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Bundle Branch Re-EntrantVentricular TachycardiaNovel Genetic Mechanisms in a Life-Threatening Arrhythmia

Jason D. Roberts, MD, MAS,a,b Michael H. Gollob, MD,c Charlie Young, MD,d Sean P. Connors, MD, DPHIL,e

Chris Gray, MD,f Stephen B. Wilton, MD, MSC,g Martin S. Green, MD,h Dennis W. Zhu, MD,i,j

Kathleen A. Hodgkinson, PHD,k Annie Poon, PHD,l Qiuju Li, MSC,c Nathan Orr, BSC,c Anthony S. Tang, MD,a

George J. Klein, MD,a Julianne Wojciak, MSC,b Joan Campagna, CCMA,b Jeffrey E. Olgin, MD,b

Nitish Badhwar, MBBS,b Vasanth Vedantham, MD, PHD,b Gregory M. Marcus, MD, MAS,b

Pui-Yan Kwok, MD, PHD,l Rahul C. Deo, MD, PHD,l,m Melvin M. Scheinman, MDb

ABSTRACT

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OBJECTIVES This study sought to investigate for an underlying genetic etiology in cases of apparent idiopathic

bundle branch re-entrant ventricular tachycardia (BBRVT).

BACKGROUND BBRVT is a life-threatening arrhythmia occurring secondary to macro–re-entry within the His-Purkinje

system. Although classically associated with dilated cardiomyopathy, BBRVT may also occur in the setting of

isolated, unexplained conduction system disease.

METHODS Cases of BBRVT with normal biventricular size and function were recruited from 6 North American centers.

Enrollment required a clinically documented wide complex tachycardia and BBRVT proven during invasive

electrophysiology study. Study participants were screened for mutations within genes associated with cardiac

conduction system disease. Pathogenicity of identified mutations was evaluated using in silico phylogenetic and

physicochemical analyses and in vitro biophysical studies.

RESULTS Among 6 cases of idiopathic BBRVT, each presented with hemodynamic compromise and 2 suffered cardiac

arrests requiring resuscitation. Putative culprit mutations were identified in 3 of 6 cases, including 2 in SCN5A

(Ala1905Gly [novel] and c.4719C>T [splice site mutation]) and 1 in LMNA (Leu327Val [novel]). Biophysical analysis of

mutant Ala1905Gly Nav1.5 channels in tsA201 cells revealed significantly reduced peak current density and positive

shifts in the voltage-dependence of activation, consistent with a loss-of-function. The SCN5A c.4719C>T splice

site mutation has previously been reported as disease-causing in 3 cases of Brugada syndrome, whereas the novel LMNA

Leu327Val mutation was associated with a classic laminopathy phenotype. Following catheter ablation, BBRVT was

noninducible in all cases and none experienced a clinical recurrence during follow-up.

CONCLUSIONS Our investigation into apparent idiopathic BBRVT has identified the first genetic culprits for this

life-threatening arrhythmia, providing further insight into its underlying pathophysiology and emphasizing a

potential role for genetic testing in this condition. Our findings also highlight BBRVT as a novel genetic etiology

of unexplained sudden cardiac death that can be cured with catheter ablation. (J Am Coll Cardiol EP 2017;3:276–88)

© 2017 by the American College of Cardiology Foundation.

m the aSection of Cardiac Electrophysiology, Division of Cardiology, Department of Medicine, Western University, London,

tario, Canada; bSection of Cardiac Electrophysiology, Division of Cardiology, Department of Medicine, University of California

n Francisco, San Francisco, California; cPeter Munk Cardiac Centre, Toronto General Hospital, University of Toronto, Toronto,

tario, Canada; dSection of Cardiac Electrophysiology, Division of Cardiology, Department of Medicine, Santa Clara Kaiser

dical Center, Santa Clara, California; eSection of Cardiac Electrophysiology, Division of Cardiology, Department of Medicine,

morial University, St. John’s, Newfoundland, Canada; fSection of Cardiac Electrophysiology, Division of Cardiology, Depart-

nt of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada; gLibin Cardiovascular Institute of Alberta, Cumming School

Medicine, University of Calgary, Calgary, Alberta, Canada; hSection of Cardiac Electrophysiology, Division of Cardiology,

partment of Medicine, University of Ottawa Heart Institute, Ottawa, Ontario, Canada; iCardiac Electrophysiology Laboratories,

AB BR E V I A T I O N S

AND ACRONYM S

BBRVT = bundle branch

re-entrant ventricular

tachycardia

BrS = Brugada syndrome

ECG = electrocardiogram

EPS = electrophysiology study

H = His bundle signal

V = ventricular activation

WT = wild-type

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B undle branch re-entrant ventricular tachy-cardia (BBRVT) is a life-threateningarrhythmia characterized by macro–re-entry

within the His-Purkinje system (1). The BBRVT circuitmost often consists of antegrade conduction alongthe right bundle branch followed by transseptal intra-myocardial conduction and retrograde conductionalong the left bundle branch (Figure 1A) (2). This man-ifests as a left bundle branch block pattern on the sur-face electrocardiogram (ECG), although a right bundlebranch block pattern may also be observed when cir-cuit propagation is in the opposite direction(Figure 1B) (3). The hallmark finding of BBRVT oninvasive electrophysiology study (EPS) distinguishingit from myocardial VT is the presence of a His bundlesignal (H) preceding ventricular activation (V), withchanges in the H-H interval driving changes in theV-V interval (Figure 1C) (3).

