Clin Genet 2013: 84: 489495 2013 John Wiley & Sons A/S.

Printed in Singapore. All rights reserved Published by John Wiley & Sons Ltd
CLINICAL GENETICS
doi: 10.1111/cge.12126

Original Article

High prevalence of genetic variants

previously associated with Brugada syndrome
in new exome data

Risgaard B, Jabbari R, Refsgaard L, Holst AG, Hauns S, Sadjadieh A, B Risgaarda,b,c , R Jabbaria,b,c ,

Winkel BG, Olesen MS, Tfelt-Hansen J. High prevalence of genetic L Refsgaarda,b , AG Holsta,b,c ,
variants previously associated with Brugada syndrome in new exome data. S Haunsa,b,c,d , A Sadjadiehe ,
Clin Genet 2013: 84: 489495. John Wiley & Sons A/S. Published by BG Winkela,b,c , MS Olesena,b
John Wiley & Sons Ltd, 2013 and J Tfelt-Hansena,b,c
More than 300 variants in 12 genes have been associated with Brugada a Danish National Research Foundation

syndrome (BrS) which has a prevalence ranging between 1:2000 and Centre for Cardiac Arrhythmia (DARC),
1:100,000. Until recently, there has been little knowledge regarding the University of Copenhagen, Copenhagen,
distribution of genetic variations in the general population. This problem Denmark, b Laboratory of Molecular
was partly solved, when exome data from the NHLI GO Exome Cardiology, Department of Cardiology,
The Heart Centre, c Department of
Sequencing Project (ESP) was published. In this study, we aimed to report
Cardiology, The Heart Centre,
the prevalence of previously BrS-associated variants in the ESP Copenhagen University Hospital
population. We performed a search in ESP for variants previously Rigshospitalet, Copenhagen, Denmark,
associated with BrS. In addition, four variants in ESP were genotyped in a d
Department of Medicine and Surgery,
second Danish control population (n = 536) with available Faculty of Health Science, University of
electrocardiograms. In ESP, we identified 38 of 355 (10%) variants, Copenhagen, Copenhagen, Denmark,
distributed on 272 heterozygote carriers and two homozygote carriers. The and e Department of Cardiology,
genes investigated were on average screened in 6258 individuals. This Copenhagen University Hospital of
corresponds to a surprisingly high genotype prevalence of 1:23 (274:6258). Bispebjerg, Copenhagen, Denmark
Genotyping the four common ESP-derived variants CACNA2D1 S709N, Key words: arrhythmia Brugada
SCN5A F2004L, CACNB2 S143F, and CACNB2 T450I in the Danish syndrome channelopathies exome
controls, we found a genotype prevalence comparable with that found in next-generation sequencing
sudden cardiac death
ESP. We suggest that exome data are used in research, as an additive tool
to predict the pathogenicity of variants in patients suspected for BrS. Corresponding author: Bjarke
Risgaard, MD, Department of
Conflict of interest Cardiology, Section 2142, Copenhagen
University Hospital, Rigshospitalet,
The authors declare no conflict of interest. Blegdamsvej 9, DK-2100 Copenhagen,
Denmark.
Tel.: +45 35 45 65 01;
fax:+45 35 45 65 00;
e-mail: bjarkerisgaard@gmail.com

Received 10 January 2013, revised

and accepted for publication 13
February 2013

Today hundreds of variants in 12 genes have been
associated with Brugada syndrome (BrS), which is
considered a rare and inherited Mendelian disorder with
a prevalence ranging between 1:2000 and 1:100,000
(15). BrS is a primary arrhythmic syndrome, charac-
terized by ST-segment elevations in the right precordial
leads (V1 V3 ), with an increased risk of sudden cardiac
death, due to malignant ventricular arrhythmias, in the
absence of a structural heart disease (6). Since BrS was
first described in 1992 (7), it has been associated with
several genetic variations affecting the cardiac sodium
current, the potassium transient outward current, and
the calcium current (810). However, in the clinical
setting SCN5A is the only recommended gene for

