DOI: 10.1161/CIRCGENETICS.114.000623
1
Evaluation of Genes Encoding for the Transient Outward Current (Ito) Identifies the KCND2 Gene as a Cause of J Wave Syndrome Associated with
Sudden Cardiac Death
Running title: Perrin et al.; KCND2 mutation in J-wave syndrome
Mark J. Perrin, MBBS, PhD1; Arnon Adler, MD1; Sharon Green, BSc1; Foad Al-Zoughool,
Msc1; Petro Doroshenko, PhD1; Nathan Orr, BSc1; Shaheen Uppal, Bsc1; Jeff S. Healey, MD2;
David Birnie, MBBS1; Shubhayan Sanatani, MD3; Martin Gardner, MD4; Jean Champagne,
MD5; Chris Simpson, MD6; Kamran Ahmad, MD7; Maarten P. van den Berg, MD, PhD8; Vijay
Chauhan, MD9; Peter H. Backx, DVM, PhD9; J. Peter van Tintelen, MD, PhD8; Andrew D.
Krahn MD3; Michael H. Gollob MD9
1Division of Cardiology, Department of Medicine, University of Ottawa Heart Institute, Ottawa; 2Population Health Research Institute, McMaster University, Hamilton, ON; 3Division of Cardiology, Department of Medicine,
University of British Columbia, Vancouver, BC, 4Division of Cardiology, Department of Medicine, Dalhousie University, Halifax, NS; 5Division of Cardiology, Department of Medicine, Laval University, Québec, QC;
6Division of Cardiology, Department of Medicine, Queens University, Kingston; 7Division of Cardiology, St Michael’s Hospital, University of Toronto, Toronto, ON, Canada; 8Department of Genetics, University of
Groningen, University Medical Center, Groningen, the Netherlands; 9Division of Cardiology, Toronto General Hospital, University of Toronto, Toronto, ON, Canada
Correspondence:
Michael H. Gollob, MD
Toronto General Hospital
University of Toronto
200 Elizabeth St.
Toronto, Ontario, M5G 2C4
Canada
Tel: +1 416-340-4800
Fax: +1 613-761-5060
E-mail: [email protected]
Journal Subject Codes: [5] Arrhythmias, clinical electrophysiology, drugs, [109] Clinical genetics, [106] Electrophysiology, [132] Arrhythmias - basic studies, [152] Ion channels/membrane transport
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DOI: 10.1161/CIRCGENETICS.114.000623
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Abstract:
Background - J-wave ECG patterns are associated with an increased risk of sudden arrhythmic
death and experimental evidence supports an Ito-mediated mechanism of J-wave formation. This
study aimed to determine the frequency of genetic mutations in genes encoding the transient
outward current (Ito) in patients with J waves on electrocardiogram (ECG).
Methods and Results - Comprehensive mutational analysis was performed on Ito-encoding
KCNA4, KCND2 and KCND3 genes, as well as the previously described J-wave associated
KCNJ8 gene, in 51 unrelated patients with ECG evidence defining a J-wave syndrome. Only
patients with a resuscitated cardiac arrest or type 1 Brugada ECG pattern were included for
analysis. A rare genetic mutation of the KCND2 gene, p.D612N, was identified in a single
patient. Co-expression of mutant and/or wild-type KCND2 with KChIP2 in HEK293 cells
demonstrated a gain-of-function phenotype, including an increase in peak Ito current density of
48% (p<0.05) in the heterozygous state. Using computer modeling, this increase in Ito resulted in
loss of the epicardial action potential dome, predicting an increased ventricular transmural Ito
gradient. The previously described KCNJ8-S422L mutation was not identified in this cohort of
patients with ECG evidence of J-wave syndrome.
Conclusions - These findings are the first to implicate the KCND2 gene as a novel cause of J
wave syndrome associated with sudden cardiac arrest. However, genetic defects in Ito-encoding
genes appear to be an uncommon cause of sudden cardiac arrest in patients with apparent J-wave
syndromes.
Key words: genetic heart disease, arrhythmia (heart rhythm disorders), sudden cardiac death, arrhythmia
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Introduction
J-wave syndromes refer to a spectrum of electrocardiographic (ECG) observations characterized
by early ST segment take-off from the terminal QRS or J-point1,2. The associated QRS segment
may demonstrate terminal slurring, representing a J-wave concealed within the QRS complex, or
present a more distinctly visible notch representing a J-wave. Brugada syndrome is the most
well characterized J wave syndrome, both in terms of clinical and genetic features. Alternative
patterns of J-wave syndromes have long been recognized, commonly involving the infero-lateral
ECG leads, and until recent years were considered a benign ECG pattern3-8.
