JAK2, CALR, and MPL mutation spectrum in

Published Ahead of Print on November 14, 2014, as doi:10.3324/haematol.2014.115113.
Copyright 2014 Ferrata Storti Foundation.
JAK2, CALR, and MPL mutation spectrum in Japanese
myeloproliferative neoplasms patients
by Shuichi Shirane, Marito Araki, Soji Morishita, Yoko Edahiro, Hiraku Takei, Yongjin Yoo,
Murim Choi, Yoshitaka Sunami, Yumi Hironaka, Masaaki Noguchi, Michiaki Koike,
Naohiro Noda, Akimichi Ohsaka, and Norio Komatsu
Haematologica 2014 [Epub ahead of print]
Citation: Shirane S, Araki M, Morishita S, Edahiro Y, Takei H, Yoo Y, Choi M, Sunami Y, Hironaka Y,
Noguchi M, Koike M, Noda N, Ohsaka A, and Komatsu N. JAK2, CALR, and MPL mutation spectrum
in Japanese myeloproliferative neoplasms patients.
Haematologica. 2014; 99:xxx
doi:10.3324/haematol.2014.115113
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JAK2, CALR,
and
MPL
mutation
spectrum
in
Japanese
myeloproliferative
neoplasms patients
Shuichi Shirane1, Marito Araki2, Soji Morishita2, Yoko Edahiro1, Hiraku Takei1,3,
Yongjin Yoo4, Murim Choi4, Yoshitaka Sunami1, Yumi Hironaka1, Masaaki Noguchi5,
Michiaki Koike6, Naohiro Noda7, Akimichi Ohsaka2, Norio Komatsu1
1Department
of Hematology, Juntendo University School of Medicine, Tokyo, Japan;
2Department
of Transfusion Medicine and Stem Cell Regulation, Juntendo University
Graduate School of Medicine, Tokyo, Japan; 3Department of Life Science and
Medical Bioscience, Waseda University, Tokyo, Japan; 4Department of Biomedical
Sciences, Seoul National University College of Medicine, Seoul, Korea; 5Department
of Hematology, Juntendo Urayasu Hospital, Chiba, Japan; 6Department of Hematology,
Juntendo Shizuoka Hospital, Shizuoka, Japan;
7Biomedical
Research Institute,
National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki,
Japan
Corresponding
Author:
Norio Komatsu, Department of Hematology, Juntendo
University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421 Japan;
e-mail: [email protected]
Letter to the Editor
Recurrent somatic mutations in the JAK2, MPL, and CALR genes have been
described in patients diagnosed with Philadelphia-negative myeloproliferative
neoplasms (MPN), including polycythemia vera (PV), essential thrombocythemia
(ET), and primary myelofibrosis (PMF). These mutations are generally mutually
exclusive, and their profiles in different disease entities are diverse. In PV,
JAK2
mutations exist in approximately 95% of patients. However, in ET and PMF patients,
,
, and
JAK2 CALR
MPL
mutations are present at frequencies of approximately 60%,
20%, and 5%, respectively1.
To further understand MPN pathogenesis associated with the ET and PMF
induced
by
different
gene
alterations, classification
and
epidemiological
examination of patients according to gene alterations have been performed. In ET,
the CALR mutation is associated with a lower hemoglobin level, higher platelet count,
lower leukocyte count, and younger age compared with the JAK2V617F mutation2-5;
similar characteristics have been observed in PMF patients6, 7. The CALR mutation is
also associated with male predominance2, 5, lower thrombosis risk4, 6, and better
overall survival6; however, these characteristics are not always evident and are
diverse in some cases. The same gender ratio has been reported in a Chinese cohort
of ET patients with JAK2 and CALR mutations4. The variation between cohorts in the
published data most likely reflects different genetic backgrounds in the different
ethnic groups that were studied. In addition, because all analyses have been
performed in Caucasian populations, with the exception of one study from China4,
the epidemiological evidence regarding the Asian population is limited.
