Download DNMT3A mutations occur early or late in patients with

Published Ahead of Print on August 6, 2015, as doi:10.3324/haematol.2015.129510.
Copyright 2015 Ferrata Storti Foundation.
DNMT3A mutations occur early or late in patients with
myeloproliferative neoplasms and mutation order influences
phenotype
by Jyoti Nangalia, Francesca L. Nice, David C. Wedge, Anna L. Godfrey, Jacob Grinfeld,
Clare Thakker, Charlie E. Massie, Joanna Baxter, David Sewell, Yvonne Silber,
Peter J. Campbell, and Anthony R. Green
Haematologica 2015 [Epub ahead of print]
Citation: Nangalia J, Nice FL, Wedge DC, Godfrey AL, Grinfeld J, Thakker C, Massie CE, Baxter J,
Sewell D, Silber Y, Campbell PJ, Green AR. DNMT3A mutations occur early or late in patients with
myeloproliferative neoplasms and mutation order influences phenotype.
Haematologica. 2015; 100:xxx
doi:10.3324/haematol.2015.129510
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DNMT3A mutations occur early or late in patients with myeloproliferative neoplasms and mutation order influences phenotype Jyoti Nangalia1,2,3, Francesca L. Nice1, David C. Wedge3, Anna L Godfrey1,2, Jacob Grinfeld1,2,3, Clare Thakker1, Charlie E. Massie1, Joanna Baxter2,4, David Sewell2,4, Yvonne Silber1, Peter J. Campbell1,2,3, and Anthony R. Green1,2 1Department of Haematology, Cambridge Institute for Medical Research and Wellcome Trust/MRC Stem Cell Institute, University of Cambridge, Cambridge, UK 2Department of Haematology, Addenbrooke’s Hospital, Cambridge, UK 3Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK 4Cambridge Blood and Stem Cell Bank, University of Cambridge, Cambridge, UK Correspondence Professor Anthony R. Green, Cambridge Institute for Medical Research, Hills Road, Cambridge, CB2 0XY, UK. E-­‐mail: [email protected] Running title: DNMT3A mutations in MPNs and influence of mutation order 1 Letter to the editor
JAK2 CALR
Somatic mutations in
,
and
MPL
are found in the majority of myeloproliferative neoplasms
(MPN) but many patients also harbor somatic mutations in epigenetic regulators of DNA methylation
TET2 DNMT3A
(
,
TET2, ASXL1
IDH1/2
and
and
EZH2
) or chromatin structure (
JAK2V617F
and
TET2
/progenitor cell biology and clinical presentation 2.
TET2,
affecting 7-10% of patients
DNMT3Amut)
mutations (
JAK2V617F
and
EZH2
). In MPN patients, mutations in
occur either prior to or following the acquisition of
order of mutation acquisition for
in MPNs after
ASXL1
3–5.
JAK2V617F 1
and recently, the
has been shown to influence hematopoietic stem
DNMT3A
is the next most frequently mutated gene
DNMT3A
However in contrast to other mutations,
have only been reported early in myeloid disease: prior to acquisition of
or in a separate clone in MPNs1,6; prior to
NPM1
or
FLT3
de novo
mutations in
acute myeloid
leukemia (AML) 7; and prior to AML transformation of MPNs or myelodysplasia 8. Here, we have used
clonal
analysis
of
hematopoietic
colonies
from
DNMT3Amut
MPN
patients
to
investigate
timing
of
mutation acquisition, subclonal evolution, and the influence of mutation order.
Thirteen
DNMT3Amut
MPN patients were identified in whom viable material was available for clonal
analysis (Table S1): 9
(WES),
1
mutated
JAK2V617F-
JAK2exon12-
mutated
patient
identified
mutated and 2
patient
by
MPL-
identified
targeted
gene
by
mutated patients identified by exome-sequencing
whole-genome
screening
(TGS).
sequencing
MPN
(WGS),
diagnoses
and
conformed
1
CALR-
to
both
British Committee for Standards in Haematology and World Health Organization 2008 classifications.
All samples were obtained following written informed consent and ethical approval. Peripheral blood
mononuclear
cells
were cultured
to
obtain BFU-Es
2
as described previously ,
and 2991 individual
colonies (average of 230 colonies/patient) were genotyped by Sanger sequencing for mutations in
DNMT3A, JAK2, CALR
DNMT3Amut
and
MPL
.
occurred prior to acquisition of
JAK2V617F
as evidenced by single-mutant colonies harboring
DNMT3Amut
and
JAK2V617F
.
