Research Article Mutation Analysis of JAK2V617F, FLT3-ITD ...downloads.hindawi.com/journals/bmri/2014/485645.pdf · blasts[ , ].FLT-ITD(internaltandemduplicationofFLT) is the most

Research ArticleMutation Analysis of JAK2V617F FLT3-ITDNPM1 and DNMT3A in Chinese Patients withMyeloproliferative Neoplasms

Min Wang Na He Tian Tian Lu Liu Shuang Yu and Daoxin Ma

Department of Hematology Qilu Hospital Shandong University 107 West Wenhua Road Jinan 250012 China

Correspondence should be addressed to Daoxin Ma daoxinmasdueducn

Received 12 December 2013 Revised 14 February 2014 Accepted 16 February 2014 Published 11 May 2014

Academic Editor Gautam Sethi

Copyright copy 2014 Min Wang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Since the discovery of JAK2V617F tyrosine kinase-activatingmutation several genes have been foundmutated inmyeloproliferativeneoplasms (MPNs) FLT3-ITD NPM1 and DNMT3A mutations frequently occurred in AML patients and have been foundconferred with myeloproliferative neoplasms in mouse model Therefore we sought to search for mutations in JAK2V617F FLT3-ITD NPM1 and DNMT3A in 129 cases including 120 classic MPN cases and 9 MDSMPN cases JAK2V617F mutation wasfound in 60 of the 120 classic MPNs However none of the patients displayed FLT3-ITD and NPM1 mutations only 2 patientsharboredDNMT3AR882mutation Further studies includingwhole-genome sequencewill be conducted to investigate the possibleinvolvement of these genes in MPN

1 Introduction

Myeloproliferative neoplasms (MPNs) are a class of stem cell-derived myeloid hematologic malignancies characterizedby expansion of one or more hematopoietic cell lineageswith resulting bone marrow hypercellularity a trend oftransformation to myelofibrosis or acute leukemia Matureand immature marrow elements readily traffic into theperipheral blood as evidenced by increased white bloodcells count hemoglobin or platelet count [1 2] Chronicmyeloid leukemia (CML) and the three nonleukemic forms(polycythemia vera (PV) essential thrombocythemia (ET)and myelofibrosis (MF)) comprise the majority of MPNs andare commonly referred to as the classical forms [1]

The underlying causes of MPN are largely unknownGenetic studies have identified that recurrent somatic andgermline alterations may be responsible for the pathogenesisof MPN [3] Since the discovery of the JAK2V617F mutationin 2005 an increasing number of novel somatic and germlinemutations have been described in MPN in recent yearsincluding myeloproliferative leukemia virus (MPL) TEToncogene family member 2 (TET2) additional sex combs-like 1 (ASXL1) casitas B-lineage lymphoma proto-oncogene

(CBL) isocitrate dehydrogenase (IDH) and IKAROS familyzinc finger 1 (IKZF1) However none of these mutationswas MPN specific displayed mutual exclusivity or could betraced back to a common ancestral clone [4] Several linesof evidence suggest that mutations in genes other than thesementioned above must be present in MPN patients and theinitiating genetic events responsible for the development ofMPN are still not totally understood [3]

FMS-like tyrosine kinase 3 (FLT3) a member of theclass III receptor tyrosine kinase family that is expressed byearly hematopoietic progenitors plays a key role in growthregulation of hematopoietic progenitor cells [5] Some studieshave reported that FLT3 is also expressed on AML leukemiccells and stimulates survival and proliferation of leukemicblasts [6 7] FLT3-ITD (internal tandemduplication of FLT3)is the most prevalent mutation found in AML and hasbeen identified in 20ndash30 of all AML patients [8] Studiessuggest that AML patients with FLT3-ITD have significantlyelevated peripheral white blood cell counts and increasedbone marrow blasts at diagnosis [9] Moreover Li et alrecently showed that knock-in of an ITD mutation intomurine FLT3 conferredmyeloproliferative disease in amousemodel [10] which indicated the potential involvement of

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 485645 8 pageshttpdxdoiorg1011552014485645

2 BioMed Research International

FLT3-ITD in MPN However to date the data about FLT3-ITD mutation in human MPN remain poorly defined

The nucleophosmin 1 (NPM1 localized on 5q35) geneencodes for a multifunctional phosphoprotein located pri-marily in the nucleolus NPM1 mutations are known to becommon inAMLand are commonly associatedwith a diploidkaryotype [11] Sportoletti et al demonstrated that NPM1acts indeed as a haploinsufficient tumor suppressor gene invivo [12] while some findings suggested that NPM1mutationin AML is likely a gain-of-function one rather than simplehaploinsufficiency [13] The most common NPM1 mutationin AML is a duplication of a TCTG tetra-nucleotide atpositions 956 to 959 of the reference sequence (GenBankaccession number NM 002520) and accounts for 75 to80 of cases [14] One ldquoconventionalrdquo knock-in model ofNPM1 mutation demonstrated that NPM1 mutation canresult in myeloproliferative disease but is insufficient forleukemogenesis [13] Nevertheless the frequency of NPM1mutation and its possible pathogenetic role in MPNs arerarely investigated until now

Alterations of epigenetic markers are thought to playan important role in myeloid malignancies In particularaberrant DNA methylation is a hallmark of these diseases[15] DNA methyltransferases (DNMTs) catalyze the methy-lation of cytosine residues of CpG dinucleotides in DNA andare encoded by the human genes DNMT1 DNMT3A andDNMT3B [16] More recently a whole-genome sequencingstudy in AML uncovered recurrent mutations of DNMT3Ain 22 of AML patients and DNMT3A mutations wereassociated with poor outcome [17] Many of the reportedDNMT3Amutations mainly occurred at codon R882 in exon23 but the occurrence of DNMT3A mutations in MPNpatients is not well clarified