Here, we studied a Japanese MPN cohort that was previously
characterized with respect to the JAK2V617F mutation; the cohort consisted of 66
PV, 112 ET, and 23 PMF patients, as defined by the 2008 World Health Organization
(WHO) criteria8. Clinical and laboratory parameters were obtained at the time of
first diagnosis or when genomic DNA samples were collected. This study was
conducted in accordance with the Declaration of Helsinki and was approved by the
ethics committee of Juntendo University School of Medicine (IRB#2012208
#2013020).
and
All patient specimens that were previously analyzed for the JAK2 mutation8
were assessed for
and
CALR
MPL
mutations using polymerase chain reaction
(PCR)-based assays and subsequent deep-sequencing. In addition, the specimens
that exhibited a
V617F mutant allele frequency below 10% in the previous
JAK2
study were re-evaluated via deep sequencing (see supplemental data). Re-evaluation
identified 1 PV and 2 ET patients as negative for the
PCR-based assay previously identified as
JAK2
mutation whom a
V617F-positive with a low allele
JAK2
frequency. Conversely, JAK2 mutations in MPN patients who were negative for JAK2,
, and
MPL
CALR
mutations by PCR-based assays were not identified by deep
sequencing (see below for MPL and CALR mutation detection). Thus, JAK2 mutations
were found in 64 (97%) PV (including 3 exon 12 mutations), 61 (54.5%) ET, and 11
(47.8%) PMF patients in this cohort (Figure 1A).
The
W515K/L mutation was assessed using a newly developed
MPL
allele-specific PCR technique called dual amplification refractory mutation system
PCR (DARMS-PCR) and subsequent capillary electrophoresis9. All identified
MPL
mutations were further verified using deep sequencing. In addition, by screening
MPN patients who were previously negative for JAK2, MPL, and CALR mutations by
PCR-based assays, we identified MPLW515K/L mutations below the detection limit
of DARMS-PCR as well as other
MPL
mutations (MPLW515R). Collectively, the
W515K/L/R mutation was identified in 7 (6.3%) ET and 1 (4.3%) PMF patients
MPL
(Figure 1A, Table 1), which was similar to the frequencies of 3-8.3% that were
found in Caucasian cohorts2, 3, 5-7, 10-13 but different from those in a Chinese cohort
with a substantially lower frequency (1.2%)4. We noted that one ET patient
exhibited both the
W515K (allele frequency 50.7%) and W515L (allele
MPL
frequency 2.5%) mutations.
The CALR mutation on exon 9 was examined using our in-house fragment
analysis method (see supplemental data). All identified
CALR
mutations were
confirmed by deep sequencing.
CALR
mutations were identified in 22 (19.6%) ET
and 7 (30.4%) PMF patients (Figure 1A), which was similar to the reported
frequency in ET (15.5 to 28%)2-6 and PMF (25%) patients7. In contrast, one study
examined a Cyprus cohort and identified a frequency of 8.7%10, but patients with
thrombocytosis were not classified according to the WHO 2008 criteria. Unlike the
MPL
mutation, the CALR mutation was present at similar frequencies in ET patients
in both Japanese (19.6%) and Chinese (22.7%)4 cohorts.
CALR
mutations (n=29) were present in the following distribution: 11 type
1 (c.1092_1143del), 8 type 2 (c.1154_1155insTTGTC), 1 type 4 (c.1102_1135del), 1
type
22
(c.1120_1123del),
1
type
28
(c.1131_1152del),
2
type
33
(c.1154_1155insATGTC), and 1 type 34 (c.1154delinsCTTGTC) mutation, as well as
four
novel
mutations
c.1153_1154insTCTGT,
(type
and
42-45;
c.1110_1133del,
c.1146_1147del;1153_1154del)
c.1126_1144del,
(summarized
in
supplemental Table 2). The CALR mutation in PMF patients is limited to types 1 and
2, and the type 1 mutation (n=6) is more frequent than the type 2 mutation (n=1),
as observed in a Caucasian PMF cohort3. All novel CALR mutations generate a frame
shift that converts the C-terminal amino acids from negatively to positively charged,
as is the case for other mutations (supplemental Table 2).
Although
,
JAK2
, and
MPL
CALR
mutations have been proposed to be
mutually exclusive, we identified 1 ET patient with
JAK2
mutations and 1 ET patient with
CALR
V617F and
JAK2
V617F and
W515L
MPL
mutations, which was
consistent with recent reports that described a rare concomitant mutation of these
gene mutations in Caucasian7, 14 and Chinese4 cohorts. Finally, patient specimens
that were negative for
V617F,
JAK2
W515K/L, and
MPL
CALR
exon 9 mutations by
PCR-based assays were analyzed by deep sequencing of all the JAK2, MPL, and CALR
exons (see supplemental data). This analysis identified 22 (19.6%) ET and 4
(17.4%) PMF “triple-negative” patients (Figure 1A).
