Hematopathology / High Percentage of JAK2 Exon 12 Mutation
High Percentage of JAK2 Exon 12 Mutation in Asian
Patients With Polycythemia Vera
Yu-Min Yeh, MD,1* Yi-Lin Chen, MS,2* Hsieh-Yin Cheng,2 Wu-Chou Su, MD,1
Nan-Haw Chow, MD,2 Tsai-Yun Chen, MD,1† and Chung-Liang Ho, MD, PhD2†
Key Words: JAK2V617F; Exon 12 mutation; Polycythemia vera; Taiwan
DOI: 10.1309/AJCPK7KGOWPHYWM0
Abstract
We examined the occurrence of JAK2V617F
and JAK2 exon 12 mutations in a clinical cohort of
polycythemia vera (PV) in Taiwan. Of 22 patients
with PV, 17 (77%) had the V617F mutation, and all
5 V617F-negative patients (23%) had the exon 12
mutation. We found 3 different exon 12 mutations:
3 N542-E543del, 1 F537-K539delinsL, and 1 novel
mutation, I540-E543delinsKK. Patients with V617F
showed significantly higher WBC and platelet counts
at diagnosis than patients with exon 12 mutations
(P = .021 and P = .038, respectively). We report a
surprisingly high incidence of exon 12 mutations in
Taiwanese patients with PV, a result quite different
from reports in the Western literature (P = .001). Our
data suggest that exon 12 mutation of JAK2 in patients
with PV may have an uneven geographic distribution.
A clinical laboratory providing the V617F test alone
may risk missing a substantial number of patients with
PV in areas with a high incidence of exon 12 mutation.
The chronic myeloproliferative disorders are clonal
hematopoietic stem cell malignancies with 3 main subtypes:
polycythemia vera (PV), essential thrombocytosis, and idiopathic myelofibrosis. PV is characterized by increased RBC
proliferation in the absence of erythropoietin and proliferation
of myeloid lineages usually is noted, too.1 A gain-of-function
mutation of Janus kinase 2 (JAK2) V617F, is identified in
about 95% of patients with PV and about 50% of patients
with essential thrombocytosis and idiopathic myelofibrosis.2-5
JAK2 belongs to the Janus family of kinases and contains 7
JAK homology domains (JH1 to JH7). The JH2 domain, the
pseudokinase domain, has no kinase activity but suppresses
JAK2 kinase activity.5 The V617F mutation, involving exon
14, which encodes a part of the JH2 autoinhibitory domain of
the JAK2 kinase, relieves autoinhibition by the pseudokinase
domain and causes constitutive tyrosine kinase activation.
Recently, Scott and coworkers6 described 4 different mutations of JAK2 exon 12 in V617F-negative patients with PV or
idiopathic erythrocytosis.6 It has been shown that JAK2 exon
12 mutations can activate erythropoietin signaling pathways.
While these findings have been confirmed by many studies
from Western countries,7,8 there are no reports from Asian
countries about the prevalence of the JAK2 exon 12 mutation in patients with PV. In the present study, we determined
the prevalence of JAK2V617F and JAK2 exon 12 mutations in
patients with PV in Taiwan.
Materials and Methods
Patients and Samples
We enrolled patients with a diagnosis of PV at National
Cheng Kung University Hospital, Tainan, Taiwan (NCKUH),
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DOI: 10.1309/AJCPK7KGOWPHYWM0
© American Society for Clinical Pathology
Hematopathology / Original Article
from 1993 to March 2009, and still followed up in our hematologic outpatient clinic. The diagnosis of PV was established
according to the 2002 World Health Organization criteria.9
The study included 22 patients. Clinical information and
the CBC data at diagnosis were obtained by retrospectively
reviewing medical records. The presence or absence of
splenomegaly at diagnosis was documented by abdominal
sonography. For 17 patients, there were posttreatment serum
erythropoietin levels. No initial erythropoietin level was
available because the test was not available to our hospital
for routine diagnosis until late 2009. This study was approved
by the institutional review board of NCKUH and proceeded
according to the Helsinki Declaration. Peripheral blood for
JAK2 mutation analysis was obtained from patients after their
informed consent was obtained.
JAK2V617F Mutation Analysis
Total DNA was isolated from buffy coat cells taken from
peripheral blood using a kit (QIAamp DNA Mini Kit, Qiagen,
Hilden, Germany) according to the manufacturer’s instructions. Allele-specific polymerase chain reaction (PCR) was
performed using 80 ng of genomic DNA as the template in a
35-cycle PCR reaction at an annealing temperature of 58°C, as
previously described.3 The mutant allele yields a 203-base-pair
(bp) PCR product (sensitivity of mutant allele detection ≤1%).