Single-mutant
colonies
in four patients (‘
DNMT3Amut
DNMT3A
-first’ patients, Fig.1A)
only and double-mutant colonies with
represented
a
substantial
proportion
of
BFU-Es
(mean 58%, range 27%-86%), consistent with mutant allele fractions from granulocyte WES, and
indicating the presence of significant ‘pre-
These results
accord with
observations
JAK2’
multi-lineage clonal hematopoiesis in these patients.
of clonal hematopoiesis
in ET patients despite low
allele
2
burdens
of
JAK2V617F 9,
and with
reports
DNMT3Amut-
of
associated
DNMT3Amut
individuals 10,11. In three patients,
and
JAK2V617F
clonal
hematopoiesis
in
normal
colonies were mutually exclusive (‘biclonal’
patients, Fig.1B). In such patients, the two mutations have arisen either in separate cells downstream
of a shared ancestral clone or in clonally unrelated cells.
DNMT3Amut
and
JAK2V617F
clones did not share
additional mutations (identified by WES) in any of the 3 patients (Fig.2 patients #25, #81, #27).
Moreover, in one of the two female patients,
DNMT3Amut
X-chromosomes (Fig.S1). These data demonstrate that
and
JAK2V617F
DNMT3Amut
clones harbored different active
and
JAK2V617F
clones in the same
patient may represent clonally-unrelated expansions.
In
three
patients,
DNMT3Amut
patients,
Fig.1C) but these
occurred
patients
after
acquisition
were more
of
mutated
JAK2
or
MPL JAK2/MPL
difficult to identify. In patient
(
#650,
the order
mutation acquisition was initially unclear as the majority of colonies were double-mutant for
and
JAK2exon12,
confirmed
wildtype
that
and
the
DNMT3A
no
antecedent
JAK2exon12
single-mutant
mutation
arose
colonies
first,
and
were
that
JAK2exon12
(Fig.1C).
heterozygous-
became undetectable following acquisition of
from an earlier timepoint showed heterozygous-
detected
DNMT3Amut,
JAK2exon12
DNMT3Amut
order, the two homozygous-
different
mitotic
antecedent
colonies
we
with
because colonies grown
colonies with wildtype
DNMT3A
(Fig.S2A).
JAK2exon12
in
in only the later timepoint (Fig.S2B). Consistent with this mutation
JAK2exon12
recombination
JAK2/MPL
of
DNMT3A
However,
Furthermore, WGS of a diagnostic sample and a later timepoint demonstrated mutated
both timepoints but
-first
subclones present in this patient (Fig.1C, h1 and h2) carried
breakpoints
(Fig.S2C).
In
two
further
JAK2/MPL-
first
patients,
single-mutant colonies were detected but were present only at low levels (~3%
of total colonies, Fig.1C). Overall, considering MPN patients with sequential acquisition of mutations
within
mutant
the
same
clone,
DNMT3Amut
single-mutant
JAK2/MPL
subclones
were
significantly
smaller
than
single-
and double-mutant subclones (p=0.03 for both comparisons, t-test, Fig.S3). To
exclude a confounding effect of mutations in other genes known to be recurrently mutated in myeloid
malignancies, WES or WGS data were interrogated for all 13 patients. Four patients had additional
mutations in
TET2, CBL
or
SH2B3
. Delineation of full phylogenetic hierarchies using Sanger sequencing
or Fluidigm SNP Genotyping (see supplementary methods) of individual colonies from these patients
did not identify any preferential association of these mutations with either single or double-mutant
subclones (Fig.2). Our data therefore suggest that
JAK2/MPL
single-mutant subclones may have a
3
competitive disadvantage compared with
DNMT3Amut
subclones, in which case
JAK2/MPL
-first patients
may be enriched among those in whom mutation order could not be determined by colony assay
(Fig.1D). To investigate competition between
JAK2/MPL-
mutated and
DNMT3Amut
subclones further,
colonies were grown from paired samples obtained at different timepoints (median separation 35
months; range 6-179 months) in 10 patients (3 biclonal, 3
unclear).