We compared the hematological and clinical features of patients who were
classified according to mutation status (Table 1), with the exception of 2 ET patients
who harbored concurrent JAK2 and MPL or CALR mutations (see above). In the ET
patients, compared with the JAK2V617F mutation, the presence of the CALR or MPL
mutation was associated with lower leukocyte and higher platelet counts (Table 1).
The
CALR
mutation was also associated with a lower red blood cell count. These
hematological features are consistent with the features reported for different ethnic
groups2-5. We observed a trend of male dominance among the ET patients with CALR
mutations (male/female=13/8) compared with the patients with
JAK2
mutations
(male/female=24/35), which is consistent with findings in Caucasian cohorts but
inconsistent with findings in a Chinese cohort4.
We determined that the triple-negative ET patients (mean age 45.0 years
old) were strikingly younger than the patients with other genotypes (Table 1, Figure
1B). Adjusted p-values for multiple comparisons of ages between triple-negative and
other genotypes such as mutated
,
, or
JAK2 CALR
MPL
by Tukey-Kramer test were
<0.001, 0.015, and 0.037, respectively. The mean age of the triple-negative ET
patients exhibited a wide variation between the cohorts, ranging from 42 to 53
years old3-5. The difference in age between the triple-negative patients and patients
with other genotypes was more than 14 years, which was uniquely observed in our
cohort. In other cohorts, the difference in the ages of the triple-negative patients
with the youngest age and patients with other genotypes with the second youngest
age was, at most, 5 years15. Despite a lack of known clonal gene mutations, the
triple-negative patients’ bone marrow biopsies indicated apparent proliferation of
megakaryocytes with a large and mature morphology, and their clinical
characteristics corresponded to the WHO 2008 criteria for ET. Although it was a
small cohort, we observed a very similar phenomenon in PMF patients; the
difference in the ages of the triple-negative patients and patients with other
genotypes was more than 10 years (Table 1). Patients exhibiting myelofibrosis with
no clonal mutations (“triple-negative”) were diagnosed as PMF by excluding other
diseases such as myelodysplasia through confirming no dysplasia in erythroid
and/or myeloid lineages on bone marrow biopsy, chronic myeloid leukemia through
defining Bcr-Abl negativity by FISH or PCR, and other diseases including
autoimmune disorders through examining clinical records. This finding suggests
that triple-negative ET and PMF patients in Japan have a distinct genetic
background that facilitates the acceleration of disease onset compared with the
Caucasian population.
In summary, we have shown that the mutation profile of our Japanese
cohort of ET and PMF patients is comparable to that of Caucasian cohorts; however,
the frequency of the
MPL
mutation differs between Asian populations, as
demonstrated by the differences found between our cohort and a Chinese cohort.
We identified four novel
CALR
mutations, all of which generate an altered
C-terminus sequence that is commonly observed in patients with other
CALR
mutations, which implies that they are genuine mutations. The triple-negative ET
and PMF patients were significantly younger than the patients with other genotypes
in our cohort. The magnitude of this difference in Japan is much larger than that in
other cohorts, which implies the presence of ethnic differences in ET and PMF
development in triple-negative cases. Further genetic analysis would aid in
elucidating the genetic factors associated with ET development other than
,
JAK2
, and CALR mutations.
MPL
Acknowledgments
We thank Kazuhiko Ikeda (Fukushima Medical University), Nobuyoshi Hanaoka
(Wakayama Medical University), Toshiro Kurokawa (Toyama Red Cross Hospital),
Hideo Harigae (Tohoku University), Takayuki Ikezoe (Kochi University), Jun
Murakami (University of Toyama), Kensuke Usuki (NTT Kanto Medical Center),
Keita Kirito (University of Yamanashi), and Takao Hirano (Juntendo Nerima
Hospital) for providing patient specimens and clinical information. We also thank
Joe Matsuoka (Clinical Research Support Center, Juntendo University Graduate
School of Medicine); Satoshi Tsuneda and Yuji Sekiguchi for their generous
support and encouragement; Kyoko Kubo, Kazuko Kawamura, Junko Enomoto, and
Megumi Hasegawa for providing secretarial assistance; and other members of the
Department of Hematology for providing support in this study. We also
acknowledge the Laboratory of Molecular and Biochemical Research, Research
Support Center, Juntendo University Graduate School of Medicine.
