- Email: [email protected]
Peptide nucleic acid probe-based fluorescence melting curve analysis for rapid screening of common JAK2, MPL, and CALR mutations
Accepted Manuscript Peptide nucleic acid probe-based fluorescence melting curve analysis for rapid screening of common JAK2, MPL, and CALR mutations
Joonhong Park, Minsik Song, Woori Jang, Hyojin Chae, Gun Dong Lee, KyungTak Kim, Heekyung Park, Myungshin Kim, Yonggoo Kim PII: DOI: Reference:
S0009-8981(16)30496-X doi: 10.1016/j.cca.2016.12.002 CCA 14588
To appear in:
Clinica Chimica Acta
Received date: Revised date: Accepted date:
10 November 2016 30 November 2016 2 December 2016
Please cite this article as: Joonhong Park, Minsik Song, Woori Jang, Hyojin Chae, Gun Dong Lee, KyungTak Kim, Heekyung Park, Myungshin Kim, Yonggoo Kim , Peptide nucleic acid probe-based fluorescence melting curve analysis for rapid screening of common JAK2, MPL, and CALR mutations. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Cca(2016), doi: 10.1016/ j.cca.2016.12.002
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ACCEPTED MANUSCRIPT Peptide nucleic acid probe-based fluorescence melting curve analysis for rapid screening of common JAK2, MPL, and CALR mutations Running title: PNA-based FMCA for MPN mutation screening
Joonhong Park, M.D.a,b, Minsik Songc, Woori Jang, M.D.a,b, Hyojin Chae, M.D.a,b, Gun Dong
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Leeb, KyungTak Kimc, Heekyung Parkc, Myungshin Kim, M.D.a,b*, Yonggoo Kim, M.D.a,b*
a
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Department of Laboratory Medicine, College of Medicine, The Catholic University of Korea,
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Seoul, Republic of Korea b
Catholic Genetic Laboratory Center, Seoul St. Mary’s Hospital, College of Medicine, The
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Catholic University of Korea, Seoul, Republic of Korea c
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SeaSun Biomaterials, Daejeon, Republic of Korea
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*Corresponding author:
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Joonhong Park and Minsik Song contributed equally as co-first authors.
Myungshin Kim, M.D., PhD., 222 Banpo-daero, Seocho-gu, Department of Laboratory Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea,
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Seoul 06591, Republic of Korea.
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Tel: +82-2-2258-1645, Fax: +82-2-2258-1719, Email: [email protected]
Yonggoo Kim, M.D., PhD., 222 Banpo-daero, Seocho-gu, Department of Laboratory Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul 06591, Republic of Korea. Tel: +82-2-2258-1642, Fax: +82-2-2258-1719, Email: [email protected]
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ACCEPTED MANUSCRIPT Abstract Background: We developed and evaluated the feasibility of peptide nucleic acid (PNA)based fluorescence melting curve analysis (FMCA) to detect common mutations in myeloproliferative neoplasms (MPNs). Methods: We have set up two separate reactions of PNA-based FMCA: JAK2 V617F &
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CALR p.Leu367fs*46 (set A) and MPL W515L/K & CALR p.Lys385fs*47 (set B). Clinical usefulness was validated with allele-specific real-time PCR, fragment analysis, Sanger
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sequencing in 57 BCR-ABL1-negative MPNs.
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Results: The limit of detection (LOD) of PNA-based FMCA was approximately 10% for each mutation and interference reactions using mixtures of different mutations were not observed.
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Non-specific amplification was not observed in normal control. PNA-based FMCA was able to detect all JAK2 V617F (n=20), CALR p.Leu367fs*46 (n=10) and p.Lys385fs*47 (n=8).
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Three of six MPL mutations were detected except three samples with low mutant concentration in out of LOD. JAK2 exon 12 mutations (n=7) were negative without
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influencing V617F results. Among six variant CALR exon 9 mutations, two were detected by
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this method owing to invading of probe binding site. Conclusions: PNA-based FMCA for detecting common JAK2, MPL, and CALR mutations is a rapid, simple, and sensitive technique in BCR-ABL1-negative MPNs with > 10% mutant
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allele at the time of initial diagnosis.
Keywords: Peptide nucleic acid; FMCA; mutation screening; JAK2; MPL; CALR
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ACCEPTED MANUSCRIPT 1. Introduction Myeloproliferative neoplasms (MPNs) are a heterogeneous group of clonal hematopoietic stem cell myeloid neoplasms with the potential to progress to acute leukemia. World Health Organization (WHO)-defined MPN is classified into eight subcategories, including three that are operationally grouped as “BCR-ABL1-negative MPNs”: polycythemia vera (PV), essential
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thrombocythemia (ET), and primary myelofibrosis (PMF). The WHO classification system uses the presence of JAK2 (Janus kinase 2) V617F or exon 12 mutations and MPL (MPL
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proto-oncogene, thrombopoietin receptor) mutations as pathognomonic clues for a diagnosis
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of BCR-ABL1-negative MPNs. JAK2 V617F is detected in approximately 97% of patients with PV and 60% of patients with ET or PMF. Additionally, exon 12 mutations in JAK2 were
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discovered in most patients with JAK2 V617F-negative PV. MPL mutations were found in 10–20% of patients with ET or PMF who were negative for JAK2 V617F [1]. The
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identification of JAK2/MPL mutations has been essential for the diagnosis and classification of BCR-ABL1-negative MPNs; however, approximately 30% of patients with ET or PMF
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lacked one of the two clonal markers, that is, they were negative for both JAK2 and MPL
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mutations. Recently, novel mutations in calreticulin (CALR) in patients with ET or PMF were described as cell-derived, mostly heterozygous, and mutually exclusive of JAK2 and MPL mutations [2, 3]. The discovery of CALR mutations represents another milestone in the
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ongoing effort to decipher the molecular pathogenesis of MPN and also provides an
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additional molecular marker for the purposes of both diagnosis and prognosis [4]. Therefore, the discovery of this subgroup of somatic CALR mutations that are unrelated to JAK/MPL mutations may provide a new tool for the molecular diagnosis of BCR-ABL1-negative MPNs and may have implications for clinical management of patients [5-8]. Therefore, there is a need to develop more generally applicable, sensitive assays to identify common JAK2, MPL, and CALR mutations for use in diagnostic screening tests in BCRABL1-negative MPNs at the same time. Among various molecular techniques, fluorescence melting curve analysis (FMCA) allows for detection of multiple targeted DNA sequences with 3
ACCEPTED MANUSCRIPT different melting temperatures (Tm) values in individual fluorescent channels [9]. In addition, artificially hybridized polymer, peptide nucleic acid (PNA) can easily hybridize with DNA and has strong binding affinity because it lacks a negatively charged phosphate group in its backbone [10, 11]. PNA probes are able to be conveniently applied dual-labeled selfquenching probe-based FMCA to screen various mutations including single nucleotide
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variant, insertion and deletion rapidly [27]. The term ‘‘dual-labeled’’ means that the probe has
refers to the single-strand base stacking of PNA [12].
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a fluorophore at the 5′ end and a quencher at the 3′ end, and the term ‘‘self-quenching’’
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In this study, we developed and evaluated the feasibility of PNA-based FMCA to detect
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common JAK2, MPL, and CALR mutations in MPNs.
2.1. Oligonucleotides and PNA probes
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2. Materials and methods
Primers, synthetic target template, and PNA probes were designed with Primer3 software
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(http://bioinfo.ut.ee/primer3/), PNA Probe Designer (Applied Biosystems; Thermo Fisher
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Scientific, Inc., Waltham, MA). All PNA probes (FAM-, HEX-, Texas Red-, or Cy5-labeled) were synthesized and HPLC-purified (Panagene Inc., Daejeon, Republic of Korea). Target oligonucleotides were synthesized and PAGE-purified (Cosmo Genetech Co., Ltd, Seoul,
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Republic of Korea). The purity of all probes was confirmed by mass spectrometry. In PNA-
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based FMCA, unwanted secondary structural characteristics of the probe should be avoided to allow for better hybridization with its target. The mutation sites are generally located in the centre of the probe so as to obtain a Tm shift around ± 3°C as estimated by the software. For detection of deletion and insertion, end position deletion/insertion and center position deletion/insertion were used to optimize Tm shift. All PNA probes used to identify the six mutation targets were designed to match with the wild type sequences, except the probe used for the detection of the CALR type 2 mutation, in which the probe for nucleotide sequence c.1154_1155insTTGTC matched with the insertion-induced frameshift mutation of 4
ACCEPTED MANUSCRIPT CALR exon 9 instead of the wild type version of CALR.
2.2. Simulation analysis of denaturation of hybrids formed between PNA probes and synthetic target template using PNA-based FMCA To analyze the Tm of PNA probe, synthetic single-stranded DNA oligomers were directly
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used as the target DNA. Thermal denaturation of hybrids formed between PNA probes and synthetic target template was carried out with the CFX96TM Real-time PCR Detection
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System (Bio-Rad Laboratories Inc., Hercules, CA). A thermal cyclic reaction was performed
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using the following conditions: the 20 µL reaction mixtures containing 0.5 µL PNA probe, 1.0 µL synthetic DNA template (0.5 µL wild type and 0.5 µL mutant type), and 1X SSB Real-Time
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FMCA™ buffer (SeaSun Biomaterials). Melting curve analysis began with a denaturation step for 3 min at 95°C, a hybridization step consisting of a temperature decrease from 75 to
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45°C at 10°C per step with a 30 sec pause between each step and lastly, a stepwise temperature increase from 20°C to 80°C at 1°C per step with a 5 sec pause between each
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step. Fluorescence was recorded at each step in the corresponding detection channel. Initial
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and post-melt fluorescence signals of all samples were normalized to relative values of 1.0 and 0. Shifting the normalized melting curves along the temperature axis allows for creation of a plot analyzing the differences in melting curve shape that is superimposed at a specific
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fluorescence intensity to enhance visualization of heteroduplex and homoduplex synthetic
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target template. The data obtained were plotted as fluorescence versus temperature as well as the negative derivative of fluorescence over temperature versus temperature. Tm values were identified by the peak position of the latter curve. Synthetic single-stranded DNA oligomers were used as target DNA for probe validation, and each target DNA contained mismatch(es), deletion(s), or insertion(s) (Table S1).
2.3. Clinical sample preparation A total of 57 clinical samples were tested: 26 PV with JAK2 V617F, 24 ET with three MPL 5
ACCEPTED MANUSCRIPT W515L/K or 21 CALR exon 9 mutations, and 7 PMF with one JAK2 V617F, three MPL W515L/K, or three CALR exon 9 mutations. Genomic DNA was prepared from bone marrow aspirates using the QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Clinical diagnosis of PV, ET, or PMF was conducted in accordance with the 2008 WHO classifications [13]. All patients in this study provided written
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informed consent, and the study protocol was approved by the Institutional Review Board of
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The Catholic University of Korea.
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2.4. Screening of common JAK2, MPL, and CALR mutations by PNA-based FMCA Two separate reaction tubes of set A and B were used to identify common JAK2, MPL, and
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CALR mutations in 57 BCR-ABL1-negative MPNs (Table 1). Although extremely rare, a double mutation within codon 617 of JAK2 can also result in the V617F mutation, that is
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1849G>T combined with 1851C>T (annonated as c.1849_1851GTC>TTT), which also encodes phenylalanine. The occurrence of this double mutation has been reported to
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produce a false negative result in an assay, which specifically targets the 1849G>T mutation
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[14]. Thus this double mutation was included as target for detecting JAK2 V617F. An asymmetric PCR [15] was carried out to generate single-stranded DNA target according to the following conditions: each 20 µL reaction contained 10 µL 2x qPCR PreMix (SeaSun
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Biomaterials), a primer/probe mix containing all three primer pairs (forward 0.05 µM /
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revserse 0.5 µM), two differently labeled PNA probes (0.5 µL each), and 1.0 µL of clinical DNA template. The primer sets are described in Table S2. PNA-based FMCA consisted of a 10 min denaturation step at 95°C, 41 cycles of 95°C for 30 sec, 58°C for 30 sec, and 78°C for 45 sec, followed by a denaturation step for 3 min at 95°C, a hybridization step of a temperature decrease from 75 to 45°C at 10°C per step with a 30 sec pause between each step, and a stepwise temperature increase from 20°C to 80°C at 1°C intervals with a 5 sec stop between each step. The results were analyzed as fluorescence versus temperature graphs by Bio-Rad CFX Manager (Bio-Rad Laboratories Inc.) with normalized, temperature6
ACCEPTED MANUSCRIPT shifted melting curves displayed as a difference plot.
