have been reported. However, only 1 single-nucleotide polymorphism (rs3820551) has been reported to involve exon 11, and no exon 10 polymorphisms have been reported.18 We detected 1 synonymous polymorphism in exon 10 and 3 additional synonymous polymorphisms in exon 11 (Table 3) (Fig. 4). The frequency of these polymorphisms was very low (0.1% to 0.25%), and although they were synonymous, we cannot rule out the possibility that they are relevant to disease development as silent polymorphisms have been reported to play a role in RNA stability and protein levels. In contrast, they may also represent tag polymorphisms for significant changes in the MPL locus that may influence this genomic region.JOURNAL/dimp/04.03/00019606-201103000-00005/table3-5/v/2021-02-17T200029Z/r/image-tiffPossible Novel MPL SNPs Detected in Patients With JAK2-negative Suspected Non-CML MPDsJOURNAL/dimp/04.03/00019606-201103000-00005/figure4-5/v/2021-02-17T200029Z/r/image-jpegSchematic representation of myeloproliferative leukemia gene (MPL) mutations. The MPL protein is depicted in blue and the junction at the cellular membrane, which is depicted in pink. The mutations are shown on the right. The mutations that have been reported but not detected in our testing are shown in rectangles. Detected single-nucleotide polymorphisms (SNPs) are shown on the left. Only the R537Q SNP has been reported earlier. All other SNPs are synonymous and nucleotides are shown.DISCUSSIONMore than any other neoplasm, chronic MPDs are defined by specific genomic abnormalities. The Philadelphia chromosome defines CML, and JAK2 mutations define PV.1,4–7 However, ET and IMF remain less defined at the molecular level. Despite the specific JAK2 V617F abnormality seen in PV and in a significant percentage of ET and IMF patients, many patients with suspected MPD present without any specific molecular abnormalities. Our earlier and this studies show that some of these patients may have mutations in JAK2 outside V617F. Our findings also show that MPL mutations can be found in certain patients with JAK2 mutation-negative MPD. In fact, MPL mutations were more common than mutations in JAK2 exons 12 to 14 in patients with suspected Philadelphia chromosome-negative MPDs lacking the V617F mutation.The aim of our study was to develop a strategy for assessing molecular abnormalities in patients with suspected MPDs. Therefore, we screened consecutive samples from patients presenting to their clinician with symptoms and signs suggestive of a non-CML MPD, leading to the ordering of tests to rule in MPD. Our results confirm that the first test in this setting should be for the JAK2 V617F mutation. Patients negative for V617F should be screened first for the MPL gene mutations and then for mutations in exons 12 to 14 of the JAK2 gene (Fig. 5). This conclusion is based on the finding that for every 100 non-CML MPD patients positive for JAK2 V617F, 12.9 had mutations in MPL whereas only 2.3 had mutations in JAK2 exon 12 and only 1.3 had mutations in exons 13 or 14. However, as our testing was sequential, the possibility of coexistence of mutations in both the JAK2 and MPL genes cannot be ruled out.JOURNAL/dimp/04.03/00019606-201103000-00005/figure5-5/v/2021-02-17T200029Z/r/image-jpegFlowchart shows a suggested sequence for molecular testing of mutations in patients suspected to have chronic myeloproliferative disorder.We found MPL mutations in codons aside from W515 and S505. The newly discovered mutations (Table 2), along with the mutations reported earlier in the Sanger database (http://www.sanger.ac.uk) (Fig. 4), indicate that most MPL mutations reside between codons 450 and 650. Therefore, we recommend screening for mutations in exons 10 and 11.W515 is a key amino acid located in a unique amphipathic domain involved in preventing the spontaneous activation of MPL. Thus, mutations in this codon most likely lead to constitutive activation. Pikman et al12 suggested that the W515 mutation in hematopoietic cells leads to a cytokine-independent proliferation capacity and results in constitutive activation of JAK-STAT signaling. Although W515 remains the most commonly mutated MPL codon, other mutations constitute a significant percentage. We expect all MPL insertion/deletion mutants to have effects similar to those of the W515 mutant, conferring a myeloid proliferative advantage leading to MPDs. However, mutations at other sites, and the 3 unreported potential polymorphisms (T496, L543, and D534) described here, need further functional studies.In conclusion, MPL mutations are much less common than JAK2 V617F in patients with suspected MPDs, but are more frequent than the mutations in exons 12 to 14. The MPL mutation profile from this study indicates that more than 20% of MPL mutations would have been missed if only W515L and W515K had been tested. On the basis of our data, we recommend analyzing the entire exons 10 and 11 region of the MPL gene.ACKNOWLEDGMENTThe authors thank Jeff Radcliff (Quest Diagnostics Nichols Institute) for editorial assistance.REFERENCES1. Panani AD. Cytogenetic and molecular aspects of Philadelphia negative chronic myeloproliferative disorders: clinical implications Cancer Lett.. 2007;255:12–25[Context Link]2. Baxter EJ, Scott LM, Campbell PJ, et al. Cancer genome project: acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders Lancet.. 