In
6
patients
who
harbored
single-mutant
JAK2/MPL
DNMT3Amut
-first, 3
colonies
DNMT3
(2
-first, 1 order
patients
receiving
hydroxycarbamide, 1 patient receiving pipobroman, 2 patients receiving interferon-alpha (IFN), and 1
patient not receiving cytoreduction), the proportions did not significantly change between timepoints
(Fig.3A, B; blue shading). Similarly, in 7 patients with double-mutant colonies (4 patients receiving
hydroxycarbamide, 1 patient receiving pipobroman and 2 patients not receiving cytoreduction) there
was no significant change in the proportions over time (Fig.3A, B; purple shading). In contrast, in all 6
patients with single-mutant
JAK2 MPL
/
colonies (1 patient receiving hydroxycarbamide, 2 patients
receiving IFN and 3 patients not receiving cytoreduction), the proportions of single-mutant colonies
fell significantly over time (Fig.3A, B; red shading, p=0.027, Wilcoxon signed rank test,). Whilst the
observed reduction of
JAK2/MPL
colonies could have been influenced by IFN treatment in 2 patients
(#81 and #25), 2 patients in whom this pattern was also observed had not received cytoreduction
(#27 and #650),
disadvantage.
supporting the notion that single-mutant
We
found
no
associations
between
JAK2/MPL
subclonal
subclones have a competitive
changes
over
time
and
treatment
responses (measured in accordance with European Leukemia Net guidelines) in patients, possibly
because individual patients harboured multiple and differing combinations of the various clones.
We
next
assessed
whether
order
of
acquisition
of
DNMT3Amut
influenced
phenotype. In the 8 patients in whom mutation order was established, 4
had
ET,
and
of
4
patients
in whom
JAK2V617F
occurred
on
a
DNMT3A
JAK2V617F-
mutated
MPN
DNMT3Amut-
first patients all
-nonmutated
background
(1
JAK2V617F
–first and 3 biclonal patients), 3 had PV and 1 had MF. To expand this cohort, an additional
918 patients were screened by TGS, and a further 33 patients with
identified.
Copy-number
corrected
variant
allele
fractions
were
DNMT3Amut
used
to
and
determine
JAK2V617F
the
were
order
of
2
mutation acquisition as recently described , and mutation order was unambiguously assigned in 8
further patients: 2
DNMT3A
MF, 1 PMF and 1 ET.
-first patients had ET, and 6
Combining both cohorts, all 6
JAK2
-first patients comprised 3 PV, 1 post PV-
DNMT3A
-first patients presented with ET. By
4
contrast, of 10 patients in whom
JAK2V617F
arose on a wildtype
DNMT3A
background, 7 presented with
PV and only 1 with ET (p=0.003, Chi squared test). There were no significant differences in other
clinical features (Table S2) in this cohort. Our results therefore indicate that mutation order influences
clinical
presentation,
not
only
in
MPN patients
,
JAK2V617F
described2 but also in patients with
TET2
and
JAK2V617F
with
DNMT3A
prior to mutation of
DNMT3A
or
TET2
mutations,
.
JAK2
previously
DNMT3A
or
JAK2V617F
. By contrast, acquisition of
TET2
is associated with PV. ‘
-first’ patients were older
at presentation in our previous study 2, but no difference in age was identified between
and
as
mutations Mutations in either
are associated with an ET phenotype when acquired prior to
JAK2V617F
TET2
and
DNMT3A
-first
-first patients. This may be due to the smaller number of patients in the current study or may
reflect a real difference in the age at which
TET2
In summary, we demonstrate that in MPNs,
JAK2/MPL
single-mutant
DNMT3Amut
subclones
have
and
DNMT3A
DNMT3Amut
a
mutations are acquired.
can follow
competitive
JAK2
and
disadvantage
subclones. This concept is consistent with observations that
MPL
mutations, and that
in vivo
DNMT3A
compared
and
TET2
with
mutations
confer an advantage to hematopoietic stem/progenitor cells 2,12–14, whereas this is not the case for
JAK2V617F
in
DNMT3Amut
some
mouse
models 15
.
Furthermore,
we
show
that
mutation
order
of
JAK2V617F
and
is associated with differences in MPN phenotype. This emphasizes the importance of the
pattern of acquisition of
phenotype of
JAK2V617F-
JAK2V617F
with respect to mutations in epigenetic modifiers in influencing the
mutated MPNs.
Acknowledgements:
We thank the Cambridge Blood and Stem cell Biobank (Cambridge University)
and
Haemato-Oncology
Anthony
Bench,
Diagnostics
Service
(Addenbrooke’s
hospital)
for
sample
collection and storage, and the Cancer Genome Project (Wellcome Trust Sanger Institute, Hinxton) for
next
generation
sequencing.
Work
in
the
Green
lab
is
supported
by
Leukemia
and
Lymphoma
Research, the Wellcome Trust, the Medical Research Council, the Kay Kendall Leukaemia Fund, the
Cambridge NIHR Biomedical Research Center, the Cambridge Experimental Cancer Medicine Centre
and the Leukemia and Lymphoma Society of America. ALG and JN were supported by Kay Kendall
Leukaemia Fund clinical fellowships. PJC is a Wellcome Trust senior clinical fellow.