2.5. Molecular analysis for confirmation of common JAK2, MPL, and CALR mutations The presence of common and other mutations of the JAK2, MPL, and CALR in 57 BCRABL1-negative MPNs was validated by different molecular methods following as: an allele-
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specific real-time PCR assay (Real-Q JAK2V617F Kit, BioSewoom, Seoul, Republic of Korea) was used to detect the JAK2 V617F mutation using the amplification refractory mutation
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system principle. To identify the JAK2 exon 12 and 14 mutation, a multiplex fragment
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analysis-based assay was used [16] and Sanger sequencing was conducted to confirm the code in the region containing the entirety of exon 12, as previously described [17]. The MPL
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W515L/K mutation was analyzed using allele-specific real-time PCR (Real-Q MPLW515L/K Screening Kit, BioSewoom). All PCR reactions were run in duplicate. Single-positive PCR
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reactions (CT 35–40) were repeated in triplicate and the samples were classified as mutation-positive only if all replicates were positive. All negative cases for MPL W515L/K
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were directly sequenced with mutant-enriched PCR to identify other MPL mutations not
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covered by PNA-based FMCA. To screen for CALR exon 9 mutations, we first performed PCR fragment analysis using PCR primers spanning exon 9 and a forward primer labeled with 6-FAM, as described previously [2]. All CALR exon9 mutations identified were confirmed
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bi-directionally by Sanger sequencing. RefSeq ID: NM_001322194.1, NM_005373.2, and
3. Results
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NM_004343.3 for JAK2, MPL, and CALR were used for cDNA nucleotide numbering.
3.1. Analytical performance of PNA-based FMCA An analytical limit of detection study was done in triplicate to show that this assay can detect serially diluted plasmids (100%, 50%, 25%, 10%, 7.25%, and 0% of the synthetic target template relative to the wild type template per reaction) with each of the two reaction sets in PNA-based FMCA. When mutant and wild type templates were mixed, mutant templates 7
ACCEPTED MANUSCRIPT were detectable when present in the range of 10 to 100% (Fig. 1). Analytical accuracy testing was performed three times to ensure that this method can precisely identify common JAK2, MPL, and CALR mutations. The assay was used to analyze genomic DNA from 20 JAK2 V617F, three MPL W/515L/K, 10 CALR p.Leu367fs*46 (type 1), and eight CALR p.Lys385fs*47 (type 2) that were confirmed by triplicate Sanger
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sequencing of the same PCR amplicons. The concordance rate between PNA-based FMCA and Sanger sequencing results was 100%. Analytical specificity testing was done in triplicate
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to show that this method can identify wild type JAK2 V617, MPL W515 and CALR exon 9
melting curves detected by PNA-based FMCA.
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residues in 100 samples from normal, healthy individuals. The results showed no abnormal
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The possibility of interference in reactions with a mixture of different mutations occurring in the same gene or with a mixture of diverse mutations occurring in different genes was
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evaluated in PNA-based FMCA (Table S3). As a result, only a single mutation in each of the three genes was identified in its corresponding fluorescent channel, regardless of whether a
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mixture of mutations occurred indifferent genes in same tube, when using probe set A or B
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(Fig. 2). These results demonstrate that PNA-based FMCA with color multiplexing can be used to identify the exact mutation type with combined PNA probes in each of the two
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reaction sets, A and B, simultaneously.
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3.2. Detection of common JAK2, MPL, and CALR mutations by PNA-based FMCA PNA-based FMCA with two separate reactions was used to screen for common JAK2, MPL, and CALR mutations in 57 BCR-ABL1-negative MPNs. The sensitivity and specificity of PNA-based FMCA are 93% (41/44) and 86% (12/14), respectively. The presence of MPL W515S was considered to estimate the specificity of PNA-based FMCA. Three false negative MPL W515L and two false positive CALR type 1 mutations were occurred. In detail, we were able to detect all JAK2 V617F positive specimens (n=20) by PNA-based FMCA. All patients with JAK2 exon 12 mutations (n=7) were negative without influencing 8
ACCEPTED MANUSCRIPT V617F results (Table 2). Second, two MPL W515L and one MPL W515K mutations were detected in six patients with MPL W515L/K (Table 3). In the three samples, two false negative finding for MPL W515L/K occurred owing to a lower concentration of the mutant allele than the limit of detection of 10%. On the other hand, an abnormal melting curve (Tm = 53°C) was observed in sample 7, which harbored a very low concentration of W515L, as
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shown by allele-specific real-time PCR which specifically targets MPL W515L/K. To resolve this issue, Sanger sequencing was conducted, which confirmed that, other than the MPL
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W515L/K mutation, c.1544G>C, producing p.Trp515Ser, was present (Fig. 3A). Third, PNA-
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based FCMA detected true common CALR mutations consisting of type 1 (n=10) and type 2 (n=8) mutations. Two false positive CALR type 1 mutation turned out to have other CALR 9
mutations
invading
parts
of
the
probe
binding
site
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exon
(c.1087_c.1101GAGGAGCAGAGGCTT); CALR exon 9 mutations, c.1092_1138del47
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(p.Gln365Glyfs*8) and c.1099_1132del34 (p.Leu367Argfs*52). Another CALR exon 9 mutations (n=4) were not detected without influencing PNA-based FCMA results.
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Interestingly, in sample 302, which had another CALR exon 9 mutation, c.1105_1156del52
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(p.Glu369Argfs*44), the false positive result for the CALR type 1 mutation occurred by fragment analysis because of an inability to discriminate the alleles that resulted from the same size difference (Δ 52bp) between the mutant and wild type alleles. No CALR type 1
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mutation was detected precisely by PNA-based FMCA with probe set A (Fig. 3B).
4. Discussion
The conventional FMCA uses double-strand DNA intercalating dyes such as SYBR Green or EvaGreen, but several studies demonstrates its disadvantages such as extensive optimization, concentration-dependent PCR inhibition, and effects on DNA melting temperature [18-21]. To overcome this limitations, molecular beacons and other hairpin probes with stem structures have been developed [22-24], However, the design of stem structure is more fastidious and complex than that of linear probe in a regard of its procedure 9
ACCEPTED MANUSCRIPT and conditions. Fortunately, designing PNA probes for FMCA is not much different from that of general dual-labeled DNA probes, and asymmetric PCR methods are easy to implement [25-27]. A PNA probe is naturally a dual-labeled, self-quenching probe owing uncharged nature and their peptide bond-linked backbone and the coiled conformation of PNA probe facilitates fluorescence self-quenching. Moreover, PNA probes can be designed shorter (9–
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13 bp) than DNA probes (20 bp or more) with the same T m and makes a large difference in ΔTm between base substitutions as well as insertion or deletion, which can contribute to
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discriminate mismatch from perfect match by changing dramatic melting shift [27].
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In this study, a clear difference was exhibited between the wild type and the mutant samples after normalization and temperature shifting in PNA-based FMCA. In principle, a
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two-tube assay is still not as efficient from workflow perspective as a single tube assay. However CALR type 1 and 2 mutations are close to each other, which cause interference
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response in a single-tube assay, PNA-based FMCA was divided inevitably into two separate tubes of set A for JAK2 and CALR type 1 and set B for MPL and CALR type 2. Among 57
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BCR-ABL1-negative MPNs, a total of 20 PV, 18 ET, and three PMF samples, each with one
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of the common JAK2, MPL, or CALR mutations, showed a clear and distinctive pattern on melting and difference curve plots compared with plots of wild type samples in PNA-based FMCA. Moreover, the common JAK2, MPL, or CALR mutations could be clearly
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distinguished in six different patterns using the Tm. No positives were found in 100 samples
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taken from normal, healthy individuals. Samples that were positive in PNA-based FMCA analyses were bidirectionally sequenced using the same PCR product. Interestingly, of the 20 CALR mutations with PNA-based FMCA-positive curves, 10 were type 1 deletions (c.1092_1143del52; p.Leu367Thrfs*46), eight were type 2 insertions c.1154_1155insTTGTC; p.Lys385Asnfs*47), and two were false type 1 deletions resulting from a small overlap with the probes and a deletion in c.1092_1138del47 (sample 513) and c.1099_1132del34 (sample Bu37). Type 1 deletions (52 bp in size) and Type 2 insertions (5 bp) showed clearly distinct PNA-based FMCA patterns. In addition, the PNA-based FMCA curves of two other 10
ACCEPTED MANUSCRIPT CALR exon 9 mutations mimicking type 1 or type 2 mutations, in samples 302 and 797, were definitely classified as uncommon CALR mutations by fragment analysis. None of sixteen wild type samples evidenced common JAK2, MPL, or CALR mutations, even those with a borderline pattern, except two false positive for CALR type 1 and two negative results for MPL W515L/K as determined by Sanger sequencing. Only sample 7, with an abnormal Tm at
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53°C detected by PNA probes, had an MPL W515S mutation. Although this sample showed a very low Ct value of 39.5 by allele-specific real-time PCR used to detect MPL W515L/K, its
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sequence revealed a heterozygous mutant allele burden, c.1544G>C (p.Trp515Ser).
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From a clinical standpoint, mutation screening for common JAK2, MPL, and CALR genes allows for confirmation of a morphologic diagnosis as well as an understanding of disease
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prognosis. During a diagnostic work-up, almost all patients with PV can be identified by the presence of a JAK2 mutation. Thus, JAK2 exon 12 mutations should be studied in cases of
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suspected PV related to a subnormal serum erythropoietin level and an absence of JAK2 V617F. However, JAK2 exon 12 mutations, occurring at low incidence in affected PV
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individual, were excluded from the target of this assay [28]. On the other hand, based on its
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high frequency and relative specificity in ET or PMF with wild-type JAK2/MPL, screening for CALR mutations should now be included in the diagnostic work-up of MPN and formally incorporated in the revised WHO classification system [8]. For patients suspected of having
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ET or PMF, initial mutation screening should start with an assessment of JAK2 V617F and
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then proceed with CALR mutation screening only in patients who are JAK2 V617F‑negative. Owing to the significantly lower incidence of MPL mutations compared with CALR mutations, screening for mutant CALR should be done first, before proceeding with MPL mutation studies in patients with JAK2 V617F-negative ET or PMF [4]. CALR mutations have been known to be mutually exclusive of JAK2 and MPL mutations, however incidental co-occurrence of CALR and other mutations has been reported in MPN [29]. Given the results of those studies and our previous study [30], it is noteworthy that our new multiplexing method can detect the coexistance of common triple mutations [30-32] or 11
ACCEPTED MANUSCRIPT other
CALR
mutations
[31-38]
involving
parts
of
the
probe
binding
site
(c.1087_c1101GAGGAGCAGAGGCTT) of CALR simultaneously identified by fragment analysis or Sanger sequencing. It is suggested that ET or PMF patients carrying both JAK2 V617F and a CALR mutation may show a unique clinical course and phenotype, and it is necessary to define the prognosis, risk factors, and outcomes for such MPN patients [31, 34,
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39]. There remain a few limitations in this PNA-based FMCA methodology. The technique will
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not be able to identify common JAK2, MPL, and CALR mutations when the mutant allele
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burden is lower than 10% mutant allele burden or when there are other kinds of mutations present. Thus, in cases that appear to be negative for common triple mutations, it is
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necessary to consider other method with higher sensitivity or confirmatory test for mutation detection. Despite of these limitations, this screening test is efficient to apply in clinically
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suspected MPN patients because common JAK2, MPL and CALR mutations were detected
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in majority of them.
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5. Conclusions
The rapid and simple PNA-based FMCA is a sensitive and specific technique for screening of common JAK2, MPL, and CALR mutations in BCR-ABL1-negative MPN at the time of
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initial diagnosis. With the combined merit of color multiplexing, design flexibility, and cross-
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platform compatibility achieved by the PNA probes, PNA-based FMCA will find increasing applications in clinical diagnostics, translational research, and even discovery of new genomic variants.
Acknowledgments This research was supported by a grant from the Korean Healthcare technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (HI14C3417).
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[17] D. Pietra, S. Li, A. Brisci, F. Passamonti, E. Rumi, A. Theocharides, M. Ferrari, H. Gisslinger, R. Kralovics, L. Cremonesi, Somatic mutations of JAK2 exon 12 in patients with JAK2 (V617F)-negative myeloproliferative disorders, Blood 111(3) (2008) 1686-1689.
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[18] K. Nath, J.W. Sarosy, J. Hahn, C.J. Di Como, Effects of ethidium bromide and SYBR®
AC
Green I on different polymerase chain reaction systems, J Biochem Biophys Methods 42(1) (2000) 15-29.