In 2008, Haissaguere and colleagues challenged the concept that infero-lateral patterns of
J-wave syndromes are a benign entity, reporting a higher prevalence of infero-lateral J-waves in
previously well individuals experiencing a sudden cardiac arrest9. Further data corroborated the
observation that infero-lateral J-wave ECG patterns are prevalent in 20-30% of survivors of
unexplained cardiac arrest, which is considerably higher than that found in healthy controls10,11.
The electrophysiologic mechanism underlying the manifestation of J-waves on the ECG
has been elegantly demonstrated utilizing ventricular wedge preparations, and has been shown to
be the result of transmural dispersion of the early repolarizing current, the transient outward
current (Ito), which mediates phase 1 of the cardiac action potential12-14. Despite insight into this
pathophysiology, knowledge of the genetic determinants of J- wave syndromes, aside from
Brugada syndrome, remains scarce.
In this study, in view of the known role of Ito in J-wave formation, we sought to
determine the frequency of genetic defects in the predominant Ito-encoding genes in a population
of unexplained cardiac arrest survivors with infero-lateral J wave ECG patterns, and additionally
in a cohort of type 1 Brugada syndrome patients with previously negative genetic testing results.
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Methods
Study Population
The study cohort consisted of 51 unrelated J-wave syndrome patients. J-wave ECG pattern was
defined as QRS slurring and/or notching associated with QRS-ST junction (J-point) elevation of
at least 0.1 mV in a minimum of 2 contiguous leads (Supplemental Figure 1). Cases with infero-
lateral J-waves (n=31) were only included for analysis if a history of sudden cardiac arrest
requiring defibrillation was documented. These cases represent a sub-group of patients enrolled
in the Cardiac Arrest Survivors with Preserved Ejection Fraction Registry (CASPER)15. The
remaining J-wave syndrome patients consisted of patients demonstrating a spontaneous or
provoked type 1 Brugada ECG pattern who had previous negative genetic testing results for the
most common gene causative for Brugada syndrome, SCN5A. Patients were excluded if any
coronary artery had stenosis > 50% or had anomalous coronary arteries, if imaging demonstrated
evidence of hypertrophic cardiomyopathy, if they experienced commotio cordis, or if IV
adrenaline or treadmill testing suggested a diagnosis of catecholaminergic polymorphic
ventricular tachycardia (CPVT) or Long QT syndrome.
All patients had documented preserved left ventricular function (ejection fraction > 50%)
and structure determined by echocardiography and/or cardiac MRI, and normal coronary arteries
based on coronary angiography. All patients provided written informed consent and the study
was approved by the Institutional Review Boards of the participating Institutions.
Mutation Analysis
Genomic DNA was extracted from peripheral lymphocytes and comprehensive open reading
frame/splice site mutational analysis of the KCNJ8, KCNA4, KCND2 and KCND3 genes was
performed using PCR and direct DNA sequencing. DNA from 100 healthy controls was
stry (CASPER)15. ThTTT
ng a sponttttaneous ss orororor
y e 1 Br ada ECG ttern who had evious n ative netic testin results fo
m y
rtery had stenosis > 50% or had anomalous coronary arteries, if imaging demons
f
or treadmill testing suggested a diagnosis of catecholaminergic polymorphic
ype e e 1111 BrBrBBrugugugadaaa a aa ECEEE G pattern who had prevevevioi us negative genenenetic testing results fo
moooon gene causaatiiive fffooor BBBBrrur gaaadda sssyyndrrroomee, SCCCN5N5N5AAA.. Paaattientts wererere exxxclclclc udddeeded ifff aany
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screened for any identified non-synonymous variant, and cross-reference to the Exome Server
Database (http://evs.gs.washington.edu/EVS/) involving over 6000 genotyped individuals was
utilized to further assess for the frequency of any identified genetic variant.
Cloning and Mutagenesis
Wild-type KCND2 (Kv4.2) and KCNIP2 (KChIP2) human cDNA clones were provided by Dr.
Peter Backx (University of Toronto). KCND2 cDNA was subcloned into pIRES-DsRed
(Clontech, Mountain View, CA, USA) expression vector, and KCNIP2 cDNA was subcloned
into pIRES-ZsGreen1 (Clontech, Mountain View, CA, USA). The KCND2 D612N mutation
was engineered from the wild-type KCND2 clone using the Quickchange XL Site-Directed
Mutagenesis Kit (Strategene, La Jolla, CA, USA). Complete DNA sequencing was undertaken to
ensure fidelity of mutant and wild-type clones.