JAK2 Exon 12 Mutation Screening
For exon 12 mutation screening, 80 ng of genomic DNA
was amplified by specific primers designed to amplify a region
of 453 bp containing the 128 bp of the exon 12 sequence of
JAK2.2 PCR products were directly sequenced in both directions
on an ABI 3730 DNA Analyzer using the BigDye Terminator
Sequencing kit (Applied Biosystems, Foster City, CA).
Statistical Analysis
A Mann-Whitney U test was used to compare the peripheral RBC, WBC, and platelet counts at diagnosis between
patients with a JAK2 V617F mutation and patients with a
JAK2 exon 12 mutation. All probability values were 2-tailed.
The rate of exon 12 mutation of patients with PV in NCKUH
was compared with those reported in the Western literature by
means of the χ2 test. All values of P less than .05 were considered significant. The analyses were performed using SPSS
13.0 statistical software (SPSS, Chicago, IL).
Results
The study included 22 patients with PV (10 women and
12 men; age range, 32-84 years; mean age, 58.5 years). Patient
age and laboratory data at diagnosis and JAK2 mutation profiles are given in ❚Table 1❚. Of the 22 patients, all except 1
❚Table 1❚
Patient Age and Laboratory Data at Diagnosis and JAK2 Mutation Profiles*
JAK2 Mutation
Case No./ Sex/Age
at Diagnosis (y)
Duration of
Follow-up (y)
Year of
Diagnosis
Hemoglobin WBC Count Platelet Count
EPO†
(g/dL)
(/μL)
(× 103/μL)
Splenomegaly (IU/L)
V617F Exon 12
1/F/67
2/M/32
3/F/57
4/M/33
5/M/63
6/F/76
7/M/77
8/F/63
9/M/59
10/M/64
11/F/35
12/M/68
13/M/43
14/F/84
15/F/66
16/F/58
17/M/80
18/M/69
19/M/55
20/F/50
21/F/63
22/M/47
9.3
7.6
1.8
4.3
1.3
3
1.6
1.6
3.9
3.9
1.1
0.6
7.3
0.2
1.1
16
0.1
1.3
4.2
2.3
1.4
4.3
1999
2001
2007
2004
2007
2006
2007
2007
2005
2005
2008
2008
2001
2009
2008
1993
2009
2006
2005
2006
2007
2004
20.5
17.1
20.6
22.7
20.3
17.4
18.4
18.1
21.7
17.7
20.4
20.9
21.8
17.9
18.8
23.2
17.9
20.1
21.9
20.6
16.9
18.4
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
–
–
–
–
–
11,000
8,800
8,000
19,700
6,600
17,100
18,300
36,000
13,200
11,400
13,400
17,000
14,200
21,900
10,600
15,000
17,000
7,100
16,400
5,900
6,500
8,100
375
342
232
317
299
1,234
289
641
984
525
547
417
468
235
689
547
501
176
194
394
217
481
+
+
–
+
–
–
–
–
–
+
+
–
+
+
–
+
+
–
+
–
–
+
1.9
6.3
2.3
1.6
NA
NA
43.4
16.5
NA
<1.0
1.2
6.4
3.3
2.7
4.3
19.3
4.4
NA
NA
1.6
3.1
3.1
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
F537-K539delinsL
N542-E543del
N542-E543del
I540-E543delinsKK
N542-E543del
EPO, erythropoietin; NA, not available; +, positive; –, negative.
* Values are given in conventional units. Conversions to Système International are as follows: hemoglobin concentration (g/L), multiply by 10.0; WBC count (× 109/L), multiply
by 0.001; platelet count (× 109/L), multiply by 1.0. The mean age and mean selected laboratory values for the JAK2V617F+ group were as follows: age, 60.3 years; hemoglobin
concentration, 19.7 g/dL; WBC count, 15,200/μL; and platelet count, 508 × 103/μL; and for the JAK2V617F+ group, 56.8, 19.6 g/dL, 8,800/μL, and 292 × 103/μL, respectively.
† Obtained after treatment. The normal range for the serum EPO level is 3.7-31.5 IU/L.
© American Society for Clinical Pathology
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(case 18) were alive and under regular follow-up as of this
writing. The mean duration of follow-up, which was defined
from the time of diagnosis until March 2009 or the time of
death, was 3.7 years (range, 0.2-16 years). Patients received
phlebotomy treatment or hydroxyurea therapy. The JAK2V617F
mutation was detected in 17 (77%) of 22 patients. All 5
patients negative for the JAK2V617F mutation (5/22 [23%])
had exon 12 mutations. Three different exon 12 mutations
were found: 3 N542-E543del, 1 F537-K539delinsL, and 1
I540-E543delinsKK ❚Figure 1❚. All patients underwent bone
marrow biopsy. ❚Image 1❚ shows the representative histologic
features of PV for case 6. The marrow is hypercellular with
proliferation in the erythroid, granulocytic, and megakaryocytic lineages. The megakaryocytes show increased polymorphism, some of which are hyperlobulated. There is no
maturation defect or excess of blasts.
