Japanese Journal of Clinical Oncology Advance Access published online on June 2, 2009
Japanese Journal of Clinical Oncology, doi:10.1093/jjco/hyp048
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© The Author (2009). Published by Oxford University Press. All rights reserved
Detection of Low Allele Burden of JAK2 Exon 12 Mutations Using TA-cloning in Patients with Erythrocytosis
1 Intractable Disease Research Center, Tokyo Medical University
2 Department of Materials and Life Science, Seikei University
3 First Department of Internal Medicine, Tokyo Medical University, Tokyo, Japan
For reprints and all correspondence: Junko H. Ohyashiki, Intractable Disease Therapeutic Research Center, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, Japan. E-mail: junko{at}hh.iij4u.or.jp
Received January 6, 2009; accepted April 20, 2009
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Abstract |
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 TOP Abstract INTRODUCTIONPATIENTS AND METHODSRESULTS AND DISCUSSIONFundingConflict of interest statementAcknowledgementsReferences |
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Objective: Polycythemia vera (PV) is a clonal myeloproliferative neoplasia associated with the activation of the Janus-activating kinase 2 (JAK2) mutation. The aim of this study is to identify clonal expansion of exon 12 mutations.
Methods: We performed DNA sequencing of the JAK2 exon 12 after TA-cloning in JAK2-V617F-negative and JAK2-V617F-positive PV patients.
Results and Conclusions: We found clonal mutations (i.e. H538-K539delinsL and D544G) in 3 of 7 JAK2-V617F-negative PV patients, however, unlike JAK2-V617F, allele burden of JAK2 exon 12 mutation was low. Since allele-specific PCR is able to amplify only the limited region which contains known mutations with gain-of-function, we need to clarify the biological implications of unknown single nucleotide substitution of the JAK2 exon 12 with low clonal burden in erythrocytosis patients.
Key Words: Janus kinase 2 • polycythemia vera • mutation
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INTRODUCTION |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTS AND DISCUSSIONFundingConflict of interest statementAcknowledgementsReferences |
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The Janus-activating kinase 2 (JAK2)-V617F mutation, due to a single-nucleotide substitution (G1849T) at the exon 14 of the gene, occurs in the vast majority of patients with polycythemia vera (PV), and the gain-of-function JAK2-V617F mutation leads to constitutive tyrosine phosphorylation of JAK2 of the erythropoietin signaling. In 2007, Scott et al. (1) demonstrated that somatic JAK2 exon 12 mutations in patients with PV or idiopathic erythrocytosis who do not carry the unique exon 14 mutation and several novel mutations within the JAK2 exon 12 have also been reported (1–8). The current detection assays are mostly based on the allele-specific polymerase chain reaction (allele-specific PCR) technique; however, unknown mutations could not be amplified by allele-specific primers. Recently, high-resolution melting analysis by Jones et al. (9) made it possible to detect a minor clone (i.e. a low clonal burden) with JAK2 exon 12 mutation in some patients with PV or erythrocytosis patients who failed to demonstrate JAK2 exon 12 mutations by allele-specific PCR technique.
In a retrospective single-center study, we previously showed that JAK2 exon 12 mutation could not be detected in seven patients with ‘isolated erythrocythemia’ by conventional PCR-direct sequencing (10). A recent report by Theocharides et al. (11) demonstrated that the allele ratios in PV patients with JAK2 exon 12 mutations were markedly lower, compared with those in PV patients with JAK2-V617F. It is therefore reasonable to re-examine our samples by a more sensitive assay to detect a low clonal burden. Since the gene-dosage effect of JAK2 exon 12 mutations is somewhat different from that in JAK2-V617F, it is still uncertain whether JAK2 exon 12 mutations with a low clonal burden shows a distinct clinical features compared with those with high mutated allele burden. We therefore performed TA-cloning and detected a low clonal burden of JAK2 exon 12 mutation by DNA sequencing.
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PATIENTS AND METHODS |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTS AND DISCUSSIONFundingConflict of interest statementAcknowledgementsReferences |
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Patients
Patients with erythrocytosis, who fulfilled all the criteria of Polycythemia Vera Study Group (PVSG) had been referred to Tokyo Medical University Hospital, were tentatively termed PV and enrolled in this study. We used the clinical data set of PV patients from our previous report on 33 patients, including 24 PV patients with JAK2-V617F mutation and 9 patients without JAK2-V617F mutation. We measured the JAK2-V617F mutation and allelic burden of mutated JAK2 by the sequence-specific primer-single molecule fluorescence detection assay, as in our previous report (10). We studied JAK2 exon 12 mutations on seven of the nine patients without JAK2-V617F, since we were unable to analyze JAK2 exon 12 mutations in the remaining two patients due to insufficient materials. We also studied JAK2 exon 12 mutations in four patients with JAK2-V617F (TT genotype) and four healthy volunteers (Table 1). Mutation analysis, using specimens from either patients or healthy volunteers, was approved by the Institutional Review of Committee of Tokyo Medical University. Informed consent was obtained from patients and volunteers in accordance with the Declaration of Helsinki.