Funding
This work was funded in part by JSPS (http://www.jsps.go.jp/english/e-grants/)
KAKENHI grant #25860416 (SM). The funders had no role in the study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests
The authors declare no competing interests.
Author Contributions
Conceived and designed the experiments: SS, MA, SM, YE, and NK. Performed the
experiments: SS, MA, SM, YE, HT, YS, and YH. Analyzed the data: SS, SM, YE, HT, YY,
and MC. Contributed reagents/materials/analysis tools: MN, MK, NN, AO, and NK.
Wrote the manuscript: SS, MA, SM, and NK.
References
1. Cazzola M, Kralovics R. From Janus kinase 2 to calreticulin: the
clinically relevant genomic landscape of myeloproliferative neoplasms.
Blood. 2014;123(24):3714-9.
2. Rumi E, Pietra D, Ferretti V, Klampfl T, Harutyunyan AS, Milosevic JD,
et al. JAK2 or CALR mutation status defines subtypes of essential
thrombocythemia with substantially different clinical course and
outcomes. Blood. 2014;123(10):1544-51.
3. Tefferi A, Lasho TL, Finke C, Belachew AA, Wassie EA, Ketterling RP, et
al. Type 1 vs type 2 calreticulin mutations in primary myelofibrosis:
differences in phenotype and prognostic impact. Leukemia.
2014 ;28(7):1568-70.
4. Fu R, Xuan M, Zhou Y, Sun T, Bai J, Cao Z, et al. Analysis of calreticulin
mutations in Chinese patients with essential thrombocythemia: clinical
implications in diagnosis, prognosis and treatment. Leukemia.
2014;28(9):1912-4.
5. Rotunno G, Mannarelli C, Guglielmelli P, Pacilli A, Pancrazzi A, Pieri L,
et al. Impact of calreticulin mutations on clinical and hematological
phenotype and outcome in essential thrombocythemia. Blood.
2014;123(10):1552-5.
6. Klampfl T, Gisslinger H, Harutyunyan AS, Nivarthi H, Rumi E,
Milosevic JD, et al. Somatic mutations of calreticulin in
myeloproliferative neoplasms. The New England journal of medicine.
2013; 369(25):2379-90.
7. Tefferi A, Lasho TL, Finke CM, Knudson RA, Ketterling R, Hanson CH,
et al. CALR vs JAK2 vs MPL-mutated or triple-negative myelofibrosis:
clinical, cytogenetic and molecular comparisons. Leukemia.
2014 ;28(7):1472-7.
8. Edahiro Y, Morishita S, Takahashi K, Hironaka Y, Yahata Y, Sunami Y,
et al. JAK2V617F mutation status and allele burden in classical
Ph-negative myeloproliferative neoplasms in Japan. International
journal of hematology. 2014;99(5):625-34.
9. Takei H, Morishita S, Araki M, Edahiro Y, Sunami Y, Hironaka Y, et al.
Detection of MPLW515L/K mutations and determination of allele
frequencies with a single-tube PCR assay. PloS one. 2014;9(8):e104958.
10. Chi J, Nicolaou KA, Nicolaidou V, Koumas L, Mitsidou A, Pierides C, et
al. Calreticulin gene exon 9 frameshift mutations in patients with
thrombocytosis. Leukemia. 2014;28(5):1152-4.
11. Pardanani AD, Levine RL, Lasho T, Pikman Y, Mesa RA, Wadleigh M, et
al. MPL515 mutations in myeloproliferative and other myeloid disorders:
a study of 1182 patients. Blood. 2006;108(10):3472-6.
12. Rumi E, Pietra D, Guglielmelli P, Bordoni R, Casetti I, Milanesi C, et al.
Acquired copy-neutral loss of heterozygosity of chromosome 1p as a
molecular event associated with marrow fibrosis in MPL-mutated
myeloproliferative neoplasms. Blood. 2013;121(21):4388-95.