The mean age at diagnosis for the JAK2V617F mutation
group was 60.3 years and for the JAK exon 12 mutation group
was 56.8 years. At diagnosis, mean hemoglobin levels were
almost identical in the 2 groups, 19.7 g/dL (197 g/L) and 19.6
g/dL (196 g/L), respectively; however, the WBC and platelet counts were higher in patients with the V617F mutation
(WBC count, 15,200/μL vs 8,800/μL [15.2 vs 8.8 × 109/L];
platelet count, 508 vs 292 × 103/μL [508 vs 292 × 109/L]).
The Mann-Whitney U test comparing WBC and platelet
counts at diagnosis between these groups showed statistically
significant differences: P = .021 and P = .038, respectively.
The findings were consistent with those in a previous report,8
showing that isolated erythrocytosis was significantly higher
in patients with exon 12 mutations than in those with V617F+
PV. No female predominance was seen in our small group of
patients with exon 12 mutations.
In 1 patient with an exon 12 mutation (case 18; F537K539delinsL), disease evolution was shown. In this patient,
myelofibrosis developed 1.3 years after the diagnosis of PV,
and the patient later died of pneumonia. There are no data that
suggest that the V617F or exon 12 mutation carries a higher
risk for disease evolution. The allele burden may be responsible for this. More research on the prognostic significance of
exon 12 mutations is necessary.
❚Figure 1❚ Sequencing data for JAK2 exon 12 in patients
with polycythemia vera or idiopathic erythrocytosis. A, DNA
sequence trace of JAK2 exon 12 from peripheral blood buffy
coat cells of a healthy person and JAK2V617F mutation–negative
patients with polycythemia vera. The traces reveal 3 mutations
within JAK2 exon 12, and arrows indicate the beginning of
the mutated codons. B, Sequence of amino acids encoded
by exon 12 without (wild-type) and with mutations.
A
AA A T A T CA A A T C T T CA T T T C T GA T T T T G T G AAA CA C CA T
Wild-type
AAA T A T CA AA T C T T CA T T T G T G A T T A AC A C C A T T T G G T T
F537-K539delinsL
(Patient 18)
AA A T A T CA A A T C T C T G A T T T T G T G AAA CA C C A T T T G G T T
N542-E543del
(Patient 19)
A AA T A T C AA A T C T T CA T T T C T G A T T T T G T G AA A C A CC A T
I540-E543delinsKK
(Patient 21)
(Anti-sense)
B
Wild-type
F537-K539delinsL
N542-E543del
I540-E543delinsKK
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ATGAACCAAATGGTGTTTCACAAAATCAGAAATGAAGATTTGATATTTAAT
M N Q M V F H K I R N E D L I F N
ATGAACCAAATGGTG
M N Q M V
TTAATCAGAAATGAAGATTTGATATTTAAT
L I R N E D L I F N
ATGAACCAAATGGTGTTTCACAAAATCAGA
M N Q M V F H K I R
ATGAACCAAATGGTGTTTCACAAAA
M N Q M V F H K K
AAA
K
GATTTGATATTTAAT
D L I F N
AAGATTTGATATTTAAT
D L I F N
© American Society for Clinical Pathology
Hematopathology / Original Article
A
B
❚Image 1❚ Representative bone marrow biopsy specimen in polycythemia vera. The specimen shows hypercellularity with
prominent proliferations of erythroid and megakaryocytic lineages. There is no maturation defect or excess of blasts (A, H&E,
×200; B, H&E, ×400).
Discussion
The JAK2V617F mutation rate (77%) that we found in
patients with PV is consistent with the rates in other reports,
which range from 65% to more than 95%,2-4 and close to those
recently reported in Taiwanese (85%)10 and mainland Chinese
patients (73.7%).11 Differences in reported rates are likely
because of differences in diagnostic precision and assay sensitivity. Different exon 12 mutation rates in V617F– PV or idiopathic erythrocytosis have been reported, ranging from 11.6%
to 100%,6,8,12-17 but the exon 12 mutation rates in all patients
with PV according to the literature were low, from about
1.9% to 5% ❚Table 2❚.6,12-14 In contrast with these low exon
12 mutation rates, we found a high percentage, 23%, of exon
12 mutations in patients with PV. Previous studies on PV in
❚Table 2❚
Exon 12 Mutation Rates in Patients With PV Reported
in the Literature
Reference
No. of
Exon 12
Mutations
Exon 12
Mutation/
V617F– PV*
Exon 12
Mutation/
Total PV*
Scott et al6
Martínez-Avilés et al16
Pardanani et al14
Pietra et al8
Kouroupi et al15
Williams et al13
Butcher et al12
Bernardi et al17
Present study
2
4
5
17
8
3
3
5
5
2/2 (100)
4/20 (20)
5/6 (83)
17/37 (46)
8/26 (31)
3/10 (30)
3/3 (100)
5/47 (11)
5/5 (100)
2/73 (3)
NA
5/220 (2.3)
NA
NA
3/157 (1.9)
3/62 (5)
5/190 (2.6)
5/22 (23)
NA, not available; PV, polycythemia vera.