2005;365:1054–1061[Context Link]3. Passamonti F, Rumi E, Pietra D, et al. Relation between JAK2 (V617F) mutation status, granulocyte activation and constitutive mobilization of CD34+ cells into peripheral blood in myeloproliferative disorders Blood.. 2006;107:3676–3682[Context Link]4. Lippert E, Boissinot M, Kralovics R, et al. The JAK2-V617F mutation is frequently present at diagnosis in patients with essential thrombocythemia and polycythemia vera Blood.. 2006;108:1865–1867[Context Link]5. Tefferi A, Lasho TL, Schwager SM, et al. The clinical phenotype of wild-type, heterozygous, and homozygous JAK2V617F in polycythemia vera Cancer.. 2006;106:631–635[Context Link]6. Scott LM, Tong W, Levine RL, et al. JAK2 exon 12 mutation mutations in polycythemia vera and idiopathic erythrocytosis N Engl J Med.. 2007;356:459–468[Context Link]7. Scott LM, Beer PA, Bench AJ, et al. Prevalence of JAK2 V617F and exon 12 mutations in polycythaemia vera Br J Haematol.. 2007;139:511–512[Context Link]8. Ma W, Kantarjian H, Zhang X, et al. Mutation profile of JAK2 transcripts in patients with chronic myeloproliferative neoplasias J Mol Diagn.. 2009;11:49–53[Context Link]9. Pietra D, Li S, Brisci A, et al. Somatic mutations of JAK2 exon 12 in patients with JAK2(V617F)-negative myeloproliferative disorders Blood.. 2008;111:1686–1689[Context Link]10. Ding J, Komatsu H, Wakita A, et al. Familial essential thrombocythemia associated with a dominant-positive activation mutation of the c-MPL gene, which encodes for the receptor for thrombopoietin Blood.. 2004;103:4198–4200[Context Link]11. Teofili L, Giona F, Martini M, et al. Markers of myeloproliferative diseases in childhood polycythemia vera and essential thrombocythemia J Clin Oncol.. 2007;25:1048–1053[Context Link]12. Pikman Y, Lee BH, Mercher T, et al. MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia PLoS Med.. 2006;3:1140–1151[Context Link]13. Pancrazzi A, Guglielmelli P, Ponziani V, et al. A sensitive detection method for MPLW515L or MPLW515K mutation in chronic myeloproliferative disorders with lockednucleic acid modified probes and real-time polymerase chain reaction J Mol Diagn.. 2008;10:435–441[Context Link]14. Beer PA, Campbell PJ, Scott LM, et al. MPL mutations in myeloproliferative disorders: analysis of the PT-1 cohort Blood.. 2008;112:141–149[Context Link]15. Kilpivaara O, Levine RL. JAK2 and MPL mutations in myeloproliferative neoplasms: discovery and science Leukemia.. 2008;22:1813–1817[Context Link]16. Siemiątkowska A, Bieniaszewska M, Hellmann A, et al. JAK2 and MPL gene mutations in V617F-negative myeloproliferative neoplasms Leuk Res.. 2009. [Epub ahead of print].[Context Link]17. Bench AJ, Pahl HL. Chromosomal abnormalities and molecular markers in myeloproliferative disorders Semin Hematol.. 2005;42:196–205[Context Link]18. Williams DM, Kim AH, Rogers O, et al. Phenotypic variations and new mutations in JAK2 V617F-negative polycythemia vera, erythrocytosis, and idiopathic myelofibrosis Exp Hematol.. 2007;35:1641–1646[Context Link]MPL; JAK2; RT-PCR; mutation; MPD; myeloproliferative neoplasm; polymorphism; diagnosis; inherited; somaticSummary of JAK2 Mutations 2790 Patients With Suspected Non-CML MPDsMPL Mutation Profile in Patients With JAK2-negative Suspected Non-CML MPDs (n=2242)Sequence analysis of myeloproliferative leukemia gene (MPL) mutations at codon W515. For both panels (A and B), direct and indirect sequencing are shown for the patient and control; the mutated nucleotides are indicated with arrows. A, Examples of various mutations detected at W515. The expected amino acids are shown. B, Examples of homozygous/hemizygous mutations at codon W515.Examples of novel mutations involving various codons in myeloproliferative leukemia gene (MPL). Both direct and indirect sequencing are shown for the patient and control. The mutated nucleotides are indicated with arrows.Examples of insertion/deletion mutations. A, Deletion of ag and insertion of t at nucleotide position 1618 (arrow), leading to an R525C mutation with frameshift and termination at amino acid 14. B, Deletion of tggcagtttcct and insertion of aaaat at nucleotide 1588 (arrow), leading to a W515-p518 deletion and insertion of 2 amino acids (KT). The first trace is a control, the middle trace is the forward sequence, and the bottom trace is the reverse direction. C, Forward and reverse insertion of 12 nucleotides (gctctggtgatc) (arrow) leading to the insertion of 4 new amino acids (ALVI) at codon T496.Possible Novel MPL SNPs Detected in Patients With JAK2-negative Suspected Non-CML MPDsSchematic representation of myeloproliferative leukemia gene (MPL) mutations. The MPL protein is depicted in blue and the junction at the cellular membrane, which is depicted in pink. The mutations are shown on the right. The mutations that have been reported but not detected in our testing are shown in rectangles. Detected single-nucleotide polymorphisms (SNPs) are shown on the left. Only the R537Q SNP has been reported earlier. All other SNPs are synonymous and nucleotides are shown.Flowchart shows a suggested sequence for molecular testing of mutations in patients suspected to have chronic myeloproliferative disorder.<em xmlns:mrws="http://webservices.ovid.com/mrws/1.0">MPL</em> Mutation Profile in <em xmlns:mrws="http://webservices.ovid.com/mrws/1.0">JAK2</em> Mutation-negative Patients With Myeloproliferative DisordersMa Wanlong BSc; Zhang, Xi BSc; Wang, Xiuqiang BSc; Zhang, Zhong MD; Yeh, Chen-Hsiung PhD; Uyeji, Jennifer BSc; Albitar, Maher MDOriginal ArticlesOriginal Articles120p 34-39