489
Risgaard et al.
targeted screening in patients with BrS, but only
1530% of all cases are caused by loss of function
in this gene (11). SCN5A encodes the alpha subunit
(Nav 1.5) of the cardiac sodium channel complex, and
was the first gene to be associated with BrS (8, 12).
SCN5A has also been associated with several other
arrhythmogenic diseases such as lone atrial fibrillation
and long QT syndrome (LQTS) (1315).
In the last years, several other genes have been
associated with BrS. Untill now, the function of the
sodium channel complex is also known to be modulated
by RANGRF , SCN1B , SCN3B and GPD1L genes
which have all been associated with BrS phenotypes
(16). Other currents important for the action potential
have also been associated with BrS, including the
inward transient potassium current and the inward
calcium current. As such, in the setting of BrS, the
inward transient potassium current has been shown
to be affected by mutations in the KCNJ8 , KCND3 ,
KCNH2 and KCNE3 genes (16). Finally, changes in
the inward calcium current has been shown to be
caused by mutations in the CACNA1C , CACNA2D1 ,
and CACNB2 genes (9).
Until recently, there has been little knowledge
regarding the distribution of genetic variations in the
general population. Potentially it could be a problem,
when rare variants are associated with a rare disease,
as some of these variants most likely just have a
modifying effect (16). In June 2011 this problem was
partly solved, when exome data from the NHLBI GO
Exome Sequencing Project (ESP) was published (17).
The aim of this study was to report the prevalence of
variants present in the ESP population, previously asso-
ciated with BrS. In addition, we aimed to establish if
multiple in silico tools could distinguish between vari-
ants represented in ESP (ESP-derived) and variants not
represented in ESP (non-ESP-derived) adding further
information to the pathogenicity of these variants.
Materials and methods
In ESP, next-generation sequencing was carried out
for all protein-coding regions in 6500 persons from
different population studies (17). Clinical data on the
ESP populations were not available even on request.
None of these studies specifically included patients with
channelopathies or other heart diseases, and at least two
studies excluded such patients (18).
In order to find all genes and variants previously
associated with BrS, a search in the Human Gene Muta-
tion database (HGMD) was conducted on 1 June 2012,
for Brugada syndrome (19). In this way 279 variants
in 11 genes were identified. Furthermore, a literature
search in the PubMed database was carried out. The
following search query was used: {(Brugada) or [Bru-
gada syndrome] or [Brugada syndrome (Mesh)]} and
{[Genetic*] or [Genetics (Mesh)]} and (mutation). We
found 390 articles that matched the search term, and
these were systematically examined. In this way, 74
SCN5A variants reported by Kapplinger et al. (11), one
SCN5A variant reported by Marangoni et al. (20), as
490
well as KCNJ8 S244L reported by Barajas-Martnez
et al. (21) were additionally included. In total, 355 vari-
ants in 12 genes, previously associated with BrS, were
included in this study (Table 1).
The ESP exome data was searched 1 July 2012,
for these missense and nonsense variants. Due to lack
of data regarding variations positioned in introns and
UTR regions in ESP, these were not included in our
search. In addition, the literature was searched for data
on functional studies and familial co-segregation on
variants represented in ESP. Familial co-segregation
was defined as at least two genotype positive family
members having the same phenotype.
To test if the ESP population harboured an overrep-
resentation of variants associated with BrS, four com-
mon variants in our own healthy control population
(n = 536) of Northern European origin were genotyped,
as described previously (22). This control population
had no history of arrhythmias or other cardiac diseases.
electrocardiograms (ECGs) were available for the entire
control population (23, 24). In those patients harbour-
ing a variant, ECGs were examined by two physicians
independently to exclude ST-segments elevations in the
right precordial leads (BrS type 1 pattern) as well as
incomplete right bundle branch block.
Phylogenetic- and/or physicochemical-based phenotype
prediction analyses
In this study, four in silico prediction tools (Grantham
values, Polyphen, sift, and Conservation across
species) were used to predict if missense variants
associated with BrS were benign or pathogenic.
The detailed use of these prediction tools has been
described in detail by Giudicessi et al. in patients with
the LQTS (25).
Grantham physicochemical values were calculated
using the Grantham amino acid difference matrix. In
this study, values above 100 were considered radical
(pathogenic), and values below 100 were considered
conservative (benign) (25, 26). Using Polyphen
predictions (version 2.2.2) (27), each variant were
labelled probably damaging, possibly damaging
or benign. Probably damaging and possibly
damaging were considered damaging (pathogenic).
sift predictions (version 4.05) were calculated (28),
and variants were classified as tolerated (benign)
or damaging (pathogenic). Finally, the degree of
Conservation Across Species was obtained from
HGMD (19), and classified as occurring at a position
with no substitutions (conserved or pathogenic) or 1
substitution (not conserved or benign). Using each in
silico tool, we calculated the percentage of variants
predicted to be pathogenic. Any observed difference
was tested with the 2 test for categorical data and a
significance level of p < 0.05 was used.
Results
In the ESP population, 38 of 355 (10%) vari-
ants previously associated with BrS were identified
 High prevalence of genetic variants previously associated with BrS
Table 1. Variants previously associated with Brugada syndrome and present in the ESP population

