T h e
n e w e n gl a n d j o u r n a l
o f
m e d i c i n e
n engl j med 356;5 www.nejm.org february 1, 2007
459
original article
JAK2 Exon 12 Mutations in Polycythemia
Vera and Idiopathic Erythrocytosis
Linda M. Scott, Ph.D., Wei Tong, Ph.D., Ross L. Levine, M.D.,
Mike A. Scott, Ph.D., Philip A. Beer, M.R.C.P., M.R.C.Path.,
Michael R. Stratton, M.D., Ph.D., P. Andrew Futreal, Ph.D.,
Wendy N. Erber, M.D., Mary Frances McMullin, F.R.C.P., F.R.C.Path.,
Claire N. Harrison, M.R.C.P., M.R.C.Path., Alan J. Warren, F.R.C.Path., F.Med.Sci.,
D. Gary Gilliland, M.D., Ph.D., Harvey F. Lodish, Ph.D.,
and Anthony R. Green, F.R.C.Path., F.Med.Sci.
From the University of Cambridge (L.M.S.,
P.A.B., A.J.W., A.R.G.) and Addenbrooke’s
National Health Service Trust (M.A.S.,
W.N.E., A.J.W., A.R.G.) — both in Cam-
bridge, United Kingdom; Whitehead In-
stitute for Biomedical Research (W.T.,
H.F.L.) and Massachusetts Institute of
Technology (H.F.L.) — both in Cambridge,
MA; Brigham and Women’s Hospital and
Dana–Farber Cancer Institute, Harvard
Medical School (R.L.L., D.G.G.), and How-
ard Hughes Medical Institute, Harvard
Medical School (D.G.G.) — all in Boston;
Wellcome Trust Sanger Institute, Hinxton,
United Kingdom (M.R.S., P.A.F.); Queen’s
University, Belfast, Northern Ireland
(M.F.M.); and St. Thomas’ Hospital, Lon-
don (C.N.H.). Address reprint requests to
Dr. Anthony R. Green at the Department
of Haematology, Cambridge Institute for
Medical Research, Hills Rd., Cambridge
CB2 2XY, United Kingdom, or at arg1000@
cam.ac.uk.
Drs. Tong and Levine contributed equally
to this article.
N Engl J Med 2007;356:459-68.
Copyright © 2007 Massachusetts Medical Society.
A b s t r a c t
Background
The V617F mutation, which causes the substitution of phenylalanine for valine at
position 617 of the Janus kinase (JAK) 2 gene (JAK2), is often present in patients with
polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. How-
ever, the molecular basis of these myeloproliferative disorders in patients without
the V617F mutation is unclear.
Methods
We searched for new mutations in members of the JAK and signal transducer and
activator of transcription (STAT) gene families in patients with V617F-negative
polycythemia vera or idiopathic erythrocytosis. The mutations were characterized
biochemically and in a murine model of bone marrow transplantation.
Results
We identified four somatic gain-of-function mutations affecting JAK2 exon 12 in
10 V617F-negative patients. Those with a JAK2 exon 12 mutation presented with
an isolated erythrocytosis and distinctive bone marrow morphology, and several
also had reduced serum erythropoietin levels. Erythroid colonies could be grown
from their blood samples in the absence of exogenous erythropoietin. All such ery-
throid colonies were heterozygous for the mutation, whereas colonies homozygous
for the mutation occur in most patients with V617F-positive polycythemia vera.
BaF3 cells expressing the murine erythropoietin receptor and also carrying exon
12 mutations could proliferate without added interleukin-3. They also exhibited in-
creased phosphorylation of JAK2 and extracellular regulated kinase 1 and 2, as com-
pared with cells transduced by wild-type JAK2 or V617F JAK2. Three of the exon 12
mutations included a substitution of leucine for lysine at position 539 of JAK2. This
mutation resulted in a myeloproliferative phenotype, including erythrocytosis, in
a murine model of retroviral bone marrow transplantation.