13. Guglielmelli P, Pancrazzi A, Bergamaschi G, Rosti V, Villani L, Antonioli
E, et al. Anaemia characterises patients with myelofibrosis harbouring
Mpl mutation. British journal of haematology. 2007;137(3):244-7.
14. Lundberg P, Karow A, Nienhold R, Looser R, Hao-Shen H, Nissen I, et al.
Clonal evolution and clinical correlates of somatic mutations in
myeloproliferative neoplasms. Blood. 2014;123(14):2220-8.
15. Tefferi A, Wassie EA, Guglielmelli P, Gangat N, Belachew AA, Lasho TL,
et al. Type 1 versus Type 2 calreticulin mutations in essential
thrombocythemia: A collaborative study of 1027 patients. American
journal of hematology. 2014;89(8):E121-4.
Table 1: Clinical and hematological characteristics according to gene mutation status
ET (n=110**)
Mutation
Number of patients
(N)
Male:Female (N)
Age (years)
WBC (×10⁹/L)
RBC (×10⁹/L)
Hct (%)
Hb (g/dL)
MCV (fL)
Platelets (×10⁹/L)
Thrombotic event*
Splenomegaly*
JAK2
59
(A)
24:35
60.2
(19-83)
11.1
（5.2-52.3）
4783
(3420-7060
)
42.0
(25.4-54.1)
13.7
(7.9-16.9)
88.6
(66.1-106.6)
890
(486-1580)
9/55
（16.4％）
8/59
（13.6％）
(C)
negative (D)
22
13:8
59.0
(33-86)
7.9
(4.1-12.7)
4421
(2590-5570)
2:6
62.0
(32-81)
8.2
(4.9-11.3)
4364
(3600-4600)
9:13
45.0
(26-81)
9.6
(4.8-21.9)
4615
(3670-6160)
41.3
(27.6-49.6)
13.6
(9.4-17.0)
93.7
(87.4-115.4)
1109
(496-2832)
1/19
（5.3％）
0/20
（0％）
39.6
(34.3-44.6)
12.7
(10.8-14.1)
90.8
(85.5-97.8)
1177
(800-1592)
1/8
（12.5％）
1/8
（12.5％）
40.9
(33.8-50.0)
13.4
(10.8-16.8)
89.3
(61.7-97.0)
910
(349-2337)
3/19
（15.8％）
1/20
（5.0％）
CALR
21
(B)
PMF (n=23)
MPL
8
JAK2
CALR
MPL
11
7
1
7：4
71.9
(52-88)
14.8
(2.2-52.8)
3876
(2230-5760
)
32.8
(20.4-40.5)
10.3
(6.6-13.0)
86.2
(60.8-98.4)
430
(70-1280)
2/6
（33.3％）
9/11
（81.8％）
5：2
63.6
(55-81)
7.2
(3.2-12.3)
3236
(2200-4111)
1：0
77
29.2
(19.4-38.7)
9.1
(5.7-12.7)
90.2
(83.5-98.5)
477
(82-1491)
0/5
（0％）
4/6
（66.7％）
24.8
4.9
2960
7.8
83.8
351
0/1
（0％）
1/1
（100％）
negative
4
2：2
53.3
(38-76)
16.4
(5.4-44.1)
3105
(1920-3680)
28.1
(17.5-34.0)
9.4
(6.1-11.5)
90.7
(87.0-92.4)
264
(2-855)
0/2
（0％）
3/4
（75％）
WBC, white blood cell count; RBC, red blood cell count; Hct, hematocrit; Hb, hemoglobin; MCV, mean corpuscular volume. Values with a range are indicated by
median values. *A subset of patients was evaluated. **The patients who exhibited mutations in two different genes were omitted.
Figure Legends
Figure 1. The
patients.
,
JAK2
, and
MPL
CALR
mutation frequencies in ET and PMF
(A) The JAK2, MPL, and CALR mutation frequencies in ET (n=112) and
PMF (n=23) patients are shown. (B) The mutation statuses of different age groups
are shown.
JAK2, CALR, and MPL mutation spectrum in Japanese myeloproliferative neoplasms patients.