In the last few years the prevalence of DNMT3A NPM1or FLT3-ITD has been individually studied in some myeloidneoplasms mainly including AML or MDS Furthermoresome studies indicate that themutagenesis of these genesmaydiffer within different races and be associated with patientrsquosage Therefore in this study we put the three genes togetheralong with JAK2V617F to determine their mutational statusin a series of well-defined adult Chinese patients with MPNand explored their clinical significance also the associationbetween JAK2V617F mutation and DNMT3A NPM1 orFLT3-ITD mutation was also investigated

2 Materials and Methods

21 Patients and Samples A total of 120 newly diagnosedclassic MPN patients and 9 MDSMPN patients accordingto the World Health Organization (WHO) criteria [18] wereincluded in this study Enrollment took place between Jan-uary 2011 and December 2012 in Department of HematologyQiLu Hospital of Shandong University China Bone marrowor peripheral blood samples were collected at diagnosis Thestudy was approved by the Institutional Review Boards ofQiLu Hospital of Shandong University Informed consentwas obtained from each patient before being included inthis study The clinical characteristics of these subjects weresummarized in Table 1

22 DNA Extraction Leukocytes were separated from bonemarrow or peripheral blood samples using erythrocyte lysingsolution Genomic DNA was isolated using the TIANGENDNA isolation kit (TIANGEN China) according to themanufacturerrsquos protocol Store the DNA samples at minus80∘C forthe following polymerase chain reaction (PCR) amplification

23 PCR and Sequencing for DNMT3A NPM1 and FLT3 Asthe recurrentmutation points exon 23 ofDNMT3A and exon12 of NPM1 as well as FLT3 exons 11 and 12 were amplifiedby PCR then the PCR products were sequenced BrieflyPCR was performed in 25 120583L volume containing 125 120583L2x Taq PCR MasterMix (TIANGEN China) 75120583L RNase-free ddH2O 1 120583L forward and reverse primer respectivelyand 3 120583L genomic DNA The primers are shown as followsDNMT3A exon 23 forward 51015840-TCC TGC TGT GTG GTTAGA CG-31015840 reverse 51015840-TAT TTC CGC CTC TGT GGT TT-31015840 NPM1 exon 12 forward 51015840-GGTCTCTGTTCTTTCTGTTGATTTCC-31015840 reverse 51015840-CAACACATTCTTGGCAATAGA ACC T-31015840 FLT3 exon 11 forward 51015840-GCA ATT TAGGTA TGA AAG CCA GC-31015840 exon 12 reverse 51015840-CTT TCAGCA TTT TGA CGG CAA CC-31015840 The amplification wasdone with a DNA thermal cycler (BIO-RAD S1000 ThermalCycler) After denaturing at 94∘C for 3min the amplificationwas conducted for 35 cycles at 94∘C for 30 s 55∘C for 30 s and72∘C for 1min followed by reextension for 5min at 72∘C

The PCR products were loaded onto a 2 agarose gelcontaining ethidium bromide and electrophoretically sepa-rated After being purified the PCR products were directlysequenced on both directions using the ABI PRISM 3730xlanalyzer (Applied Biosystems Inc Foster City CA USA)to screen for the presence of mutations The samples withmutated FLT3-ITD mutated NPM1 or mutated DNMT3Awere used as positive controls

24 JAK2V617F Mutation Analysis We used the TaqManMGB probe joint real-time PCR to detect JAK2V617F muta-tion The TaqMan MGB probes and Mix were purchasedfrom Applied Biosystems PCR amplifications of DNA weredone in a total volume of 10120583L PCR mixture containing5 120583L TaqMan Universal PCR Master Mix 04 120583L forwardand reverse primer respectively 02 120583L FAM and VICfluorophore respectively 28 120583L RNase-free ddH2O and1 120583L genomic DNA The primer and TaqMan MGB probesequences are as follows forward primer 51015840-AAG CTT TCTCAC AAG CAT TTG GTT G-31015840 reverse primer 51015840-AGAAAG GCA TTA GAA AGC CTG TAG TT-31015840 probe1 FAM-TCC ACA GAA ACA TAC-MGB probe2 VIC-TCT CCACAG ACA TAC-MGB PCR amplification conditions were50∘C 10min 95∘C 30 s 95∘C 15 s and 62∘C 1min 45 cyclesPCR was performed with ABI 7500 Real-Time PCR system(Applied Biosystems)

25 Statistical Analysis Statistical analysis was performedusing the SPSS Statistical Analysis Software Differencesin JAK2V617F percentage age and peripheral blood cellscountswere accessed byChi-squared tests Fisherrsquos exact tests

BioMed Research International 3

Table 1 Basic characteristic of JAK2V617F-positive and negative MPN patients

All patients JAK2V617F mutation119875 value

Negative PositiveNumber ()

All patients 120 48 (40) 72 (60)PV patients 25 6 (24) 19 (76)ET patients 55 22 (40) 33 (60)MF patients 24 15 (625) 9 (375)MPN-u patients 15 5 (333) 10 (667)CNL patients 1 0 (0) 1 (100)

Age (years) 119909 plusmn 119904All patient 55 plusmn 16 48 plusmn 16 60 plusmn 11 0000PV patients 53 plusmn 23 40 plusmn 15 63 plusmn 8 0011ET patients 53 plusmn 3 47 plusmn 17 57 plusmn 12 0016MF patients 58 plusmn 12 56 plusmn 14 62 plusmn 4 0184MPN-u patients 53 plusmn 0 43 plusmn 20 58 plusmn 12 0183CNL patients 68 mdash 68

Gender (MaleFemale)All patients 5664 2127 3537 0709PV patients 1510 51 109 0345ET patients 2035 818 1217 0575MF patients 159 65 94 0675MPN-u patients 69 23 46 1000CNL patients 01 mdash 01119875 value refers to the comparison of JAK2V617F-positive versus -negative subjectsMPN myeloproliferative neoplasms PV polycythemia vera ET essential thrombocythemia MF myelofibrosis MPN-u MPN-unclassifiable CNL chronicneutrophilic leukemia

and 119905-tests respectively 119875 values less than 005 (two tailed)are considered significantly different