European Americans African Americans

genotype genotype All genotype
Amino Minor /Minor/ Major/ Minor /Minor/ Major/ Minor /Minor/ Major/
Gene Variant acid minor major major minor major major minor major major

CACNA1C c.1468G>A p.G490R 0 5 4170 0 0 2010 0 5 6180

c.5510G>A p.C1837Y 0 1 4169 0 1 2014 0 2 6183
c.5639G>A p.R1880Q 0 6 4158 0 1 1994 0 7 6152
c.6040G>A p.V2014I 0 4 4092 0 1 1916 0 5 6008
CACNA2D1 c.2126G>A p.S709N 1 45 4249 0 1 2201 1 46 6450
c.2751A>T p.q917H 0 4 4288 0 0 2199 0 4 6487
CACNB2 c.428C>T p.S143F 0 12 4288 0 0 2203 0 12 6491
c.1195C>T p.L399F 0 4 4296 0 1 2202 0 5 6498
c.1349C>T p.T450I 0 18 4282 0 1 2202 0 19 6484
c.1614C>A p.D538E 0 3 4297 0 1 2202 0 4 6499
GPD1L c.839C>T p.A280V 0 1 4299 0 0 2203 0 1 6502
KCND3 c.1798G>C p.G600R 0 1 4299 0 0 2203 0 1 6502
KCNH2 c.2617G>A p.G873S 0 0 4300 0 4 2199 0 4 6499
SCN1Bb c.641G>A p.R214Q 0 21 2216 0 2 1239 0 23 3455
SCN3B c.29T>C p.L10P 0 0 4299 0 1 2201 0 1 6500
KCNJ8 c.1265C>T p.S422L 0 19 4281 0 1 2202 0 20 6483
SCN5A c.103G>A p.G35S 0 1 4131 0 0 1957 0 1 6089
c.481G>A p.E161K 0 1 4247 0 0 2139 0 1 6386
c.647C>T p.s216L 0 11 4205 0 1 2044 0 12 6249
c.659C>T p.T220I 0 4 4200 0 0 2016 0 4 6216
c.694G>A p.V232I 0 0 4163 0 3 1935 0 3 6098
c.1127G>A p.R376H 0 1 4200 0 0 2046 0 1 6246
c.1577G>A p.R526H 0 1 4191 0 1 2026 0 2 6217
c.1844G>A p.G615E 0 5 4198 0 0 2063 0 5 6261
c.1855C>T p.L619F 0 2 4204 0 0 2056 0 2 6260
c.1981C>T p.R661W 0 0 4298 0 1 2198 0 1 6496
c.2150C>T p.P717L 0 0 4264 0 1 2152 0 1 6416
c.3718G>C p.E1240Q 0 1 4299 0 0 2203 0 1 6502
.3727G>A p.D1243N 0 1 4299 0 0 2203 0 1 6502
c.3878T>C p.F1293S 0 4 4202 0 0 2046 0 4 6248
c.3922C>T p.L1308F 0 0 4239 0 17 2088 0 17 6327
c.3956G>T p.G1319V 0 0 4237 0 1 2099 0 1 6336
c.4573G>A p.V1525M 0 1 4184 0 0 2013 0 1 6197
c.5494C>G p.Q1832E 0 0 4248 0 3 2133 0 3 6381
c.5770G>A p.A1924T 0 1 4204 0 0 2112 0 1 6316
c.5851G>T p.V1951L 0 5 4192 0 19 2060 0 24 6252
c.5903T>G p.I1968S 0 0 4189 0 1 2063 0 1 6252
c.6010T>C p.F2004L 1 24 4159 0 2 2012 1 26 6171