[19] M. Jung, J.M. Muche, A. Lukowsky, K. Jung, S.A. Loening, Dimethyl sulfoxide as additive in ready-to-use reaction mixtures for real-time polymerase chain reaction analysis with SYBR Green I dye, Anal Biochem 289(2) (2001) 292-295. [20] A. Karsai, S. Müller, S. Platz, M.-T. Hauser, Evaluation of a homemade SYBR® Green I reaction mixture for real-time PCR quantification of gene expression, Biotechniques 32(4) (2002) 790. 14
ACCEPTED MANUSCRIPT [21] S. Giglio, P.T. Monis, C.P. Saint, Demonstration of preferential binding of SYBR Green I to specific DNA fragments in real‐time multiplex PCR, Nucleic Acids Res 31(22) (2003) e136-e136. [22] G. Goel, A. Kumar, A. Puniya, W. Chen, K. Singh, Molecular beacon: a multitask probe, J Appl Microbiol 99(3) (2005) 435-442.
PT
[23] S.A. Marras, S. Tyagi, F.R. Kramer, Real-time assays with molecular beacons and other fluorescent nucleic acid hybridization probes, Clin Chim Acta 363(1) (2006) 48-60.
RI
[24] K. Huang, A.A. Martí, Recent trends in molecular beacon design and applications, Anal
SC
Bioanal Chem 402(10) (2012) 3091-3102.
[25] J.J. Ahn, Y. Kim, S.Y. Lee, J.Y. Hong, G.W. Kim, S.Y. Hwang, Fluorescence melting
NU
curve analysis using self-quenching dual-labeled peptide nucleic acid probes for simultaneously identifying multiple DNA sequences, Anal Biochem 484 (2015) 143-147.
MA
[26] J.J. Ahn, S.Y. Lee, J.Y. Hong, Y. Kim, G.W. Kim, S.Y. Hwang, Application of fluorescence melting curve analysis for dual DNA detection using single peptide nucleic acid probe,
D
Biotechnol Prog 31(3) (2015) 730-735.
PT E
[27] D. Hur, M.S. Kim, M. Song, J. Jung, H. Park, Detection of genetic variation using duallabeled peptide nucleic acid (PNA) probe-based melting point analysis, Biol Proced Online 17(1) (2015) 1.
AC
(2011) 668-676.
CE
[28] L.M. Scott, The JAK2 exon 12 mutations: a comprehensive review, Am J Hematol 86(8)
[29] R.Z. Ahmed, M. Rashid, N. Ahmed, M. Nadeem, T.S. Shamsi, Coexisting JAK2V617F and CALR Exon 9 Mutations in Myeloproliferative Neoplasms-Do They Designate a New Subtype?, Asian Pac J Cancer Prev 17(3) (2015) 923-926. [30] Y. Kim, J. Park, I. Jo, G.D. Lee, J. Kim, A. Kwon, H. Choi, W. Jang, H. Chae, K. Han, Genetic-pathologic characterization of myeloproliferative neoplasms, Exp Mol Med 48(7) (2016) e247. [31] L. Zamora, B. Xicoy, M. Cabezón, C. Fernandez, S. Marcé, P. Velez, M. Xandri, D. 15
ACCEPTED MANUSCRIPT Gallardo, F. Millá, E. Feliu, Co-existence of JAK2 V617F and CALR mutations in primary myelofibrosis, Leuk Lymphoma 56(10) (2015) 2973-2974. [32] Y. Lin, E. Liu, Q. Sun, J. Ma, Q. Li, Z. Cao, J. Wang, Y. Jia, H. Zhang, Z. Song, The Prevalence of JAK2, MPL, and CALR Mutations in Chinese Patients With BCR-ABL1– Negative Myeloproliferative Neoplasms, Am J Clin Path 144(1) (2015) 165-171.
PT
[33] R. Fu, M. Xuan, Y. Zhou, T. Sun, J. Bai, Z. Cao, L. Zhang, H. Li, D. Zhang, X. Zhang, Analysis of calreticulin mutations in Chinese patients with essential thrombocythemia: clinical
RI
implications in diagnosis, prognosis and treatment, Leukemia 28(9) (2014) 1912.
SC
[34] G. McGaffin, K. Harper, D. Stirling, L. McLintock, JAK2 V617F and CALR mutations are not mutually exclusive; findings from retrospective analysis of a small patient cohort, Br J
NU
Haematol 167(2) (2014) 276-278.
[35] A. Tefferi, T. Lasho, C. Finke, R. Knudson, R. Ketterling, C. Hanson, M. Maffioli, D.
MA
Caramazza, F. Passamonti, A. Pardanani, CALR vs JAK2 vs MPL-mutated or triple-negative myelofibrosis: clinical, cytogenetic and molecular comparisons, Leukemia 28(7) (2014) 1472-
D
1477.
PT E
[36] C. Al Assaf, F. Van Obbergh, J. Billiet, E. Lierman, T. Devos, C. Graux, A.-S. Hervent, T. Tousseyn, P. De Paepe, P. Papadopolous, Analysis of phenotype and outcome in Essential Thrombocythemia with CALR and JAK2 mutations, Haematologica, Il Pensiero Scientifico,
CE
2015, pp. 535-535.
AC
[37] J.-S. Ha, Y.-K. Kim, Calreticulin exon 9 mutations in myeloproliferative neoplasms, Ann Lab Med 35(1) (2015) 22-27. [38] N. Xu, L. Ding, C. Yin, X. Zhou, L. Li, Y. Li, Q. Lu, X.-l. Liu, A report on the co-occurrence of JAK2V617F and CALR mutations in myeloproliferative neoplasm patients, Ann Hematol 94(5) (2015) 865. [39] K. Lim, Y. Chang, C.G.-S. Chen, H. Lin, W. Wang, Y. Chiang, H. Cheng, N. Su, J. Lin, Y. Chang,
Frequent
CALR
exon
9
alterations
in
JAK2
V617F-mutated
essential
thrombocythemia detected by high-resolution melting analysis, Blood Cancer J 5(3) (2015) 16
ACCEPTED MANUSCRIPT e295.
Figure legends Fig. 1. Melting curve analysis of common JAK2, MPL, and CALR mutations with varied
PT
percentages (100%, 50%, 25%, 10%, 7.25%, and 0%) of each mutant allele relative to the wild type templates. Both mutant and wild type templates were from cloned artificial
RI
sequences. (A) JAK2 V617F-1 (B) JAK2 V617F-2 (C) MPL W515L (D) MPL W515K (E)
SC
CALR type 1 (p.Leu367fs*46) and (F) CALR type 2 (p.Lys385fs*47).
NU
Fig. 2. Evaluation of the possibility of interference caused by a mixture of diverse mutations occurring in the different genes in PNA-based FMCA. The identified mutations are indicated
MA
by black solid arrows in each melting curve plot.
(A) The JAK2 V617F-1 mutation was analyzed alone (first left). Then, MPL W515L, CALR
D
type 1 (p.Leu367fs*46), and CALR type 2 (p.Lys385fs*47) mutations were admixed with
PT E
JAK2 V617F-1 and analyzed in order from second left, respectively in a single tube with probe set A.
(B) The MPL W515L mutation was analyzed alone (first left). Then, JAK2 V617F-1, CALR
CE
type 1, and CALR type 2 mutations were admixed with MPL W515L and analyzed in order
AC
from second left, respectively, in a single tube with probe set B. (C) The CALR type 1 mutation was analyzed alone (first left). Then, JAK2 V617F-1 and MPL W515L mutations were admixed with CALR type 1 and analyzed in order from second left, respectively, in a single tube with probe set A. (D) The CALR type 2 mutation was analyzed alone (first left). Then, JAK2 V617F-1 and MPL W515L mutations were admixed with CALR type 2 and analyzed in order from second left, respectively, in a single tube with probe set B.
17
ACCEPTED MANUSCRIPT Fig. 3. The technically outstanding results of PNA-based FMCA compared to allele-specific real-time PCR or fragment analysis in two BCR-ABL1-negative MPNs with MPN W515S or other CALR exon 9 mutation. (A) The MPL W515L/K mutation, representing MPL W515S in sample 7. Abnormal melting curve (Tm = 53°C) identified by PNA-based FMCA with probe set B (upper left). False
PT
negative result of MPL W515L/K owing to a lower concentration of the mutant allele than the limit of detection of 10%. Very weakly positive result (Ct 39.5) for MPL W515L/K from allele-
RI
specific real-time PCR (upper right). Electropherogram from Sanger DNA sequencing,
SC
showing c.1544G>C (p.Trp515Ser) in the MPL gene (lower).
(B) A different CALR mutation, annotated as c.1105_1156del52 (p.Glu369Argfs*44),
NU
identified in sample 302. No CALR type 1 (p.Leu367fs*46) mutation was detected by PNAbased FMCA with probe set A (upper). False positive result of CALR type 1 mutation owing
MA
to problems with discrimination resulting from the same size difference (Δ 52bp) between the mutant alleles and the wild type allele by fragment analysis (middle). Electropherogram from
AC
CE
PT E
D
Sanger DNA sequencing, showing another exon 9 mutation in CALR (lower).
18
ACCEPTED MANUSCRIPT Figure 1 A
B
100%
100%
50% 25% 10% 7.25%
50% 25% 10%
PT
0% 7.25%
D
NU
C
MA
100%
PT E
50% 25% 10% 7.25%
F
CE
E
100%
0%
D
50%
25% 10% 7.25% 0%
SC
RI
0%
100%
AC
100%
50%
50%
25% 10%
25% 10%
7.25%
7.25%
0%
0%
19
ACCEPTED MANUSCRIPT
AC
CE
PT E
D
MA
NU
SC
RI
PT
Figure 2
20
ACCEPTED MANUSCRIPT Figure 3
NU
SC
RI
PT
A
AC
CE
PT E
D
MA
B
21
ACCEPTED MANUSCRIPT
RI
PT
CALR Type1 mutation in Sample 305
AC
CE
PT E
D
MA
NU
SC
Other CALR mutation in Sample 302
22
ACCEPTED MANUSCRIPT Table 1. Design and utilization of PNA-based FMCA probes for the detection of common JAK2, MPL, and CALR mutations in 57 BCR-ABL1-negative MPNs Set
Target mutation
Target sequence
Tm (℃)
5'
to
3'
Probe
sequence
(fluorescence)
A
JAK2 V617F-1
c.1849_1851TTC
46 ± 3
Set
JAK2 V617F-2
c.1849_1851TTT
30 ± 3
c.1849_1851GTC
60 ± 3
Dabcyl-GTATGTGTCTGTGG-O-K(Cy5)
(WT) 50 ± 3
Dabcyl-AAGCCTCTGCTCCTC-O-
WT-1
62 ± 3
K(HEX)
Dabcyl-CTGAGGTGGCAGT-O-K(FAM)
MPL W515L
c.1543_1545TTG
58 ± 3
Set
MPL W515K
c.1543_1545AAG
44 ± 3
c.1543_1545TGG
70 ± 3
SC
B
(WT) WT-2
55 ± 3
Dabcyl-CAATTGTCCTCTGCC-O-
c.1154_1155insTTGTC
63 ± 3
K(TexR)
NU
CALR Type2
PT
c.1092_1143del52
RI
CALR Type1
AC
CE
PT E
D
MA
WT, wild type; Tm, melting temperature; CALR type 1, p.Leu367fs*46; CALR type 2, p.Lys385fs*47
23
ACCEPTED MANUSCRIPT
Table 2. Comparison between allele-specific real-time PCR confirmed by Sanger sequencing and PNA-based FMCA in 27 JAK2 mutations Sam ple
Se
WBC 9
Hb
PLT 9
x
(×10
(g/d
(×10
/A
/L)
L)
/L)
Karyoty
Diagn
Allele-
ping
osis
specific
based
real-
FMCA
ge
Sanger sequencing
PNA-
time PCR
16.7 2
19. 3
333
27.4 6
17. 5
236
7.06
19. 2
181
10.9 4
20. 5
246
18.2 4
22. 1
829
43.8 6
19. 9
211
9.63
21
320
M/ 66
323
M/ 78
350
M/ 66
393
M/ 55
610
M/ 54
714
F/4 7
747
F/7 6 F/6
11.43
0 2001
M/
7.65
50 F/7 8 Bu23
M/ 66
In03
M/
F/6 8
Ue13
F/7
17.9 1
F/3
535
123
18. 2
315
10.3 3
19
617
9.84
18. 2
188
21.2 2
17. 2
287
11.07
18. 9
289
21. 4
283
6 Yo12
na
PV
na
PV
46,XY
46,XY
PV
PV
47,XX,+ 8 46,XX
46,XX
na
PV
PV
PV
PV
8
51 In07
18.