Expression of Kv4.2 and KChIP2 in HEK293 cells
Heterologous expression of Kv4.2 in HEK293 cells was achieved by co-transfecting 0.5 ug
mutant or wild-type clone with 1.5 ug of wild-type KCNIP2 clone using 2 ul of Lipofectamine
2000 transfection reagent (Invitrogen, Carlsbad, CA, USA) in 50 ul OPTI-MEM media
(Invitrogen, Carlsbad, CA, USA). For analysis in the heterozygous state, equal quantity of
mutant and wild-type clone were mixed (total 0.5 ug) and co-transfected with KCNIP2.
Electrophysiological Studies and Analysis
Following 24-48 hours post-transfection, cells emitting both red and green fluorescence were
selected for whole cell patch clamp recordings. Patch clamp experiments and analysis were
performed blinded to KCND2 transfected clones. Patch clamp recordings were made using low
resistance electrodes (<3 M ), and a routine series resistance compensation by an Axopatch
200B amplifier (Axon Instruments Inc, Foster City, CA, USA) was performed to minimize
CND2 D612N muttatatio
nge XXLL SiSitet -DiDirectcteed
is Kit (Strat ene, La Jolla, CA, USA). Complete DNA se enci was underta
l
n
us expression of Kv4.2 in HEK293 cells was achieved by co-transfecting 0.5 ug
wild type clone with 1 5 ug of wild type KCNIP2 clone using 2 ul of Lipofectam
is KiKit t (S(Strtratateggenene, La Jolla, CA, USA). CCoomplete DNA seeququencing was underta
litity y of mutant annd wiwildd--tytype ccllonenes.
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voltage-clamp errors. The patch pipette solution contained (mmol/L): 110 KCl, 10 EDTA, 1.42
MgCl2, 4 MgATP, 5.17 CaCl2, and 10 HEPES, pH adjusted to 7.2 with Tris-OH. The
extracellular bath solution contained (mmol/L): 148 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES,
pH adjusted to 7.4 using NaOH. Whole cell current was generated by 500 ms-long voltage-clamp
command pulses from a holding potential of -80 mV to a voltage of 40 mV in 10 mV increments
using pClamp 10.3 software (Axon Instruments Inc). Currents were filtered at 5 KHz and
sampled at 10 KHz. Specific voltage-clamp protocols used to determine voltage-dependence of
activation, inactivation and recovery from inactivation are illustrated in the figure legend. All
experiments were carried out at room temperature. Data was digitally stored and analyzed using
pClamp 10.3 and Prism 3.03 (GraphPad Software Ins, San Diego, CA, USA) software. The
voltage-dependent inactivation curve was fitted with the Boltzmann's equation: Im = Imin + (Imax -
Imin)/((1 + exp(V50-Vm)/k)), where V50 is the membrane potential of half-maximal inactivation
and k - is the slope of the inactivation curve.
Recovery from inactivation curves were fitted with the one-phase exponential association
equation: Im = Imax x (1 - exp(-t/ )), where is the time constant of the exponent.
Simulated Transmural Right Ventricular Action Potential Propagation
We simulated action potential propagation across the right ventricular (RV) wall in a theoretical
1.45 cm fiber using a modified Luo Rudy II myocyte model adjusted to incorporate Ito16. For
control simulations, the conductance of RV Ito was set to 1.1 mS/μF in the epicardium and 0.93
mS/μF in the midmyocardial layer; Ito was not expressed in the endocardium17. For Kv4.2-
WT/D612N, Ito current was increased by 48% in agreement with our experimental results with
heterozygous expression. For mutant and control simulations, the conductance of ICaL was
reduced in the midmyocardium and epicardium by 25% to compensate for a decrease in action
n the figure legendddd.. A
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.3 and Prism 3.03 (Gr hPad Software Ins, San Die , CA, USA) software. Th
p (
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covery from inactivation curves were fitted with the one phase exponential assoc
.3 ananand d d PrPrPPrisisism mmm 3.3.3.03000 (GraphPad Software InInIns,, San Diego,, CAAA,,, USA) software. Th
peeeenddddent inactivavaation currrrvevvv waasas fffittted wwithh tthe BBololololtzzmmmannn's eeequuuatatation:::: IIImmm = Imiin + (
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potential duration associated with high Ito expression16. The density of IKr (rapid delayed rectifier
potassium current) to IKs (slow delayed rectifier potassium current) varied across the fiber: 11:1
(endocardium), 4:1 (midmyocardium) and 35:1 (epicardium)17. Extracellular ion concentrations
were: [Nao] = 150 mM/L, [Ko] = 4.0 mM/L and [Cao] = 1.8 mM/L. The fiber was paced with a 1
to allow equilibration of intracellular calcium. Simulations were performed in Mathematica 9.01
(Wolfram Technologies, Champaign, IL, USA).