ESP, NHLBI GO Exome Sequencing Project.

(Tables 1 and 2). All variants in ESP were missense.
When available, functional characterization and familial
co-segregation data was recorded (Table 3).
In ESP there were 272 heterozygote carriers and
two homozygote carriers of one of these variants. The
genes investigated were screened on average in 6258
individuals, corresponding to a genotype prevalence of
1:23 (274:6258; Table 3). ESP harboured 22 of 303
(7%) variants in SCN5A. In other words, 93% of the
variants in SCN5A were not found among nearly 6500
individuals.
When genotyping the four common ESP-derived
variants CACNA2D1 S709N, SCN5A F2004L,
CACNB2 S143F, and CACNB2 T450I in a healthy
Danish control population, 18 heterozygote carriers
were found corresponding to a genotype prevalence of
1:30 (18:536). That is comparable with the genotype
prevalence of these variants in ESP (1:30 vs 1:61).
The mean age of these Danish patients were 63 years
(range: 5671) and none of them had any clinical
manifestations of BrS. None of the examined ECGs
had ST-segment elevations or incomplete right bundle
branch block.
Prediction analyses of ESP-derived vs non-ESP-derived
variants
Using the Grantham chemical values, 24% (9/38) of
ESP-derived variants were predicted to be pathogenic
compared with 32% (74/232) of the non-ESP-derived