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TA-cloning and DNA Sequencing
Genomic DNA obtained from whole blood by an automated system (12) was amplified as follows. The primer set for the amplification of a JAK2 exon 12 DNA was designed according to GenBank AL161450 [GenBank] , using forward primers at the intron 11 region: 5'-gac aag agc ccg ggc ttc tcc-3' and reverse primers at the intron 12 region: 5'-tgg tct aga gct agc cta ggc tcg aga a-3'. The PCR conditions were 95°C for 30 s, 60°C for 40 s and 72°C for 30 s for 35 cycles using Expand High FidelityPLUS PCR system (Roche Diagnostics, Mannheim, Germany). The PCR products of the JAK2 exon 12 were purified using a High Pure PCR Product Purification Kit (Roche Molecular Biochemicals Diagnostics, Indianapolis, IN, USA) and cloned into a pCR2.1 vector (Invitrogen, Carlsbad, CA, USA). We obtained 5–18 clones in each individual. Theoretically, it would be logical to obtain at least 10 clones in each individual; however, in practical terms, it was difficult to obtain 10 clones in some samples. We therefore tried to sequence clones as much as possible, using a BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster city, CA, USA) with an ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems). Finally, the sequence was compared with the JAK2 sequence. We used both forward and reverse sequencing to avoid misreading of nucleotides. Since the number of clones analyzed differs among samples, we could not quantify the allele burden precisely. We therefore arbitrarily categorized clonal change when identical mutated sequence was detected in two or more clones in single individuals.
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RESULTS AND DISCUSSION |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTS AND DISCUSSIONFundingConflict of interest statementAcknowledgementsReferences |
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DNA sequencing analysis after TA-cloning demonstrated that four of seven patients without JAK2-V617F had some exon 12 mutations (Fig. 1). In detail, one patient (JAK2_0096) had two of eight clones with H538-K539delinsL (Fig. 2A), whereas one of eight clone had single-nucleotide substitution without amino acid conversion. JAK2_0109 patient had 6 of 18 clones with H538-K539delinsL, JAK2_0111 showed 2 of 9 clones with D544G (Fig. 2B) and JAK2_0113 patient did 1 of 7 clone with L545S (Fig. 2C). The remaining three JAK2-V617F-negative patients did not have any clonal or non-clonal mutation in the JAK2 exon 12 region within the indicated region in Fig. 1. In conclusion, we detected clonal JAK2 exon 12 mutations in three patients (JAK2_0096, JAK2_0109 and JAK2_0111) with the predictive amino acid substitution. Of note is that allele-specific PCR with previously utilized primer setting is unable to detect these single-nucleotide mutations found in some JAK2-V617F-negative patients. In contrast, we did not find any clonal JAK2 exon 12 mutation in PV patients with JAK2-V617F and normal individuals: we analyzed >10 clones in all the normal individuals to verify that there was no clonal JAK2 exon 12 mutation in normal individuals. Although the biological significance was unclear, non-clonal nucleotide substitutions were also detected in one JAK2-V617F-positive patient (K539E) and in two normal subjects (N542S and Q534R), respectively (Table 1).
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Previous reports indicate that N542-E543del, F537-K539delinsL and E543-D544del are frequently detected (8). Pietra et al. (8) reported two PV patients with novel duplications involving a substitution F547 using PCR-direct sequencing. DNA sequencing in combination with allele-specific PCR might be useful in detecting certain known mutations; however, it appears difficult to identify new single-nucleotide mutations, since allele-specific PCR using certain primers could cover only the deletions or abnormalities within the setting primer itself. For example, Martinez-Aviles et al. (2) found K539L mutation (AAA
CTA) by direct sequencing, although they did not achieve a positive result by allele-specific PCR technique; K539L substitution due to nucleotide mutation (AAA TTA) has been reported by other investigators using allele-specific PCR, using primers containing TTA sequence. Another important issue we should address is that it is possible to underestimate single-nucleotide mutations as the wild-type JAK2 exon 12, in cases with only minor portion of neoplastic cells, contains JAK2 exon 12 mutation. In this study, we found clusters of single-nucleotide mutations located within the region of N542 to L545 amino acids of the JAK2 exon 12, which conserved amino acid alignment across multiple species. Although we could not completely rule out the possibility of artifacts due to TA-cloning, we used a High Fidelity Taq system to avoid inappropriate complementation during the process of PCR. Despite the fact that TA-cloning is a time-consuming procedure and is not suitable for clinical practice, it is of note that we shed light on the existence of very minor clones with JAK2 exon 12 mutations. High-through-output screening using melting analysis by Jones et al. (9) might be a powerful useful approach, and the use of enriched leukocyte fraction will improve the delectability of JAK2 exon 12 mutations. Unlike JAK2-V617F, allele burden of JAK2 exon 12 mutations is somehow low; therefore, gene-dosage effect of JAK2 exon 12 mutation has not been clarified. In addition, low allele burden may exist in condition of clonal hematopoiesis, even in the pre-JAK2 phase. Although the number of patients is too small to draw a final conclusion, we need to clarify the biological implications of poor clonal burden of neoplastic cells containing JAK2 exon 12 mutations in patients with erythrocytosis.