Shirane, et al
Supplemental data Materials and Methods Mutation analysis The JAK2V617F mutation profile was first obtained in a previous study1. The patient specimens that exhibited a JAK2V617F mutant allele frequency below 10% in the previous study were re-‐evaluated via deep sequencing (see below). The MPLW515K/L mutation was assessed using a newly developed allele-‐specific polymerase chain reaction (PCR), called dual amplification refractory mutation system PCR (DARMS-‐PCR), and a subsequent capillary electrophoresis method using ABI3130xl (Life Technologies, Carlsbad, USA)2. The CALR mutation on exon 9 was examined using our in-‐house fragment analysis method. The CALR fragment including exon 9 was PCR-‐amplified from 20 ng of genomic DNA using Titanium Taq (TOYOBO, Osaka, Japan) with a FAM (5-‐carboxyfluorescein hydrate)-‐labeled forward primer (TGGTCCTGGTCCTGATGTC) and a reverse primer (GTGGATTTTGGTTTTGTTCC). The PCR conditions were as follows: an initial denaturation at 94°C for 3 min; 30 cycles of denaturation at 95°C for 30 sec, annealing at 68.5°C for 30 sec, extension at 72°C for 30 sec; and a final extension at 72°C for 2 min. The PCR products were diluted with formamide and then analyzed using an ABI3730 DNA Analyzer (Life Technologies) and Gene mapper 4.0 (Life Technologies). Deep-‐sequencing analysis The patient specimens that exhibited a JAK2V617F mutation rate of <10%, were positive for MPLW515K/L, or were positive for a CALR mutation were subjected to deep-‐sequencing analysis. According to the identified mutation, we PCR-‐amplified JAK2 exons 12 and 14, MPL exon 10, or CALR exons 1-‐9 from genomic DNA using KOD plus Neo (TOYOBO); the primers are listed in supplemental Table 1. The specimens were negative for JAK2V617F, MPLW515K/L, and CALR exon 9 mutations based on the previously described PCR-‐based assays; 1
JAK2, CALR, and MPL mutation spectrum in Japanese myeloproliferative neoplasms patients.
Shirane, et al
follow-‐up analyses were conducted by deep sequencing all exons of the three genes. PCR-‐amplified fragments were purified using an Agencourt AMPure XP kit (Beckman Coulter), and the concentration of each aliquot was subsequently measured using a Quantus fluorometer (Promega, Madison, US). The purified amplicons were mixed together in an equal molecular ratio and were then fragmented to approximately 200 bp using a M220 Forced-‐ultrasonicator (Covaris, Woburn, USA). The sample library was prepared using TruSeq DNA LT Sample prep kits Sets A and B (Illumina, San Diego, USA) according to the manufacturer’s instructions. The libraries were deep-‐sequenced using a MiSeq bench-‐top sequencer (Illumina). For the identification of the JAK2, MPL, and CALR mutations, the data were analyzed using CLC Genomics Workbench software version 6.5 (CLC Bio, Aarhus, Denmark). In addition, an alternative algorithm was used for the identification of CALR mutations. Sequence reads were mapped to the reference genome (hg19) using the BWA program3. Reads containing over 5 uncalled bases and unmapped reads were discarded from subsequent analyses. From the reads aligned to the CALR region, CIGAR strings from the reads in SAM/BAM format were parsed to call potential insertions or deletions using perl scripts. Although mutation calls were not always consistent between two algorithms, at least one algorithm produced mutation calls that matched fragment analysis data. Conversely, deep-‐sequencing analysis revealed low-‐frequency CALR mutations in some specimens, whereas no mutations were detected using fragment analysis. In this case, we determined that no CALR mutations were present based on the fragment analysis data. Statistical analysis Adjusted p-‐values for multiple comparisons of laboratory parameters between patient groups with different mutations were determined by Tukey-‐Kramer test with R3.1.1 (Free Software Foundation, Boston, USA). p-‐values below 0.05 were considered significant. 2
JAK2, CALR, and MPL mutation spectrum in Japanese myeloproliferative neoplasms patients.