Conclusions
JAK2 exon 12 mutations define a distinctive myeloproliferative syndrome that af-
fects patients who currently receive a diagnosis of polycythemia vera or idiopathic
erythrocytosis.
The New England Journal of Medicine
Downloaded from nejm.org at UQ Library on March 26, 2017. For personal use only. No other uses without permission.
Copyright © 2007 Massachusetts Medical Society. All rights reserved.
T h e
n e w e n gl a n d j o u r n a l
o f
m e d i c i n e
n engl j med 356;5 www.nejm.org february 1, 2007
460
T
he myeloproliferative disorders
comprise a spectrum of chronic hemato-
logic diseases that are likely to arise from
a mutant multipotent hematopoietic stem cell.
1,2
The V617F somatic mutation in the Janus kinase
(JAK) 2 gene (JAK2), which causes the substitution
of phenylalanine for valine at position 617, has
recently been found in the majority of patients
with polycythemia vera and in many with essen-
tial thrombocythemia or idiopathic myelofibro-
sis.
3-7
This gene encodes a cytoplasmic tyrosine
kinase. The mutation, which occurs in the JAK
homology 2 (JH2) negative regulatory domain,
increases JAK2 kinase activity and causes cyto-
kine-independent growth of cell lines and cul-
tured bone marrow cells. Mutant JAK2 transfected
into murine bone marrow cells produces eryth-
rocytosis and subsequent myelofibrosis in recipi-
ent animals,
3,8,9
suggesting a causal role for the
mutation.
Allele-specific polymerase chain reaction (PCR)
can be used to detect the V617F mutation in ap-
proximately 95% of patients with polycythemia vera
and in 50 to 60% of patients with essential throm-
bocythemia or idiopathic myelofibrosis.
4,10,11
The
mutation is also present in hematopoietic pro-
genitors committed to granulocytic or erythroid
differentiation
4,12
and in purified hematopoietic
stem cells from patients with polycythemia vera.
13
Many patients with polycythemia vera or idio-
pathic myelofibrosis are homozygous for the V617F
mutation, as a result of mitotic recombination af-
fecting chromosome 9p,
3-6
but homozygosity is
rare in patients with essential thrombocythemia.
12
The mutation occurs infrequently in patients with
myelodysplasia or acute myeloid leukemia but does
not occur in those with lymphoid tumors, epithe-
lial cancers, or sarcomas.
14-18
The JAK2 mutation allows for a distinction be-
tween two subtypes of idiopathic myelofibrosis
and essential thrombocythemia.
19-21
The phe-
notype of V617F-positive, but not V617F-nega-
tive, essential thrombocythemia resembles that
of polycythemia vera.
20
However, patients with
V617F-negative essential thrombocythemia do
have cytogenetic abnormalities, dysplastic mega-
karyocytes, and a risk of transformation to myelo-
fibrosis or acute myeloid leukemia, all of which
are features of a myeloproliferative disorder.
20
Activating mutations in the thrombopoietin re-
ceptor have been reported in 10% of patients with
V617F-negative idiopathic myelofibrosis
22
and in
a few patients with essential thrombocythemia.
23
However, the molecular basis of V617F-negative
polycythemia vera is unknown.
M e t h od s
Patient s
We recruited patients from Addenbrooke’s Hos-
pital in Cambridge, St. Thomas’ Hospital in Lon-
don, and Belfast City Hospital in Belfast (all in
the United Kingdom) and from those enrolled in
the Myeloproliferative Disorders Study of Harvard
University in Boston.
5
Diagnoses assigned by local
physicians were reviewed centrally and revised ac-
cording to established criteria for polycythemia
vera,
24
essential thrombocythemia,
25
and idiopath-
ic myelofibrosis.
26
The Addenbrooke’s National
Health Service Trust Research Ethics Committee
approved this study. Written informed consent was
obtained from each patient.
Mutation screening
The isolation of granulocytes and T lymphocytes
and hematopoietic colony assays were performed
as previously described.
4
Individual burst-forming
units and erythropoietin-independent erythroid
colonies were harvested into water and boiled.