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Funding |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTS AND DISCUSSIONFundingConflict of interest statementAcknowledgementsReferences |
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This work was supported in part by the ‘High-Tech Research Center’ Project from the Ministry of Education, Culture, Sports and Technology (MEXT) and by the ‘University-Industry Joint Research Project’ from MEXT. This work has been also supported by Foundation for Promotion of Cancer Research in Japan (JHO).
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Conflict of interest statement |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTS AND DISCUSSIONFundingConflict of interest statementAcknowledgementsReferences |
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None declared.
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Acknowledgements |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTS AND DISCUSSIONFundingConflict of interest statementAcknowledgementsReferences |
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Thanks are due to Professor J. Patrick Barron for his review of this manuscript.
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References |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTS AND DISCUSSIONFundingConflict of interest statementAcknowledgementsReferences |
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1 Scott LM, Tong W, Levine RL, Scott MA, Beer PA, Stratton MR, et al. JAK2 exon 12 mutations in polycythemia vera and idiopathic erythrocytosis. N Engl J Med (2007) 356:459–68.
2 Martinez-Aviles L, Besses C, Alvarez-Larrán A, Cervantes F, Hernández-Boluda JC, Bellosillo B. JAK2 exon 12 mutations in patients with polycythemia vera or idiopathic erythrocytosis. Haematologica (2007) 92:1717–8.
3 Pardanani A, Lasho TL, Finke C, Hanson CA, Tefferi A. Prevalence and clinicopathologic correlates of JAK2 exon 12 mutations in JAK2 V617F-negative polycythemia vera. Leukemia (2007) 21:1960–3.[CrossRef][Web of Science][Medline]
4 Percy MJ, Scott LM, Erber WN, Harrison CN, Reilly JT, Jones FG, et al. The frequency of JAK2 exon 12 mutations in idiopathic erythrocytosis patients with low serum erythropoietin levels. Haematologica (2007) 92:1607–14.
5 Kouroupi E, Zoi K, Parquet N, Zoi C, Kiladjian JJ, Grigoraki V, et al. Mutations in exon 12 of JAK2 are mainly found in JAK2 V617F-negative polycythaemia vera patients. Br J Haematol (2008) 142:676–9.[CrossRef][Web of Science][Medline]
6 Butcher CM, Hahn U, To LB, Gecz J, Wilkins EJ, Scott HS, et al. Two novel JAK2 exon 12 mutations in JAK2-V617F-negative polycythemia vera patients. Leukemia (2008) 22:870–3.[CrossRef][Web of Science][Medline]
7 Li S, Kralovics R, De Libero G, Theocharides A, Gisslinger H, Skoda RC. Clonal heterogeneity in polycythemia vera patients with JAK2 exon 12 and JAK2-V617F mutations. Blood (2008) 111:3863–6.
8 Pietra D, Li S, Brisci A, Passamonti F, Rumi E, Theocharides A, et al. Somatic mutations of AK2 exon 12 in patients with JAK2(V617F)-negative myeloproliferative disorders. Blood (2008) 111:1686–9.
9 Jones AV, Cross NCP, White HE, Green AR, Scott LM. Rapid identification of JAK2 exon 12 mutations using high resolution melting analysis. Haematologica (2008) 93:1560–4.
10 Ohyashiki K, Kiguchi T, Ito Y, Gotoh A, Tauchi T, Miyazawa K, et al. Isolated erythrocythemia: a distinct entity or a sub-type of polycythemia vera? Jpn J Clin Oncol (2008) 38:230–2.
11 Theocharides A, Passweg JR, Medinger M, Looser R, Li S, Hao-Shen H, et al. The allele burden of JAK2 mutations remains stable over several years in patients with myeloproliferative disorders. Haematologica (2008) 93:1890–3.
12 Ohyashiki K, Hori K, Makino T, Ohyashiki JH. Automated JAK2V617F quantification using a magnetic filtration system and sequence-specific primer-single molecule fluorescence detection. Cancer Genet Cytogenet (2007) 179:19–24.[CrossRef][Medline]
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