3 Results

31 JAK2V617F Mutation in MPN

311 JAK2V617F Mutation In the 120 classic MPNs studiedJAK2V617F mutation was found in 72120 (60) The fre-quency of JAK2V617Fmutationwas 76amongpatientswithPV (19 of 25) 60 among patients with ET (33 of 55) 375among patients with MF (9 of 24) and 667 amongMPN-upatients (10 of 15) There was significant difference betweenthese four groups (119875 = 0046 119875 lt 005) and the positiveincidence in PV group was remarkably higher than that inthe other three groups In addition the only one chronicneutrophilic leukemia (CNL) patient was also observed withJAK2V617F mutation Among the 9 MDSMPN patientsonly one had JAK2V617F mutation

312 The Association of JAK2V617F Mutation with Clini-cal Characteristics As shown in Table 1 the patients withJAK2V617F mutation were much older than those withoutmutation (119875 = 0000) In PV and ET group the JAK2V617Fmutant patients were older than those JAK2V617F-negativepatients while no significant difference was found in MFand MPN-u group As shown in Table 2 compared with

younger patients aged lt60 years 469 (30 of 64) thefrequency of JAK2V617F mutation was significantly higherin older patients aged ⩾60 years 731 (41 of 56) (119875 =0003) JAK2V617Fmutation rate was higher in older patientswith PV while not in ET MF and MPN-u patients Inolder patients JAK2V617F mutation rate in PV patients washigher than in ET MF and MPN-u patients however thisphenomenonwas not seen in younger patients As for genderthere was no statistical significance of JAK2V617F mutationincidence between male 625 (35 of 56) and female 578(37 of 64) patients (119875 = 0709)

We analyzed the peripheral hemogram of the 85 hospi-talized patients The number of white blood cells (WBCs)red blood cells (RBCs) and hemoglobin (HB) of JAK2V617F-positive patients was significantly higher than those ofJAK2V617F-negative patients in contrast no significant dif-ference was found when comparing platelet (PLT) countwhile in PV patients with JAK2V617F mutation the PLTcount was higher than those without mutation In ET andMF patients the number of WBCs of JAK2V617F-mutatedpatients was much higher besides the JAK2V617F-mutatedET patients also had higher HB counts (as shown in Table 3)

32 DNMT3AMutation inMPN By sequence analysis of theDNMT3A gene we found DNMT3Amutations in 2 patientsThe two mutations were heterozygous and missense one wasa MF patient (c2644CgtT pR882C JAK2V617F-positive)the other one was a MDSMPN patient (c2645GgtA


Table 2 The relationship between JAK2V617F mutation and age atdiagnosis

ge60 years 119899 = (56) lt60 years 119899 = (64)119875 value

Positive Negative Positive NegativePV patients 14 (100) 0 5 (45) 6 0003ET patients 17 (74) 6 16 (50) 16 0098MF patients 6 (46) 7 3 (27) 8 0423MPN-u patients 4 (80) 1 6 (60) 4 06lowast

119875 value 0016 0471All patients 41 14 30 34 0003119875 value refers to the comparison of JAK2V617F mutation rate betweenpatients gt60 years old and lt60 years old within all patients and everysubgrouplowast

119875 value refers to the JAK2V617F mutation rate difference between foursubgroups in patients gt60 years old and patients lt60 years oldMPN myeloproliferative neoplasms PV polycythemia vera ET essentialthrombocythemia MF myelofibrosis MPN-u MPN-unclassifiable CNLchronic neutrophilic leukemia

pR882H JAK2V617F-negative) (Figure 1) Hence the fre-quency of DNMT3A exon 23 mutation in MF was 4 (124)and the overall frequency in ourMPNpatientswas close to 1(1120) Both the 2 DNMT3A-mutant patients were wild typefor FLT3-ITD and NPM1 Moreover the DMNT3A-mutatedMF patient also had an abnormal karyotype 46XYminus3+marThis patient was a 60-year-old man who was diagnosed withMPN 9 years ago and later developed into postpolycythemiavera MF And the patient also had spleen infarct complica-tions and died ultimately with serious infections and systemicorgan failure

33 FLT3-ITD and NPM1 Mutation in MPN We analyzedthe mutations of FLT3-ITD and NPM1 by PCR followed bysequencing However our present study did not reveal anysequence variation in the 120MPNpatients and 9MDSMPNpatients we studied as shown in Figures 2(a) and 2(c)while Figures 2(b) and 2(d) showed the chromatograms ofpositive controls (samples fromAMLpatientswith FLT3-ITDor NPM1 mutation Figure 2(b) showed the FLT3 internaltandem duplications mutation in exons 11 and 12 Figure 2(d)showed the insertion of a TCTG tetra nucleotide at positions956 to 959 of the reference sequence) All these resultssuggested that the studied FLT3-ITD or NPM1 mutationpoints are unlikely the candidate factors for human MPNdevelopment

4 Discussion

In this current study we have mainly screened for thethree putative candidate genes along with well-determinedJAK2V617F in a cohort of Chinese MPN patients Thefrequency of JAK2V617Fwas 60 inMPNpatients Howevernone of the 120 MPN patients and 9 MDSMPN patientsdisplayed known FLT3-ITD mutation at the juxtamembrane(JM) coding sequences and NPM1 mutation Two kinds ofmutations of DNMT3A were observed

C C C C T TA T G G G GA

(a) DNMT3A wild type

C C C C T TA T G G GA A

(b) c2645GgtA pR882H

C C C C T TA T G G GGA

(c) c2644CgtT p R882c

Figure 1The DNMT3Amutation and wild type (a)The DNMT3Awild type (b) the MF patient who has a DNMT3A mutation(c2644CgtT pR882C) and (c) the MDSMPN patient who has aDNMT3A mutation (c2645GgtA pR882H)