5
Authorship contributions:
JN designed experiments, analysed data and wrote the paper. JN, ALG,
FLN, CT and YS performed colony work. Microsatellite screening was by JN and ALG. X chromosome
inactivation assessment was by JN. FLN performed Fluidigm SNP Genotyping and colony work. JN and
CEM analysed WES and WGS data. EJB and DS prepared patient samples. DCW performed analysis of
mutation
order.
JN
and
JG
screened
TGS
data
and
assessed
clinical
correlates.
ARG
directed
the
research. All authors reviewed the manuscript.
Disclosure of conflicts of interest: The authors declare no competing financial interests.
References
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7
Figure Legends
Figure 1. Timing of acquisition of DNMT3A mutations
Single cell derived hematopoietic erythroid colonies (BFU-Es) were grown
in vitro
from peripheral
blood derived mononuclear cells and individually genotyped for mutations using Sanger sequencing
Plots in (A), (B), (C) and (D) show colony genotyping results for mutations in
or
CALR
for each patient and the order of mutation acquisition
.
DNMT3A
JAK2/MPL/CALR
JAK2, MPL
Within each plot, each dot represents a
single colony and its quadrant placement shows the corresponding genotype of
axis) and
and
.
DNMT3A
(vertical
(horizontal axis). Solid red arrows within quadrants show the confirmed
path of clonal evolution. Wt, wildtype; het, heterozygous mutation; hom, homozygous mutation; PV,
polycythemia vera; ET,
myelofibrosis
(A)
essential
DNMT3A-
PMF, primary myelofibrosis; PPV-MF,
. (B) Biclonal patients: 3 patients in whom
separate clones. (C)
JAK2/MPL-
to the acquisition of mutated
DNMT3A
and
DNMT3A
first patients: 4 patients in whom mutated
JAK2V617F
acquisition of
acquisition of
thrombocythemia;
DNMT3Amut
first patients: 3 patients in whom mutated
DNMT3A
JAK2
post-PV
occurred prior to the
and
or
JAK2V617F
MPL
were in
occurred prior
. (D) Order unknown: 3 patients in whom the order of mutation
JAK2/MPL/CALR
could not be delineated as only wildtype colonies and/or
double mutant colonies were detected.
Figure 2. Clonal structures of DNMT3A-mutated MPNs
Individual
BFU-Es
from
6
patients
were
genotyped
using
Sanger
sequencing
or
Fluidigm
SNP
Genotyping for their respective somatic variants identified previously by whole exome sequencing.
Genotyping results from individual colonies were then used to construct phylogenetic trees. Circles
represent
the
subclones;
wildtype
(white);
mutated
(brown).
The
earliest
detectable
clone
is
represented at the top of each structure, with subsequent subclones shown below. Somatic mutations
acquired
in
each
subclone
are
indicated
beside
respective
circles,
and
represent
those
that
are
acquired in addition to mutations present in earlier clones. Numbers of colonies identified for each
subclone are shown inside circles. Mutations in
Additional
mutations
in
genes
known
to
be
JAK2, MPL, CALR
recurrently
and
mutated
DNMT3A
in
are highlighted in red.
myeloid
malignancies
are
8
highlighted in green. ET, essential thrombocythemia; PMF, primary myelofibrosis; PV, polycythemia
vera; het, heterozygous mutation; hom, homozygous mutation.
Figure 3 Evolution of subclones in DNMT3A-mutated MPNs
(A) Colonies grown
months;
6-179
from paired samples
months)
in
10
patients.