491
Risgaard et al.
Table 2. Genes and variants associated with Brugada average age of diagnostic of BrS. In this population,
syndrome the prevalence of these variants was comparable with
that found in ESP (1:30 vs 1:61; Table 4). This
Number of % of variants
Genes variants in ESP
is pointing towards that ESP does not harbour an
overrepresentation of these variants.
RANGRF 1 0 The ability to distinguish rare pathogenic variants
KCNE3 1 0 from similarly rare yet non-pathogenic variants has
SCN5A 325 7 been a challenge for several years. With the increasingly
SCN1B 3 33 widespread use of genetic testing we might see that
CACNA2D1 4 50 more and more rare variants will be established in the
KCND3 2 50 disease causation of certain rare diseases (25). In this
KCNH2 2 50 regard, large scale exome population data may help
CACNA1C 7 57 permit discrimination between low-frequency genetic
CACNB2 7 57 variants and disease causing variants. This is especially
GPD1L 1 100 applicable in low prevalence diseases such as BrS,
SCN3B 1 100
where it is expected to find only very few cases in
KCNJ8 1 100
the general population. Furthermore, large-scale exome
ESP; NHLBI GO Exome Sequencing Project. data on the general population might also be needed
per ethnic group or geographical region because of the
occurrence of such rare variants and their frequency
variants (p = 0.9). The ESP-derived variants were might be region-dependent.
predicted pathogenic in 50% (19/38) of the cases, using Besides using exome data, in silico tools have
Polyphen, compared with 85% (197/232) of the non- been developed to assess the phylogenetic- and/or
ESP-derived variants (p < 0.0001). Finally, using both physicochemical properties of amino acid changes
sift and Conservation Across Species we found that altered by rare single nucleotide variants. Thus, these
42% (16/38) and 47% (18/38) of the ESP-derived tools are thought to increase the likelihood of predicting
variants were predicted pathogenic compared with 81% the true pathogenicity of certain variants. Recently,
(189/232) and 83% (192/232) of the non-ESP-derived Giudicessi et al. published results supporting the
variants, respectively (p < 0.0001). potential clinical utility of the synergistic use of these
The frequencies of the ESP and non-ESP-derived tools. The classification of rare variants, in patients
variants were also calculated when 3 tools pre- with the LQTS, was enhanced when 3 tools predicted
dicted pathogenicity. In this synergistic use of the pathogenicity (25). In this study, using 3 of these
prediction analyses, 47% (15/38) of the ESP-derived in silico tools it was found that 47% and 75% of
and 75% (174/232) of the non-ESP-derived vari- the ESP-derived and non-ESP-derived variants were
ants were predicted pathogenic (p < 0.0001; Fig. 1). predicted pathogenic, respectively, p < 0.0001. This
Analyses were performed on all missense mutations was the case even though several non-ESP-derived
found in ESP, but the prediction analyses could not variants in SCN5A were not predicted because stop
be conducted on 219 SCN5A variants and on one codons were introduced.
SCN1B variant, as deletions and stop codons were The ESP most likely represents the general popu-
introduced. lation, and even there was clinical data regarding the
phenotypic presentation on the cohort; it is not likely
Discussion
that there would be significantly different conclusions
considering the reduced penetrance and variable expres-
In this study, 355 variants associated with BrS were sivity seen in BrS. Even though the presence of vari-
identified. Searching the ESP population, 38 variants ants in the ESP population might raise questions about
distributed on 272 heterozygote carriers and two disease causation, it does not definitively exclude the
homozygote carriers were found which corresponds to a possibility of pathogenicity. It is conceivable that some
genotype prevalence of 1:23 (Table 1). The prevalence variants present in ESP could in fact be disease caus-
of BrS in the general population has been estimated to ing for instance, the finding of GPD1L A280V that
range between 1:2000 and 1:100,000. was initially identified by London et al. in 2007. They
The ESP represents the general population in this studied a large family consisting of several members
study, but we cannot exclude the possibility that there diagnosed with BrS (29). In this study, it was con-
might be an overrepresentation of BrS in ESP, although vincingly demonstrated that the variant A280V affects
the prevalence is much higher than expected. To the intracellular localization of GPD1L as well as it
test this, we genotyped four common variants found decreases the surface expression of SCN5A and thereby
in ESP in our own healthy control population with probably decreases the inward sodium current (29). In
available ECGs (23, 24). It is noteworthy although, other words, even though this variant was present in
that none of these controls had a flecainide or ajmaline ESP in one individual, it might still play the pivotal
test performed. Hence, we cannot exclude that some role in the pathogenicity.
patients might be asymptomatic carriers although the Screening for inherited cardiac diseases in family
mean age were >60 years and thus, well above the members has become an important tool in the family

492
 High prevalence of genetic variants previously associated with BrS
Table 3. Functional data and family co-segregation for genes and variants in the ESP population

Gene Amino acid Genotype frequency Functional data Family co-segregation

CACNA1C p.G490R 5 Loss of function No

p.C1837Y 2 No data available Yes
p.R1880Q 7 No data available No data available
p.V2014I 5 Loss of function No
CACNA2D1 p.S709N 47 No data available No data available
p.q917H 4 No data available No data available
CACNB2 p.S143F 12 No data available No
p.L399F 5 No data available Yes
p.T450I 19 No data available Yes
p.D538E 4 No data available No data available
GPD1L p.A280V 1 Loss of function Yes
KCND3 p.G600R 1 Gain of function No data available
KCNH2 p.G873S 4 Gain of function No data available
SCN1B p.R214Q 23 Loss of function No data available
SCN3B p.L10P 1 loss of function No
KCNJ8 p.S422L 20 Loss of function No data available
SCN5A p.G35S 1 No data available No
p.E161K 1 No data available No data available
p.s216L 12 Loss of function No
p.T220I 4 No data available No data available
p.V232I 3 No data available No data available
p.R376H 1 Loss of function Yes
p.R526H 2 No data available No data available
p.G615E 5 No data available No data available
p.L619F 2 No data available No data available
p.R661W 1 No data available No data available
p.P717L 1 No data available No data available
p.E1240Q 1 No data available No data available
p.D1243N 1 No data available No data available
p.F1293S 4 No data available No data available
p.L1308F 17 No data available No data available
p.G1319V 1 Loss of function No data available
p.V1525M 1 No data available No data available
p.Q1832E 3 No data available No data available
p.A1924T 1 Gain of function No data available
p.V1951L 24 No data available No data available
p.I1968S 1 Loss of function No data available
p.F2004L 27 Loss of function No