Shirane, et al
References 1. Edahiro Y, Morishita S, Takahashi K, Hironaka Y, Yahata Y, Sunami Y, et al. JAK2V617F mutation status and allele burden in classical Ph-‐negative myeloproliferative neoplasms in Japan. International journal of hematology. 2014 May;99(5):625-‐34. 2. Takei H, Morishita S, Araki M, Edahiro Y, Sunami Y, Hironaka Y, et al. Detection of MPLW515L/K mutations and determination of allele frequencies with a single-‐tube PCR assay. PloS one. 2014;9(8):e104958. 3. Li H, Durbin R. Fast and accurate short read alignment with Burrows-‐Wheeler transform. Bioinformatics. 2009 Jul 15;25(14):1754-‐60. 4. Klampfl T, Gisslinger H, Harutyunyan AS, Nivarthi H, Rumi E, Milosevic JD, et al. Somatic mutations of calreticulin in myeloproliferative neoplasms. The New England journal of medicine. 2013 Dec 19;369(25):2379-‐90. 3
forward
GAAGTGGGAGTGGTGTGGG
CCCCTGGATTTATGTGGTAGTAG
CCAGCCCATTTGTAACTTTATTG
CGACTGCTATTACATTTTGTTCC
CGTTTGTATTTGAACTATTTGGAAGC
TGGCCAATTTGTATCTTGTAAATG
TGTATGTGCTTTTTTATCCCTAGC
AATGGCTCTGTAAATTCTACCCG
TAATTCATATTGAGTACTGAGCCA
TGATTGTTTTAGATGACACTTGGTC
GATGTCCATTGTGACTATCCCTC
TGGAGCAATTCATACTTTCAGTG
TCCTACTTCGTTCTCCATCTTTAC
GAGAAAGTGCATCTTTATTATGGC
TTGTTTAGACTCCTACTCTTGCTG
TTCTTCTTTAAATCTGTTTTGGGG
TTGGTTTACTTGTGAATTATTTAACCC
AAGAAGGTTGGTGTGGCATTAC
TTTTGGTCAACTTGAATGTATATCAG
JAK2 exon 1
JAK2 exon 2
JAK2 exon 3
JAK2 exon 4
JAK2 exon 5
JAK2 exon 6
JAK2 exon 7
JAK2 exon 8
JAK2 exon 9
4
JAK2 exon 10
JAK2 exon 11
JAK2 exon 12
JAK2 exon 13
JAK2 exon 14
JAK2 exon 15
JAK2 exon 16
JAK2 exon 17
JAK2 exon 18
JAK2 exon 19
CAAGCACTCCTTAAAATGTTGTAG
CCCAAATGACATCAAGAAAATG
CCTTCTTTTTAAAATTAGATGGGC
TTTACACCACTGCCCAAGTAAAG
TTTGTTTCCAGGTAAAGATAATTTG
TACACTGACACCTAGCTGTGATCC
TCTTTAAACAGCATAAACTACATGAAC
AACACAAGGTTGGCATATTTTTC
TACTTCACGCCACATAAACAATC
TTTGTACAGAATCTCTGAGGAAATTAAG
CAGCATTTACTTTTCTAAAAATTTACTT
TTTAAAACTACAATCAAAATTCCTACC
CTTTGTTTTTATTTCCATTGTACTTA
TCACACTTATGTGTAAGGATTTGC
CAAGACAGAACTGCAATTTTCC
TTTGCCTTTTAGCATTAAGTGAG
AAAACAATCCATGCATAAAAAGG
CAGCGATAAAGGACAGCACAC
AAGCCAGGCAGAGATAACACC
reverse
!"##$%&%'()$*+),$%*-.*/01&%02*"2%3*450*%65'*)&#$1417)(15'8*
target
!
!
!
JAK2, CALR, and MPL mutation spectrum in Japanese myeloproliferative neoplasms patients.
