Supplemental Methods:
Gene selection for screening: Like SCN5A, SCN10A (Nav1.8) encodes a TTX-resistant sodium
channel and is located adjacent to SCN5A on human chromosome 3p21–22. SCN10A expression
has been suggested to contribute to the late sodium current (INa) (1) and more recently the locus
has been shown to act as an enhancer of SCN5A gene expression (2). TBX5 and TBX3 encode
transcription factors which regulate SCN5A expression (3). HAND1 encodes a transcription
factor essential for cardiac morphogenesis, and over-expression in the adult mouse leads to QRS
prolongation and predisposition to ventricular arrhythmia (4). CASQ2 encodes the cardiac form
of calsequestrin, a calcium-binding protein located in the lumen of the sarcoplasmic reticulum.
Mutations in CASQ2 are associated with catecholaminergic polymorphic ventricular tachycardia
(5). Phospholamban (PLN) is a phosphoprotein found in cardiac sarcoplasmic reticulum that is a
reversible regulator of the Ca2+-ATPase activity and cardiac contractility. TKT, which encodes
transketolase, an ubiquitous enzyme used in multiple metabolic pathways (6), was selected as the
most
likely
functional
candidate
gene
within
the
original
QRS-associated
locus
(TKT/CACNA1D/PRKCD). Expression analysis in blood (7) revealed very strong cis-eQTL
associations (P = 5.87×10−70) between the top QRS signal at this locus (rs4687718) and an
eSNP for TKT (rs9821134) which are in moderate linkage disequilibrium (r2 = 0.47) (8).
Basic procedure for ND7/23 transient transfection: The cells were cultured in Dulbecco
modified Eagle’s medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum
(Invitrogen), 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 g/mL streptomycin at 37C
under 5% CO2. After growth to ~ 70% confluence, cells were transiently transfected with 3 g of
the cDNA encoding human WT or variant Nav1.8 (V1073A, A200V, or I671V) and 0.5 g of a
1
plasmid encoding green fluorescent protein (GFP) were mixed with 10 L of FuGENE6
(Omega) in 0.8 mL serum-free DMEM medium and incubated for 30 min at room temperature.
The mixture was then added to the culture dish and the cells were incubated at 37C for 48 hr
before the electrophysiology studies were conducted.
Voltage-clamp recordings: We studied the biophysical properties of the Nav1.8 channels using
whole-cell voltage-clamp protocols as previously described (1). Briefly, whole-cell currents were
elicited by 100-ms pulses from -80 mV up to +50 mV in 10-mV increments from a holding
potential of -120 mV. Peak inward currents were measured and plotted as a function of the test
potential. Voltage dependence of Nav1.8 activation was calculated by normalizing the peak
currents to the largest measured current, plotting the values as a function of voltage and fitting
the data with a Boltzmann function (I/Imax = [1 + exp((V – V1/2) / k)] –1) to obtain the half
maximal activation potential (V1/2) and slope factor (k). Voltage dependence of inactivation was
studied using 500-ms pre-pulses from -120 to -20 mV in 10-mV increments, followed by a 100ms test pulse to -20 mV. Peak inward currents recorded at -20 mV were normalized to the peak
current and plotted as a function of the pre-pulse potential. The data were then fit to a Boltzmann
function to obtain V1/2 and k. The sodium window current, defined as the current range between
complete inactivation and starting of activation, was also compared in this study. The pipette
resistance used in this study was, on average, between 2 and 3 M, and the average access
resistance was below 10 M (5-10 M). Sodium currents were recorded at room temperature
using an Axopatch 200B series amplifier (Molecular Devices Corp., Sunnyvale, CA, USA).
Whole-cell currents were acquired at a sampling interval of 10 kHz and filtered at 1 kHz. Pulse
generation and data collection were done with Clampex 9.2.
Solutions and drugs: NaV1.8 currents in transiently transfected ND7/23 cells were recorded
2
using an external potassium-free solution that contained (in mmol/L): 135 NaCl, 1.8 CaCl2, 1.0
MgCl2, 20 TEA-Cl, 10 HEPES and 10 glucose, with a pH of 7.4, adjusted with NaOH.