ESP, NHLBI GO Exome Sequencing Project.

cascade screening. In a recently published HRS/EHRS
consensus document screening is recommended in
family members and appropriate relatives following
the identification of a BrS-causative mutation in an
index case (12). Furthermore, it is recommended
that SCN5A targeted testing can be useful in cases
where a cardiologist suspects BrS following a clinical
examination. This is only recommended or possible
because of a low prevalence (2%) of variants of
uncertain significance in controls with a signal to noise
ratio of 1 in 10 (12).
The ESP only harboured 22 of 303 (7%) variants in
SCN5A previously associated with BrS. That is, 93%
of the variants in SCN5A, associated with BrS, were
not found among nearly 6500 controls confirming the
important role and usefulness of this gene in cascade
screening in families. Nevertheless, it is important to
keep in mind that the absence of variants in ESP in
itself, do not establish disease causation, but certainly
strengthen the possibility of one.
Lack of properly sized control populations is a
problem when dealing with rare genetic variations in the
context of rare monogenetic diseases. Without this we
might misdiagnose family members undergoing genetic
testing, and this may have great consequences for the
treatment, clinical advice and follow-up. We suggest
that exome data, like ESP, are used in research and
in the everyday clinical practice as a tool alongside
with other known prediction tools, to get a better
understanding of the pathogenicity of the variants
associated with BrS or other rare inherited diseases.
In conclusion, 38 of 355 (10%) variants, associated
with BrS, were identified in the ESP population
distributed on 272 heterozygote carriers and two
homozygote carriers corresponding to a genotype
prevalence of 1:23. Importantly, 93% of the variants

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Risgaard et al.

Fig. 1. Percentage of variants predicted to be pathogenic, using different predictions analyses on variants present and not present in NHLBI GO
Exome Sequencing Project (ESP).

Table 4. The prevalence of four variants in ESP and in the Danish control population

Danish control population ESP Population

Found in number Found I number
Genes Amino acid of patients Prevalence Mean age (year) of patients Prevalence

CACNA2D1 p.S709N 6 1:89 47 1:137

CACNB2 p.S143F 3 1:178 12 1:540
CACNB2 p.T450I 6 1:89 19 1:314
SCN5A p.F2004L 3 1:178 27 1:237
Overall 18 1:30 (18:536) 63 105 1:61 (105:6399)

ESP, NHLBI GO Exome Sequencing Project.

in SCN5A were not represented among nearly 6500 Acknowledgement

individuals confirming the pivotal role of this gene in
cascade screening in families.
Genotyping four common ESP-derived variants in
a healthy Danish control population, 18 heterozygote
carriers were identified corresponding to a comparable
genotype prevalence with that found in ESP. These
results suggests that exome data be used together with
other known prediction tools, to better understand the
pathogenicity of variants associated with BrS or other
rare inherited diseases.
The authors would like to thank the NHLBI GO Exome Sequencing
Project and its ongoing studies which produced and provided
exome variant calls for comparison: the Lung GO Sequencing
Project (HL-102923), the WHI Sequencing Project (HL-102924),
the Broad GO Sequencing Project (HL-102925), the Seattle GO
Sequencing Project (HL-102926) and the Heart GO Sequencing
Project (HL-103010).
The work was supported by The Danish Heart Foundation (12-
04-R91-A3790-22689), The Danish National Research Foundation
Centre for Cardiac Arrhythmia (DARC), The John and Birthe

494
 High prevalence of genetic variants previously associated with BrS
Meyer Foundation, The Research Foundation at the Heart Centre,
Rigshospitalet, The foundation of Edith and Henrik Henriksens
mindelegat (50892) and The A.P. Mller foundation for the
Advancement of Medical Science.
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