obtained at different timepoints
Vertical
axis
shows
the
(median separation
percentage
of
columns are shaded to represent the proportions of the different genotypes (red,
DNMT3A
total
colonies
JAK2/MPL
35
and
-only; blue,
-only; purple, double mutant). Numbers of colonies genotyped per patient are shown above
columns and the timings of sample acquisition (months from diagnosis) are shown below. (B) Changes
in subclonal proportions over time for the 10 patients in (A) for a total of 19 subclones. red,
only;
blue,
DNMT3A
-only;
purple,
double
mutant;
T1
and
T2
represent
the
earliest
JAK2/MPLand
latest
timepoints sampled for the patients. The median interval between T1 and T2 did not significantly
differ between the different subclones (1-way analysis of variance). * <0.05 Wilcoxon ranked sum test
9
Figure 1
ET #98
ET #97
het
hom
JAK2 V617F
wt
DNMT3A R882H
het
wt
wt
het hom
JAK2 V617F
wt
het hom
JAK2 V617F
het hom
JAK2 V617F
wt
het hom
JAK2 V617F
PMF #27
PV #81
DNMT3A S770L
wt
het
DNMT3A Y660F
wt
het
PV #25
DNMT3A c.2597+1G>A
wt
het
B Biclonal
wt
het hom
JAK2 V617F
wt
het hom
JAK2 V617F
C JAK2/MPL-first
DNMT3A Y908*
wt
het
wt
het hom
JAK2 V617F
ET #50
h2
h1
wt
het hom
JAK2 F537_K539delinsL
DNMT3A F731V
wt
het hom
PV #650
PV #152
DNMT3A I705T
wt
het
wt
het hom
MPL W515L
D Order unknown
ET #78
PMF #42
wt
het hom
MPL W515L
PPV-MF #28
DNMT3A R882H
wt
het
DNMT3A D279FS*1
wt
het hom
wt
ET #03
DNMT3A R882H
wt
het
DNMT3A R882H
wt
het
ET #052
DNMT3A R882H
wt
het
DNMT3A c.1123-1_1144del23
wt
het
A DNMT3A-first
wt
het hom
CALR R376fs*55
wt
het hom
JAK2 V617F
Figure 2
PV #25
Wild type
Wild type
58
JAK2 V617F
TET2 N281fs*0
2 POLK C769R
L1044L
PLD1
15
DNMT3A
PV #81
Y660F
JAK2 V617F hom
JAK2
24
DNMT3A
S770L
V617F het
DNMT3A
SLC6A6 L675V
2
V617F
JAK2
V617F hom
GRID1
ET #052
Wild type
2
DNMT3A R882H
APLP2 V359V
5
12
GABRB3
R283W
28
V617F
A487T
25
JAK2
C10orf71 T295T
CD3E G170S
R1178C
ENSG0218819
LRRC6B F514S
NBAS E1326D
R55H
OR2S2
RPGRIP1l R321H
ZSCAN5C S568S
SCNSA T413T
N972N
12
PMF #42
4
DNMT3A splice
L818P
RICTOR
JAK2 V617F
RFTN2 R89*
I65V
TMEM87A
10
15
8
1
21
44
splice
1
ET #03
Wild type
Wild type
62
4
MRPL49 Q28H
PMF #27
TET2 Q744fs*10
JAK2
KIF4B
Y2125*
MYO9A
G641R
COL9A2
L383L
PRKAG3
Mutation in
JAK2, DNMT3A
5
GCAT V409M
XIRP1 R1275C
SLC4A9 I514N
ENSG218819
RBM44 G1028D
RENBP L144L
ZNF143 S286R
MUL1 G70R
CALR R376fs*55 het
TET2 p.K1785* het
p.D279fs*1
DNMT3a
hom
splice
SH2B3
hom
N727S
79
CALR
R376fs*55
C401S
64
42
CBL
SCN11A
M1706T
Additional mutations
associated with myeloid
malignancies
Other mutations
hom
Figure 3
A
ET #052
JAK2-mt
100
ET #98
ET #03
JAK2-mt JAK2-mt
ET #78
MPL-mt
197
93
79
76
PV #81
JAK2-mt
70
141
133
91
59
76
153
72
45
59
73 139 151 99 105 54
78
0
172 179
PV #25 PMF #27 ET #50
JAK2-mt JAK2-mt MPL-mt
PV #152
JAK2-mt
91
134
112
185 138
0
31
91 132 32 152 20
174 76
82
PV #650
JAK2-mt
6
178 120 Colonies (n)
Colonies (% of total)
80
60
40
20
0
Months from Diagnosis
JAK2/MPL only
DNMT3A only
B
Colonies (% of total)
Wildtype
80
60
40
20
0
T1
JAK2/MPL
only (n=6)
T2
T1
DNMT3A
only (n=6)
T2
13 152 183
Double mutant
*
100
45
T2
Double
mutant (n=7)
T1