SEE PAGE 289

Findings common to patients with BBRVT in initialstudies included the presence of a prolonged HV-interval, along with structural heart disease, mostoften in the form of dilated cardiomyopathy (3).Subsequent reports emerged documenting BBRVT inthe absence of structural heart disease, suggesting thatconduction system disease in isolation may be suffi-cient for arrhythmia development (4,5). Notably,manyof the patients with BBRVT and structurally normalhearts were young and otherwise healthy, spawningcuriosity into the underlying etiology responsible fortheir predisposition to the arrhythmia (4).

Conduction system disease, a feature common toall cases of BBRVT, is increasingly appreciated tohave an underlying genetic etiology, particularlyamong individuals younger than 60 years of age(6–8). Given that His-Purkinje disease may provide asufficient substrate for BBRVT in isolation, wehypothesized that mutations within genes impli-cated in conduction system disease may serve asthe underlying culprits for cases of apparent idio-pathic BBRVT.

Regions Hospital, St. Paul, Minnesota; jDepartment of Medicine, University

Medicine, Memorial University, St. John’s, Newfoundland, Canada; lCardio

San Francisco, San Francisco, California; and the mDepartment of Medicine,

Institute for Human Genetics, University of California-San Francisco, San Fra

George Mines Travelling Fellowship Grant from the Canadian Heart Rhythm S

Mid-Career Investigator Award from the Heart and Stroke Foundation of O

anthropic fund. All other authors have reported that they have no relationsh

All authors attest they are in compliance with human studies committe

institutions and Food and Drug Administration guidelines, including patien

visit the JACC: Clinical Electrophysiology author instructions page.

Manuscript received September 2, 2016; revised manuscript received Septem

METHODS

STUDY POPULATION. Individuals youngerthan 60 years of age with BBRVT in theabsence of cardiomyopathy were recruitedfrom 6 North American medical centers.Enrollment required a clinically documentedwide complex tachycardia and confirmationof normal biventricular size and functionwith echocardiography and/or cardiac mag-netic resonance imaging. BBRVT had to besuccessfully induced at the time of invasive

EPS. Individuals with a medical disorder known tocause conduction system disease, including musculardystrophies associated with heart block, specificforms of myocarditis (Lyme and giant cell), andsarcoidosis, were excluded. Participant demographicsand medical details were obtained through review ofmedical records. Individuals who did not have a ge-netic culprit identified as part of their clinical careunderwent genetic testing as part of a research pro-tocol and provided signed informed consentapproved by the University of California, San Fran-cisco Committee on Human Research.

ELECTROPHYSIOLOGY STUDY AND CATHETER

ABLATION. Invasive EPS and BBRVT ablations wereperformed as described previously (9,10). In the eventthat standard programmed extrastimulation was un-successful for induction, long-short extra-stimuliwere used. Full details regarding the EPS, criteria toconfirm the diagnosis of BBRVT, and ablation pro-cedure are provided in the Online Appendix.

GENETIC ANALYSIS. Two of the 6 patients hadcommercial genetic testing (Ambry Genetics, AlisoViejo, California, and DNA Diagnostic Laboratory,University of Colorado, Denver, Colorado) as part oftheir clinical work-up. The remaining 4 individualswere screened for mutations using a next-generationsequencing panel containing 12 genes previouslyimplicated in conduction system disease (SCN5A,SCN10A, SCN1B, TRPM4, GJA1, LMNA, TBX5, NKX2-5,

of Minnesota, Minneapolis, Minnesota; kFaculty of

vascular Research Institute, University of California

California Institute for Quantitative Biosciences and

ncisco, California. Dr. Roberts was supported by the

ociety during this study. Dr. Gollob is supported by a

ntario. Dr. Scheinman is supported by the Deb phil-

ips relevant to the contents of this paper to disclose.

es and animal welfare regulations of the authors’

t consent where appropriate. For more information,

ber 27, 2016, accepted September 29, 2016.

FIGURE 1 BBRVT Circuit With Surface Electrocardiogram and Intracardiac Findings

(A) BBRVT circuit with antegrade conduction down the right bundle and retrograde conduction along the left bundle resulting in a surface

electrocardiogram with a left bundle branch block morphology. (B) BBRVT circuit with antegrade conduction down the left bundle and

retrograde conduction along the right bundle resulting in a surface electrocardiogram with a right bundle branch block morphology.

(C) Intracardiac tracing of BBRVT revealing the H-H interval preceding and predicting the subsequent ventricular-ventricular interval.