Amounts of molecular and clinical evidences have shownthat JAK2V617F mutation has a direct causal role in thepathogenesis of MPNs JAK2V617F mutations were foundin approximately 70ndash90 of patients with PV in 35ndash70 with ET and in 30ndash50 with MF which was in linewith our results Studies have suggested that JAK2V617Fmutation is more common in old than in young patientswith MPN [19] In our study the presence of JAK2V617Fwas found to be significantly correlative with advanced age(ge60 years) at diagnosis Besides JAK2V617F mutation wascommon in old patients with PV Complete blood cell count(CBC) is essential in diagnosis of MPN several studieshave showed that hemogram is altered in patients withJAK2V617F mutation However thus far the relationshipbetween JAK2V617F mutation and blood cell counts iscontroversial and the impact of JAK2V617F mutation on thepatientsrsquo hemogram variation remains not very clear [20]


Table 3 JAK2V617F mutation and peripheral hemogram

JAK2V617F Number WBC (109L) RBC (1012L) HB (gL) PLT (109L)PV 21

Mutation 16 112 plusmn 45 68 plusmn 11 1856 plusmn 324 3622 plusmn 2223Wild type 5 9 plusmn 57 62 plusmn 17 1902 plusmn 374 1857 plusmn 712119875 value 0383 0543 0813 0015

ET 35Mutation 20 194 plusmn 155 44 plusmn 08 1332 plusmn 247 10468 plusmn 6083Wild type 15 93 plusmn 5 38 plusmn 08 1101 plusmn 219 8718 plusmn 2969119875 value 0012 0052 0013 0272

MF 22Mutation 8 187 plusmn 128 34 plusmn 13 835 plusmn 214 188 plusmn 512Wild type 14 81 plusmn 92 26 plusmn 10 74 plusmn 265 999 plusmn 895119875 value 0035 0179 0372 0135

MPN-u 6Mutation 4 238 plusmn 60 57 plusmn 2 141 plusmn 438 5498 plusmn 2215Wild type 2 274 plusmn 178 35 plusmn 15 954 plusmn 433 11255 plusmn 8393119875 value 0821 0251 0293 0507

CNL 1Mutation 1 2677 plusmn 0 422 plusmn 0 1385 plusmn 0 233 plusmn 0

Total 85Mutation 49 173 plusmn 122 52 plusmn 17 143 plusmn 451 6398 plusmn 5544Wild type 36 98 plusmn 85 37 plusmn 16 1062 plusmn 472 4904 plusmn 4676119875 value 0002 0000 0001 0197119875 value refers to the comparison of JAK2V617F-positive versus -negative subjectsMPN myeloproliferative neoplasms PV polycythemia vera ET essential thrombocythemia MF myelofibrosis MPN-u MPN-unclassifiable CNL chronicneutrophilic leukemia WBC white blood cell RBC red blood cell HB hemoglobin PLT platelet

So we determined the relationship between the JAK2V617Fmutation rate and hemogram in adult Chinese classic MPNsour data demonstrated that patients harboring JAK2V617Fmutation had higher leukocyte counts red blood cell countsand hemoglobin levels Our JAK2V617F-mutated ET patientswere found with advanced age and remarkably higher leuko-cytes which was consistent with previous studies [21] whilein PV andMF group the association of JAK2V617Fmutationwith hemogramvariationswas relatively notwell determinedAs far as we know our research is the first one to study therelationship between JAK2V617Fmutation and hemogram aswell as age factor in adult Chinese classic MPNs

The majority of FLT3 mutations are ITDs in the JMdomain encoded by exons 11 and 12 and were first reported inpatients with AML in 1996 [6] Xu et al detected the patientswith various malignant hematologic diseases and found thatFLT3-ITD mutation mainly occurred in AML patients andmight be a strong prognostic factor [22] In another studyFLT3 mutations were also observed in patients with MDSor CMML but at a much lower frequency than AML anddid not predict poor outcome [9] However the data aboutFLT3 mutations in MPN patients and their relationship withJAK2V617F mutations were limited

Several studies in animal models uncovered the impor-tance of FLT3-ITD in MPN four studies have indicatedthat FLT3-ITD could induce myeloproliferative disease usingtransgenic mouse models respectively [23ndash26] Because of

the putative involvement of FLT3-ITD inMPN developmentwe detected the total 129 MPN and MDSMPN cases usingPCR followed by sequencingmethodHowever no FLT3-ITDpatients were found Our negative result is similar to Par-danani et al [27] report that no FLT3 mutations were foundin a cohort of patients with chronic myeloid disorders whilebeing in contrast to Linrsquos study that FLT3 mutations occur inapproximately 10of Philadelphia (Ph) chromosome-CMPDand CMPDMDS [5] In a recent article FLT3 mutationanalysis was performed on 90 cases of JAK2-negative MPNsor MDSMPNs and 62 cases of JAK2V617F-positive MPNsOne FLT3-ITD mutation was identified in the JAK2V617F-negative group (11) and none were identified in theJAK2V617F-positive group confirming the absence of FLT3mutations in JAK2V617F-positive specimens [28] which isbasically the same as our results that FLT3 mutation was rarein the usual types ofMPNand the twomutations aremutuallyexclusiveThese differences may be due to the diversity of thestudied diseases or population

NPM1mutations first identified by the aberrant cytoplas-mic localization of NPM1 protein were found to be frequentevents in AML [14] Some animal models bearing enforcedhuman NPM1-mutant expression showed an expansion ofhematopoietic cells and developed myeloproliferation indi-cating a pathogenic role of mutant NPM1 protein in myeloiddisorders [29 30] Some studies showed that NPM1mutationoccurred with low frequencies in patients with MDS [11 31]