Primers for the coding exons of JAK1, JAK2, JAK3,
the tyrosine kinase 2 gene (TYK2), and of two sig-
nal transducer and activator of transcription genes
(STAT5A and STAT5B) are listed at www.sanger.
ac.uk/genetics/CGP; all additional primers used
are listed in
Table 1
in the Supplementary Appen-
dix (available with the full text of this article at
www.nejm.org). We performed allele-specific PCR
using DNA from granulocytes or from total pe-
ripheral blood, an annealing temperature of 62°C,
JAK2 exon 12 control primers, and primers spe-
cific for the alleles containing the K539L muta-
tion (leading to the replacement of lysine at posi-
tion 539 with a leucine), the N542-E543del mutation
(causing the deletion of asparagine at position
542 and glutamic acid at position 543), the F537-
K539delinsL mutation (leading to the replace-
ment of phenylalanine at position 537 through
lysine at position 539 by a single leucine), or the
H538QK539L mutation (causing a substitution of
glutamine for histidine at position 538 and leu-
cine for lysine at position 539). We amplified DNA
from in vitro colonies using exon 12 primers and
sequenced or genotyped the PCR products using
digestion with AseI.
The New England Journal of Medicine
Downloaded from nejm.org at UQ Library on March 26, 2017. For personal use only. No other uses without permission.
Copyright © 2007 Massachusetts Medical Society. All rights reserved.
New JAK2 Mutations and Er ythrocy tosis
n engl j med 356;5 www.nejm.org february 1, 2007
461
Bone Marrow Biopsy
Bone marrow biopsy specimens from the iliac
crest were fixed in neutral buffered formalin. Some
were processed in paraffin and others in methyl-
methacralate after decalcification in 5.5% EDTA.
Sections (1 to 3 μm thick) were cut and visualized
using hematoxylin and eosin or Wright–Giemsa
stain. All stained sections were viewed under a
light microscope (Olympus-BX51) equipped with
a 10×-H26.5 ocular lens. Low-power (20×) and
high-power (40×) images were obtained with a
digital camera (Pixera Pro150ES) and Studio 3.0.1
software (Adobe Systems).
Site-directed mutagenesis and produc tion
of retrovirus
We introduced the mutations V617F, H538QK539L,
K539L, N542-E543del, and F537-K539delinsL into
murine Jak2 complementary DNA in a bicistron-
ic retroviral vector encoding green fluorescent
protein (MSCViresGFP),
8
using QuikChange site-
directed mutagenesis (Stratagene). The complete
nucleotide sequence of each retroviral vector was
confirmed before use. For the production of each
retrovirus, equal amounts of Jak2 retroviral vec-
tor and packaging plasmids (Ecopak) were com-
bined, incubated with FuGene (Roche) for 15 min-
utes, and then added to the human embryonic
kidney-cell line, 293T. The supernatants were har-
vested 48 hours later and were used to transduce
BaF3 cells expressing the murine erythropoietin
receptor (BaF3/EpoR cells)
27
or murine bone mar-
row cells.
BaF3- cell proliferation assays and Western
blot ting
BaF3/EpoR cells were maintained in RPMI-1640
medium containing 10% fetal-calf serum and
10% medium conditioned with WEHI-3B cells, as
a source of interleukin-3, and infected with ret-
roviral supernatants containing MSCViresGFP
vectors encoding mutant or wild-type Jak2. The
green fluorescent protein–positive population
from each transduction was purified by flow-
cytometric sorting 2 days later and was then ex-
panded in RPMI-1640 medium with 10% fetal-calf
serum and 10% WEHI-3B–conditioned medium
for 3 to 8 days. To assay for growth-factor hyper-
sensitivity, transduced BaF3/EpoR cells were cul-
tured in the absence of interleukin-3, and the num-
ber of viable cells was measured at days 2 and
4 with the use of trypan-blue exclusion. Data from
four independent experiments were combined in
analyses.