Shirane, et al
TGCAGGATTTGGGTCAAACAG
CGAAGTCTGACCCTTTTTGTCT
CCTGCCAATCCACTGCCA
TGGTCCTGGTCCTGATGTC
MPL exon 9
MPL exon 10
MPL exon 11–12
CALR exon 9
TGCAGCAGGTTAAGAATTTTTTC
AGGCCTGATTCAATGACTCT
TGGTGAATGTGTTTTTTAAATGG
MPL exon 7–8
TTTCACATAAAGGGAACAAATGTC
GTTGGAGGCTCTCTCAGCTG
CCTTCAACCCAAAATCAGAAGTC
TTTCAACTCAGCTTTTTGAGACC
MPL exon 5–6
CAAAACCCACCATATTTTATTTTAGC
TTCTGAGACCAAAGTAGATTTACAGAAC
GTGGTACTCAGAGTTCTGATGTG
TTTATGGCTTAAGCTTAAATTTAGT
GCCCTTAGTGTTCATTTAATTTTG
JAK2 exon 25
MPL exon 4
CCACAAATAGATACAAGGGAAACC
TTGACTGGAGGAAATTGAGAAAG
JAK2 exon 24
CTGTATCTGACAGGAACCTGAGG
GCGATATAAATGAACAAGGACAC
TTTGCAGGTAAAATCAAGAGTCC
JAK2 exon 23
MPL exon 1–3
TTTTCAGGTCTCAAAAAGCTGAG
AATAAAGGGAATATATAGGGTTAAGACC
JAK2 exon 22
5
!
!
!
!
GGAACAAAACCAAAATCCAC
TTACCTTAATCCCATGCCAGC
GGTCACAGAGCGAACCAAGA
GGCTTGCCTCACCGGTCT
TGAGGTCTGTGGGCATTTGTTG
AACCTCAATCAGCAGTTCAG
CAAGATTGAAGGTAGGAGATAAGA
GTGACAGGAGGATGGCTCT
CAGGAAGTGAACTGAAGTCCTTG
GGTGGATACCCTAAAAGCTCTG
CAATAAAGTTTGAAGTCTGTGCTC
TTCCTAATGTCTACTTCAACACGG
GCAGAGTAAAACATTATTTCCACC
JAK2 exon 21
TTTAAAATAGGTTTCAATGGGCAG
CTTGAAAACTTGGTATTTCCATCC
JAK2 exon 20
JAK2, CALR, and MPL mutation spectrum in Japanese myeloproliferative neoplasms patients.
Shirane, et al
6
c.1153_1154insTCTGT
c.1146_1147del;1153_1154del
44
45
* types 1, 2, 4, 22, 28, 33, 344
Total
c.1126_1144del
43
c.1131_1152del
28
c.1110_1133del
c.1120_1123del
22
42
c.1102_1135del
4
c.1154delinsCTTGTC
c.1154_1155insTTGTC
2
34
c.1092_1143del
1
c.1154_1155insATGTC
wild type
–
33
Nucleotide
Type
11 (5/6)
24 (20/4)
(ET/PMF)
Pts.
1 (1/0)
1 (1/0)
1 (1/0)
1 (1/0)
1 (1/0)
AAEKQMKDKQDEEQRLKEEEEDKKRKEEEEAGRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTEA-
53 (42/11)
1 (1/0)
AAEKQMKDKQDEEQRLKEEEEDKKRKEEEEAEDICRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTEA- 1 (1/0)
AAEKQMKDKQDEEQRLKEEEEDKKQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTEA-
AAEKQMKDKQDEEQRRRRRQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTEA-
AAEKQMKDKQDEEQRLKEEEEDKKRKEEEEAEDTCRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTEA- 1 (1/0)
AAEKQMKDKQDEEQRLKEEEEDKKRKEEEEAEDCRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTEA- 2 (2/0)
AAEKQMKDKQDEEQRLKEEEEDKKRKRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTEA-
AAEKQMKDKQDEEQRLKEEEEDNAKRRRRQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTEA-
AAEKQMKDKQDEEQRLRRRQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTEA-
AAEKQMKDKQDEEQRLKEEEEDKKRKEEEEAEDNCRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTEA- 8 (7/1)
AAEKQMKDKQDEEQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTEA-
AAEKQMKDKQDEEQRLKEEEEDKKRKEEEEAEDKEDDEDKDEDEEDEEDKEEDEEEDVPGQAKDEL-
Amino Acid
*
!"##$%&%'()$*+),$%*9.*:12(*54*!"#$*&"()(15'2*13%'(141%3*1'*(;%*#0%2%'(*2("3<*
100
1.9
1.9
1.9
1.9
1.9
3.8
1.9
1.9
1.9
15.1
20.8
45.3
%
JAK2, CALR, and MPL mutation spectrum in Japanese myeloproliferative neoplasms patients.
Shirane, et al

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