T T TT T TT TTT TT T TCCC CG G G G G G G GA AA A A A A A A A AAA A A

(a) FLT3 wild type

T T T T T T T T T T T TT T T TC CG G G G G G G GGA AAA A A A A A A A A A A

(b) FLT3-ITD mutation

T T TT T T T T T T T T T TT TC C C C CC CG G G G G GAA A A A A

(c) NPM1 wild type

T T TT T T T T T T T T T TT TC C C C CC CG G G G G GA A A A A

(d) NPM1 mutation

Figure 2 FLT3 and NPM1 wild type and mutation The sequencingchromatograms of FLT3-ITD and NPM1 (a) FLT3 wild type (b) theFLT3-ITD mutation in exon 11 and 12 (c) the NPM1 wild type and(d) the NPM1 mutation in exon 12

while others found no mutations in MDS [32 33] Ernstet al showed that NPM1 mutations occurred in 6187 (3)MDSMPN patients and the 6 patients were all CMMLpatients indicating that NPM1 mutation may be associatedwith a poor prognosis [34] Schnittger et al found that NPM1mutation was observed in 667 secondary AML (s-AML)patients with a history ofMPN and concluded that the NPM1mutations are not only a key factor in the initiation of denovoAMLbutmay contribute to s-AML followingMPN [35]However relatively few data regarding the presence of NPM1mutations are available for classic MPN cases One study ina small cohort of classic MPNs (14 PV 7 ET and 9 MF)reported that NPM1 mutations were not observed in thesepatients [32] In our relative larger cohort of Chinese MPNpatients noNPM1mutationwas found indicating thatNPM1mutation might not be prevalent in MPN

Mutations in DNMT3A were shown to be one of theearly initiating events in AML pathogenesis [36] DNMT3Amutations were also noted in patients with MDS and s-AML Walter et al found DNMT3A mutations in 8 ofMDS patients similar to the trend noted in AML The majormutant point was at amino acid R882 and has a significantlypoorer outcome [37] The studies about DNMT3A mutationin MPN were limited and inconsistent Stegelmann et alreported DNMT3A mutations in 7 PV 15 MF and 143s-AML and indicatedDNMT3A alterations occurred concur-rentlywith JAK2 [38] Abdel-Wahab et al delineated that total3 DNMT3A-positive cases in 46 primary MF patients werealso found to have cooccurring JAK2V617F mutation [39]However other studies identified that DNMT3A mutationswere rare or absent [15 40 41] We explored the mutationfrequency of DNMT3A in 120 Chinese MPN patients andmutation was only observed in a MF patient concurrentlywith JAK2V617F mutation Moreover this positive patienthad an abnormal karyotype and spleen infarct complicationsand died ultimately with serious infections and systemicorgan failure Therefore DNMT3A mutation may not bea frequent characteristic of MPN but could be a poorprognostic indicator always concurrently with JAK2V617Fmutation However larger cohort of patients are needed todetermine the exact frequency of DNMT3A mutations inChinese MPN patients and to clarify its role in the molecularpathogenesis of MPN

So far more and more genetic events which may con-tribute to the pathogenesis of MPN have been elucidatedHowever despite significant insight into the role of specificmutations including the JAK2V617F mutation the precisemechanisms in MPN remain elusive It is very likely thatadditional mutations in MPN will be described soon butpractical relevance in terms of either disease prognosticationor value as drug targets has so far been limited [42]

MPN genes belong to two major pathways intracellularmetabolism and epigenetic regulation [43] The molecularpathogeneses of these three candidate genes mutations westudied here are different (1) DNMT3A alterations areinvolved in epigenetic regulation of gene transcriptionmdashaberrant DNA methylation (2) FLT3 mutation is associatedwith signaling pathways and proliferation FLT3 as well asJAK2V617F abnormity can activate tyrosine kinase and result


in aberrant activation of tyrosine kinase signaling [34] (3)NPM1 mutation in exon 12 results in loss of its nuclearlocalization signal the altered protein concentrates in thecytoplasm where it dimerizes to wild-type NPM1 blockingits activity in the nucleus [43] So burgeoning insight intothe role of these genes mutations in the pathogenesis ofmyeloid malignancies has prompted increased interest indevelopment of novel targeted therapies Methyltransferaseinhibitors and JAK2 inhibitors are commonly used in clinicaltrials FLT3 kinase inhibitors and NMP1 targeted therapyare reported to have made exciting progress recently [44]However further studies that explore their precise roles inhematopoiesis and in the pathogenesis ofMPNaswell as theirprognostic impact and potential as a therapeutic target areneeded

5 Conclusion

In conclusion we first studied the mutations of JAK2V617FFLT3-ITD NPM1 and DNMT3A together in Chinese adultMPN patients However the DNMT3A R882 amino acidresidue which is a mutation hotspot in AML was onlymutated once in our series of MPNs (in a MF patient) andthe other 2 mutations were not found suggesting that thesegenes may be not involved in the pathogenesis of MPN andof course this hypothesis should be further validated in thefuture researches while hotspot mutations in the DNMT3AFLT3 and NPM1 genes are not common in MPN patientsmaybe due to that these selected genes harbor activatingmutations in other regions which were not examined This isa limitation in our study So further studies including whole-genome sequence will be conducted to clarify the compre-hensive mutational status as well as possible involvement ofthese genes in Chinese MPNs

Conflict of Interests

The authors declare that they have no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

MinWang and Na He contributed equally to this paper

Acknowledgments

This work was supported by Grants from the Tai ShanScholar Foundation National Natural Science Foundationof China (no 81070407 no 81170515 and no 81070422)Outstanding Young Scientist Research Award Foundation ofShandong Province (BS2009SW014) and Independent Inno-vation Foundation of Shandong University (2009TS063)

References

[1] J M Klco R Vij F H Kreisel A Hassan and J L FraterldquoMolecular pathology of myeloproliferative neoplasmsrdquo

American Journal of Clinical Pathology vol 133 no 4pp 602ndash615 2010

[2] L Martınez-Aviles C Besses A Alvarez-Larran E Torres SSerrano and B Bellosillo ldquoTET2 ASXL1 IDH1 IDH2 andc-CBL genes in JAK2- and MPL-negative myeloproliferativeneoplasmsrdquo Annals of Hematology vol 91 no 4 pp 533ndash5412012

[3] O Abdel-Wahab ldquoGenetics of the myeloproliferative neo-plasmsrdquo Current Opinion in Hematology vol 18 no 2 pp 117ndash123 2011

