Supplementary information
Novel recurrent mutations in ethanolamine kinase 1 (ETNK1) gene in systemic mastocytosis with
eosinophilia and chronic myelomonocytic leukemia
Lasho TL et al.
Pages 2-3: Supplementary Table 1. List of somatic coding region single nucleotide variants identified in
index case of aggressive systemic mastocytosis with eosinophilia
Pages 4-5: Methods for whole exome-sequencing of index case for mutation identification and targeted
resequencing of additional cases for mutation confirmation
Page 6: Supplementary Figure 1. Nucleotide sequence conservation between species for ETNK1 gene in
vicinity of region encoding for amino acid residues N244 and G245
1
Supplementary Table 1. List of somatic coding region single nucleotide variants identified in index
case of aggressive systemic mastocytosis with eosinophilia
Annotated Gene
Mutation
Type
Amino Acid
Change
Chromosom
e
Position
Allele
Change
ACAD10
AMMECR1
ATXN2L
AVPR1B
BMPR2
BRPF1
C4orf26
CDK19
CEACAM1
CERCAM
CLIP1
COL27A1
EP400
ETNK1
EZH2
F7
FAT4
GRAMD1A
GRM6
KBTBD4
KIT
LCT
LPHN3
MMRN2
NCAPD2
NEK8
NR5A2
PGM2
PLIN4
PLIN4
RP1L1
SCN4A
SELPLG
SLC17A4
SLC26A9
SLC6A2
SLFN11
STAT5A
STK36
SYNE1
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
D404A
S118L
H871P
T12P
T606P
L975F
H97P
V16G
T298P
Y232S
E966K
K452N
T2216P
N244S
H694R
V285G
D2022A
C456G
Y487D
V78G
D816V
T687P
H1340P
V433G
Y1325S
A306T
R187G
V341G
C819S
C819S
E1300A
E872G
P161R
I147F
P746R
T63P
T707P
V707E
D1073A
F8509L
chr12
chrX
chr16
chr1
chr2
chr3
chr4
chr6
chr19
chr9
chr12
chr9
chr12
chr12
chr7
chr13
chr4
chr19
chr5
chr11
chr4
chr2
chr4
chr10
chr12
chr17
chr1
chr4
chr19
chr19
chr8
chr17
chr12
chr6
chr1
chr16
chr17
chr17
chr2
chr6
112165822
109560947
28846647
206224474
203420204
9786696
76489546
111136293
43025485
131186822
122812709
116931191
132529360
22811995
148506431
113772775
126336183
35510328
178413880
47599367
55599321
136570175
62936235
88703243
6640096
27064863
200017395
37848566
4511475
4511474
10467709
62029022
109017650
25770436
205887987
55705945
33679962
40461400
219563485
152457885
A>C
G>A
A>C
A>C
A>C
A>C
A>C
A>C
T>G
A>C
C>T
A>C
A>C
A>G
T>C
T>G
A>C
T>G
A>C
A>C
A>T
T>G
A>C
A>C
A>C
G>A
C>G
T>G
A>T
C>G
T>G
T>C
G>C
A>T
G>C
A>C
T>G
T>A
A>C
G>T
Tlymphocyte
allele
fraction
(control)¶
Eosinophil
allele
fraction
(tumor)
1.90%
0.00%
2.50%
0.60%
3.00%
2.50%
0.00%
1.20%
2.50%
3.10%
3.00%
2.20%
2.90%
1.40%
3.10%
2.70%
1.40%
1.80%
1.50%
1.80%
0.00%
2.70%
2.90%
1.60%
2.90%
0.80%
0.80%
3.40%
0.90%
0.90%
1.70%
3.70%
1.30%
2.00%
0.00%
2.80%
2.20%
0.00%
0.90%
1.40%
6.20%
42.60%
5.30%
3.00%
1.60%
1.40%
2.60%
2.20%
9.20%
7.90%
2.00%
3.40%
7.40%
50.00%
97.40%
6.70%
2.70%
4.40%
2.70%
6.20%
47.10%
7.60%
1.60%
5.20%
5.40%
7.00%
1.70%
6.30%
2.70%
2.10%
0.70%
12.10%
4.90%
3.70%
35.90%
1.10%
4.10%
67.00%
2.20%
52.60%
2
TAS2R30
Missense
I289T
chr12
TECPR2
Missense
N138S
chr14
TMEM170A
Missense
S12A
chr16
TRIM24
Missense
N554T
chr7
UMOD
Missense
L630F
chr16
WDR6
Missense
V884G
chr3
XKR8
Missense
T187P
chr1
XPC
Missense
A425G
chr3
ZEB2
Missense
H95P
chr2
¶Only cases with T-lymphocyte allele fraction <4% displayed
11285978
102874889
75498465
138252356
20344671
49051528
28293082
14200109
145187383
A>G
A>G
A>C
A>C
G>A
T>G
A>C
G>C
T>G
3.00%
0.00%
3.70%
1.80%
2.70%
3.40%
0.30%
3.00%
3.40%
5.10%
53.80%
9.90%
7.20%
46.20%
5.90%
2.80%
1.60%
1.60%
3
Supplementary Methods
Illumina HiSeq Sure Select XT Custom Capture and Paired End Sequencing
Paired-end indexed libraries were prepared using the Agilent Bravo liquid handler following the
manufacturer’s protocol (Agilent Technologies, Santa Clara, CA). Briefly, 3 µg of target DNA was
fragmented using the Covaris E210 Sonicator. The settings of duty cycle 10%, intensity 5, cycles 200,
time 360 seconds generated double-stranded DNA fragments with blunt or sticky ends with a fragment
size mode of between 150-200bp. The ends were repaired and phosphorylated using Klenow, T4
polymerase, and T4 polynucleotide kinase, after which an “A” base was added to the 3’ ends of doublestranded DNA using Klenow exo- (3’ to 5’ exo minus). Paired end Index DNA adaptors (Agilent) with a
single “T” base overhang at the 3’ end were ligated, and the resulting constructs were purified using
AMPure SPRI beads from Agencourt (Beckman Coulter Genomics, Danvers, MA). The adapter-modified
DNA fragments were enriched by 4 cycles of PCR using SureSelect forward and SureSelect Pre-Capture
indexing reverse (Agilent) primers. The concentration and size distribution of the libraries was
determined via Agilent Bioanalyzer DNA 1000 chip.