Endogenous tetrodotoxin (TTX)-sensitive INa and L- and T-type calcium currents were
eliminated with TTX 200 nM, nisoldipine 1 μM and NiCl2 200 μM, respectively, as previously
reported (9, 10). Figure S1 (Supplementary data) shows the effect of these three drugs on a nontransfected ND7/23 cell. The drugs completely abolished the endogenous inward currents in
ND7/23 cells. The pipette electrodes were filled with the internal solution that contained (in
mmol/L): 10 NaF, 110 CsF, 20 CsCl, 5 EGTA, 10 HEPES and 5 Mg2-ATP, with a pH of 7.3
adjusted with CsOH.
Supplemental data
Clinical pedigree and case analysis:
SCN10A E19K: The carrier of this variant presented with syncope at the age of 26 during a
febrile illness and demonstrated an intermittent type 1 pattern. Ajmaline provocation was
positive and atrial fibrillation, as well as polymorphic VT, were inducible at electrophysiological
study. He was treated with an ICD implant. There was no family history of sudden death. No
additional family was available for assessment.
SCN10A R1121C: This 52 year old man was identified with the type 1 pattern following an
ajmaline test due to a strong family history of premature SCD. His past medical history included
atrial fibrillation. No other family members were available for assessment.
TKT R148Q: This 48 year old man presented with syncope. A type 1 pattern was diagnosed on
ajmaline provocation testing. Two affected relatives were available for cascade testing but did
3
not carry the putative mutation.
TKT S427C: This 45 year old man presented with syncope. A type 1 pattern was diagnosed upon
ajmaline provocation testing. No additional family members were available for cascade testing.
TBX5 G145R: The index case was a 10 year old boy incidentally found to carry a type 1 pattern.
His 38 year old mother was asymptomatic with a family history of premature SCD. She
presented with a spontaneous type 1 pattern. Both were heterozygous carriers for the G145R
variant. Six additional asymptomatic family members were available. None showed a type 1
pattern on standard 12 lead ECGs and five carried the G145R variant. They have not undergone
testing with a sodium channel blocker.
Supplemental Text References:
1. Yang T, Atack TC, Stroud DM, Zhang W, Hall L, Roden DM. Blocking SCN10A channels in
heart reduces late sodium current and is antiarrhythmic. Circ. Res. 2012;111:322–332.
2. Van den Boogaard M, Smemo S, Burnicka-Turek O, et al. A common genetic variant within
SCN10A modulates cardiac SCN5A expression. J. Clin. Invest. 2014;124:1844–1852.
3. Arnolds DE, Liu F, Fahrenbach JP, et al. TBX5 drives Scn5a expression to regulate cardiac
conduction system function. J. Clin. Invest. 2012;122:2509–2518.
4. Breckenridge RA, Zuberi Z, Gomes J, et al. Overexpression of the transcription factor Hand1
causes predisposition towards arrhythmia in mice. J. Mol. Cell. Cardiol. 2009;47:133–141.
5. Bezzina CR, Barc J, Mizusawa Y, et al. Common variants at SCN5A-SCN10A and HEY2 are
associated with Brugada syndrome, a rare disease with high risk of sudden cardiac death. Nat.
Genet. 2013;45:1044–1049.
6. Zhao J, Zhong C-J. A review on research progress of transketolase. Neurosci. Bull.
2009;25:94–99.
7. Dubois PCA, Trynka G, Franke L, et al. Multiple common variants for celiac disease
influencing immune gene expression. Nat. Genet. 2010;42:295–302.
8. Sotoodehnia N, Isaacs A, de Bakker PIW, et al. Common variants in 22 loci are associated
with QRS duration and cardiac ventricular conduction. Nat. Genet. 2010;42:1068–1076.
4
9. Holm H, Gudbjartsson DF, Arnar DO, et al. Several common variants modulate heart rate, PR
interval and QRS duration. Nat. Genet. 2010;42:117–122.
10. Savio-Galimberti E, Weeke P, Muhammad R, et al. SCN10A/Nav1.8 modulation of peak and
late sodium currents in patients with early onset atrial fibrillation. Cardiovasc. Res. 2014.