[4] A Tefferi ldquoNovel mutations and their functional and clinicalrelevance in myeloproliferative neoplasms JAK2 MPL TET2ASXL1 CBL IDHand IKZF1rdquoLeukemia vol 24 no 6 pp 1128ndash1138 2010

[5] R Luthra F Vega-Vazquez W Chen et al ldquoActivatingFLT3 mutations are detectable in chronic and blast phasesof chronic myeloproliferative disorders other than chronicmyeloid leukemiardquo American Journal of Clinical Pathology vol126 no 4 pp 530ndash533 2006

[6] M Nakao S Yokota T Iwai et al ldquoInternal tandem duplicationof the flt3 gene found in acutemyeloid leukemiardquo Leukemia vol10 no 12 pp 1911ndash1918 1996

[7] G Yoshimoto T Miyamoto S Jabbarzadeh-Tabrizi et alldquoFLT3-ITD up-regulates MCL-1 to promote survival of stemcells in acute myeloid leukemia via FLT3-ITD-specific STAT5activationrdquo Blood vol 114 no 24 pp 5034ndash5043 2009

[8] H Nakajima F Shibata H Kumagai K Shimoda and T Kita-mura ldquoTyk2 is dispensable for induction of myeloproliferativedisease by mutant FLT3rdquo International Journal of Hematologyvol 84 no 1 pp 54ndash59 2006

[9] N Daver P Strati E Jabbour et al ldquoFLT3 mutationsin myelodysplastic syndrome and chronic myelomonocyticleukemiardquo American Journal of Hematology vol 88 no 1 pp56ndash59 2013

[10] L Li O Piloto H B Nguyen et al ldquoKnock-in of an internaltandem duplication mutation into murine FLT3 confers myelo-proliferative disease in a mouse modelrdquo Blood vol 111 no 7 pp3849ndash3858 2008

[11] A Bains R Luthra L J Medeiros and Z Zuo ldquoFLT3 andNPM1mutations in myelodysplastic syndromes frequency andpotential value for predicting progression to acute myeloidleukemiardquo American Journal of Clinical Pathology vol 135 no1 pp 62ndash69 2011

[12] P Sportoletti S Grisendi S MMajid et al ldquoNpm1 is a haploin-sufficient suppressor of myeloid and lymphoid malignancies inthe mouserdquo Blood vol 111 no 7 pp 3859ndash3862 2008

[13] S H Chou B S Ko J S Chiou et al ldquoA knock-in Npm1mutation in mice results in myeloproliferation and implies aperturbation in hematopoietic microenvironmentrdquo PloS ONEvol 7 no 11 Article ID e49769 2012

[14] B Falini C Mecucci E Tiacci et al ldquoCytoplasmic nucleophos-min in acute myelogenous leukemia with a normal karyotyperdquoThe New England Journal of Medicine vol 352 no 3 pp 254ndash266 2005

[15] M Brecqueville N Cervera V Gelsi-Boyer et al ldquoRaremutations in DNMT3A in myeloproliferative neoplasms andmyelodysplastic syndromesrdquo Blood Cancer Journal vol 1 no 5article e18 2011

[16] O Abdel-Wahab and A T Fathi ldquoMutations in epigeneticmodifiers in myeloid malignancies and the prospect of novelepigenetic-targeted therapyrdquoAdvances in Hematology vol 2012Article ID 469592 12 pages 2012


[17] T J Ley L Ding M J Walter et al ldquoDNMT3A mutations inacute myeloid leukemiardquoTheNew England Journal of Medicinevol 363 no 25 pp 2424ndash2433 2010

[18] J W Vardiman J Thiele D A Arber et al ldquoThe 2008 revisionof the World Health Organization (WHO) classification ofmyeloid neoplasms and acute leukemia rationale and impor-tant changesrdquo Blood vol 114 no 5 pp 937ndash951 2009

[19] M L Randi E Ruzzon F Tezza et al ldquoJAK2V617F mutationis common in old patients with polycythemia vera and essentialthrombocythemiardquoAgingmdashClinical and Experimental Researchvol 23 no 1 pp 17ndash21 2011

[20] J Zhou Y Ye S Zeng et al ldquoImpact of JAK2 V617F mutationon hemogram variation in patients with non-reactive elevatedplatelet countsrdquo PLoSONE vol 8 no 2 Article ID e57856 2013

[21] A Carobbio G Finazzi V Guerini et al ldquoLeukocytosis isa risk factor for thrombosis in essential thrombocythemiaInteraction with treatment standard risk factors and Jak2mutation statusrdquo Blood vol 109 no 6 pp 2310ndash2313 2007

[22] B Xu G Chen X Luo and J Tang ldquoDetection of FLT3ITDgene mutations in patients with hematologic malignancy andtheir clinical significancerdquo The Chinese-German Journal ofClinical Oncology vol 8 no 2 pp 100ndash103 2009

[23] R Grundler C Miething CThiede C Peschel and J DuysterldquoFLT3-ITD and tyrosine kinase domain mutants induce 2distinct phenotypes in a murine bone marrow transplantationmodelrdquo Blood vol 105 no 12 pp 4792ndash4799 2005

[24] L M Kelly Q Liu J L Kutok I R Williams C L Boultonand D G Gilliland ldquoFLT3 internal tandem duplication muta-tions associated with human acute myeloid leukemias inducemyeloproliferative disease in a murine bone marrow transplantmodelrdquo Blood vol 99 no 1 pp 310ndash318 2002

[25] B H Lee I R Williams E Anastasiadou et al ldquoFLT3 internaltandem duplication mutations induce myeloproliferative orlymphoid disease in a transgenic mouse modelrdquo Oncogene vol24 no 53 pp 7882ndash7892 2005

[26] L Li E Bailey S Greenblatt D Huso andD Small ldquoLoss of thewild-type allele contributes to myeloid expansion and diseaseaggressiveness in FLT3ITD knockin micerdquo Blood vol 118 no18 pp 4935ndash4945 2011

