Deep sequencing reveals double mutations in cis of MPL exon 10 in myeloproliferative neoplasms

Abstract

Somatic mutations of MPL exon 10, mainly involving a W515 substitution, have been described in JAK2 (V617F)-negative patients with essential thrombocythemia and primary myelofibrosis. We used direct sequencing and high-resolution melt analysis to identify mutations of MPL exon 10 in 570 patients with myeloproliferative neoplasms, and allele specific PCR and deep sequencing to further characterize a subset of mutated patients. Somatic mutations were detected in 33 of 221 patients (15%) with JAK2 (V617F)-negative essential thrombocythemia or primary myelofibrosis. Only one patient with essential thrombocythemia carried both JAK2 (V617F) and MPL (W515L). High-resolution melt analysis identified abnormal patterns in all the MPL mutated cases, while direct sequencing did not detect the mutant MPL in one fifth of them. In 3 cases carrying double MPL mutations, deep sequencing analysis showed identical load and location in cis of the paired lesions, indicating their simultaneous occurrence on the same chromosome.

Introduction

The JAK2 (V617F) mutation is found in more than two-thirds of patients with a myeloproliferative neoplasm (MPN), and more specifically in about 95% of patients with polycythemia vera (PV) and 50–60% of those with essential thrombocythemia (ET) or primary myelofibrosis (PMF).1 Activating somatic mutations of MPL exon 10, mainly involving a W515 substitution, have been detected in JAK2 (V617F)-negative patients with ET and PMF.2–11 Germline mutations of MPL have been found to be responsible for hereditary thrombocytosis,12–15 and patients with the familial thrombocytosis associated with MPL (S505N) have been reported to have a high risk of developing splenomegaly and bone marrow fibrosis, i.e. typical myeloproliferative features.16 In addition, a functional MPL polymorphism (G1238T, K39N) may be associated with a clinical phenotype of thrombocytosis in African Americans.17

The optimal approach to the detection of MPL mutations in MPN has not yet been defined. In this work we used direct sequencing and high resolution melt (HRM) analysis to identify somatic mutations of MPL exon 10 in 570 patients with MPN. A subset of mutated patients was further characterized using allele specific PCR and deep sequencing.

Design and Methods

We evaluated patients with myeloproliferative neoplasms followed at the Department of Hematology Oncology, Fondazione IRCCS Policlinico San Matteo and University of Pavia, Pavia, Italy. This study was approved by the institutional ethics committee (Comitato di Bioetica, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy). The procedures followed were in accordance with the Helsinki Declaration of 1975, as revised in 2000, and samples were obtained after patients provided written informed consent.

Diagnosis of PV, ET and PMF was initially made according to the criteria in use at the time of first observation, and patients were then reclassified according to the revised World Health Organization (WHO) criteria using the available data and revising the existing bone marrow biopsy.18 The criteria of the International Working Group for Myelofibrosis Research and Treatment (IWG-MRT) were employed for the diagnosis of post-PV MF and post-ET MF.19 Overall 570 patients with sporadic MPN were investigated for MPL mutations, as summarized in Table 1.

Table 1.WHO classification of the 570 cases of MPN studied and information on mutational status of JAK2 and MPL.

Peripheral blood granulocytes and T lymphocytes were isolated as previously described.20 DNA extraction was performed using the Puregene Blood DNA isolation kit (Qiagen, Germantown, MD, USA) according to the manufacturer’s procedure. Granulocyte JAK2 (V617F) mutation burden was assessed using a quantitative polymerase chain reaction (qPCR)-based allelic discrimination assay21 and applying the standard curve method.

MPL mutation scanning was performed on granulocyte and T lymphocyte gDNA using both HRM analysis and direct sequencing. These methods are described in the Online Supplementary Appendix. Patients who were found to be MPL mutated at the HRM analysis but not at sequencing were further investigated using the Ipsogen MPL MutaScreen kit (Ipsogen, Marseille, France) for the detection of MPL (W515L) and (W515K) mutations. These investigations were conducted on the Rotor-Gene 6000 real-time analyzer. The sensitivity of this TaqMan allelic discrimination assay is approximately 1.5% mutant alleles.