5
Supplemental Table S1
Gene
TBX5
Exon
1(Part1)
1(Part2)
2
3
4
5
6
7
8
9(Part1)
9(Part2)
TBX3
1
2
3
4
5
6
7
PLN
1
2
SCN10A 1
2
3
4
5
6
Forward primer 5’to3’
GGTATTCATTTGCCCAGAGC
CCAGCCAAACGTGACAGC
TTTCTCTCGTTCTCTCTCTGTCC
GTGTTTTGGGGGAGTTTGG
GAGGCTGCCTTAAAATACTGG
CTGGTGCGTGAACTGAAGC
GGGAGCAGGGTTTTATCTGG
TGGCTTAATTTGCTTCTTTTGG
TCTCTCACACCTGGTTCAGC
TTGGCCAAATAACTGTCTCC
ACTTCTCCGCTCACTTCACC
AGCAGCTGCGGACTTGT
AAGATTACGGGTGGTTTAT
ATCCCGTTTGTTCTGCTAA
ACACCCTCAAAACATTCTAGA
AGGGTGTGAGGTATGTGTGT
TTGGCTCATACTGGAGAT
AGAGGAGAGGGATGAGATAAGC
AGACTGAGACTGTGGCTAACC
ATTGTATTTTTTGTTCTGAGG
AAGGGCTGTTCTGACAATC
CCCTTCTTGCTCATAAGCCT
ATATTTATACCCACCTGTAGATCAG
GTCAGAGCAGGTCATCCCTCC
TCAGTCTATAGATGGCAGTGTCACTAGATTC
ATGAAGAAGTGTGCTCCG
Reverse primer 5’to3’
CCCAGTAAAATAAAGAGGCAACC
GCCAAGTGCAAAGAGAAACC
CAGACTCTGACTTTGATCTCTGC
GCCACCTTTTCTTCTTCACC
AACTTTTTGGGAGAAGGTTCC
GAGGACAAGAGGGAGACAAGG
TGCAAAAGAAAGAGCAGACG
GGTTGCTGCTGGCTTACC
ATACTCCTCACCCCCTCACC
GCTGGAACATTCCCTCTCC
TTTTTAAAATTGTGGTTTCAAGC
ACCGACCAACCGACTGTTCT
ACTCATGAAATGGGAAGCACT
ATCCTGACTTAAAGCAGCTT
ACACTTCAGAGTTGGATCCTA
TTCAACTCTTCCAGGCCA
ATTATTACAGCTACTAGGCCA
CTGCAAAAGGAAGGGCTAAC
AGAATTACCAAAGTCAGCGAA
AATATTGTTTTCCTGTCTGCA
GGGTGGGACCGAGAC
GGGCTCTGTTGCTAACCTCTA
AGACCTGGTTATTACAAAGACATATAG
GGACCTGCATGTTAGCCCTGT
ATAGTCTTTGCCCTGGAACCTTACAG
TCCCCTGTCCCTATATGATAC
Product size(bp)
478
390
297
243
248
282
280
294
390
465
474
529
398
279
212
320
839
641
234
405
458
319
452
307
338
326
Annealing temperature
59.0°C
59.0°C
59.0°C
59.0°C
59.0°C
59.0°C
59.0°C
59.0°C
59.0°C
59.0°C
59.0°C
60.0°C
58.0°C
58.0°C
50.0°C
59.0°C
60.0°C
58.0°C
61.0°C
61.0°C
54.0°C
54.0°C
51.0°C
56.4°C
55.5°C
54.0°C
6
HAND1
7
8
9
10
11
12
13
14
15(Part1)
15(Part2)
16(Part1)
16(Part2)
17
18
19
20
21
22
23
24
25
26
27(Part1)
27(Part2)
27(Part3)
1(Part1)
1(Part2)
2
TGCTGGGGGAGATTGAG
ACATAAAACTCTGTGGTTGTCTCGC
GGAGGCTGGTGGGTCA
CCTGGCACCCCATGTC
AAATGGGAGTAGGTAGAGG
TAAGAGGCTGAATGATCCAAC
GTCTAGAGGATGACCGCAGAA
GCTGCCATTTTTATCATTGC
TTTCCAGGCAAAACTTTAGA
GCCATCATTGTCTTTGTC
AGCTGCTAACCCGGTAGGC
CAGGTCTTTGGCCATCGTAC
CCTTCAAATTTAGAACTGGTC
TGAACCTGCAGTGACCGTGT
GGACAGGGACCACTGGTGTCATC