[27] A Pardanani T L Reeder T K Kimlinger et al ldquoFlt-3 and c-kit mutation studies in a spectrum of chronic myeloid disordersincluding systemic mast cell diseaserdquo Leukemia Research vol27 no 8 pp 739ndash742 2003

[28] L Williams H H Kelley X Meng A Prada and D CrisanldquoFLT3 mutations in myeloproliferative neoplasms the Beau-mont experiencerdquo Diagnostic Molecular Pathology vol 22 no3 pp 156ndash160 2013

[29] K Cheng P Sportoletti K Ito et al ldquoThe cytoplasmic NPMmutant induces myeloproliferation in a transgenic mousemodelrdquo Blood vol 115 no 16 pp 3341ndash3345 2010

[30] N Bolli E M Payne C Grabher et al ldquoExpression of thecytoplasmic NPM1 mutant (NPMc+) causes the expansion ofhematopoietic cells in zebrafishrdquoBlood vol 115 no 16 pp 3329ndash3340 2010

[31] Y Zhang M Zhang L Yang and Z Xiao ldquoNPM1 mutations inmyelodysplastic syndromes and acute myeloid leukemia withnormal karyotyperdquo Leukemia Research vol 31 no 1 pp 109ndash111 2007

[32] Y Oki J JelinekM Beran S Verstovsek HM Kantarjian andJ J Issa ldquoMutations and promoter methylation status of NPM1in myeloproliferative disordersrdquo Haematologica vol 91 no 8pp 1147ndash1148 2006

[33] J Rocquain N Carbuccia V Trouplin et al ldquoCombinedmutations of ASXL1 CBL FLT3 IDH1 IDH2 JAK2 KRASNPM1 NRAS RUNX1 TET2 andWT1 genes in myelodysplas-tic syndromes and acute myeloid leukemiasrdquo BMC Cancer vol10 article 401 2010

[34] T Ernst A Chase K Zoi et al ldquoTranscription factor mutationsin myelodysplasticmyeloproliferative neoplasmsrdquo Haemato-logica vol 95 no 9 pp 1473ndash1480 2010

[35] S Schnittger U Bacher C Haferlach et al ldquoCharacterizationof NPM1-mutated AML with a history of myelodysplasticsyndromes ormyeloproliferative neoplasmsrdquo Leukemia vol 25no 4 pp 615ndash621 2011

[36] J S Welch T J Ley D C Link et al ldquoThe origin and evolutionof mutations in acutemyeloid leukemiardquoCell vol 150 no 2 pp264ndash278 2012

[37] M JWalter L Ding D Shen et al ldquoRecurrentDNMT3Amuta-tions in patients with myelodysplastic syndromesrdquo Leukemiavol 25 no 7 pp 1153ndash1158 2011

[38] F Stegelmann L Bullinger R F Schlenk et al ldquoDNMT3Amutations in myeloproliferative neoplasmsrdquo Leukemia vol 25no 7 pp 1217ndash1219 2011

[39] O Abdel-Wahab A Pardanani R Rampal T L Lasho RL Levine and A Tefferi ldquoDNMT3A mutational analysis inprimary myelofibrosis chronic myelomonocytic leukemia andadvanced phases of myeloproliferative neoplasmsrdquo Leukemiavol 25 no 7 pp 1219ndash1220 2011

[40] J Lin D Yao J Qian et al ldquoRecurrent DNMT3A R882mutations in Chinese patients with acute myeloid leukemia andmyelodysplastic syndromerdquo PLoS ONE vol 6 no 10 Article IDe26906 2011

[41] M Brecqueville J Rey F Bertucci et al ldquoMutation analysisof ASXL1 CBL DNMT3A IDH1 IDH2 JAK2 MPL NF1SF3B1 SUZ12 and TET2 in myeloproliferative neoplasmsrdquoGenes Chromosomes andCancer vol 51 no 8 pp 743ndash755 2012

[42] A Tefferi T L Lasho O Abdel-Wahab et al ldquoIDH1 and IDH2mutation studies in 1473 patients with chronic- fibrotic- orblast-phase essential thrombocythemia polycythemia vera ormyelofibrosisrdquo Leukemia vol 24 no 7 pp 1302ndash1309 2010

[43] LMGutiyamaD F CoutinhoMV Lipkin and I R ZalcbergldquoMyeloid neoplasias what molecular analyses are telling usrdquoISRN Oncology vol 2012 Article ID 321246 7 pages 2012

[44] E Hatzimichael G Georgiou L Benetatos and E BriasoulisldquoGene mutations and molecularly targeted therapies in acutemyeloid leukemiardquo American Journal of Blood Research vol 3no 1 pp 29ndash51 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


FLT3-ITD in MPN However to date the data about FLT3-ITD mutation in human MPN remain poorly defined

The nucleophosmin 1 (NPM1 localized on 5q35) geneencodes for a multifunctional phosphoprotein located pri-marily in the nucleolus NPM1 mutations are known to becommon inAMLand are commonly associatedwith a diploidkaryotype [11] Sportoletti et al demonstrated that NPM1acts indeed as a haploinsufficient tumor suppressor gene invivo [12] while some findings suggested that NPM1mutationin AML is likely a gain-of-function one rather than simplehaploinsufficiency [13] The most common NPM1 mutationin AML is a duplication of a TCTG tetra-nucleotide atpositions 956 to 959 of the reference sequence (GenBankaccession number NM 002520) and accounts for 75 to80 of cases [14] One ldquoconventionalrdquo knock-in model ofNPM1 mutation demonstrated that NPM1 mutation canresult in myeloproliferative disease but is insufficient forleukemogenesis [13] Nevertheless the frequency of NPM1mutation and its possible pathogenetic role in MPNs arerarely investigated until now