For deep sequencing, PCR was performed in 50 …L containing 100 ng DNA, 1 unit of Platinum Taq DNA polymerase high fidelity together with 1x buffer and 2 mM MgSO₄ (Invitrogen, Carlsbad, CA, USA), 200 …M dNTPs and 200 nM of each primer (Online Supplementary Table S2). Cycling conditions were as follows: 95° C for 2 min (one cycle); 95°C for 30 sec, 62°C for 30 sec, 72°C for 30 sec (35 cycles); 72°C for 10 min (one cycle). Aliquots of 500–1000 ng of amplified regions were purified by MinElute columns (Qiagen, Valencia, CA, USA) in order to remove the fragments less than 70 bp. Ligation of the purified samples to specific adaptors, preparation of the single-stranded libraries (ssDNA) and their amplification in emulsion were performed following the manufacturer’s instructions (Roche, Basel, Switzerland). The reactions were recovered by isopropanol emulsion breaking and enriched for positive reaction beads. Each enriched sample was separately loaded onto one-sixteenth of the PicoTiterPlate (PTP) and was sequenced according to the 454 GS-FLX Titanium protocol. The resulting data were analyzed with the GS-FLX amplicon variant analyzer (AVA) software (Roche), version 2.0.01.12.

Results and Discussion

Overall 570 patients with MPN were studied for MPL exon 10 mutations, and results are reported in Tables 1 and 2. Based on the detection of abnormal melting curve profiles in granulocytes but not in T lymphocytes, 34 patients were found to carry a somatic mutation of MPL exon 10. While no MPL mutation was detected in 57 patients with PV, somatic mutations were detected in 34 of 513 (6.6%) of all patients with ET or PMF. The prevalence of these mutations was equal to 33 of 221 (14.9%) in patients with these conditions who were JAK2 (V617F)-negative. One patient with ET carried both JAK2 (V617F) and MPL (W515L) in circulating granulocytes.

The 34 patients with abnormal HRM profiles were studied by direct sequencing on both granulocyte and T-lymphocyte DNA. As shown in Table 2, 27 out of these 34 patients carried somatic mutations of MPL identified by direct sequencing. The remaining 7 patients were studied by means of the TaqMan allelic discrimination assay, and 6 of them were found to carry MPL (W515L). The remaining patient (MPC07_336) was scored as wild type, because neither the W515L nor W515K alleles were detected, suggesting that other lesions, different from W515L and W515K, were present at a mutant burden higher than 1% but lower than 5–10%, which are the limit of sensitivity of HRM and sequencing, respectively, as reported in the Online Supplementary Appendix.

In 3 patients (PV06_799, PV03_110 and MPC07_349) HRM analysis generated abnormal melting plots clearly distinguishable from patterns associated with a single mutation (Figure 1A shows 2 of the 3 patients with double MPL mutations in comparison with the patient PV05_558 carrying the MPL W515L mutation). Direct sequencing showed double mutations in these cases: W515L combined with the novel S505C (A1513T) mutation in patient PV06_799, W515L combined with the novel V501A (T1502C) mutation in patient PV03_110, and W515R combined with V501A in patient MPC07_349 (Figure 1A and Table 2).

Considering the MPL mutations identified in this study (Table 2), MPL (W515L) was the most common (67% of cases, including double mutations); MPL (W515K) and MPL (W515A) were found in 15% of cases each, and W515R in just one case (3%).

In order to investigate the 3 patients found to carry two different MPL mutations (Table 2), we used the GS-FLX Titanium sequencing technology. The reads generated were aligned to the reference genomic sequences, and were then grouped into 4 categories according to the possible cis or trans location of the 2 mutations: 1) Wt/Wt if no sequence variations were present; 2) Mut/Mut if the 2 mutations were detected on the same strand; 3) Wt/Mut if the mutations were present only at codon 515; 4) Mut/Wt if the sequence variation was detected only at codon 501 or 505. As shown in Figure 1B, in all cases deep sequencing clearly detected the Wt/Wt and Mut/Mut fragments (blue and green bars, respectively), while the Wt/Mut and Mut/Wt reads were present at very low or negligible percentages (orange and yellow bars, respectively) not distinguishable from the background. Thus, in these MPN patients carrying two different MPL lesions, both sequence variations were found to be located in cis on the same allele and to have the same mutation load, and no significant percentages of reads with a single mutation could be detected.

This study defines a diagnostic strategy for detecting MPL mutations in MPN. Our findings indicate that the HRM analysis of granulocyte DNA represents an optimal approach for scanning for MPL exon 10 mutations in patients with MPN, confirming the recent observations by Boyd et al.10 Although the most common MPL mutations are associated with distinct HRM profiles, a second test is required to properly identify the underlying mutation in a patient with abnormal HRM plot. Direct sequencing is clearly useful, but has a low sensitivity, and in this study it did not detect about one fourth of cases carrying MPL (W515L). TaqMan allele discrimination assays have a much better sensitivity but our findings indicate that they should include not only PCR for MPL (W515L) and MPL (W515K), but also a PCR for MPL (W515A) and possibly (W515R). The availability of a deep sequencing technology allows not only the characterization of any MPL mutation, but also precise assessment of the mutant allele burden as shown in Figure 1B, and might become a diagnostic procedure in the near future.