Supplementary Appendix Construction of phylogenetic hierarchies by genotyping of individual haematopoietic colonies Haematopoietic colonies (BFU-­‐E) were grown and individually genotyped using Sanger sequencing or Fluidigm SNP Genotyping for all the mutations that had been previously identified by next-­‐generation sequencing in each patient. Colony genotyping for patients #81, #03, #42 and #25 was by PCR and Sanger sequencing. Colony genotyping for #052 and #27 was by Fluidigm SNP Genotyping. Fluidigm genotyping primers were designed according to the manufacturer’s recommendations, and obtained from Fluidigm (Fluidigm Corp, CA, USA). The Fluidigm SNPType Genotyping Reagent Kit (Fluidigm Corp, CA, USA) was used according to the manufacturer’s instructions. Colony DNA was first amplified using Specific Target Amplification as per the manufacturer’s instructions, to enrich for the DNA sequences for subsequent PCR. Genotyping reactions were then performed on a nanofluidic 192.24 Dynamic Array Integrated Fluid Circuit (IFC; Fluidigm Corp, CA, USA), which automatically assembles PCR reactions. End-­‐point fluorescent images were acquired on a Biomark system and analysed using the Biomark SNP Genotyping Analysis software version 3.1.2. Colonies with ambiguous genotype results underwent repeat PCR and Sanger sequencing. Primer sequences for Sanger sequencing were as follows: Gene V617F JAK2
exon12 JAK2
R882 DNMT3A
Y660F DNMT3A
splice(#81) DNMT3A
splice(#052) DNMT3A
S770L DNMT3A
Y908* DNMT3A
D279fs*1 DNMT3A
DNMT3A
F731V
I705T
DNMT3A
N281fs*0 TET2
Q744fs*10 TET2
K1785* TET2
CALR MPL POLK PLD1 MRPL49 APLP2 TMEM87A RFTN2 SH2B3 LANCL3 Forward primer TTTCCTTAGTCTTTCTTTGAAGCAGC CTCCTCTTTGGAGCAATTCA AATACTCCTTCAGCGGAGCGAAGA GTCCCCGACGTACATGATCT CAGGACGTTTGTGGAAAACA TGCCCTCATTTACCTTCTGG ATGAAGCAGCAGTCCAAGG CGCCTCTGTGGTTTTTGTTT CACCTCGTACTCTGGCTCGT ATGAAGCAGCAGTCCAAGG CAAGGAGGAAGCCTATGTG CACATGGTGAACTCCTGGAA GAGCAGATTCCCAAACTGAAA TGGTGAACATCATTCACCTTCT CCTGCAGGCAGCAGAGAAAC TGACCGCTCTGCATCTAGT TGTTCTAGTCTCCCAAGCAAGTC CATCCCCAGGAAGTCACTGT CGCCCAGCCTTTTATTTATG GACGTCACTGCCTCTGTCCT AATGCCAGACTATTAGATTTCAGAAC TAAAACCCCCATGTGTCCTT ACCACCTTTGCTGCTACCAC GCTCACGGCTTGTCGTCTAT Reverse Primer TAGTTTACACTGACACCTAGCTGTGATCC CATCTAACACAAGGTTGGCATA AAGATTCGGCAGAACTAAGCAGGC GACTTGGGCCTACAGCTGA CTCCATAAAGCAGGGCAAA CTCAGAGTCTGGCCTTGAGC TTGAGTTCTACCGCCTCCTG AGTCATCCGCCACCTCTTC CAGGAATGAATGCTGTGGAA GGCTTTCTCTTCCGACCTCT CTTCCTGTCTGCCTCTGTCC TCAGCATCATCAGCATCACA CTTTGGGGGTGAGGAAAAGT TGGTGAACATCATTCACCTTCT ACAGAGACATTATTTGGCGCG TACAGGCCTTCGGCTCCA AAGCAAACATCCACATGCAC CATTCGAGCTGAGGAGGAAC GGGATCTGGGATCCTGGTAG GCGAGGCAGGACTTACTCAT TTGAAGCCATCTGAGGCTAA CGGGGCTATTCATCCTGTTA CCCACCTTGGTTAAGGGAAT CAGTGCACCAGCTCATTCTC Microsatellite marker analysis
Microsatellite analysis was used to determine the extent of chromosome 9p loss-of-heterozygosity.
A panel of microsatellite markers along chromosome 9p were selected from the GeneLoc database.
The PCR reaction contained 1.25µl 10x ReddyMix PCR buffer IV, 0.75µl 25mM magnesium
chloride solution, 0.125µl Thermoprime plus polymerase (Thermo Scientific, MA, USA), 0.1µl
100mM dNTPs, 0.06µl 100µM forward primer, 0.06µl 100µM reverse primer, and 0.5-2µl DNA, in
a total reaction volume of 12.5µl. PCR conditions were 11 minutes at 95 oC, followed by 38 cycles
of 30 seconds at 95oC, 30 seconds at 57oC and 1 minute at 72oC, followed by a final extension at
72oC for 10 minutes. The PCR reaction was diluted between 1:5 and 1:40 in sterile water. Fragment
detection was performed on a 3730xl Genomic analyser and results were analysed using the Peak
Scanner software version 1.0 (Applied Biosytems, CA, USA). Heterozygosity for these markers
was initially analysed using constitutional DNA obtained from a buccal swab. Informative markers
were then taken forwards for testing on tumour DNA from individual homozygous JAK2exon12mutated colonies to ascertain the extent of loss-of-heterozygosity in heterozygous microsatellite
markers.