AC
Bu17
20. 8
PV
15.6 1
c.1849G>T;
p.Val61
7Phe
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
7Phe
CE
835
46,XY
p.Val61
46,XX
46,XY
na
46,XX
46,XX
46,XX
p.Val61
PV
PV
PV
PV
PV
PV 24
p.Val617Phe
7Phe
c.1849G>T;
p.Val61
SC
225
PV
7Phe
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
7Phe
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
7Phe
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
7Phe
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
7Phe
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
7Phe
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
7Phe
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
7Phe
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
7Phe
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
7Phe
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
7Phe
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
7Phe
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
7Phe
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
NU
46
46,XY
PT
229
RI
18. 9
MA
6.9
D
M/
PT E
38
ACCEPTED MANUSCRIPT
M/
11.52
17. 3
291
20.8 3
15. 7
302
14.3
20. 8
337
16.1 6
16. 5
594
5.61
18. 8
63
69 Yo14
F/7 4
Yo15
F/8 8
Yo28
F/6 9
72
F/6 6
na
PV
na
PV
na
PV
46,XX
na
PV
PV
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
7Phe
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
7Phe
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
7Phe
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
7Phe
p.Val617Phe
7Phe
Not
c.1614_1616delinsAT
Not
Detecte
T;
Detecte
d
PT
Yo13
7Phe
RI
0
p.His538_Lys539delin
d
139
F/6
6.17
4
453
M/
8.2
499
F/4
8.14
6
F/7 6
In04
F/7 1
13.6 3
13. 7
15. 9
AC
776
18. 3
281
136
na
PV
na
na
PV
PV
D
68
16. 5
290
7.29
55
17.
112
204
na
Not
c.1609_1622delinsGT
Not
Detecte
AATCAA;
Detecte
d
p.Phe537_Arg541deli
d
NU
58
19. 6
MA
8.92
PT E
M/
CE
122
SC
sGlnLeu
nsValIleLys
Not
c.1627_1632delGAA
Not
Detecte
GAT;
Detecte
d
p.Glu543_Asp544del
d
Not
c.1613_1622delinsTG
Not
Detecte
ATCAT;
Detecte
d
p.His538_Arg541delin
d
sLeuIleIle PMF
Not
c.1620_1627delinsGA
Not
Detecte
;
Detecte
d
p.Ile540_Glu543delin
d
sMetLys na
na
PV
PV
7
Not
c.1624_1629delAATG
Not
Detecte
AA;
Detecte
d
p.Asn542_Glu543del
d
Not
c.1622_1627delGAAA
Not
Detecte
TG;
Detecte
d
p.Arg541_Glu543deli
d
nsLys WBC, white blood cell; Hb, hemoglobin; PLT, platelet; PV, polycythemia vera; PMF, primary myelofibrosis; na, not available
25
ACCEPTED MANUSCRIPT Table 3. Comparison between allele-specific real-time PCR confirmed by Sanger sequencing and PNA-based FMCA in six MPL mutations
ple
Se
WBC 9
Hb
PLT 9
x
(×10
(g/d
(×10
/A
/L)
L)
/L)
Karyoty
Diagno
Allele-
Sanger
PNA-
ping
sis
specific
sequencing
based
real-time
ge 7*
M/
FMCA
PCR 3.8
7
293
Na
PMF
64
p.Trp515Le
c.1544G>T;
Not
u/Lys
p.Trp515Leu
Detected
(low
(with
burden)
mutant-
enriched PCR)
Abnorm
c.1544G>C;
al curve
RI
Not
PT
Sam
targeted
p.Trp515Ser
at 53°C
359*
F/7
2.23
7.9
1256
46,XX
ET
836
M/
8.24
13.1
802
46,XY
1020
M/
10.99
7.6
343
46,XY
3
Yo38
F/6
7.81
9
5.4
11.3
166
na
PT E
*
4.39
678
CE
F/5
D
81
1029
ET
MA
69
46,XX
p.Trp515Le
c.1544G>T;
Not
u/Lys
p.Trp515Leu
Detected
NU
1
SC
(high burden)
PMF
(with
mutant-
enriched PCR)
p.Trp515Le
c.1544G>T;
p.Trp515
u/Lys
p.Trp515Leu
Leu
p.Trp515Le
c.1543_1544del
p.Trp515
u/Lys
insAA;
Lys
p.Trp515Lys PMF
p.Trp515Le
c.1544G>T;
Not
u/Lys
p.Trp515Leu
Detected
(with
mutant-
enriched PCR) ET
p.Trp515Le
c.1544G>T;
p.Trp515
u/Lys
p.Trp515Leu
Leu
WBC, white blood cell; Hb, hemoglobin; PLT, platelet; PMF, primary myelofibrosis; ET, essential
AC
thrombocythemia; na, not available * Discrepant results
26
ACCEPTED MANUSCRIPT
Table 4. Comparison between fragment analysis confirmed by Sanger sequencing and PNAbased FMCA in 24 CALR mutations
ple
Se
WBC
Hb
9
PLT
Karyotyping 9
x
(×10
(g/d
(×10
/A
/L)
L)
/L)
Diagno
Fragm
Sanger
PNA-
sis
ent
sequencing
base
ge
305
M/
9.12
15.1
1739
46,XY
ET
d
is
FMC
c.1092_1143del52;
Type
mutati
p.Leu367fs*46
1
M/
9.47
14.1
836
46,XY
ET
494
F/6
7.77
9.2
781
na
13.7
13.6
1222
M/
10.41
61
F/6 7
4.84
10.2
AC
792
13
892
CE
683
ET
PT E
2
46,XX
D
F/7
ET
MA
5
573
NU
38
SC
on
483
899
A
Type 1
RI
67
analys
PT
Sam
(Type 1)
mutati on
Type 1
c.1092_1143del52;
Type
mutati
p.Leu367fs*46
1
on
(Type 1)
mutati on
Type 1
c.1092_1143del52;
Type
mutati
p.Leu367fs*46
1
on
(Type 1)
mutati on
Type 1
c.1092_1143del52;
Type
mutati
p.Leu367fs*46
1
on
(Type 1)
mutati on
46,XY
ET
Type 1
c.1092_1143del52;
Type
mutati
p.Leu367fs*46
1
on
(Type 1)
mutati on
46,XX
ET
Type 1
c.1092_1143del52;
Type
mutati
p.Leu367fs*46
1
on
(Type 1)
mutati on
1072
M/
5.81
12.5
146
46,XY
PMF
60
Type 1
c.1092_1143del52;
Type
mutati
p.Leu367fs*46
1
on
(Type 1)
mutati on
1091
F/2 0
11.94
10.8
897
46,XX,add(5)(q1 5), der(10)t(5;10)(q 33;p13)
27
ET
Type 1
c.1092_1143del52;
Type
mutati
p.Leu367fs*46
1
ACCEPTED MANUSCRIPT on
(Type 1)
mutati on
Yo54
F/3
14.95
12.1
1172
46,XX
ET
0
Type 1
c.1092_1143del52;
Type
mutati
p.Leu367fs*46
1
on
(Type 1)
mutati on
F/7
9.19
11.1
1501
46,XX
ET
0
F/5
7.11
12.6
772
46,XX
ET
7.24
13.1
1227
46,XX
6.82
13.4
1071
na
M/
13.08
11
68
M/
25.29
F/3 8
AC
72
777
7.9
99
CE
753
1529
6.55
11.8
na
ET
PT E
594
p.Leu367fs*46
1
on
(Type 1)
mutati
c.1154_1155insTT
1384
on Type2
mutati
GTC;
mutati
on
p.Lys385fs*47
on
(Type 2)
Type 2
c.1154_1155insTT
Type2
mutati
GTC;
mutati
on
p.Lys385fs*47
on
(Type 2) Type 2
c.1154_1155insTT
Type2
mutati
GTC;
mutati
on
p.Lys385fs*47
on
(Type 2)
D
3
ET
MA
F/4
ET
NU
9
542
mutati
SC
F/5
Type
Type 2
6
540
c.1092_1143del52;
RI
172
Type 1
PT
Yo56
Type 2
c.1154_1155insTT
Type2
mutati
GTC;
mutati
on
p.Lys385fs*47
on
(Type 2)
na
PMF
Type 2
c.1154_1155insTT
Type2
mutati
GTC;
mutati
on
p.Lys385fs*47
on
(Type 2) 46,XX
ET
Type 2
c.1154_1155insTT
Type2
mutati
GTC;
mutati
on
p.Lys385fs*47
on
(Type 2) 1102
F/4
7.06
13
838
46,XX
ET
7
Type 2
c.1154_1155insTT
Type2
mutati
GTC;
mutati
on
p.Lys385fs*47
on
(Type 2)
28
ACCEPTED MANUSCRIPT Yo57
F/5
8.94
13.6
1540
46,XX
ET
8
Type 2
c.1154_1155insTT
Type2
mutati
GTC;
mutati
on
p.Lys385fs*47
on
(Type 2) 513*
M/
8.99
13.4
1108
na
ET
79
Other
c.1092_1138del47;
Type1
exon 9
p.Gln365Glyfs*8
mutati
mutati
on
on M/
*
74
15.36
14.2
1441
46,XY
ET
Other
exon 9
Type1
p.Leu367Argfs*52
mutati
RI
mutati
c.1099_1132del34;
PT
Bu37
on
on M/
8.43
10.1
49
46,XY
PMF
56
Type1
SC
302*
mutati
c.1105_1156del52;
Not
p.Glu369Argfs*44
Detec
on
M/
12.54
12.6
1294
46,XY
517
F/6
7.6
8.9
819
na
ET
D
8
MA
41
ET
NU
351
F/5
5.66
12.5
na
ET
CE
2
762
Other
c.1120_1143delins
Not
exon 9
TGCGT;
Detec
mutati
p.Lys374Cysfs*50
ted
Other
c.1123_1125delins
Not
exon 9
TGTTT;
Detec
mutati
p.Lys375Cysfs*56
ted
Other
c.1131_1151delins
Not
exon 9
GTGCCTCCTCCT
Detec
mutati
GGAGG;
ted
on
p.Glu378Cysfs*51
on
on
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797
ted
WBC, white blood cell; Hb, hemoglobin; PLT, platelet; PMF, primary myelofibrosis; ET, essential thrombocythemia; na, not available
AC
* Discrepant results
29
ACCEPTED MANUSCRIPT Highlights
•
Detection of JAK2, MPL, and CALR mutations is essential in BCR-ABL1-negative MPN. Peptide nucleic acid-based fluorescence melting curve analysis is develped.
•
The limit of detection (LOD) was approximately 10% for each mutation.
•
Interference reactions using mixtures of different mutations were not observed.
•
The rapid and simple PNA-based FMCA is a sensitive and specific technique.
AC
CE
PT E
D
MA
NU
SC
RI
PT
•
30
Joonhong Park, Minsik Song, Woori Jang, Hyojin Chae, Gun Dong Lee, KyungTak Kim, Heekyung Park, Myungshin Kim, Yonggoo Kim PII: DOI: Reference:
S0009-8981(16)30496-X doi: 10.1016/j.cca.2016.12.002 CCA 14588
To appear in:
Clinica Chimica Acta
Received date: Revised date: Accepted date:
10 November 2016 30 November 2016 2 December 2016
Please cite this article as: Joonhong Park, Minsik Song, Woori Jang, Hyojin Chae, Gun Dong Lee, KyungTak Kim, Heekyung Park, Myungshin Kim, Yonggoo Kim , Peptide nucleic acid probe-based fluorescence melting curve analysis for rapid screening of common JAK2, MPL, and CALR mutations. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Cca(2016), doi: 10.1016/ j.cca.2016.12.002
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Peptide nucleic acid probe-based fluorescence melting curve analysis for rapid screening of common JAK2, MPL, and CALR mutations Running title: PNA-based FMCA for MPN mutation screening
Joonhong Park, M.D.a,b, Minsik Songc, Woori Jang, M.D.a,b, Hyojin Chae, M.D.a,b, Gun Dong
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Leeb, KyungTak Kimc, Heekyung Parkc, Myungshin Kim, M.D.a,b*, Yonggoo Kim, M.D.a,b*
a
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Department of Laboratory Medicine, College of Medicine, The Catholic University of Korea,
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Seoul, Republic of Korea b
Catholic Genetic Laboratory Center, Seoul St. Mary’s Hospital, College of Medicine, The
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Catholic University of Korea, Seoul, Republic of Korea c
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SeaSun Biomaterials, Daejeon, Republic of Korea
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*Corresponding author:
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Joonhong Park and Minsik Song contributed equally as co-first authors.