For immunoprecipitation and Western blot
studies, BaF3/EpoR cells expressing wild-type
or mutant Jak2 were starved for 4 to 5 hours in
RPMI-1640 medium containing 1% bovine serum
albumin and were then pelleted and frozen for
subsequent analysis. Cells stimulated with 10 U
per milliliter of erythropoietin for 10 minutes
served as a positive control. For the analysis of
Jak2 and Stat5, 3×10
7
cells were lysed in 10 mM
TRIS–hydrochloric acid (pH 7.4) with 150 mM
sodium chloride and 0.5% NP-40 buffer contain-
ing phosphatase and protease inhibitors. The pro-
tein supernatant was precipitated with anti-Jak2
antibody (Upstate Cell Signaling Solutions) or
anti-Stat5 antibody (Santa Cruz Biotechnology).
Precipitates were blotted with antibodies against
phosphorylated Stat5 (phosphotyrosine at posi-
tion 694) (Cell Signaling Technology), phospho-
tyrosine (4G10) (Upstate Cell Signaling Solu-
tions), Jak2, or Stat5 (Santa Cruz Biotechnology).
Alternatively, total cell lysates were resuspend-
ed in lithium dodecyl sulfate sample buffer (Invi-
trogen) and then blotted with antibodies against
phosphorylated extracellular regulated kinase 1
and 2 (Erk1 and Erk2) (phosphothreonine at po-
sition 202 and phosphotyrosine at position 204
in Erk) or against total Erk (Cell Signaling Tech-
nology).
Bone marrow tr ansplantation a ssay in mice
Bone marrow transplantation was performed as
previously described.
28
Briefly, retroviral superna-
tants were titrated by determining the percentage
of BaF3 cells that were positive for green fluores-
cent protein 48 hours after the introduction of the
retroviral vector. Supernatants containing equal
titers of wild-type Jak2 or V617F or K539L Jak2
were used to transfect bone marrow cells. BALB/c
donor mice were treated with 150 mg of 5-fluo-
rouracil per kilogram of body weight, and cells
harvested from femurs and tibias 7 days later were
cultured for 24 hours in transplantation medium
(RPMI-1640 medium, 10% fetal-calf serum, 6 ng
of murine interleukin-3 per milliliter, 10 ng of hu-
man interleukin-6 per milliliter, and 10 ng of mu-
rine stem-cell factor per milliliter). Bone marrow
cells were centrifuged at 2500 rpm for 90 min-
utes in the presence of 1 ml of retroviral superna-
tant and 10 μg of polybrene per 4×10
6
cells. Expo-
sure to retroviral supernatant and centrifugation
The New England Journal of Medicine
Downloaded from nejm.org at UQ Library on March 26, 2017. For personal use only. No other uses without permission.
Copyright © 2007 Massachusetts Medical Society. All rights reserved.
T h e
n e w e n gl a n d j o u r n a l
o f
m e d i c i n e
n engl j med 356;5 www.nejm.org february 1, 2007
462
were repeated 1 day later. Aliquots of 1×10
6
bone
marrow cells were resuspended in 0.7 ml of Hank’s
balanced salt solution and then injected into lethal-
ly irradiated BALB/c mice. Peripheral-blood counts
and cell morphology were evaluated for each re-
cipient 38 days after transplantation.
Statistical Analysis
We used an unpaired Student’s t-test to compare
demographic and laboratory features at the time
of diagnosis between patients with a V617F JAK2
mutation and those with a JAK2 exon 12 mutation
and to compare peripheral-blood counts among
AUTHOR:
FIGURE:
JOB: ISSUE:
4-C
H/T
RETAKE
SIZE
ICM
CASE
EMail
Line
H/T
Combo
Revised
AUTHOR, PLEASE NOTE:
Figure has been redrawn and type has been reset.
Please check carefully.