Statistical Methods
All electrophysiological data are expressed as mean ± standard error of the mean (S.E.M.).
Determinations of statistical significance of differences between means in control (WT) and
mutant channel constructs (D612N and WT/D612N mutants) under the various experimental
conditions were performed using an unpaired, two-tailed Student t-test (GraphPad Prism).
Differences were deemed significant at a P value <0.05.
Results
Clinical and Genetic Data
A total of 51 patients with a J-wave syndrome were screened for genetic mutations in genes
encoding the potassium subunits responsible for Ito current in the heart (KCND2, KCND3 and
KCNA4). These patients were also screened for genetic defects in the cardiac IK-ATP channel
(KCNJ8), a channel previously attributed to being a cause of J- wave syndrome19-22. To avoid
screening presumably benign forms of J-wave syndrome, we restricted our inclusion to patients
with inferior, lateral or infero-lateral J wave patterns who had previously experienced an
otherwise unexplained cardiac arrest requiring defibrillation for resuscitation. This group
comprised 61% of our cohort, all were Caucasian, 81% were male and the average age at the
f the mean (S(S(S(S EEE.E.MMMM ))).).
ions of statistical si ificance of differences between means in control (WT) and
nnel constructs (D612N and WT/D612N mutants) under the various experiment
w
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s were deemed siiiigngg ififififiici ant at a PPPP value <0.000 05050505.PPP
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time of cardiac arrest was 43 +/- 12 years. An additional 20 patients with spontaneous or drug-
provoked type 1 Brugada ECG pattern and previous negative genetic testing for the SCN5A gene
were also screened. The average age of this group was 43 +/- 15 years, 90% were male, and 2
patients were non-Caucasian (1 African, 1 Asian). Sporadic cases represented 75% of this group.
A history of resuscitated sudden cardiac arrest was present in 4 patients (20%), and syncope in 6
patients (30%).
In a single patient, a rare missense mutation was identified in the Ito-encoding KCND2
gene (Fig. 1A), a gene not previously described to be a cause of inherited arrhythmia syndromes.
The identified mutation, denoted c.1834 G>A, leads to the substitution of a highly conserved
aspartate (D) residue for asparagine (N) at position 612 in the protein (p.D612N) (Fig 1B). This
variant was absent from 200 alleles of local, healthy control samples. Reference to the exome
server database indicates that KCND2-D612N is a very rare allele in individuals of undefined
clinical background, observed in 2/13,004 alleles, 1/3 of which are of African decent.
The affected patient, previously well and of African decent, experienced a sudden cardiac
arrest at age 51 while eating in a restaurant and received 2 defibrillation shocks by paramedics
with ultimate return to normal sinus rhythm. Coronary angiography, echocardiography and
cardiac magnetic resonance imaging were all normal. 12-lead ECG demonstrated large J waves
across the anterior precordial leads, evidence for right bundle conduction delay with shallow S
waves in lead I and V6, and notable broad, fractionated QRS complexes in leads V1/V2 (Fig 2).
Intravenous procainamide provocation (1 gm) did not change the QRS pattern but resulted in a
ventricular couplet and triplet at 30 minutes of infusion. Clinically, it was felt that this ECG
pattern was not consistent with the Brugada ECG pattern but rather represented an unusual J
wave syndrome. However, due to the similar ECG localization of J waves the patient was
ted arrhythmia syyndndndndr
n of a hihihihi hhhghlylll conseseseservrvrvrve
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s absent from 200 alleles of local, healthy control samples. Reference to the exo
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e affected patient previously well and of African decent experienced a sudden c
D) rereresisisidudududue ee fofofofor asasasparagine (N) at positionnn 6666121 in the protein nn (p(p(p(p.D612N) (Fig 1B).
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screened for genetic defects in reported Brugada syndrome susceptibility genes, including
SCN5A, GPD1L, CACNA1C, CACNB2, SCN1B, KCNE3 and SCN3B. No rare genetic variants
were identified. The patient underwent placement of an implantable cardioverter-defibrillator.