GGGTCCTTCCCTATCAGTCTGG
CATTGGAGTTCCGAATAA
TGGGAGGTTCCAGGTCTTTCTCTATC
CTTGGGAAGGGTCCAGTAT
CACCCAGGTCCATGTCTTA
TGGGCTTGGTTTGTCTATCTG
TGGAAGAGGTAAGGATAGTATTTCG
ATCACAGCCAACTTTGTCACG
TGTGGGAGCCCAGCCGTAGG
CACTGTCATTCAAAAGGCCTATCG
TGGGCTGCGCGTCTCATTTT
CGGCTGGAGGCGCTTGG
TACGAGCCTGTTTGCCTATTT
TTTTGGGGTCATTTGTTACAC
GAAAGAAGAGAAACCTGTACCCATAGC
ACGGTGCCCTAATTGAAGTG
TGATCCACTTCCCTAAGGTGT
ACAGGATGATGGCTAAAG
AAGATGGTGGGGCCTAAG
CCACCCCACCCGAACT
CTCCCTCCCACAAGGAC
AAAGGATGAGGCATATGGAT
GCCATCATTGTCTTTGTC
AGCTGCTAACCCGGTAGGC
CCTTTTGCCCTTTGTCACTC
CCTTCAAATTTAGAACTGGTC
GGATCCCAGGTGAGTGTTCG
AAGGTTGGGGACTTTCAGCTCAGTAG
AAACTTTTCTTTGGAGTAGCCCTTGAG
GGTGGTTTTGAACTCCTAA
ACATAGCATCTTCTGGGAACCTAGACTCTC
TGCAAGGAGGGACAGTGT
GTGATGGGCTGTGAGTAGTGT
ACCCTCATCCCAACGTCA
CAGGGTTCTTCTAACATAAACACG
TGAGGAAGGAGATGATGATGTAGG
GAGAGTGCCATGGAGCGGTGC
GCATTGGTGAGGCTGTAGCTGG
TCTCCGCTCCTTCTTGGGT
GTGTGTGCATTTTTGGAGGG
GCTGCCGGCCGCGCCGCCA
477
358
401
472
491
345
400
360
401
374
425
411
398
283
542
370
387
404
257
342
366
532
546
548
411
468
560
347
54.5°C
57.2°C
55.8°C
55.4°C
55.3°C
53.8°C
57.6°C
55.0°C
54.7°C
53.7°C
58.8°C
58.1°C
55.5°C
59.0°C
58.9°C
58.1°C
54.7°C
57.3°C
55.4°C
54.1°C
56.9°C
54.7°C
57.6°C
58.8°C
57.0°C
64.0°C
60.0°C
60.0°C
7
CASQ2
TKT
1(Part1)
1(Part2)
2
3
4
5
6
7
8
9
10
11
1
2
3
4
5
6
7
8
9
10
11F & 12R
13
14
CAGCCTGTCTGCTCTCTCCT
GGGCTTAATTTCCCCACATA
CCCTTCCATTGATACATGAGG
AGAAACCCTCAGCCAGACCT
ACAACTTCCCTCCAATGCAC
TTGGCTTTCTACTCTCTACACCA
TGCCGGAAAACATACACACT
GCAAAGGAAGCATCATCTCA
TCCAGCTTCTGAGTCCCTGT
TGAGCACCAAATGTCCTAAGAAG
TGGTGACCTAGTTACTGTGATGG
AGCTATTCAGCACGGCTATC
TGAAGGCCTGGGGTGATCC
CTGTGTGACTCCTCAGGACAT
GTCAGTGTGAAGTAGGCACAG
GCAGAGGAAGGGCTTGCTG
CAGGCTCCTTGGATGGGTG
GCAGACCTTTCCCTGGAAGG
TAGAGGGCCAGTCTCCTGGA
CTTCTCTGAAAGCAGGGCTC
CTCCACAAAAGGGGTCCTAG
CAGGATTCCAGCCCATGGAT
GATGGCCTCTCTAGAAAGACC
CCTCAGGATGTCCCACCAG
CTAAGGCTGACAGAGGGCTG
AAGACACCGGCTCATGGTAG
ATTCTCTGCATCCTCGTTCC
AAGATAGTCCGCTTATGCAAGA
CCAGCTTGTCCTTTCTTTGG
TGCTGAAGACCCATCCTCTT
GGCAAGGGAGGATGGTTAAT
GCTCTTGGCAGGTCTGAAAT
GGTTTGAGTTTGGGGACTTAAT
TGTGGATGGGATGTGAGAGA
CCCCTGCCTATTTCACCTC
CTTCTGAAAAGGCTGGGCTA