REG F
Enon
1st
2nd
3rd
Scott
1 of 4
02-01-07
ARTIST: ts
35605
33p9
A
B
F537-K539delinsL
H538QK539L
K539L
N542-E543del
Wild Type
Human
Mouse
Chicken
Xenopus
Zebrafish
JH2 domain
ATGAACCAAATGGTGTTTCACAAAATCAGAAATGAAGATTTGATATTTAAT
ATGAACCAAATGGTG––––––TTAATCAGAAATGAAGATTTGATATTTAAT
M N Q M V F H K I R N E D L I F N
ATGAACCAAATGGTGTTTCAATTAATCAGAAATGAAGATTTGATATTTAAT
M N Q M V – – L I R N E D L I F N
ATGAACCAAATGGTGTTTCACTTAATCAGAAATGAAGATTTGATATTTAAT
M N Q M V F Q L I R N E D L I F N
ATGAACCAAATGGTGTTTCACAAAATCAGA––––––GATTTGATATTTAAT
...PTSPTLQRPTHMNQMVFHKIRNEDLIFNESLGQGTF...
...QTSPTLQRHNNVMQMVFHKIRNEDLIFNESLGQGTF...
...PSSPTLQRHNHVMQSVFHKIRNEDLIFEESLGQGTF...
...SASPTLQRNNNVNQMVFHKIRNEDLHFLENLGQGTF...
...CAPSSEHRLL–VNQMIFHKIHREDLQTTEGLGQGTF...
M N Q M V F H L I R N E D L I F N
M N Q M V F H K I R – – D L I F N
T G TT N NN CA N AA N NN NA N
T G TT T CA NN N AA T CA GA A
T G TT T CA CN N AA T CA GA A
A T CA G AN AT N NN N NN TT N
T G TT T CA CA A AA T CA GA A
T G TT T CA CA A AA T CA GA A
T G TT T CA CA A AA T CA GA A
A T CA G AA AT G AA G AT TT G
T G TT N NN CA N AA N NN NA N
T G TT T CA NN N AA T CA GA A
T G TT T CA CN N AA T CA GA A
Granulocyte T Lymphocyte
Erythropoietin-
Independent Colony
F537-K539delinsL
(Patient 1)
H538QK539L
(Patient 4)
K539L
(Patient 5)
N542-E543del
(Patient 9)
Figure 1. Somatic Mutations of JAK2 Exon 12 in Patients with Polycythemia Vera or Idiopathic Erythrocytosis.
Panel A shows DNA-sequence traces from peripheral-blood granulocytes and T lymphocytes and from erythropoie-
tin-independent erythroid colonies. Nucleotides are indicated by capital letters, with N representing sites at which
wild-type and mutant nucleotides are apparent at the same position. The traces reveal four acquired mutations within
JAK2 exon 12 (indicated by arrowheads), often with low-level involvement in granulocytes. Panel B (top) shows the
alignment of wild-type and mutant exon 12 JAK2 alleles (shown in red) (nucleotides are indicated by capital letters and
amino acids by bold capital letters; dashes indicate the positions of deleted nucleotides). The amino acid alignment
across multiple species (Panel B, bottom) shows conservation of the mutated amino acids, indicated in red.
The New England Journal of Medicine
Downloaded from nejm.org at UQ Library on March 26, 2017. For personal use only. No other uses without permission.
Copyright © 2007 Massachusetts Medical Society. All rights reserved.
New JAK2 Mutations and Er ythrocy tosis
n engl j med 356;5 www.nejm.org february 1, 2007
463
mouse recipients of bone marrow cells express-
ing wild-type, V617F, or K539L Jak2. Fisher’s exact
test was used to compare frequencies of muta-
tion-positive erythroid colonies and of colonies ho-
mozygous for the mutation between patients with
the V617F mutation and patients with an exon 12
mutation.
R e s ult s
Somatic Mutations Affec ting JAK2 Exon 12
Of the 73 patients with polycythemia vera in our
original cohort, 2 did not have the V617F muta-
tion
4
and were studied further. In these two pa-
tients, mutations were not found in the coding
exons of JAK1, JAK3, TYK2, STAT5A, or STAT5B. How-
ever, both patients had alterations in JAK2 exon 12
that affected residues lying approximately 80 amino
acids before V617. One patient had a 6-bp in-frame
deletion affecting positions 1611 to 1616, resulting
in an F537-K539delinsL mutation. The second pa-
tient had a CAA→ATT mutation at positions 1614
through 1616, resulting in an H538QK539L mu-
tation (Fig. 1A). These mutations were acquired,
since they could be detected in peripheral-blood
granulocytes but not in T lymphocytes.