* Numbers in parentheses are the percentages.
Chinese descendants showed 85%10 and 73.7%11 positive rates
for the JAK2V617F mutation. It would be interesting to know
the exon 12 status for their V617F– cases. Although more data
from the general population of Asia are needed to confirm their
high exon 12 mutation rate, speculating about the reasons for
different exon 12 mutation rates between Asian and Western
populations is interesting. Whether this difference is attributable simply to ethnic differences, as is the higher epidermal
growth factor receptor somatic mutation rate in Asians with
non–small cell lung cancer,18 or to some as yet unidentified
environmental or acquired factors is worth further study. The
high mutation rate in the present study not only shows exon 12
mutation data in 1 Asian population, but also provides evidence
for the necessity of the concomitant screening for V617F and
exon 12 mutations of JAK2 in patients with PV.
In this study, we identified 5 patients with 3 different mutations of JAK2 exon 12: N542-E543del, F537K539delinsL, and I540-E543delinsKK. The first 2 mutations
had been reported before,6,8 whereas the third has not yet been
described. Since the first report of gain-of-function mutations
of JAK2 exon 12 in patients with V617F– PV in 2007,6 12
different JAK2 exon 12 mutations in 65 patients have been
reported17; the most frequently detected are N542-E543del,
F537-K539delinsL, and E543-D544del.8 The most common
of the 5 JAK2 exon 12 mutations in the present study (3 of
5 patients [60%]) was N542-E543del, which was consistent
with previous reports.8 One of our patients had a new mutation, I540-E543delinsKK, which belongs to the category with
a deletion of glutamic acid 543 (E543del), similar to I540E543delinsMK.12,19 Our data confirmed the exon 12 mutation
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cluster within the region covered by codons 537-543 and that
deletions are far more frequent in cases of exon 12 mutation,
as previously reported.6,14
Our study had several limitations. First, the number of
cases examined was relatively small, and all patients were
from Taiwan. However, we would like to point out that the
positive rate of exon 12 mutations in our patients with PV is
very high (5 of 22) compared with the cumulative ratio from
the literature (18 of 702).6,12,14,17,20 The P value is .001, and it is
unlikely that our observation is merely a sampling bias due to
a relatively small sample. Nevertheless, more cases and more
reports from different parts of East Asia are needed to confirm
the high mutation rate of exon 12 in Taiwan and other parts
of East Asia. Second, instead of quantitative real-time PCR,
allele-specific PCR amplification of the mutant allele was
used to detect the V617F mutation. The advantage was higher
sensitivity, but this precluded the availability of homozygosity
data for the V617F mutation. Third, although we used direct
sequencing to detect exon 12 mutations, buffy coat cells from
peripheral blood were used to extract DNA without isolating
granulocytes. Homozygosity data were still lacking in these
cases. Because exon 12 mutations are irregularly allocated
among several nucleotides, it is difficult to detect all mutations
with allele-specific PCR. Although allele-specific PCR is more
sensitive than direct sequencing, it did not limit the sensitivity
for detecting mutations, because no patient in our study was
negative for V617F and exon 12 mutations.
We reported the JAK2V617F mutation rate and described
the high frequency of exon 12 mutation in patients with PV
in Taiwan. The data indicated an uneven geographic distribution of exon 12 mutation and exemplified the importance
of JAK2 exon 12 analysis in a clinical laboratory for the
diagnosis of PV.
From the 1Section of Hematology/Oncology, Department of
Internal Medicine; and 2Department of Pathology, College of
Medicine, National Cheng Kung University, Tainan, Taiwan.
Supported by grants NCKU-20070048 and NCKU-20100189
from National Cheng Kung University Hospital (Y.-L. Chen).
Address reprint requests to Dr T.-Y. Chen: Dept of
Internal Medicine, National Cheng Kung University Hospital,
138 Sheng-Li Rd, Tainan 704, Taiwan; or Dr C.-L. Ho, Dept
of Pathology, National Cheng Kung University Hospital, 138
Sheng-Li Rd, Tainan 704, Taiwan.
* Contributed equally to this manuscript.
† Contributed equally to this manuscript.
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© American Society for Clinical Pathology