Evaluation of Genes Encoding for the Transient Outward ...circgenetics.ahajournals.org/content/circcvg/early/2014/09/11/CIRC...DOI: 10.1161/CIRCGENETICS.114.000623 1 Evaluation of

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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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

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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

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wild type clone with 1 5 ug of wild type KCNIP2 clone using 2 ul of Lipofectam

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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

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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

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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

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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

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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

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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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rms J-W-W-W-Wavavavave e e e sysysysyndndndndrorororomemarization. J Am Coll Cardiol 2011; 57:1587 1590

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BBBB, KKKlK ein HAAA. NoNN rmrmmalalala vvvarara iaiaiatitititioonsss iin mmmuultiplple prpreecoroordididialalal llleaeaeadsdsds. AmAmAmm HHHHeaeaeartrtrt JJJJ.JJ5–5–5–88808 8.

n MJ. RS-T segmgmgmg ent elevevevatation in mid- ananand d d left prererecocordial leaeaadsddd as a normal variaJ.JJ 1953;46:817–8–8–8–82020200..

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Survivors with prprrresesesese

ugadaaaa JJJJ etetetet aaaallll IoIoIoIoninininicccm a

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errent (Ito) gain-of-function mutations in the KCND3-encoded Kv4.3 channel an

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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

http://circgenetics.ahajournals.org/D

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24. Liu D-W, Gintant GA, Antzelevitch C. Ionic bases for electrophysiological distinctions among epicardial, midmyocardial, and endocardial myocytes from the free wall of the canine left ventricle. Circulation Res. 1993;72:671-687.

25. Gaborit N, Le Bouter S, Szuts V, Varro A, Escande D, Nattel S, et al. Regional and tissue specific transcript signatures of ion channel genes in the non-diseased human heart. J Physiol.2007;582:675-693.

26. Morita H, Kusano KF, Miura D, Nagase S, Nakamura K, Morita ST, et al. Fragmented QRS as a marker of conduction abnormality and a predictor of prognosis of Brugada syndrome. Circulation. 2008;118:1697-1704.

27. Jerng HH, Covarrubias M. K+ channel inactivation mediated by the concerted action of the cytoplasmic N- and C-terminus domains. Biophys J. 1997;72:163-74.

28. Veeramah KR, Karafet TM, Wolf D, Samson RA, Hammer MF. The KCNJ8-S422L variant previously associated with J-wave syndromes is found at an increased frequency in Ashkenazi Jews. Eur J Hum Genet. 2014;22:94-98.

29. Sinner MF, Porthan K, Noseworthy PA, Havullinna AS, Tikkanen JT, Muller-Nurasyid M, et al. A meta-analysis of genome-wide association studies of the electrocardiographic early repolarization pattern. Heart Rhythm. 2012;9:1627-1634.

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

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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

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TX 75231is published by the American Heart Association, 7272 Greenville Avenue, Dallas,Circulation: Cardiovascular Genetics

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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.

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