Primer sequences were as follows:
Microsatellite marker D9S288 D9S1852 D9S235 D9S925 D9S162 D9S161 D9S43 D9S1817 D9S1791 D9S2148 D9S176 Primer name D9S288_F D9S288_R D9S1852_F D9S1852_R D9S235_F D9S235_R D9S925_F D9S925_R D9S162_F D9S162_R D9S161_F D9S161_R D9S43_F D9S43_R D9S1817_F D9S1817_R D9S1791_F D9S1791_R D9S2148_F D9S2148_R D9S176_F D9S176_R Sequence (5’-­‐3’) GTTTCTTAGCAACCTCAACAGGG 6-­‐FAM-­‐AATCATCCAGAAAGGCCA GTTTCTTGAATCACAACATACACCCAC 6-­‐FAM-­‐GAAACATTCTTTTACAAGTAACATT 6-­‐FAM-­‐CTGTATGGAGAGAGAATACG GTTTCTTGGTCTCTCCGGTATACTCA 6-­‐FAM-­‐TGTGAGCCAAGGCCTTATAG GTCTGGGTTCTCCAAAGAAA 6-­‐FAM-­‐GCAATGACCAGTTAAGGTTC AATTCCCACAACAAATCTCC 6-­‐FAM-­‐TGCTGCATAACAAATTACCAC GTTTCTTCATGCCTAGACTCCTGATCC 6-­‐FAM-­‐TTCTGATATCAAAACCTGGC AAGGATATTGTCCTGAGGA 6-­‐FAM-­‐AGCTGTAGTGAGCCCTGAT CGTTAGGAGCCTTGAGACTT 6-­‐FAM-­‐GTAATCTTGGGCAACCTATGTATG TCAAAATAAGTCTGGGACAAAACC 6-­‐FAM-­‐TCAATCAACATCTGTCTATTCATC ACATCTGGCACTCTGGAGAG 6-­‐FAM-­‐AGCTGGCTGTTGGAGAAA TGACCAATGGCAGGGTAT Table S1: Clinical features of MPN patients with DNMT3A mutations MPN Patient At Diagnosis Sex Age Hb g/dl WCC Plts Spleen Karyotype x109/l x109/l FISH normal Karyotype normal At Follow-­‐up Therapy during study Past therapy Disease (yrs) DNMT3A mutation JAK2/MPL/CALR status Death Thrombosis HU HU 7 c.1123-­‐
1_1144del23$ JAK V617F N N P HU 14 p.R882H JAK V617F Y N ET #052 F 76 14.5 7.7 804 N ET #98 M 84 15.0 10.7 1285 N ET #8697 F 66 12.0 36.3^ 1034 N 13q del HU HU 5 p.R882H JAK V617F N N ET #03 M 54 14.3 16.4 953 N FISH normal HU IFN 11 p.R882H JAK V617F N Y PV #152 M 72 18.6 18.8 750 N Del Y HU HU 15 p.I705T JAK V617F Y Y nil nil 19 p.Y908* JAK2 exon 12$$$ N N PV #650 F 53 18.5 5.1 308 N Karyotype normal ET #50 M 75 11.9 7.1 488 N NA nil nil 5 p.F731V MPL W515L N N ET #78 M 66 14.8 9.1 983 N NA HU HU 6 p.R882H MPL W515L N N PPV
-­‐MF #28 F 45 10.4 10.4 229 Y NA nil nil 8 p.R882H JAK V617F N Y PV #25 F 45 16.2 17.6 654 Y NA IFN IFN 4 p.Y660F JAK V617F N Y PV #81 M 33 17.7 11.2 853 Y IFN IFN 16 G>A, c.2597+1$$ JAK V617F N N PMF #27 F 57 10.7 5.2 293 N nil Thal 12 p.S770L JAK V617F N N PMF #42 F 62 12.6 15 238 Y Rux Imatinib 1 p.D279fs*1 CALR R376fs*55 N N Karyotype normal FISH normal NA MPN, myeloproliferative neoplasm; Hb, hemoglobin; WCC, white cell count; Plts, platelet count; PV, polycythemia vera; PMF, primary myelofibrosis; ET, essential thrombocythemia; PPV-­‐MF, post PV myelofibrosis; HU, hydroxyurea; P, pipobroman; IFN, interferon alpha; Rux, Ruxolitinib; NA, not available; Y, yes; N, no, NA, not available; $ cDNA annotation of DNMT3A deletion affecting intron-­‐exon boundary; $$ cDNA annotation of DNMT3A mutation affecting an essential splice site; $$$ JAK2 exon 12 mutation p.F537_539_delinsL; * stop codon. No transformation events to myelodysplasia, acute myeloid leukemia or polycythemia vera occured during the follow-­‐up period.^ Significant leucocytosis due to concurrent chronic lymphocytic leukemia. Table S2 Total
PV (n)
ET (n)
MF (n)
Median age (years)
Mean WBC (x109/l)
Thrombosis (n)
Progression (n)
Death (n)
Median follow-up
(days)
JAK2V617F-first*
10
6
1
3**
54.6
9.6
2
0
1
4170 (863-5866)
DNMT3A-first
6
0
6
0
70.5
14.4
1
0
1
3149 (777-5036)
P-value
0.002
0.40
0.57
NP
NP
NP
0.52
Clinical data for JAK2V617F and DNMT3Amut patients with an established order of mutation acquisition (8 patients in
whom order was determined from initial colony analysis and 8 further patients in whom mutation order was
determined using data from targeted next-generation sequencing). Data show number of patients or events (n) in
the different mutation hierarchy groups. *includes three biclonal patients where JAK2V617F was also acquired on a
DNMT3A-wildtype background. **includes one patient with post-PV MF. P-values show results of Chi-squared
analysis for categorical variables and t-test for continuous variables. NP, not performed.