Family history was negative for known premature (age < 55 yrs) sudden cardiac death and
cascade family screening has been declined. Over a clinical follow-up of 7 years, the patient has
remained off medical therapy and has not received any device therapy for recurring arrhythmias.
Cellular Electrophysiological Analysis
To functionally characterize the biophysical consequences of KCND2-D612N, we co-expressed
this mutant clone and KCND2-WT along with KChIP2-WT in HEK293 cells to reconstitute
Kv4.2 mediated Ito current in vitro. In the homozygous state, Kv4.2-D612N significantly
increased Ito current density over the voltage range from -10 mV to +40 mV as compared to
Kv4.2-WT (n=13 and 17, respectively; p<0.05)(Fig. 3A and B). To re-capitulate the
heterozygous state, equal quantities of Kv4.2-WT and Kv4.2-D612N were co-expressed with
KChIP2-WT and similarly demonstrated a significant increase in Ito current density, including a
50% increase in peak current density at 0 mV (n=17 and 11, respectively; p<0.05)(Fig 3C).
Further studies evaluating the kinetics of WT or mutant channels demonstrated that Kv4.2-
WT/D612N had a significantly slower decay rate (tau) over the voltage range of 30 mV-40 mV
as compared to Kv4.2-WT channels (Fig. 4A) (p<0.05). No significant difference was observed
in the inactivation of Ito or recovery from inactivation between WT and heterozygote channels
(Fig 4B/C). However, Kv4.2-WT/D612N significantly increased Ito total charge over the range of
-30 mV to 40 mV in comparison to Kv4.2-WT (p<0.05) (Fig. 4D).
Right Ventricular Action Potential Propagation of Kv4.2-WT and Kv4.2-WT/D612N
We simulated action potential propagation across the RV myocardium with and without the
-D612N, we co-exxxxprppp e
93 celllllllls tttto reconststttitititituuuut
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WT and similarly demonstrated a significant increase in I current density includi
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experimentally observed gain in function in KCND2- Ito using a modified Luo-Rudy II model16.
In control simulations at a pacing cycle length of 1000 ms, a deep notch (spike and dome) was
observed in the epicardial layer (Figure 5A). In contrast, a 48% increase in Ito, as predicted by
heterozygote Kv4.2-WT/D612N channels, resulted in stable loss of the dome of the action
potential in the epicardial layer (Figure 5B).
Discussion
We evaluated the role of Ito-encoding genes in a cohort of patients with J-wave syndromes, the
majority of individuals having experienced a sudden cardiac arrest. Our data suggests a low
yield of genetic abnormalities in the major genetic contributors of the Ito current for patients with
established cardiac arrest and evident infero-lateral or anterior ECG J-waves. However, we did
observe the novel association of a rare, gain-of-function mutation in the KCND2 gene in a patient
with sudden cardiac arrest and an anterior J-wave ECG pattern.
The transient outward current (Ito) in the heart mediates early repolarization of the cardiac
action potential (phase 1) and is characterized by a transmural gradient in current density across
ventricular myocardium, particularly within the right ventricular outflow tract12. Exacerbation of
this natural epicardial to endocardial gradient, either by increased outward current (Ito, IK-ATP) or
decreased inward current (INa, ICa), results in the manifestation of the ECG J-wave and creates an
arrhythmia substrate for arrhythmia (phase 2 re-entry)13,14. Based principally on remote gene
expression studies using canine left ventricular tissue, the molecular correlates for Ito have been
deemed to predominantly involve the KCND3-encoded Kv4.3 channel, and KCHIP2, which
encodes an accessory subunit required for channel function23,24. Traditionally, the role of the
pore forming Kv4.2 subunit encoded by KCND2 has not been considered significant within
human myocardium in light of these previous observations. However, in a unique study
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evaluating regional gene expression of 79 ion channel genes within non-diseased human hearts,
KCND2 gene expression demonstrated the highest differential expression pattern between right
ventricular epicardium and endocardium, the gradient exceeding but mirrored by KCHIP2
expression25. Conversely, KCND3 did not exhibit a transmural gradient in expression within the
RV or LV25. These data suggest that Kv4.2 channels, along with KCHIP2, may represent the
molecular correlate for Ito current gradient across the RV myocardium.