JAK2 exon 12 mutations were identified in eight
of an additional nine patients who received a di-
agnosis of V617F-negative polycythemia vera from
their local physicians. The mutations were fre-
quently present at low levels in granulocyte DNA
but were readily identifiable in clonally derived
erythropoietin-independent erythroid colonies
(Fig. 1A). In total, four exon 12 alleles were identi-
fied, all of which had changes affecting conserved
residues between K537 and E543 (
Fig. 1
); three
of the alleles (in Patients 1 through 6) contained
a K539L substitution (Fig. 1B). JAK2 exon 12 muta-
tions were not detected by sequencing granulo-
cyte DNA from 55 patients with V617F-positive
polycythemia vera, 25 patients with V617F-nega-
tive essential thrombocythemia, and 12 patients
with V617F-negative cases of idiopathic myelo-
fibrosis
14
(and data not shown). Since mutation-
bearing granulocytes may represent only a minor-
ity of peripheral blood granulocytes,
4,10,29
DNA
from an additional 90 patients with V617-negative
essential thrombocythemia was screened using
sensitive allele-specific PCR assays for each exon
12 mutation, but no mutations were detected (data
not shown). These results indicate that JAK2 exon
12 mutations occur only in patients with a myelo-
Table 1. Clinical Features of Patients with JAK2 Exon 12 Mutations at Diagnosis.*
Patient
No. Sex Age JAK2 Mutation
Hemoglobin
Level
White-Cell
Count†
Platelet
Count
Serum
Erythropoietin
Level‡
Cytogenetic
Karyotype Splenomegaly
Erythroid
Colonies
Indpendent
of Erythropoietin Diagnosis
yr g/liter ×10
−3
/mm
3
IU/liter
1 M 50 F537-K539delinsL 234 7.9 450 ND Deletion in 20q Present on palpation ND PV
2 F 59 F537-K539delinsL 179 14.4 294 9.9 Deletion in 20q Absent ND PV
3 F 50 F537-K539delinsL 201 5.1 308 <2.5 Normal Absent Yes IE
4 M 27 H538QK539L 211 11.1 286 <5.0 Normal Present on ultraso-
nography
Yes PV
5 F 62 K539L 181 7.5 301 <2.5 Normal Absent Yes IE
6 M 54 K539L 207 8.6 Normal 8.4 ND Absent ND IE
7 F 17 N542-E543del 220 5.6 310 <1.0 Normal Present on palpation Yes PV
8 F 29 N542-E543del 198 5.8 285 2.0 Normal Absent Yes IE
9 F 57 N542-E543del 199 4.4 204 5.0 ND Present on ultraso-
nography
Yes PV
10 M 33 N542-E543del 225 12.1 425 ND Normal Present on palpation ND PV
* The normal range of serum erythropoietin before therapy is 5 to 25 IU per liter. The diagnosis was determined on the basis of criteria of the Polycythemia Vera Study Group.
24
The pres-
ence or absence of erythroid colonies independent of erythropoietin were assessed in 2005 or 2006. The deletion in chromosome 20q was confirmed by microsatellite polymerase chain
reaction of DNA from peripheral-blood granulocytes, bone marrow cells, or both (data not shown). PV denotes polycythemia vera, IE idiopathic erythrocytosis, and ND not determined.
† Neutrophil counts were 8960 per cubic millimeter for Patient 2, 9000 per cubic millimeter for Patient 4, and 7300 per cubic millimeter for Patient 10.
‡ The serum erythropoietin level was measured after venesection therapy in Patient 2 and before therapy in all other patients.
The New England Journal of Medicine
Downloaded from nejm.org at UQ Library on March 26, 2017. For personal use only. No other uses without permission.
Copyright © 2007 Massachusetts Medical Society. All rights reserved.