Myungshin Kim, M.D., PhD., 222 Banpo-daero, Seocho-gu, Department of Laboratory Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea,
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Seoul 06591, Republic of Korea.
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Tel: +82-2-2258-1645, Fax: +82-2-2258-1719, Email: [email protected]
Yonggoo Kim, M.D., PhD., 222 Banpo-daero, Seocho-gu, Department of Laboratory Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul 06591, Republic of Korea. Tel: +82-2-2258-1642, Fax: +82-2-2258-1719, Email: [email protected]
1
ACCEPTED MANUSCRIPT Abstract Background: We developed and evaluated the feasibility of peptide nucleic acid (PNA)based fluorescence melting curve analysis (FMCA) to detect common mutations in myeloproliferative neoplasms (MPNs). Methods: We have set up two separate reactions of PNA-based FMCA: JAK2 V617F &
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CALR p.Leu367fs*46 (set A) and MPL W515L/K & CALR p.Lys385fs*47 (set B). Clinical usefulness was validated with allele-specific real-time PCR, fragment analysis, Sanger
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sequencing in 57 BCR-ABL1-negative MPNs.
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Results: The limit of detection (LOD) of PNA-based FMCA was approximately 10% for each mutation and interference reactions using mixtures of different mutations were not observed.
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Non-specific amplification was not observed in normal control. PNA-based FMCA was able to detect all JAK2 V617F (n=20), CALR p.Leu367fs*46 (n=10) and p.Lys385fs*47 (n=8).
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Three of six MPL mutations were detected except three samples with low mutant concentration in out of LOD. JAK2 exon 12 mutations (n=7) were negative without
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influencing V617F results. Among six variant CALR exon 9 mutations, two were detected by
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this method owing to invading of probe binding site. Conclusions: PNA-based FMCA for detecting common JAK2, MPL, and CALR mutations is a rapid, simple, and sensitive technique in BCR-ABL1-negative MPNs with > 10% mutant
AC
CE
allele at the time of initial diagnosis.
Keywords: Peptide nucleic acid; FMCA; mutation screening; JAK2; MPL; CALR
2
ACCEPTED MANUSCRIPT 1. Introduction Myeloproliferative neoplasms (MPNs) are a heterogeneous group of clonal hematopoietic stem cell myeloid neoplasms with the potential to progress to acute leukemia. World Health Organization (WHO)-defined MPN is classified into eight subcategories, including three that are operationally grouped as “BCR-ABL1-negative MPNs”: polycythemia vera (PV), essential
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thrombocythemia (ET), and primary myelofibrosis (PMF). The WHO classification system uses the presence of JAK2 (Janus kinase 2) V617F or exon 12 mutations and MPL (MPL
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proto-oncogene, thrombopoietin receptor) mutations as pathognomonic clues for a diagnosis
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of BCR-ABL1-negative MPNs. JAK2 V617F is detected in approximately 97% of patients with PV and 60% of patients with ET or PMF. Additionally, exon 12 mutations in JAK2 were
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discovered in most patients with JAK2 V617F-negative PV. MPL mutations were found in 10–20% of patients with ET or PMF who were negative for JAK2 V617F [1]. The
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identification of JAK2/MPL mutations has been essential for the diagnosis and classification of BCR-ABL1-negative MPNs; however, approximately 30% of patients with ET or PMF
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lacked one of the two clonal markers, that is, they were negative for both JAK2 and MPL
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mutations. Recently, novel mutations in calreticulin (CALR) in patients with ET or PMF were described as cell-derived, mostly heterozygous, and mutually exclusive of JAK2 and MPL mutations [2, 3]. The discovery of CALR mutations represents another milestone in the
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ongoing effort to decipher the molecular pathogenesis of MPN and also provides an
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additional molecular marker for the purposes of both diagnosis and prognosis [4]. Therefore, the discovery of this subgroup of somatic CALR mutations that are unrelated to JAK/MPL mutations may provide a new tool for the molecular diagnosis of BCR-ABL1-negative MPNs and may have implications for clinical management of patients [5-8]. Therefore, there is a need to develop more generally applicable, sensitive assays to identify common JAK2, MPL, and CALR mutations for use in diagnostic screening tests in BCRABL1-negative MPNs at the same time. Among various molecular techniques, fluorescence melting curve analysis (FMCA) allows for detection of multiple targeted DNA sequences with 3
ACCEPTED MANUSCRIPT different melting temperatures (Tm) values in individual fluorescent channels [9]. In addition, artificially hybridized polymer, peptide nucleic acid (PNA) can easily hybridize with DNA and has strong binding affinity because it lacks a negatively charged phosphate group in its backbone [10, 11]. PNA probes are able to be conveniently applied dual-labeled selfquenching probe-based FMCA to screen various mutations including single nucleotide
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variant, insertion and deletion rapidly [27]. The term ‘‘dual-labeled’’ means that the probe has
refers to the single-strand base stacking of PNA [12].
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a fluorophore at the 5′ end and a quencher at the 3′ end, and the term ‘‘self-quenching’’
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In this study, we developed and evaluated the feasibility of PNA-based FMCA to detect
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common JAK2, MPL, and CALR mutations in MPNs.
2.1. Oligonucleotides and PNA probes
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2. Materials and methods
Primers, synthetic target template, and PNA probes were designed with Primer3 software
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(http://bioinfo.ut.ee/primer3/), PNA Probe Designer (Applied Biosystems; Thermo Fisher
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Scientific, Inc., Waltham, MA). All PNA probes (FAM-, HEX-, Texas Red-, or Cy5-labeled) were synthesized and HPLC-purified (Panagene Inc., Daejeon, Republic of Korea). Target oligonucleotides were synthesized and PAGE-purified (Cosmo Genetech Co., Ltd, Seoul,
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Republic of Korea). The purity of all probes was confirmed by mass spectrometry. In PNA-
AC
based FMCA, unwanted secondary structural characteristics of the probe should be avoided to allow for better hybridization with its target. The mutation sites are generally located in the centre of the probe so as to obtain a Tm shift around ± 3°C as estimated by the software. For detection of deletion and insertion, end position deletion/insertion and center position deletion/insertion were used to optimize Tm shift. All PNA probes used to identify the six mutation targets were designed to match with the wild type sequences, except the probe used for the detection of the CALR type 2 mutation, in which the probe for nucleotide sequence c.1154_1155insTTGTC matched with the insertion-induced frameshift mutation of 4
ACCEPTED MANUSCRIPT CALR exon 9 instead of the wild type version of CALR.
2.2. Simulation analysis of denaturation of hybrids formed between PNA probes and synthetic target template using PNA-based FMCA To analyze the Tm of PNA probe, synthetic single-stranded DNA oligomers were directly
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used as the target DNA. Thermal denaturation of hybrids formed between PNA probes and synthetic target template was carried out with the CFX96TM Real-time PCR Detection
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System (Bio-Rad Laboratories Inc., Hercules, CA). A thermal cyclic reaction was performed
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using the following conditions: the 20 µL reaction mixtures containing 0.5 µL PNA probe, 1.0 µL synthetic DNA template (0.5 µL wild type and 0.5 µL mutant type), and 1X SSB Real-Time
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FMCA™ buffer (SeaSun Biomaterials). Melting curve analysis began with a denaturation step for 3 min at 95°C, a hybridization step consisting of a temperature decrease from 75 to
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45°C at 10°C per step with a 30 sec pause between each step and lastly, a stepwise temperature increase from 20°C to 80°C at 1°C per step with a 5 sec pause between each
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step. Fluorescence was recorded at each step in the corresponding detection channel. Initial
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and post-melt fluorescence signals of all samples were normalized to relative values of 1.0 and 0. Shifting the normalized melting curves along the temperature axis allows for creation of a plot analyzing the differences in melting curve shape that is superimposed at a specific
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fluorescence intensity to enhance visualization of heteroduplex and homoduplex synthetic
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target template. The data obtained were plotted as fluorescence versus temperature as well as the negative derivative of fluorescence over temperature versus temperature. Tm values were identified by the peak position of the latter curve. Synthetic single-stranded DNA oligomers were used as target DNA for probe validation, and each target DNA contained mismatch(es), deletion(s), or insertion(s) (Table S1).
2.3. Clinical sample preparation A total of 57 clinical samples were tested: 26 PV with JAK2 V617F, 24 ET with three MPL 5
ACCEPTED MANUSCRIPT W515L/K or 21 CALR exon 9 mutations, and 7 PMF with one JAK2 V617F, three MPL W515L/K, or three CALR exon 9 mutations. Genomic DNA was prepared from bone marrow aspirates using the QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Clinical diagnosis of PV, ET, or PMF was conducted in accordance with the 2008 WHO classifications [13]. All patients in this study provided written
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informed consent, and the study protocol was approved by the Institutional Review Board of
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The Catholic University of Korea.
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2.4. Screening of common JAK2, MPL, and CALR mutations by PNA-based FMCA Two separate reaction tubes of set A and B were used to identify common JAK2, MPL, and
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CALR mutations in 57 BCR-ABL1-negative MPNs (Table 1). Although extremely rare, a double mutation within codon 617 of JAK2 can also result in the V617F mutation, that is
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1849G>T combined with 1851C>T (annonated as c.1849_1851GTC>TTT), which also encodes phenylalanine. The occurrence of this double mutation has been reported to
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produce a false negative result in an assay, which specifically targets the 1849G>T mutation
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[14]. Thus this double mutation was included as target for detecting JAK2 V617F. An asymmetric PCR [15] was carried out to generate single-stranded DNA target according to the following conditions: each 20 µL reaction contained 10 µL 2x qPCR PreMix (SeaSun
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Biomaterials), a primer/probe mix containing all three primer pairs (forward 0.05 µM /
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revserse 0.5 µM), two differently labeled PNA probes (0.5 µL each), and 1.0 µL of clinical DNA template. The primer sets are described in Table S2. PNA-based FMCA consisted of a 10 min denaturation step at 95°C, 41 cycles of 95°C for 30 sec, 58°C for 30 sec, and 78°C for 45 sec, followed by a denaturation step for 3 min at 95°C, a hybridization step of a temperature decrease from 75 to 45°C at 10°C per step with a 30 sec pause between each step, and a stepwise temperature increase from 20°C to 80°C at 1°C intervals with a 5 sec stop between each step. The results were analyzed as fluorescence versus temperature graphs by Bio-Rad CFX Manager (Bio-Rad Laboratories Inc.) with normalized, temperature6
ACCEPTED MANUSCRIPT shifted melting curves displayed as a difference plot.
2.5. Molecular analysis for confirmation of common JAK2, MPL, and CALR mutations The presence of common and other mutations of the JAK2, MPL, and CALR in 57 BCRABL1-negative MPNs was validated by different molecular methods following as: an allele-
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specific real-time PCR assay (Real-Q JAK2V617F Kit, BioSewoom, Seoul, Republic of Korea) was used to detect the JAK2 V617F mutation using the amplification refractory mutation
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system principle. To identify the JAK2 exon 12 and 14 mutation, a multiplex fragment
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analysis-based assay was used [16] and Sanger sequencing was conducted to confirm the code in the region containing the entirety of exon 12, as previously described [17]. The MPL
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W515L/K mutation was analyzed using allele-specific real-time PCR (Real-Q MPLW515L/K Screening Kit, BioSewoom). All PCR reactions were run in duplicate. Single-positive PCR
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reactions (CT 35–40) were repeated in triplicate and the samples were classified as mutation-positive only if all replicates were positive. All negative cases for MPL W515L/K
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were directly sequenced with mutant-enriched PCR to identify other MPL mutations not
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covered by PNA-based FMCA. To screen for CALR exon 9 mutations, we first performed PCR fragment analysis using PCR primers spanning exon 9 and a forward primer labeled with 6-FAM, as described previously [2]. All CALR exon9 mutations identified were confirmed
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bi-directionally by Sanger sequencing. RefSeq ID: NM_001322194.1, NM_005373.2, and
3. Results
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NM_004343.3 for JAK2, MPL, and CALR were used for cDNA nucleotide numbering.