Our data is the first to implicate the KCND2 gene as a cause of an atypical anterior J-
wave pattern associated with sudden cardiac death. Although the ECG features were not typical
of the more classically recognized anterior J wave pattern of Brugada syndrome, the observations
of right bundle conduction delay and notable QRS fractionation have been recognized features in
some cases of Brugada syndrome26. Giudicessi et al have reported the identification of mutations
within KCND3 in 2 patients with more classic type 1 Brugada ECG patterns. Similar to our
observation, the observed genetic variants in KCND3, Kv4.3-L450F and Kv4.3-G600R, occurred
within the C-terminus of the channel and were highly conserved across mammals17. All of these
mutants result in a significant increase in Ito current, while Kv4.3-G600R and our described
Kv4.2-D612N mutant share the observation of a significant decrease in current decay rate.
Interestingly, site-directed mutagenesis studies in vitro of the highly homologous Kv4.1 channel
indicate that deletion of C-terminal residues 422-651 results in a significant delay of current
decay, suggesting a major role of C-terminal residues in channel inactivation27.
The possible in vivo effect of the KCND2 mutant was confirmed in simulated cardiac
action potential propagation across the right ventricular wall using a modified Luo-Rudy II
human myocyte model16. With an increase in Ito (48%) in line with our experimental results from
heterozygous expression of Kv4.2-WT/D612N, we observed complete and stable loss of the
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dome of the action potential in the epicardial layer. This observation predicts the formation of
ECG J-waves, as demonstrated by Antzelevitch et al, and creates the risk of propagation of the
action potential dome from sites where it is maintained to sites where it is lost, producing local
re-excitation (phase 2 reentry) and generation of polymorphic ventricular arrhythmia12-14.
Further, the facilitation of heterogeneous action potential durations within the RVOT induced by
Kv4.2-D612N may lead to relative delayed activation of epicardial regions, delaying
depolarization in some regions and manifesting as late potentials or a fractionated QRS, as
observed in our patient. This phenomenon has been elegantly demonstrated by Morita and
colleagues using a canine, RV transmural myocardial preparation. Delayed pacing of the
epicardial tissue relative to endocardium reproduced increasing QRS duration and fractionation,
dependent upon the degree of epicardial activation delay.26
In addition to evaluating genes that specifically encode the subunits responsible for Ito,
we screened the KCNJ8 gene which encodes IK-ATP. Previous studies, using a candidate gene
approach, have implicated the gain-of-function mutation KCNJ8-S422L mutation as a
susceptibility mutation for infero-lateral and anterior J waves in 1-2% of patients19-22. In a
manner similar to the direct evidence demonstrated for enhanced Ito current, increased early
repolarizing IK-ATP current is speculated to accentuate phase 1 of the cardiac action potential,
leading to loss of the epicardial action potential dome and ECG J-wave formation. In our cohort
of 51 J-wave patients, the majority of which had experienced cardiac arrest, we did not identify
this specific mutation or other rare variants within KCNJ8. Recent data from the exome server
database in over 4,000 Caucasians suggests a frequency of 0.5% of KCNJ8-S422L. Further,
Veerameh et al report a 4% frequency of this variant in Ashkenazi Jews, including a homozygote
12 year male with apparent normal ECG28. These observations do not preclude the role of
trated by Morita anananand d
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KCNJ8 in J-wave syndromes. However, cautious interpretation and consideration of other
modifying genes in the presence of KCNJ8-S422L should be considered.
Overall, genetic interpretation based on candidate gene studies for J-wave syndromes
remains a challenge in view of the often sporadic, non-familial nature of these cases and
relatively common frequencies of these ECG patterns in otherwise healthy individuals. A recent
genome wide association study in J wave syndromes did not succeed in identifying a definitive
genetic locus, likely reflecting the considerable genetic heterogeneity and possible polygenic
nature of the phenotype.29
Study Limitations
Our cohort represents patients with persistent J wave patterns and therefore due to study design
has excluded cases of sudden death that may have occurred as a result of dynamic J wave
changes. Such a population may represent a genetically unique cohort. We did not screen the
comprehensive list of reported susceptibility genes for Brugada syndrome in all of our cases of
Brugada syndrome. However, our goal was not to re-assess the already known low frequency of
non-SCN5A mutations in this cohort, but rather to assess novel genes responsible for Ito current
in the heart in patients with various J-wave patterns. We have not provided the most robust
genetic evidence supporting KCND2 as a disease-causing gene, which is best exemplified by
segregation of the mutation with multiple affected individuals within a family. However, our
data are consistent with previous reports implicating altered Ito physiology in the heart as a cause
for J-wave formation and arrhythmogenesis. Follow-up data in larger cohorts will better clarify
the role of the KCND2 gene in sudden death associated with J-wave ECG patterns.
