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Double somatic mosaic mutations in TET2 and DNMT3A – origin of peripheral T-cell lymphoma in a case
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Tran B. Nguyen1, Mamiko Sakata-Yanagimoto1,2,3, Rie Nakamoto-Matsubara1, Terukazu Enami3, Yufu Ito3,
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Toshikata Kobayashi3, Naoshi Obara1,2,3 , Yuichi Hasegawa 1,2,3, Shigeru Chiba1,2,3,4
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Tsukuba, Ibaraki 305-8575, Japan
2.
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Department of Hematology, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba,
Ibaraki 305-8575, Japan
3.
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Department of Hematology, Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai,
Department of Hematology, University of Tsukuba Hospital, 2-1-1 Amakubo, Tsukuba, Ibaraki 3058576, Japan
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Life Science center, Tsukuba Advanced Research Alliance, University of Tsukuba, University of
Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Japan
Correspondence should be addressed to:
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Shigeru Chiba (schiba-t@md.tsukuba.ac.jp)
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Department of Hematology, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba,
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Ibaraki 305-8575, Japan. Telephone number: (+81) 298533103, Fax number: (+81) 298538079.
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Supplementary methods
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Patient and samples.
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Samples were obtained with written informed consent after approval by the ethics committee in the University
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of Tsukuba Hospital. Genomic DNA was extracted from paraffin-embedded formalin-fixed (FFPE) samples of
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lymph node, bone marrow mononuclear cells (MCs), buccal mucosal cells, and nails. Mutations in TET2 and
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DNMT3A in the tumor sample of this patient were shown in the previous reports: TET2 c.A3443G:p.Y1148C
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and DNMT3A c.T2264C:p.F755S [1].
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Sorting of tumor cell-enriched fraction and other fractions.
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MNCs were isolated from peripheral blood (PB) of the patient by Ficoll-Paque density gradient centrifugation.
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MNCs were stained by fluorescein isothiocyanate (FITC)-conjugated anti-CD4, anti-CD14 antibody (BD
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Biosciences, Cat. 555397), and phycoerythrin (PE)-conjugated anti-CD8, anti-CD19 antibody (Dako, clone
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HD37), and allphycocyanin (APC)-conjugated anti-CD279/PD1 antibody (Bio legend, clone EH12.2H7), and
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then fractionated by FACS Aria (BD Biosciences).
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Cell lysate was obtained by adding lysis buffer (0.3% Tween 20, 0.3% NP-40, 10mg/ml proteinase K and pH
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8.0 Tris- EDTA buffer). The lysate was incubated at 55oC for 1 hour and then at 80oC for 10 minutes. One µL of
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cell lysate which contained approximately 100 cells was used for polymerase chain reaction (PCR) under the
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following conditions: 94oC for 2 minutes, 35 cycles of 98oC for 10 seconds and 68oC for 30 seconds by KOD -
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Plus- Neo kit (TOYOBO) with each primer set (supplementary Table 4). PCR amplicons were used for
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amplicon-based sequencing and Sanger sequencing.
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Amplicon-based sequencing to determine the allele frequencies of the mutations in various cells and
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tissues by Ion Torrent PGM.
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The libraries were made by using the Ion Plus Fragment Library kit according to the protocol for preparing short
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amplicon libraries (Life technologies). Briefly, PCR amplicons were ligated to barcode adapters and P1
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adapters, and then amplified. Quantitation of the amplified libraries was performed by quantitative PCR with the
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Ion Library Quantitation kit according to manufacturer’s instruction (Life technologies). The libraries were then
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subjected to deep sequencing on Ion Torrent PGM according to the standard protocol for 300 base pair single-
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end reads (Life technologies). The data was analyzed by Variant caller 3.4 (Life technologies). The single
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nucleotide variant at nucleotide position A to G at c.A3443 of TET2 and that from T to C at c.T2264 of
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DNMT3A comprising equal to or more than 0.5% were adopted as the mutations (supplementary Table 2).
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Colony forming analysis from PB MCs.
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PB MCs were cultured in MethoCult H4435 Enriched (STEMCELL Technologies) for 15 days. Each colony
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was harvested. Genomic DNA of the colonies was directly amplified using Repli G single cell kit (QIAGEN).
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The DNA solution was diluted 100 times and 2ul of these were used for Sanger sequencing.
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Supplementary Figure 1: Protein structure of TET2 and DNMT3A [2]
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Cys-rich: Cystein-rich domain; Core-DSBH: Core double-strain-beta-helix domain; PWWP: PWWP domain;
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ADD: ADD domain; SALMDM: S-adenosyl-L-methionine-dependent-methyltransferase domain
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Supplementary Table 1. Mutation profile
Annotated
Mutation
Nucleotide
Amino acid
genes
Type
Change
Transcript
CACNA1D
NM_000720
Missense
c.C992T
p.T331M
0.04
EBF2
NM_022659
Missense
c.G1186A
p.A396T
0.03
NAV2
NM_001111018
Missense
c.G2315T
p.S772I
0.06
DNMT3A
NM_175629
Missense
c.T2264C
p.F755S
0.27
DNMT3A
NM_175629
Frameshift
c.177_178insC
p.P59fs
0.04
TET2
NM_001127208
Missense
c.A3443G
p.Y1148C
0.18
TET2
NM_001127208
Frameshift
c.5252_5253insT
p.Y1751fs
0.06
Genes
VAF*
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*: Variant Allele Frequency
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Supplementary Table 2. Allele frequencies analyzed by amplicon-based sequencing
Annotated
Mutation
Variant
gene
type
TET2
Missense
DNMT3A
Missense
Allele frequency (%)
times
Mean
SD**
NM_001127208:
Nail
3
0.93
0.20
c.A3443G:p.Y1148C
Buccal mucosa
3
11.94
1.36
CD4+PD1-
3
13.80
4.47
CD8+
3
19.12
1.13
CD14+
4
24.93
7.05
CD19+
3
23.72
5.51
Bone marrow
6
23.08
1.72
CD4+PD1+
3
46.13
4.94
Lymph node
3
24.80
1.94
NM_175629:
Nail
3
0.82
0.35
c.T2264C:p.F755S
Buccal mucosa
4
8.13
6.09
CD4+PD1-
3
20.73
0.71
CD8+
5
20.75
3.50
CD14+
4
17.86
2.20
CD19+
6
22.22
3.66
Bone marrow
5
21.24
18.70
CD4+PD1+
3
50.00
3.65
Lymph node
3
23.93
5.16
3
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Run
Sample
**: Standard deviation
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Supplementary Table 3. Allele frequency and coverage of random single nucleotide variant read analyzed
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by amplicon-based sequencing
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Control (n=9)
Nail (n=3)
Average allele
Average
Average allele
Average
frequency (%)
Coverage
frequency (%)
Coverage
0.01
24206.11
0.90
64415.50
<0.01
0.02
37191.89
0.82
31519.33
<0.05
P
TET2
c.A3443G:p.Y1148C
DNMT3A
c.T2264C:p.F755S
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Supplementary Table 4. Primer list for amplicon-based sequencing (p1) and Sanger sequencing (p2)
Primer
Forward
Reverse
TET2_p1
AGCCCTTAATGTGTAGTTGGGG
GCTTTGTGTGTGAAGGCTGG
DNMT3A _p1
AGTGAGCTGGCCAAACCAAG
GGCCGGCTCTTCTTTGAGTT
TET2_p2
TTGGGGGTTAAGCTTTGTGGA
GCACAGTGTGTAGTGTTGGC
DNMT3A _p2
CAGGATGAAGCAGCAGTCCA
CCCAGCTGATGGCTTTCTCT
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