3.1. Analytical performance of PNA-based FMCA An analytical limit of detection study was done in triplicate to show that this assay can detect serially diluted plasmids (100%, 50%, 25%, 10%, 7.25%, and 0% of the synthetic target template relative to the wild type template per reaction) with each of the two reaction sets in PNA-based FMCA. When mutant and wild type templates were mixed, mutant templates 7
ACCEPTED MANUSCRIPT were detectable when present in the range of 10 to 100% (Fig. 1). Analytical accuracy testing was performed three times to ensure that this method can precisely identify common JAK2, MPL, and CALR mutations. The assay was used to analyze genomic DNA from 20 JAK2 V617F, three MPL W/515L/K, 10 CALR p.Leu367fs*46 (type 1), and eight CALR p.Lys385fs*47 (type 2) that were confirmed by triplicate Sanger
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sequencing of the same PCR amplicons. The concordance rate between PNA-based FMCA and Sanger sequencing results was 100%. Analytical specificity testing was done in triplicate
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to show that this method can identify wild type JAK2 V617, MPL W515 and CALR exon 9
melting curves detected by PNA-based FMCA.
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residues in 100 samples from normal, healthy individuals. The results showed no abnormal
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The possibility of interference in reactions with a mixture of different mutations occurring in the same gene or with a mixture of diverse mutations occurring in different genes was
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evaluated in PNA-based FMCA (Table S3). As a result, only a single mutation in each of the three genes was identified in its corresponding fluorescent channel, regardless of whether a
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mixture of mutations occurred indifferent genes in same tube, when using probe set A or B
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(Fig. 2). These results demonstrate that PNA-based FMCA with color multiplexing can be used to identify the exact mutation type with combined PNA probes in each of the two
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reaction sets, A and B, simultaneously.
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3.2. Detection of common JAK2, MPL, and CALR mutations by PNA-based FMCA PNA-based FMCA with two separate reactions was used to screen for common JAK2, MPL, and CALR mutations in 57 BCR-ABL1-negative MPNs. The sensitivity and specificity of PNA-based FMCA are 93% (41/44) and 86% (12/14), respectively. The presence of MPL W515S was considered to estimate the specificity of PNA-based FMCA. Three false negative MPL W515L and two false positive CALR type 1 mutations were occurred. In detail, we were able to detect all JAK2 V617F positive specimens (n=20) by PNA-based FMCA. All patients with JAK2 exon 12 mutations (n=7) were negative without influencing 8
ACCEPTED MANUSCRIPT V617F results (Table 2). Second, two MPL W515L and one MPL W515K mutations were detected in six patients with MPL W515L/K (Table 3). In the three samples, two false negative finding for MPL W515L/K occurred owing to a lower concentration of the mutant allele than the limit of detection of 10%. On the other hand, an abnormal melting curve (Tm = 53°C) was observed in sample 7, which harbored a very low concentration of W515L, as
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shown by allele-specific real-time PCR which specifically targets MPL W515L/K. To resolve this issue, Sanger sequencing was conducted, which confirmed that, other than the MPL
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W515L/K mutation, c.1544G>C, producing p.Trp515Ser, was present (Fig. 3A). Third, PNA-
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based FCMA detected true common CALR mutations consisting of type 1 (n=10) and type 2 (n=8) mutations. Two false positive CALR type 1 mutation turned out to have other CALR 9
mutations
invading
parts
of
the
probe
binding
site
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exon
(c.1087_c.1101GAGGAGCAGAGGCTT); CALR exon 9 mutations, c.1092_1138del47
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(p.Gln365Glyfs*8) and c.1099_1132del34 (p.Leu367Argfs*52). Another CALR exon 9 mutations (n=4) were not detected without influencing PNA-based FCMA results.
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Interestingly, in sample 302, which had another CALR exon 9 mutation, c.1105_1156del52
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(p.Glu369Argfs*44), the false positive result for the CALR type 1 mutation occurred by fragment analysis because of an inability to discriminate the alleles that resulted from the same size difference (Δ 52bp) between the mutant and wild type alleles. No CALR type 1
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mutation was detected precisely by PNA-based FMCA with probe set A (Fig. 3B).
4. Discussion
The conventional FMCA uses double-strand DNA intercalating dyes such as SYBR Green or EvaGreen, but several studies demonstrates its disadvantages such as extensive optimization, concentration-dependent PCR inhibition, and effects on DNA melting temperature [18-21]. To overcome this limitations, molecular beacons and other hairpin probes with stem structures have been developed [22-24], However, the design of stem structure is more fastidious and complex than that of linear probe in a regard of its procedure 9
ACCEPTED MANUSCRIPT and conditions. Fortunately, designing PNA probes for FMCA is not much different from that of general dual-labeled DNA probes, and asymmetric PCR methods are easy to implement [25-27]. A PNA probe is naturally a dual-labeled, self-quenching probe owing uncharged nature and their peptide bond-linked backbone and the coiled conformation of PNA probe facilitates fluorescence self-quenching. Moreover, PNA probes can be designed shorter (9–
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13 bp) than DNA probes (20 bp or more) with the same T m and makes a large difference in ΔTm between base substitutions as well as insertion or deletion, which can contribute to
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discriminate mismatch from perfect match by changing dramatic melting shift [27].
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In this study, a clear difference was exhibited between the wild type and the mutant samples after normalization and temperature shifting in PNA-based FMCA. In principle, a
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two-tube assay is still not as efficient from workflow perspective as a single tube assay. However CALR type 1 and 2 mutations are close to each other, which cause interference
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response in a single-tube assay, PNA-based FMCA was divided inevitably into two separate tubes of set A for JAK2 and CALR type 1 and set B for MPL and CALR type 2. Among 57
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BCR-ABL1-negative MPNs, a total of 20 PV, 18 ET, and three PMF samples, each with one
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of the common JAK2, MPL, or CALR mutations, showed a clear and distinctive pattern on melting and difference curve plots compared with plots of wild type samples in PNA-based FMCA. Moreover, the common JAK2, MPL, or CALR mutations could be clearly
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distinguished in six different patterns using the Tm. No positives were found in 100 samples
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taken from normal, healthy individuals. Samples that were positive in PNA-based FMCA analyses were bidirectionally sequenced using the same PCR product. Interestingly, of the 20 CALR mutations with PNA-based FMCA-positive curves, 10 were type 1 deletions (c.1092_1143del52; p.Leu367Thrfs*46), eight were type 2 insertions c.1154_1155insTTGTC; p.Lys385Asnfs*47), and two were false type 1 deletions resulting from a small overlap with the probes and a deletion in c.1092_1138del47 (sample 513) and c.1099_1132del34 (sample Bu37). Type 1 deletions (52 bp in size) and Type 2 insertions (5 bp) showed clearly distinct PNA-based FMCA patterns. In addition, the PNA-based FMCA curves of two other 10
ACCEPTED MANUSCRIPT CALR exon 9 mutations mimicking type 1 or type 2 mutations, in samples 302 and 797, were definitely classified as uncommon CALR mutations by fragment analysis. None of sixteen wild type samples evidenced common JAK2, MPL, or CALR mutations, even those with a borderline pattern, except two false positive for CALR type 1 and two negative results for MPL W515L/K as determined by Sanger sequencing. Only sample 7, with an abnormal Tm at
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53°C detected by PNA probes, had an MPL W515S mutation. Although this sample showed a very low Ct value of 39.5 by allele-specific real-time PCR used to detect MPL W515L/K, its
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sequence revealed a heterozygous mutant allele burden, c.1544G>C (p.Trp515Ser).
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From a clinical standpoint, mutation screening for common JAK2, MPL, and CALR genes allows for confirmation of a morphologic diagnosis as well as an understanding of disease
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prognosis. During a diagnostic work-up, almost all patients with PV can be identified by the presence of a JAK2 mutation. Thus, JAK2 exon 12 mutations should be studied in cases of
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suspected PV related to a subnormal serum erythropoietin level and an absence of JAK2 V617F. However, JAK2 exon 12 mutations, occurring at low incidence in affected PV
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individual, were excluded from the target of this assay [28]. On the other hand, based on its
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high frequency and relative specificity in ET or PMF with wild-type JAK2/MPL, screening for CALR mutations should now be included in the diagnostic work-up of MPN and formally incorporated in the revised WHO classification system [8]. For patients suspected of having
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ET or PMF, initial mutation screening should start with an assessment of JAK2 V617F and
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then proceed with CALR mutation screening only in patients who are JAK2 V617F‑negative. Owing to the significantly lower incidence of MPL mutations compared with CALR mutations, screening for mutant CALR should be done first, before proceeding with MPL mutation studies in patients with JAK2 V617F-negative ET or PMF [4]. CALR mutations have been known to be mutually exclusive of JAK2 and MPL mutations, however incidental co-occurrence of CALR and other mutations has been reported in MPN [29]. Given the results of those studies and our previous study [30], it is noteworthy that our new multiplexing method can detect the coexistance of common triple mutations [30-32] or 11
ACCEPTED MANUSCRIPT other
CALR
mutations
[31-38]
involving
parts
of
the
probe
binding
site
(c.1087_c1101GAGGAGCAGAGGCTT) of CALR simultaneously identified by fragment analysis or Sanger sequencing. It is suggested that ET or PMF patients carrying both JAK2 V617F and a CALR mutation may show a unique clinical course and phenotype, and it is necessary to define the prognosis, risk factors, and outcomes for such MPN patients [31, 34,
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39]. There remain a few limitations in this PNA-based FMCA methodology. The technique will
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not be able to identify common JAK2, MPL, and CALR mutations when the mutant allele
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burden is lower than 10% mutant allele burden or when there are other kinds of mutations present. Thus, in cases that appear to be negative for common triple mutations, it is
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necessary to consider other method with higher sensitivity or confirmatory test for mutation detection. Despite of these limitations, this screening test is efficient to apply in clinically
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suspected MPN patients because common JAK2, MPL and CALR mutations were detected
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in majority of them.
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5. Conclusions
The rapid and simple PNA-based FMCA is a sensitive and specific technique for screening of common JAK2, MPL, and CALR mutations in BCR-ABL1-negative MPN at the time of
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initial diagnosis. With the combined merit of color multiplexing, design flexibility, and cross-
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platform compatibility achieved by the PNA probes, PNA-based FMCA will find increasing applications in clinical diagnostics, translational research, and even discovery of new genomic variants.
Acknowledgments This research was supported by a grant from the Korean Healthcare technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (HI14C3417).
12
ACCEPTED MANUSCRIPT References [1] P.J. Campbell, A.R. Green, The myeloproliferative disorders, N Engl J Med 355(23) (2006) 2452-2466. [2] T. Klampfl, H. Gisslinger, A.S. Harutyunyan, H. Nivarthi, E. Rumi, J.D. Milosevic, N.C. Them, T. Berg, B. Gisslinger, D. Pietra, Somatic mutations of calreticulin in myeloproliferative
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neoplasms, N Engl J Med 369(25) (2013) 2379-2390. [3] J. Nangalia, C.E. Massie, E.J. Baxter, F.L. Nice, G. Gundem, D.C. Wedge, E. Avezov, J.
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nonmutated JAK2, N Engl J Med 369(25) (2013) 2391-2405.
[4] A. Tefferi, A. Pardanani, Genetics: CALR mutations and a new diagnostic algorithm for
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MPN, Nat Rev Clin Oncol 11(3) (2014) 125-126.
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[7] J.J. Michiels, Z. Berneman, W. Schroyens, H. De Raeve, Changing concepts of diagnostic criteria of myeloproliferative disorders and the molecular etiology and classification of myeloproliferative neoplasms: from Dameshek 1950 to Vainchenker 2005
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and beyond, Acta haematologica 133(1) (2014) 36-51.
AC
[8] A. Tefferi, J. Thiele, A. Vannucchi, T. Barbui, An overview on CALR and CSF3R mutations and a proposal for revision of WHO diagnostic criteria for myeloproliferative neoplasms, Leukemia 28(7) (2014) 1407-1413. [9] K.S. Elenitoba-Johnson, S.D. Bohling, C.T. Wittwer, T.C. King, Multiplex PCR by multicolor fluorimetry and fluorescence melting curve analysis, Nat Med 7(2) (2001) 249-253. [10] P. E. Nielsen, O. Buchardt, M. Egholm, R. H. Berg, Peptide nucleic acids, U.S. patent 5539082, 1996. [11] R. G. Wells, Nucleic Acids: Protocols and Applications, Bioscience, Norfolk, UK, (2004). 13
ACCEPTED MANUSCRIPT [12] K.J. Livak, S.J. Flood, J. Marmaro, K.B. Mullah, Self-quenching fluorescence probe. US Patent No 5723591, 1998. [13] H. Steven, E.C. Swerdlow, N.L. Harris, E.S. Jaffe, S.A. Pileri, H. Stein, J. Thiele, J.W. Vardiman, WHO classification of tumours of haematopoietic and lymphoid tissues, IARC press: Lyon, France, (2008).