Conclusions
This study provides clinical, molecular and functional evidence implicating the KCND2 gene as
r resents tients with rsistent J wave tterns and therefore due to stud de
e
uch a population may represent a genetically unique cohort. We did not screen th
sive list of reported susceptibility genes for Brugada syndrome in all of our case
yndrome However our goal was not to re assess the already known low frequen
reppprerereseesentntnts s pppatititieeents with persistent J wavvveee ppatterns and theeerererefore due to study de
edddd ccccases of suddddden dddeathththh tthat tt mamaayy haaavee occccurrered d dd asasaa a reesultt ofofof dddyynammmmiici J wavvvee
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sive list of repopp rtedddd susceptptptibibibi iili ititii y yy gegg nes fofff r BBBrB uggg dadda syyy ddnddrome iiiin all of our case
ynddrome HHowever our go lal was n tot tto re assess tthhe allreaddy kknown llow ffrequen by guest on June 28, 2018http://circgenetics.ahajournals.org/
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a novel susceptibility gene contributing to an anterior J-wave ECG pattern associated with SCD.
Acknowledgments: The authors want to thank Yvonne Hoedemaekers MD, PhD for her help in collecting patient data. Dr. Gollob is supported by the Peter Munk Chair in Cardiovascular Molecular Medicine at the University of Toronto and a Heart and Stroke Foundation of Ontario Mid-Career Scientist Award.
Conflict of Interest Disclosures: None.
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Survivors with prprrresesesese
ugadaaaa JJJJ etetetet aaaallll IoIoIoIoninininicccm a
e
errent (Ito) gain-of-function mutations in the KCND3-encoded Kv4.3 channel an
y
K, Ru Y. Ionic current basis of electrocardio hic waveforms: a modle stu yn Res 2002;90:889 896
ms resesespopoponsnsnsibibibblel fffooor the electrocardiographihihihiccc php enotype of thehehe Brugada syndrome ayye dededeeppendennnnttt.t CiCiCiC rcrrr ulululu atataatioioioon ReReReR ssss... 11119999999 9;;;;8585858 :80303-80909909....
essisisi JJJR, Ye D,DD TTTestterrr DJJJ, CrCC otttit LLL,, MuMMuggioone A,A,A, Nesestererrenkoo VVVVV, et alll. Trraaansieenntrrent ttt (I(I(IItotototo))) gaaaininin oo-of-ff fufufunncn tiononon mmmmutatatttiooioionsnsss iiin nn thththeee KCKCKCCNDNDNDND3333-enenenncocoodededed ddd KvKvKvKv4.44 3333 chhhananannenenelll aana
yndrome. Hearttt t RhRhRhytytytythmhmhmm. 222010101011;1;11;8:8:8:8:1010101 24242424-1-1- 030032.2.2..
KKK, Rudy YY.. IoIoIoIonininic c cc cucucuurrrrrrrenenennt tt t babababasisisisis ss offoff eeeleleelectcctctroroorocaccacardrddrdioioioi grgrrgrapaapaphihiihicccc wawawawavevevevefofofoformrmmrms:s:ss: aaa mmmmodoo le studyyn RRes 22000022;9090 8:88989 889696 by guest on June 28, 2018
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Figure Legends
Figure 1: KCND2 gene mutation and biological conservation of Kv4.2 amino acid structure. (A)
DNA sequence chromatogram indicating the mutation within the KCND2 gene. (B) D612 is
highly conserved across species.
Figure 2: Representative 12-ECG of index case harboring the Kv4.2-D612N mutation with V1
and V2 in standard position.
TTTThehehehe KKKKCNCNCNCNJ8J8J8J8-S-S-S-S424242422L2L2LL vvvaaafrequency iiiin AAAAshkhkhkhkeeene
MF, Porthan K, Noseworthy PA, Havullinna AS, Tikkanen JT, Muller-Nurasyid-i
MMMMF,F,F,, Porthanananan KKKK, Noooosesessewowww rttthyhyhyhy PPPA,A,A,A HHHavavava uluu linnnna ASASSAS,,,, TiTT kkkkkkk anananennn JJT, MMMMulululllelll r-NuNuNNurararaasysysys id-aana aaala ysis of geennnomme-wiidededed assssoociaiiationnn studiies oof f f f thththt ee eeeleectrococcarrrdididiograaaaphphphiic earrrlyy ionnn pppattern. HHHeaaart RRRhythmhmhm. 2010 222;99:16227-16634.4.4.