Table 2.Molecular features of the 34 patients who carried somatic mutations of MPL exon 10 in circulating granulocytes.*

Using a sensitive allele specific PCR for JAK2 (V617F) and the described HRM assay for MPL exon 10 mutations, we detected a pathogenetic clonal marker in 64% of patients with ET, post-ET MF or PMF. This figure is lower than the 71% obtained by Boyd et al.10 in a study on a smaller patient population (175 patients vs. 513 in the current study). As both studies used the same diagnostic criteria for MPN and very similar diagnostic assays, the above difference can only reflect a different composition of the 2 study populations. Apart from JAK2 (V617F) and somatic mutations of MPL exon 10, acquired mutations in LNK have been found in ET,22 but they may occur only infrequently. Our study also confirms the observation by Beer et al.6 that the frequency of MPN patients carrying both JAK2 (V617F) and MPL mutations is negligible, supporting the concept of different genetic backgrounds underlying the occurrence of JAK2 and MPL molecular lesions.

Figure 1.Mutation analysis of MPL in patients with MPN. (A) Abnormal HRM profiles in 2 of 3 patients carrying double mutations in MPL exon 10 (PV06_799 and PV03_110) compared to wild-type (healthy subject) and W515L (PV05_558) controls. These profiles were generated by the simultaneous presence of the W515L mutation in combination with the novel V501A or S505C mutations, as shown by sequencing chromatograms. (B) Deep sequencing analysis of MPN patients carrying double mutations of MPL. In order to investigate the 3 patients carrying two different MPL mutations (Table 2), we used the GS-FLX Titanium sequencing technology. In this platform individual template molecules are amplified within droplets of an emulsion, allowing the sequencing of a single DNA strand and, consequently, the characterization of the cis or trans allelic location of each set of the two MPL sequence variants. The large set of reads generated (PV03_110 n=3846, MPC07_349 n=2082, PV06_799 n=7070) was aligned to the reference genomic sequences, yielding a highly redundant representation of the target regions. The reads were grouped into 4 categories according to the possible cis or trans location of the 2 mutations: 1) Wt/Wt if no sequence variations were present; 2) Mut/Mut if the 2 mutations were detected on the same strand; 3) Wt/Mut if the mutations were present only at codon 515; 4) Mut/Wt if the sequence variation was detected only at codon 501 or 505. Results are presented as % of reads over the totals, providing the mutant allele burden.

We identified 3 patients with double mutations in MPL exon 10. Two similar patients have been recently described by Boyd et al.10 who genotyped hematopoietic colonies in a patient with ET carrying both MPL (W515L) and (S505N), and concluded that these mutations had arisen on different chromosomes. Our deep sequencing studies provided opposite results, clearly indicating that the paired mutations arose on the same chromosome (Figure 1B). The probability for a double mutation in the same gene to occur spontaneously is clearly extremely low, but also the probability for two mutations to occur by chance alone in the two alleles of the same individual is very low. This suggests that patients with MPL mutated MPN may have a particular genetic predisposition to acquiring MPL exon 10 mutations, which is not related to JAK2 nor to MPL itself.23 Interestingly, a double L611V/V617F in cis mutation of JAK2 has been described in patients with polycythemia vera.24

Acknowledgments

The authors thank the patients of the Department of Hematology Oncology, Fondazione IRCCS Policlinico San Matteo, who contributed blood samples and participated in this observational study. The study was funded in part by a grant from Associazione Italiana per la Ricerca sul Cancro (AIRC, Milano) “Special Program Molecular Clinical Oncology 5x1000” to AGIMM (AIRC-Gruppo Italiano Malattie Mieloproliferative): a detailed description of the AGIMM project is available at http://www.progettoagimm.it. Additional financial support was provided by Fondazione Cariplo, Regione Lombardia and MIUR PRIN (grants to MC), and by FIRB MIUR (grant RBLA03ER38 to GDeB).

Footnotes

• DP and AB contributed equally to this work.
• Authorship and Disclosures The information provided by the authors about contributions from persons listed as authors and in acknowledgments is available with the full text of this paper at www.haematologica.org.
• Financial and other disclosures provided by the authors using the ICMJE (www.icmje.org) Uniform Format for Disclosure of Competing Interests are also available at www.haematologica.org.

• Received October 4, 2010.
• Revision received December 27, 2010.
• Accepted December 31, 2010.