Supplementary Figure Legends Figure S1. X chromosome inactivation status assessment by analysis of expression patterns of heterozygous SNPs in LANCL-­‐3 and MPP1 in the two female patients (#25 and #27). Hematopoietic colonies were pooled by mutation status for JAK2 and DNMT3A. Pooled colonies underwent cDNA extraction followed by SNP genotyping. Sequencing traces are of cDNA derived from colonies sharing the same genotype of JAK2V617F and DNMT3A. #27 (upper panel) has the same X chromosome inactivation status in all subclones and is not informative. #25 (lower panel) has differing expression patterns in JAK2V617F-­‐mutated and DNMT3A-­‐mutated colonies confirming their independent origins. Figure S2 (a) Colony genotyping from an early sample from #650 showing colonies with heterozygous mutated JAK2 and wildtype DNMT3A (b) Whole genome sequencing reads from #650. Forward and reverse DNA reads are shown in blue and yellow, respectively, and mismatched bases (compared with the reference genome) are shown in red. Left panels shows a region in JAK2 exon 12 and right panels shows a region in DNMT3A. Lower panels show an early bone marrow sample from diagnosis and upper panels show a peripheral blood sample from 14 years later. JAK2 exon 12 mutation (p. F537_K539delinsL) is present in both early and late timepoints, however, DNMT3A mutation (p. Y990*) is not present in the earlier time point. (c) Microsatellite mapping of 9p loss-­‐of-­‐heterozygosity (LOH) in individual hematopoietic colonies (BFU-­‐Es) to determine the breakpoint region leading to acquired uniparental disomy in colonies with homozygous mutations in JAK2 exon 12 from patient #650. The panel of microsatellite markers is shown along the bottom in relation to their position from the telomere and centromere on chromosome 9. Non-­‐
informative markers are shaded grey, heterozygous markers are shaded in black and markers showing LOH are shaded in red. Colony numbers genotyped in the two homozygous clones h1 and h2 are shown on the right. Differing lengths of LOH in h1 and h2 confirm distinct breakpoints in the two homozygous clones. Figure S3 Differences in the proportions of JAK2/MPL/CALR-­‐only, DNMT3A-­‐only and double-­‐mutant subclones in patients where mutations have been acquired sequentially (ie. excluding biclonal patients). * p<0.05 Students t-­‐test performed on pairwise comparisons. Figure S1
Wildtype
subclone
JAK2 V617F
subclone
DNMT3A mutated
subclone
#27 MPP1
#25 LANCL-3
DNMT3a Y908*
wt
het hom
Figure S2
wt
a
het hom
JAK2 Exon 12
JAK2
DNMT3A p.Y908*
(ii)
(iii)
(iv)
At
diagnosis
14 years
post diagnosis
(i)
b
JAK2
h2
n=5
h1
n=11
D9S176
Centromere
D9S43
D9S1817
D9S1791
D9S2148
33.9
D95161
D9S235
D95925
D95162
18.3
6.2
D9S288
D9S1810
D9S1852
c
Telomere
T cell
LOH
Het
Not
informative
Mb from Telomere
Figure S3 Colonies (% of total)
*
60
*
40
20
0
JAK2/MPL DNMT3A Double-mutant
(n=3)
(n=4)
(n=10)