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Figure 3: Kv4.2-D612N plus KChIP2 increase Ito current in heterologously transfected cells. A:
Representative whole-cell Kv4.2-WT plus KChIP2-WT (left), Kv4.2-WT/D612N plus KChIP2-
WT (middle) and Kv4.2-D612N traces recorded in HEK293 cells in response to a series of
depolarizing step voltage commands of 500 ms duration, shifting the membrane potential from a
holding potential of –80 mV to +40 mV in 10 mV increments. B: The current-voltage
relationships for Kv4.2-WT (n = 17) and Kv4.2-WT/D612N (n = 11) channels co-expressed with
KChIP2-WT. Each experimental data point represents mean +/- SEM. C: Bar graph showing
peak current density at 0 mV for WT (n = 17), WT/D612N (n = 11), and D612N (n=13) Kv4.2
channels co-expressed with KChIP2-WT. *P < 0.05.
Figure 4: Kinetic characteristics of Kv4.2-WT+KChIP2 (closed symbols) and Kv4.2-
WT/D612N+KChIP2 (open symbols) channels A: Inactivation time constants were determined
by fitting a mono-exponential function to the current decay for each voltage step. Each
experimental data point represents mean +/- SEM. B: Time course of recovery from inactivation
of Kv4.2-WT+KChIP2 (n=13) and Kv4.2- WT/D612N+KChIP2 (n=11) channels determined
using a two-step voltage protocol. The curves were fitted with a mono-exponential function.
Each data point represents mean +/- SEM.C: Steady-state inactivation curves of Kv4.2-
WT+KChIP2 (n=6) and Kv4.2-WT/D612N+KChIP2 (n=6) channels determined using a two-
step voltage protocol, in which the 0.5-s long test pulse from a holding potential of –100 mV to
+20 mV was preceded by a series of 0.5-s long prepulses whose amplitude increased in 5 mV
increments (see insert). The curves were fitted with a Boltzmann function. D: Total electric
charge carried by Kv4.2-WT+KChIP2-WT (n=17) and Kv4.2-D612N+KChIP2-WT (n=13)
nd D612N (n=13) ) ) KvKKK
K
N m
al data point represents mean +/ SEM B: Time course of recovery from inactiv
Kiiiineeeetic characteteeristticccs oof ff f KKv4.44 2-2--WWT+K++KChhIIP22 (c(c(cclolololosseddd ssymbboools)s)s and KKKKv444.222-
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mono-exppponen itiitiallll ffffunctioii n to thhehh current ddddecayyy ffffor eachh hh voltlll aggge steppp. Each
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channels as function of voltage obtained by measuring the area under the corresponding current
curve during the first 50 ms of each voltage step (p<0.05).
Figure 5: Simulation of action potential propagation across the right ventricular wall using a
modified Luo Rudy II model. In (A) strong Ito expression in the midmyocardial and epicardial
layers produces a prominent notch in the action potential (spike and dome) – two views of the
same simulation shown. In (B) a 25% increase in Ito (in accord with a 48% gain in function of
KCND2 mutant and heterozygous expression) results in loss of the dome of the action potential
in the epicardial layer (basic cycle length 1000 ms).
me of the action ppototototen
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van Tintelen, Andrew D. Krahn and Michael H. GollobChris Simpson, Kamran Ahmad, Maarten P. van den Berg, Vijay Chauhan, Peter H. Backx, J. Peter
Champagne,Shaheen Uppal, Jeff S. Healey, David Birnie, Shubhayan Sanatani, Martin Gardner, Jean Mark J. Perrin, Arnon Adler, Sharon Green, Foad Al-Zoughool, Petro Doroshenko, Nathan Orr,
Gene as a Cause of J Wave Syndrome Associated with Sudden Cardiac DeathKCND2) Identifies the toEvaluation of Genes Encoding for the Transient Outward Current (I
Print ISSN: 1942-325X. Online ISSN: 1942-3268 Copyright © 2014 American Heart Association, Inc. All rights reserved.
TX 75231is published by the American Heart Association, 7272 Greenville Avenue, Dallas,Circulation: Cardiovascular Genetics
published online September 11, 2014;Circ Cardiovasc Genet.
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Supplemental Material
Supplemental Figure 1A
Most common ECG of cohort demonstrating infero-lateral J wave pattern in a patient who experienced a sudden cardiac arrest while sleeping. The patient was successfully resuscitated.
Supplemental Figure 1B
ECG of case subject showing predominant lateral J wave pattern. The patient experienced a sudden cardiac arrest while eating dinner.