PT
[14] I. Warshawsky, F. Mularo, C. Hren, M. Jakubowski, Failure of the Ipsogen MutaScreen kit to detect the JAK2 617V> F mutation in samples with additional rare exon 14 mutations:
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implications for clinical testing and report of a novel 618C> F mutation in addition to 617V> F,
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Blood 115(15) (2010) 3175-3176.
[15] J.A. Sanchez, K.E. Pierce, J.E. Rice, L.J. Wangh, Linear-After-The-Exponential (LATE)–
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PCR: An advanced method of asymmetric PCR and its uses in quantitative real-time analysis, Proc Natl Acad Sci U S A 101(7) (2004) 1933-1938.
MA
[16] L.V. Furtado, H.C. Weigelin, K.S. Elenitoba-Johnson, B.L. Betz, A multiplexed fragment analysis-based assay for detection of JAK2 exon 12 mutations, J Mol Diagn 15(5) (2013)
D
592-599.
PT E
[17] D. Pietra, S. Li, A. Brisci, F. Passamonti, E. Rumi, A. Theocharides, M. Ferrari, H. Gisslinger, R. Kralovics, L. Cremonesi, Somatic mutations of JAK2 exon 12 in patients with JAK2 (V617F)-negative myeloproliferative disorders, Blood 111(3) (2008) 1686-1689.
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[18] K. Nath, J.W. Sarosy, J. Hahn, C.J. Di Como, Effects of ethidium bromide and SYBR®
AC
Green I on different polymerase chain reaction systems, J Biochem Biophys Methods 42(1) (2000) 15-29.
[19] M. Jung, J.M. Muche, A. Lukowsky, K. Jung, S.A. Loening, Dimethyl sulfoxide as additive in ready-to-use reaction mixtures for real-time polymerase chain reaction analysis with SYBR Green I dye, Anal Biochem 289(2) (2001) 292-295. [20] A. Karsai, S. Müller, S. Platz, M.-T. Hauser, Evaluation of a homemade SYBR® Green I reaction mixture for real-time PCR quantification of gene expression, Biotechniques 32(4) (2002) 790. 14
ACCEPTED MANUSCRIPT [21] S. Giglio, P.T. Monis, C.P. Saint, Demonstration of preferential binding of SYBR Green I to specific DNA fragments in real‐time multiplex PCR, Nucleic Acids Res 31(22) (2003) e136-e136. [22] G. Goel, A. Kumar, A. Puniya, W. Chen, K. Singh, Molecular beacon: a multitask probe, J Appl Microbiol 99(3) (2005) 435-442.
PT
[23] S.A. Marras, S. Tyagi, F.R. Kramer, Real-time assays with molecular beacons and other fluorescent nucleic acid hybridization probes, Clin Chim Acta 363(1) (2006) 48-60.
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[24] K. Huang, A.A. Martí, Recent trends in molecular beacon design and applications, Anal
SC
Bioanal Chem 402(10) (2012) 3091-3102.
[25] J.J. Ahn, Y. Kim, S.Y. Lee, J.Y. Hong, G.W. Kim, S.Y. Hwang, Fluorescence melting
NU
curve analysis using self-quenching dual-labeled peptide nucleic acid probes for simultaneously identifying multiple DNA sequences, Anal Biochem 484 (2015) 143-147.
MA
[26] J.J. Ahn, S.Y. Lee, J.Y. Hong, Y. Kim, G.W. Kim, S.Y. Hwang, Application of fluorescence melting curve analysis for dual DNA detection using single peptide nucleic acid probe,
D
Biotechnol Prog 31(3) (2015) 730-735.
PT E
[27] D. Hur, M.S. Kim, M. Song, J. Jung, H. Park, Detection of genetic variation using duallabeled peptide nucleic acid (PNA) probe-based melting point analysis, Biol Proced Online 17(1) (2015) 1.
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(2011) 668-676.
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[28] L.M. Scott, The JAK2 exon 12 mutations: a comprehensive review, Am J Hematol 86(8)
[29] R.Z. Ahmed, M. Rashid, N. Ahmed, M. Nadeem, T.S. Shamsi, Coexisting JAK2V617F and CALR Exon 9 Mutations in Myeloproliferative Neoplasms-Do They Designate a New Subtype?, Asian Pac J Cancer Prev 17(3) (2015) 923-926. [30] Y. Kim, J. Park, I. Jo, G.D. Lee, J. Kim, A. Kwon, H. Choi, W. Jang, H. Chae, K. Han, Genetic-pathologic characterization of myeloproliferative neoplasms, Exp Mol Med 48(7) (2016) e247. [31] L. Zamora, B. Xicoy, M. Cabezón, C. Fernandez, S. Marcé, P. Velez, M. Xandri, D. 15
ACCEPTED MANUSCRIPT Gallardo, F. Millá, E. Feliu, Co-existence of JAK2 V617F and CALR mutations in primary myelofibrosis, Leuk Lymphoma 56(10) (2015) 2973-2974. [32] Y. Lin, E. Liu, Q. Sun, J. Ma, Q. Li, Z. Cao, J. Wang, Y. Jia, H. Zhang, Z. Song, The Prevalence of JAK2, MPL, and CALR Mutations in Chinese Patients With BCR-ABL1– Negative Myeloproliferative Neoplasms, Am J Clin Path 144(1) (2015) 165-171.
PT
[33] R. Fu, M. Xuan, Y. Zhou, T. Sun, J. Bai, Z. Cao, L. Zhang, H. Li, D. Zhang, X. Zhang, Analysis of calreticulin mutations in Chinese patients with essential thrombocythemia: clinical
RI
implications in diagnosis, prognosis and treatment, Leukemia 28(9) (2014) 1912.
SC
[34] G. McGaffin, K. Harper, D. Stirling, L. McLintock, JAK2 V617F and CALR mutations are not mutually exclusive; findings from retrospective analysis of a small patient cohort, Br J
NU
Haematol 167(2) (2014) 276-278.
[35] A. Tefferi, T. Lasho, C. Finke, R. Knudson, R. Ketterling, C. Hanson, M. Maffioli, D.
MA
Caramazza, F. Passamonti, A. Pardanani, CALR vs JAK2 vs MPL-mutated or triple-negative myelofibrosis: clinical, cytogenetic and molecular comparisons, Leukemia 28(7) (2014) 1472-
D
1477.
PT E
[36] C. Al Assaf, F. Van Obbergh, J. Billiet, E. Lierman, T. Devos, C. Graux, A.-S. Hervent, T. Tousseyn, P. De Paepe, P. Papadopolous, Analysis of phenotype and outcome in Essential Thrombocythemia with CALR and JAK2 mutations, Haematologica, Il Pensiero Scientifico,
CE
2015, pp. 535-535.
AC
[37] J.-S. Ha, Y.-K. Kim, Calreticulin exon 9 mutations in myeloproliferative neoplasms, Ann Lab Med 35(1) (2015) 22-27. [38] N. Xu, L. Ding, C. Yin, X. Zhou, L. Li, Y. Li, Q. Lu, X.-l. Liu, A report on the co-occurrence of JAK2V617F and CALR mutations in myeloproliferative neoplasm patients, Ann Hematol 94(5) (2015) 865. [39] K. Lim, Y. Chang, C.G.-S. Chen, H. Lin, W. Wang, Y. Chiang, H. Cheng, N. Su, J. Lin, Y. Chang,
Frequent
CALR
exon
9
alterations
in
JAK2
V617F-mutated
essential
thrombocythemia detected by high-resolution melting analysis, Blood Cancer J 5(3) (2015) 16
ACCEPTED MANUSCRIPT e295.
Figure legends Fig. 1. Melting curve analysis of common JAK2, MPL, and CALR mutations with varied
PT
percentages (100%, 50%, 25%, 10%, 7.25%, and 0%) of each mutant allele relative to the wild type templates. Both mutant and wild type templates were from cloned artificial
RI
sequences. (A) JAK2 V617F-1 (B) JAK2 V617F-2 (C) MPL W515L (D) MPL W515K (E)
SC
CALR type 1 (p.Leu367fs*46) and (F) CALR type 2 (p.Lys385fs*47).
NU
Fig. 2. Evaluation of the possibility of interference caused by a mixture of diverse mutations occurring in the different genes in PNA-based FMCA. The identified mutations are indicated
MA
by black solid arrows in each melting curve plot.
(A) The JAK2 V617F-1 mutation was analyzed alone (first left). Then, MPL W515L, CALR
D
type 1 (p.Leu367fs*46), and CALR type 2 (p.Lys385fs*47) mutations were admixed with
PT E
JAK2 V617F-1 and analyzed in order from second left, respectively in a single tube with probe set A.
(B) The MPL W515L mutation was analyzed alone (first left). Then, JAK2 V617F-1, CALR
CE
type 1, and CALR type 2 mutations were admixed with MPL W515L and analyzed in order
AC
from second left, respectively, in a single tube with probe set B. (C) The CALR type 1 mutation was analyzed alone (first left). Then, JAK2 V617F-1 and MPL W515L mutations were admixed with CALR type 1 and analyzed in order from second left, respectively, in a single tube with probe set A. (D) The CALR type 2 mutation was analyzed alone (first left). Then, JAK2 V617F-1 and MPL W515L mutations were admixed with CALR type 2 and analyzed in order from second left, respectively, in a single tube with probe set B.
17
ACCEPTED MANUSCRIPT Fig. 3. The technically outstanding results of PNA-based FMCA compared to allele-specific real-time PCR or fragment analysis in two BCR-ABL1-negative MPNs with MPN W515S or other CALR exon 9 mutation. (A) The MPL W515L/K mutation, representing MPL W515S in sample 7. Abnormal melting curve (Tm = 53°C) identified by PNA-based FMCA with probe set B (upper left). False
PT
negative result of MPL W515L/K owing to a lower concentration of the mutant allele than the limit of detection of 10%. Very weakly positive result (Ct 39.5) for MPL W515L/K from allele-
RI
specific real-time PCR (upper right). Electropherogram from Sanger DNA sequencing,
SC
showing c.1544G>C (p.Trp515Ser) in the MPL gene (lower).
(B) A different CALR mutation, annotated as c.1105_1156del52 (p.Glu369Argfs*44),
NU
identified in sample 302. No CALR type 1 (p.Leu367fs*46) mutation was detected by PNAbased FMCA with probe set A (upper). False positive result of CALR type 1 mutation owing
MA
to problems with discrimination resulting from the same size difference (Δ 52bp) between the mutant alleles and the wild type allele by fragment analysis (middle). Electropherogram from
AC
CE
PT E
D
Sanger DNA sequencing, showing another exon 9 mutation in CALR (lower).
18
ACCEPTED MANUSCRIPT Figure 1 A
B
100%
100%
50% 25% 10% 7.25%
50% 25% 10%
PT
0% 7.25%
D
NU
C
MA
100%
PT E
50% 25% 10% 7.25%
F
CE
E
100%
0%
D
50%
25% 10% 7.25% 0%
SC
RI
0%
100%
AC
100%
50%
50%
25% 10%
25% 10%
7.25%
7.25%
0%
0%
19
ACCEPTED MANUSCRIPT
AC
CE
PT E
D
MA
NU
SC
RI
PT
Figure 2
20
ACCEPTED MANUSCRIPT Figure 3
NU
SC
RI
PT
A
AC
CE
PT E
D
MA
B
21
ACCEPTED MANUSCRIPT
RI
PT
CALR Type1 mutation in Sample 305
AC
CE
PT E
D
MA
NU
SC
Other CALR mutation in Sample 302
22
ACCEPTED MANUSCRIPT Table 1. Design and utilization of PNA-based FMCA probes for the detection of common JAK2, MPL, and CALR mutations in 57 BCR-ABL1-negative MPNs Set
Target mutation
Target sequence
Tm (℃)
5'
to
3'
Probe
sequence
(fluorescence)
A
JAK2 V617F-1
c.1849_1851TTC
46 ± 3
Set
JAK2 V617F-2
c.1849_1851TTT
30 ± 3
c.1849_1851GTC
60 ± 3
Dabcyl-GTATGTGTCTGTGG-O-K(Cy5)
(WT) 50 ± 3
Dabcyl-AAGCCTCTGCTCCTC-O-
WT-1
62 ± 3
K(HEX)
Dabcyl-CTGAGGTGGCAGT-O-K(FAM)
MPL W515L
c.1543_1545TTG
58 ± 3
Set
MPL W515K
c.1543_1545AAG
44 ± 3
c.1543_1545TGG
70 ± 3
SC
B
(WT) WT-2
55 ± 3
Dabcyl-CAATTGTCCTCTGCC-O-
c.1154_1155insTTGTC
63 ± 3
K(TexR)
NU
CALR Type2
PT
c.1092_1143del52
RI
CALR Type1
AC
CE
PT E
D
MA
WT, wild type; Tm, melting temperature; CALR type 1, p.Leu367fs*46; CALR type 2, p.Lys385fs*47
23
ACCEPTED MANUSCRIPT
Table 2. Comparison between allele-specific real-time PCR confirmed by Sanger sequencing and PNA-based FMCA in 27 JAK2 mutations Sam ple
Se
WBC 9
Hb
PLT 9
x
(×10
(g/d
(×10
/A
/L)
L)
/L)
Karyoty
Diagn
Allele-
ping
osis
specific
based
real-
FMCA
ge
Sanger sequencing
PNA-
time PCR
16.7 2
19. 3
333
27.4 6
17. 5
236
7.06
19. 2
181
10.9 4
20. 5
246
18.2 4
22. 1
829
43.8 6
19. 9
211
9.63
21
320
M/ 66
323
M/ 78
350
M/ 66
393
M/ 55
610
M/ 54
714
F/4 7
747
F/7 6 F/6
11.43
0 2001
M/
7.65
50 F/7 8 Bu23
M/ 66
In03
M/
F/6 8
Ue13
F/7
17.9 1
F/3
535
123
18. 2
315
10.3 3
19
617
9.84
18. 2
188
21.2 2
17. 2
287
11.07
18. 9
289
21. 4
283
6 Yo12
na
PV
na
PV
46,XY
46,XY
PV
PV
47,XX,+ 8 46,XX
46,XX
na
PV
PV
PV
PV
8
51 In07
18.