Custom capture of 3.69 Mb was carried out using the Agilent Bravo liquid handler following the protocol
for Agilent’s SureSelect XT. 750 ng of the prepped library was incubated with whole exon biotinylated
RNA capture baits supplied in the kit for 24 hours at 65 °C. The captured DNA:RNA hybrids were
recovered using Dynabeads MyOne Streptavidin T1 (Life Technologies, Carlsbad, CA). The DNA was
eluted from the beads and purified using Ampure XP (Beckman Coulter Genomics). The purified capture
products were then amplified using the SureSelect Post-Capture indexing forward and index PCR reverse
primers (Agilent) for 12 cycles. Libraries were validated and quantified on the Agilent Bioanalyzer.
Libraries were pooled at equimolar concentrations in batches of 96 samples and loaded onto paired end
flow cells at concentrations of 7-8 pM to generate cluster densities of 600,000-800,000/mm2 following
Illumina’s standard protocol using the Illumina cBot and HiSeq paired-end cluster kit version 3 (Illumina,
San Diego, CA).
The flow cells were sequenced as 101 X 2 paired end reads on an Illumina HiSeq 2000 using TruSeq SBS
sequencing kit version 3 and HiSeq data collection version 1.4.8 software. Base-calling was performed
using Illumina’s RTA version 1.12.4.2 (Illumina).
Analysis
We used Genesifter® software (PerkinElmer, Danvers, Massachusetts) to analyze the data. Within this
program, raw reads from the sequencing above in fastq format were first trimmed to remove
adaptor/primer sequences. Trimmed reads were then aligned using BWA against the genomic reference
sequence for Homo sapiens (Build 37.2). The Homo sapiens (Build 37.2) reference sequence and
annotation were pulled from the NCBI database (http://www.ncbi.nlm.nih.gov/). An additional
alignment, post-processing set of tools were then used to do local realignment, duplicate marking, and
score recalibration to generate a finale genomic aligned set of reads. Reads mapping to the genome were
characterized as exon, intron, or intergenic (outside any annotated gene) using Build 37.2 for the Homo
4
sapiens reference. Nucleotide variants were called using GATK (Broad Institute, Cambridge, MA),
which identified single nucleotide and small insertion/deletion (indel) events using default settings.
PCR Validation
We used both patient eosinophil (CD16-) and T-lymphocyte DNA for validation of somatic ETNK1
mutations in every case. Primers for amplification of ETNK1 exon 3 (ENST00000266517) were: ETNK1FP: 5’-AAGAAGATTCGGGAGA-3’, and ETNK1-RP: 5’-CAGCCAAAGAATCAATGC-3’. The 50µL
reaction contained 20ng of DNA, and also included 5µL 10X reaction buffer (Roche, Indianapolis,
Indiana), 1.5µL of a 25mM mixture of dNTPs, 2µL each of a 10mM concentration of both forward and
reverse primers, 0.5µL of Taq polymerase (Roche), and dH20. Amplification included an initial
denaturation at 94oC for 2 min, followed by: 35 cycles of a melt at 94oC for 30sec, annealing at 52oC for
45sec and extension at 72oC for 40sec, and ended with a final extension at 72oC for 3 min. Primers for
screening ETNK2 exon 3 (ENST00000367202) were as follows: ETNK2-FP: 5’GGCCTGGAGGGTCCCTATCTGG-3’, ETNK2-RP: 5’-CCCGAGACCTAAGCCCTTAAAC-3’.
Amplification for ETNK2 included the same master mix preparation as above and protocol as follows:
denaturation at 94oC for 2 min, followed by: 40 cycles of 94oC for 20sec, 54oC for 15sec, 72oC for 45sec,
with a final extension at 72oC for 1 min 30 sec. All products were visualized via 1.3% agarose gel and
ETNK1 and ETNK2 mutational status confirmed by standard bi-directional Sanger sequencing.
5
Supplementary Figure 1. Nucleotide sequence conservation between species for ETNK1 gene in
vicinity of region encoding for amino acid residues N244 and G245
6