Alterations of epigenetic markers are thought to playan important role in myeloid malignancies In particularaberrant DNA methylation is a hallmark of these diseases[15] DNA methyltransferases (DNMTs) catalyze the methy-lation of cytosine residues of CpG dinucleotides in DNA andare encoded by the human genes DNMT1 DNMT3A andDNMT3B [16] More recently a whole-genome sequencingstudy in AML uncovered recurrent mutations of DNMT3Ain 22 of AML patients and DNMT3A mutations wereassociated with poor outcome [17] Many of the reportedDNMT3Amutations mainly occurred at codon R882 in exon23 but the occurrence of DNMT3A mutations in MPNpatients is not well clarified

In the last few years the prevalence of DNMT3A NPM1or FLT3-ITD has been individually studied in some myeloidneoplasms mainly including AML or MDS Furthermoresome studies indicate that themutagenesis of these genesmaydiffer within different races and be associated with patientrsquosage Therefore in this study we put the three genes togetheralong with JAK2V617F to determine their mutational statusin a series of well-defined adult Chinese patients with MPNand explored their clinical significance also the associationbetween JAK2V617F mutation and DNMT3A NPM1 orFLT3-ITD mutation was also investigated

2 Materials and Methods

21 Patients and Samples A total of 120 newly diagnosedclassic MPN patients and 9 MDSMPN patients accordingto the World Health Organization (WHO) criteria [18] wereincluded in this study Enrollment took place between Jan-uary 2011 and December 2012 in Department of HematologyQiLu Hospital of Shandong University China Bone marrowor peripheral blood samples were collected at diagnosis Thestudy was approved by the Institutional Review Boards ofQiLu Hospital of Shandong University Informed consentwas obtained from each patient before being included inthis study The clinical characteristics of these subjects weresummarized in Table 1

22 DNA Extraction Leukocytes were separated from bonemarrow or peripheral blood samples using erythrocyte lysingsolution Genomic DNA was isolated using the TIANGENDNA isolation kit (TIANGEN China) according to themanufacturerrsquos protocol Store the DNA samples at minus80∘C forthe following polymerase chain reaction (PCR) amplification

23 PCR and Sequencing for DNMT3A NPM1 and FLT3 Asthe recurrentmutation points exon 23 ofDNMT3A and exon12 of NPM1 as well as FLT3 exons 11 and 12 were amplifiedby PCR then the PCR products were sequenced BrieflyPCR was performed in 25 120583L volume containing 125 120583L2x Taq PCR MasterMix (TIANGEN China) 75120583L RNase-free ddH2O 1 120583L forward and reverse primer respectivelyand 3 120583L genomic DNA The primers are shown as followsDNMT3A exon 23 forward 51015840-TCC TGC TGT GTG GTTAGA CG-31015840 reverse 51015840-TAT TTC CGC CTC TGT GGT TT-31015840 NPM1 exon 12 forward 51015840-GGTCTCTGTTCTTTCTGTTGATTTCC-31015840 reverse 51015840-CAACACATTCTTGGCAATAGA ACC T-31015840 FLT3 exon 11 forward 51015840-GCA ATT TAGGTA TGA AAG CCA GC-31015840 exon 12 reverse 51015840-CTT TCAGCA TTT TGA CGG CAA CC-31015840 The amplification wasdone with a DNA thermal cycler (BIO-RAD S1000 ThermalCycler) After denaturing at 94∘C for 3min the amplificationwas conducted for 35 cycles at 94∘C for 30 s 55∘C for 30 s and72∘C for 1min followed by reextension for 5min at 72∘C

The PCR products were loaded onto a 2 agarose gelcontaining ethidium bromide and electrophoretically sepa-rated After being purified the PCR products were directlysequenced on both directions using the ABI PRISM 3730xlanalyzer (Applied Biosystems Inc Foster City CA USA)to screen for the presence of mutations The samples withmutated FLT3-ITD mutated NPM1 or mutated DNMT3Awere used as positive controls

24 JAK2V617F Mutation Analysis We used the TaqManMGB probe joint real-time PCR to detect JAK2V617F muta-tion The TaqMan MGB probes and Mix were purchasedfrom Applied Biosystems PCR amplifications of DNA weredone in a total volume of 10120583L PCR mixture containing5 120583L TaqMan Universal PCR Master Mix 04 120583L forwardand reverse primer respectively 02 120583L FAM and VICfluorophore respectively 28 120583L RNase-free ddH2O and1 120583L genomic DNA The primer and TaqMan MGB probesequences are as follows forward primer 51015840-AAG CTT TCTCAC AAG CAT TTG GTT G-31015840 reverse primer 51015840-AGAAAG GCA TTA GAA AGC CTG TAG TT-31015840 probe1 FAM-TCC ACA GAA ACA TAC-MGB probe2 VIC-TCT CCACAG ACA TAC-MGB PCR amplification conditions were50∘C 10min 95∘C 30 s 95∘C 15 s and 62∘C 1min 45 cyclesPCR was performed with ABI 7500 Real-Time PCR system(Applied Biosystems)

25 Statistical Analysis Statistical analysis was performedusing the SPSS Statistical Analysis Software Differencesin JAK2V617F percentage age and peripheral blood cellscountswere accessed byChi-squared tests Fisherrsquos exact tests









3 Results















4 Discussion


























(a) FLT3 wild type




(c) NPM1 wild type


(d) NPM1 mutation








5 Conclusion






Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









3 Results















4 Discussion


























(a) FLT3 wild type




(c) NPM1 wild type


(d) NPM1 mutation








5 Conclusion






Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









4 Discussion


























(a) FLT3 wild type




(c) NPM1 wild type


(d) NPM1 mutation








5 Conclusion






Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

















(a) FLT3 wild type




(c) NPM1 wild type


(d) NPM1 mutation








5 Conclusion






Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



(a) FLT3 wild type




(c) NPM1 wild type


(d) NPM1 mutation








5 Conclusion






Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



5 Conclusion






Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Mutation Analysis of JAK2V617F, FLT3-ITD ...downloads.hindawi.com/journals/bmri/2014/485645.pdf · blasts[ , ].FLT-ITD(internaltandemduplicationofFLT) is the most