AC
Bu17
20. 8
PV
15.6 1
c.1849G>T;
p.Val61
7Phe
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
7Phe
CE
835
46,XY
p.Val61
46,XX
46,XY
na
46,XX
46,XX
46,XX
p.Val61
PV
PV
PV
PV
PV
PV 24
p.Val617Phe
7Phe
c.1849G>T;
p.Val61
SC
225
PV
7Phe
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
7Phe
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
7Phe
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
7Phe
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
7Phe
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
7Phe
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
7Phe
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
7Phe
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
7Phe
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
7Phe
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
7Phe
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
7Phe
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
7Phe
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
NU
46
46,XY
PT
229
RI
18. 9
MA
6.9
D
M/
PT E
38
ACCEPTED MANUSCRIPT
M/
11.52
17. 3
291
20.8 3
15. 7
302
14.3
20. 8
337
16.1 6
16. 5
594
5.61
18. 8
63
69 Yo14
F/7 4
Yo15
F/8 8
Yo28
F/6 9
72
F/6 6
na
PV
na
PV
na
PV
46,XX
na
PV
PV
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
7Phe
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
7Phe
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
7Phe
p.Val617Phe
7Phe
p.Val61
c.1849G>T;
p.Val61
7Phe
p.Val617Phe
7Phe
Not
c.1614_1616delinsAT
Not
Detecte
T;
Detecte
d
PT
Yo13
7Phe
RI
0
p.His538_Lys539delin
d
139
F/6
6.17
4
453
M/
8.2
499
F/4
8.14
6
F/7 6
In04
F/7 1
13.6 3
13. 7
15. 9
AC
776
18. 3
281
136
na
PV
na
na
PV
PV
D
68
16. 5
290
7.29
55
17.
112
204
na
Not
c.1609_1622delinsGT
Not
Detecte
AATCAA;
Detecte
d
p.Phe537_Arg541deli
d
NU
58
19. 6
MA
8.92
PT E
M/
CE
122
SC
sGlnLeu
nsValIleLys
Not
c.1627_1632delGAA
Not
Detecte
GAT;
Detecte
d
p.Glu543_Asp544del
d
Not
c.1613_1622delinsTG
Not
Detecte
ATCAT;
Detecte
d
p.His538_Arg541delin
d
sLeuIleIle PMF
Not
c.1620_1627delinsGA
Not
Detecte
;
Detecte
d
p.Ile540_Glu543delin
d
sMetLys na
na
PV
PV
7
Not
c.1624_1629delAATG
Not
Detecte
AA;
Detecte
d
p.Asn542_Glu543del
d
Not
c.1622_1627delGAAA
Not
Detecte
TG;
Detecte
d
p.Arg541_Glu543deli
d
nsLys WBC, white blood cell; Hb, hemoglobin; PLT, platelet; PV, polycythemia vera; PMF, primary myelofibrosis; na, not available
25
ACCEPTED MANUSCRIPT Table 3. Comparison between allele-specific real-time PCR confirmed by Sanger sequencing and PNA-based FMCA in six MPL mutations
ple
Se
WBC 9
Hb
PLT 9
x
(×10
(g/d
(×10
/A
/L)
L)
/L)
Karyoty
Diagno
Allele-
Sanger
PNA-
ping
sis
specific
sequencing
based
real-time
ge 7*
M/
FMCA
PCR 3.8
7
293
Na
PMF
64
p.Trp515Le
c.1544G>T;
Not
u/Lys
p.Trp515Leu
Detected
(low
(with
burden)
mutant-
enriched PCR)
Abnorm
c.1544G>C;
al curve
RI
Not
PT
Sam
targeted
p.Trp515Ser
at 53°C
359*
F/7
2.23
7.9
1256
46,XX
ET
836
M/
8.24
13.1
802
46,XY
1020
M/
10.99
7.6
343
46,XY
3
Yo38
F/6
7.81
9
5.4
11.3
166
na
PT E
*
4.39
678
CE
F/5
D
81
1029
ET
MA
69
46,XX
p.Trp515Le
c.1544G>T;
Not
u/Lys
p.Trp515Leu
Detected
NU
1
SC
(high burden)
PMF
(with
mutant-
enriched PCR)
p.Trp515Le
c.1544G>T;
p.Trp515
u/Lys
p.Trp515Leu
Leu
p.Trp515Le
c.1543_1544del
p.Trp515
u/Lys
insAA;
Lys
p.Trp515Lys PMF
p.Trp515Le
c.1544G>T;
Not
u/Lys
p.Trp515Leu
Detected
(with
mutant-
enriched PCR) ET
p.Trp515Le
c.1544G>T;
p.Trp515
u/Lys
p.Trp515Leu
Leu
WBC, white blood cell; Hb, hemoglobin; PLT, platelet; PMF, primary myelofibrosis; ET, essential
AC
thrombocythemia; na, not available * Discrepant results
26
ACCEPTED MANUSCRIPT
Table 4. Comparison between fragment analysis confirmed by Sanger sequencing and PNAbased FMCA in 24 CALR mutations
ple
Se
WBC
Hb
9
PLT
Karyotyping 9
x
(×10
(g/d
(×10
/A
/L)
L)
/L)
Diagno
Fragm
Sanger
PNA-
sis
ent
sequencing
base
ge
305
M/
9.12
15.1
1739
46,XY
ET
d
is
FMC
c.1092_1143del52;
Type
mutati
p.Leu367fs*46
1
M/
9.47
14.1
836
46,XY
ET
494
F/6
7.77
9.2
781
na
13.7
13.6
1222
M/
10.41
61
F/6 7
4.84
10.2
AC
792
13
892
CE
683
ET
PT E
2
46,XX
D
F/7
ET
MA
5
573
NU
38
SC
on
483
899
A
Type 1
RI
67
analys
PT
Sam
(Type 1)
mutati on
Type 1
c.1092_1143del52;
Type
mutati
p.Leu367fs*46
1
on
(Type 1)
mutati on
Type 1
c.1092_1143del52;
Type
mutati
p.Leu367fs*46
1
on
(Type 1)
mutati on
Type 1
c.1092_1143del52;
Type
mutati
p.Leu367fs*46
1
on
(Type 1)
mutati on
46,XY
ET
Type 1
c.1092_1143del52;
Type
mutati
p.Leu367fs*46
1
on
(Type 1)
mutati on
46,XX
ET
Type 1
c.1092_1143del52;
Type
mutati
p.Leu367fs*46
1
on
(Type 1)
mutati on
1072
M/
5.81
12.5
146
46,XY
PMF
60
Type 1
c.1092_1143del52;
Type
mutati
p.Leu367fs*46
1
on
(Type 1)
mutati on
1091
F/2 0
11.94
10.8
897
46,XX,add(5)(q1 5), der(10)t(5;10)(q 33;p13)
27
ET
Type 1
c.1092_1143del52;
Type
mutati
p.Leu367fs*46
1
ACCEPTED MANUSCRIPT on
(Type 1)
mutati on
Yo54
F/3
14.95
12.1
1172
46,XX
ET
0
Type 1
c.1092_1143del52;
Type
mutati
p.Leu367fs*46
1
on
(Type 1)
mutati on
F/7
9.19
11.1
1501
46,XX
ET
0
F/5
7.11
12.6
772
46,XX
ET
7.24
13.1
1227
46,XX
6.82
13.4
1071
na
M/
13.08
11
68
M/
25.29
F/3 8
AC
72
777
7.9
99
CE
753
1529
6.55
11.8
na
ET
PT E
594
p.Leu367fs*46
1
on
(Type 1)
mutati
c.1154_1155insTT
1384
on Type2
mutati
GTC;
mutati
on
p.Lys385fs*47
on
(Type 2)
Type 2
c.1154_1155insTT
Type2
mutati
GTC;
mutati
on
p.Lys385fs*47
on
(Type 2) Type 2
c.1154_1155insTT
Type2
mutati
GTC;
mutati
on
p.Lys385fs*47
on
(Type 2)
D
3
ET
MA
F/4
ET
NU
9
542
mutati
SC
F/5
Type
Type 2
6
540
c.1092_1143del52;
RI
172
Type 1
PT
Yo56
Type 2
c.1154_1155insTT
Type2
mutati
GTC;
mutati
on
p.Lys385fs*47
on
(Type 2)
na
PMF
Type 2
c.1154_1155insTT
Type2
mutati
GTC;
mutati
on
p.Lys385fs*47
on
(Type 2) 46,XX
ET
Type 2
c.1154_1155insTT
Type2
mutati
GTC;
mutati
on
p.Lys385fs*47
on
(Type 2) 1102
F/4
7.06
13
838
46,XX
ET
7
Type 2
c.1154_1155insTT
Type2
mutati
GTC;
mutati
on
p.Lys385fs*47
on
(Type 2)
28
ACCEPTED MANUSCRIPT Yo57
F/5
8.94
13.6
1540
46,XX
ET
8
Type 2
c.1154_1155insTT
Type2
mutati
GTC;
mutati
on
p.Lys385fs*47
on
(Type 2) 513*
M/
8.99
13.4
1108
na
ET
79
Other
c.1092_1138del47;
Type1
exon 9
p.Gln365Glyfs*8
mutati
mutati
on
on M/
*
74
15.36
14.2
1441
46,XY
ET
Other
exon 9
Type1
p.Leu367Argfs*52
mutati
RI
mutati
c.1099_1132del34;
PT
Bu37
on
on M/
8.43
10.1
49
46,XY
PMF
56
Type1
SC
302*
mutati
c.1105_1156del52;
Not
p.Glu369Argfs*44
Detec
on
M/
12.54
12.6
1294
46,XY
517
F/6
7.6
8.9
819
na
ET
D
8
MA
41
ET
NU
351
F/5
5.66
12.5
na
ET
CE
2
762
Other
c.1120_1143delins
Not
exon 9
TGCGT;
Detec
mutati
p.Lys374Cysfs*50
ted
Other
c.1123_1125delins
Not
exon 9
TGTTT;
Detec
mutati
p.Lys375Cysfs*56
ted
Other
c.1131_1151delins
Not
exon 9
GTGCCTCCTCCT
Detec
mutati
GGAGG;
ted
on
p.Glu378Cysfs*51
on
on
PT E
797
ted
WBC, white blood cell; Hb, hemoglobin; PLT, platelet; PMF, primary myelofibrosis; ET, essential thrombocythemia; na, not available
AC
* Discrepant results
29
ACCEPTED MANUSCRIPT Highlights
•
Detection of JAK2, MPL, and CALR mutations is essential in BCR-ABL1-negative MPN. Peptide nucleic acid-based fluorescence melting curve analysis is develped.
•
The limit of detection (LOD) was approximately 10% for each mutation.
•
Interference reactions using mixtures of different mutations were not observed.
•
The rapid and simple PNA-based FMCA is a sensitive and specific technique.
AC
CE
PT E
D
MA
NU
SC
RI
PT
•
30