ATTGCTTGCTGCCACCTT
CTTCCTCGTCTAGGCATCTC
AGACCACTCACCATTCCAGG
CCACATGTCTGGGAAATAGGAAG
CGATGGTGAAGGGTGGGTC
GCACCTGCACCTGCAAAGC
GGTCAGAGCTACGTAGCCAC
GCTCTCCTCCCTTTCCCAG
GCTCTGCCAACATGGCTGC
ATCCATGGGCTGGAATCCTG
TGGCCTCCTCTTCTGTTTCC
AACCCCAAGACCTAGCATGC
TCCTGGGGCTCAGATAGAAG
GTACATCTTTGAGCACCTTTCCC
287
271
278
274
292
278
317
250
237
299
275
345
405
306
253
278
355
267
381
333
329
269
448
297
324
54.0°C
54.0°C
54.0°C
54.0°C
54.0°C
58.0°C
60.0°C
57.0°C
60.0°C
54.0°C
54.0°C
54.0°C
62.0°C
64.0°C
64.0°C
67.0°C
67.0°C
64.0°C
64.0°C
64.0°C
62.0°C
62.0°C
62.0°C
60.0°C
64.0°C
8
Supplemental Table S2: Weights used in SKAT and SKAT-O tests
SNP
Position on
chromosome3
rs116353929
38739494
rs77804526
38739622
rs6599242
38739845
rs78425180
38740001
chr3:38740051 38740051
rs142217269
38743419
rs6790627
38748833
rs11711062
38753732
rs145032037
38753882
rs138832868
38755450
rs6771157
38763863
rs12632942
38764998
rs6795970
38766675
rs6791171
38766701
rs73062575
38766760
rs59468016
38768247
rs57326399
38768300
rs7374804
38768334
rs6800541
38774832
rs6599250
38784029
rs7630989
38793940
rs7617919
38793989
rs62244070
38798171
rs74717885
38805069
chr3:38812770 38812770
rs141207048
38835461
Minor Allele
frequency
0.035
0.010
0.089
0.011
0.001
0.003
0.144
0.003
0.001
0.003
0.228
0.228
0.430
0.144
0.030
0.226
0.226
0.061
0.431
0.432
0.027
0.216
0.216
0.014
0.001
0.001
CADD score
(scaled)
3.258
3.048
7.553
0.047
3.616
23.600
8.745
5.860
33
33
2.227
7.645
2.315
4.203
9.835
8.749
10.050
8.171
0.451
11.870
3.919
6.551
7.646
9.947
34.000
16.790
Allele frequency weight
in SKAT test (default)
10.683
19.437
2.679
18.952
24.581
23.165
0.598
23.408
24.265
23.380
0.051
0.050
3.488E-05
0.602
12.066
0.054
0.053
5.527
3.333E-05
3.237E-05
13.020
0.072
0.073
17.682
24.561
24.176
Allele frequency in SKAT
test x CADD score weights
34.804
59.245
20.231
0.891
88.886
546.700
5.230
137.171
800.743
771.536
0.113
0.383
8.075E-05
2.532
118.673
0.472
0.531
45.160
1.503E-05
3.843E-04
51.024
0.471
0.556
175.882
835.079
405.910
9
Supplemental Table S3: The classification of the 7 putative pathogenic BrS variants.
GeneVariant
SCN10A
E19K*
A200V
R1121C
G1299A
G590R
I671V
TKT
R148Q
S427C
TBX5
G145R
PLN
R25C
*rs141810266
Predicted
Classification
Risk/Nonrisk Prediction Tools-Classification
alleleles
GERP
Grantham score
PolyPhen2
SIFT
pathogenic
pathogenic
pathogenic
pathogenic
benign
benign
A/G
T/C
T/C
C/G
T/C
A/G
conserved
conserved
not conserved
conserved
conserved
conserved
benign
benign
damaging
benign
damaging
benign
possibly damaging
probably damaging
probably damaging
probably damaging
benign
benign
damaging
damaging
damaging
damaging
benign
N/A
pathogenic
pathogenic
A/G
G/C
conserved
conserved
benign
damaging
probably damaging
probably damaging
damaging
damaging
pathogenic
A/G
conserved
damaging
probably damaging
damaging
benign
C/T
conserved
benign
probably damaging
benign
10
Supplemental Table S4: Collapsed common and rare variant analysis results for SCN10A
Performed
test
MAF threshold
for inclusion
P-value using standard
frequency weights
P-value using combined
frequency and CADD weights
# Variants used in
test
# Common markers
SKAT
SKAT-O
SKAT
SKAT-O
SKAT
SKAT-O
All
All
< 5%
< 5%
< 1%
< 1%
0.2190605
0.3109835
0.2527421
0.3541483
0.5819197
0.1220259
0.4576156
0.6509294
0.492582
0.4476532
0.4465203
0.1826234
26
26
13
13
7
7
13
13
0
0
0
0
11
Supplemental Table S5: Common synonymous and non-synonymous SCN10A SNPs present in BrS patients.
GeneVariant
SCN10A
D1739D
V1697I
S1622S
T1570T
K1441K
T1131T
L1092P
V1073A
T1064T
P1045T
G979G
I962V
K950K
S509P
L492L
E428E
I206M
rsID
Predicted
Minor/ Genotype Frequencies
Minor Allele
Odds
Classification Major
Frequency*
ratio(95%CI)
alleles*
Cases
UK10KControls
Cases Controls
Hom
Het Hom
Hom
Het Hom
(minor)
(major) (minor)
(major)
P-value
rs116353929
rs77804526
rs6599242
rs78425180
rs6790627
rs6771157
rs12632942
rs6795970
rs6791171
rs73062575
rs59468016
rs57326399
rs7374804
rs7630989
rs7617919
rs62244070
rs74717885
benign
benign
benign
benign
benign
benign
benign
benign
benign
benign
benign
benign
benign
benign
benign
benign
benign
0.54
0.18
6.27x10-03
0.42
0.4
3.82x10-08
7.81x10-08
8.07x10-19
5.31x10-07
0.41
3.16x10-08
1.32x10-07
0.45
0.1
5.95x10-08
5.69x10-07
0.44
A/G
T/C
A/G
T/C
C/T
C/G
G/A
A/G
T/C
T/G
A/G
C/T
C/T
G/A
A/G
T/C
C/T
0
0
0
0
4
3
3
74
1
0
3
3
1
0
6
3
0
9
1
15
5
32
27
28
58
14
7
26
28
14
4
18
27
6
147
155
141
151
120
126
125
22
141
149
127
125
141
152
132
126
150
2
0
7
0
26
65
66
200
23
1
68
68
3
1
65
65
0
87
29
226
28
321
489
489
627
351
77
479
480
153
71
461
457
35
1190
1250
1046
1251
932
724
724
453
905
1201
731
731
1123
1207
753
757
1244
0.029
0.003
0.048
0.016
0.128
0.106
0.109
0.669
0.051
0.022
0.103
0.109
0.051
0.013
0.096
0.106
0.019
0.036
0.011
0.094
0.011
0.146
0.242
0.243
0.401
0.155
0.031
0.241
0.241
0.062
0.029
0.231
0.229
0.014
0.81(0.40-1.62)
0.28(0.04-2.07)
0.49(0.29-0.83)
1.48(0.57-3.85)
0.86(0.61-1.22)
0.37(0.25-0.54)
0.38(0.26-0.55)
3.02(2.35-3.87)
0.29(0.18-0.49)
0.72(0.33-1.58)
0.36(0.25-0.53)
0.39(0.27-0.56)
0.82(0.48-1.38)
0.44(0.16-1.22)
0.35(0.24-0.52)
0.40(0.27-0.58)
1.41(0.59-3.38)
12
13
14
15