BBRVT ¼ bundle branch re-entrant ventricular tachycardia; H ¼ His; V ¼ ventricular.

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PRKAG2, KCNK3, KCNK17, and HCN4). Detailsregarding genomic DNA extraction, gene capture, li-brary preparation, sequencing, and bioinformaticanalysis are provided in the Online Appendix.

IN SILICO MUTATION ANALYSIS. Prevalence ofidentified mutations was assessed using the Exome

Aggregation Consortium (11). Evaluation of sequenceconservation across species was performed using theNCBI HomoloGene database and the UCSC GenomeBrowser (12). In silico prediction of the functional ef-fects of missense mutations was examined usingPolymorphism Phenotyping v2 (13), Sorting Intolerant

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From Tolerant (14), and Mutation Taster (15). Theimpact of the putative SCN5A splice site mutationwas evaluated using the Splice Site Prediction byNeural Network, Human Splicing Finder tools (16),and Mutation Taster.

ELECTROPHYSIOLOGICAL STUDIES OF SCN5A

Ala1905Gly. The SCN5A Ala1905Gly mutation wasintroduced into a wild-type (WT) SCN5A pcDNA1 cloneusing the QuickChange site directed mutagenesis kit(Stratagene, La Jolla, California) and confirmed withbidirectional Sanger sequencing. TransmembraneNav1.5 currents were recorded using the whole cellpatch clamp technique at room temperature using anAxopatch 200B amplifier (Axon Instruments, UnionCity, California), whereas voltage-clamp commandpulses were generated using pCLAMP software v8.0(Axon Instruments). Data were acquired and analyzedwith the pClamp10 and Clampfit software programs,respectively (Axon Instruments). Additional detailsregarding WT and mutant SCN5A expression in tsA201cells and electrophysiological analysis are provided inthe Online Appendix.

STATISTICAL ANALYSIS. Normally distributedcontinuous variables are presented as mean � SD forclinical data and mean � SE for the electrophysio-logical measurements from patch clamping. Com-parison of continuous variables was performed usingStudent t tests. Statistical analyses were performedusing GraphPad Prism5 (GraphPad Software, Inc., LaJolla, California). Two-tailed p values <0.05 wereconsidered statistically significant.

RESULTS

CLINICAL CASES. Cl in ica l features . Six cases ofBBRVT were identified in individuals younger than60 years of age with normal biventricular size andfunction. The mean age at diagnosis was 26.8 � 9.3years (range 17 to 38 years) and 4 were male (Table 1).Genetic culprits were identified in Case 1 (SCN5AAla1905Gly), Case 2 (SCN5A c.4719C>T), and Case 3(LMNA Leu327Val). Two of the 6 cases initially pre-sented with intermittent third-degree atrioventric-ular block and subsequently developed BBRVT withindays of their sentinel event (Cases 4 and 5; the clinicalaspects of Case 5 have been previously reported [17])(Table 1). Notably, both had bicuspid aortic valvesdocumented on echocardiography and heavy calcifi-cation was observed in Case 5, whereas only valvularthickening was present in Case 4. A single patient hada longstanding history of persistent atrial fibrillationdiagnosed at 28 years of age (Case 3) (Table 1). None of

the remaining cases had any evidence of structuralheart disease or prior cardiac history. Of the 4 casesthat presented with BBRVT, 2 suffered cardiac arrestsrequiring resuscitation, 1 had syncope, and 1 hadpalpitations (Table 1). Case 3 had a brother thatrequired permanent pacemaker insertion at 52 yearsof age for sinus node dysfunction, whereas there wasno family history of other arrhythmic issues,including unexplained premature sudden cardiacdeath, in any of the remaining cases.Surface ECG. Evidence of conduction system diseaseon surface ECG was present in 4 of the 6 patients atbaseline in the absence of BBRVT (Cases 3 to 6)(Table 1, Figure 2). Although Case 1 exhibited mildQRS prolongation (120 ms) and Case 2 had an inde-terminate axis, neither had definitive findingsconsistent with conduction system disease. As high-lighted previously, Cases 4 and 5 presented withthird-degree atrioventricular block and had wide-complex infranodal escape rhythms (Figure 3). Pa-tient 3 had a history of persistent atrial fibrillation inassociation with a nonspecific intraventricular con-duction delay, whereas Case 6 had incomplete rightbundle branch block (Table 1, Figure 2). Aside from thecalcification observed in association with the bicuspidaortic valve in Case 5, which may predispose to Levdisease, there were no other known etiologies thatcould account for the observed conduction systemdisease in the study participants.Elect rophys io logy study . All patients underwentinvasive EPS and all had a prolonged HV at baseline(mean: 69.2 � 9.1 ms) (Table 1). BBRVT was induced inall cases with programmed extrastimulation(Figure 4A). BBRVT during EPS had right and leftbundle branch block morphologies in Cases 1 and 5(Online Figure 1), whereas only a left bundle branchblock morphology was observed in the remaining 4individuals (Online Figure 1). The cycle length ofBBRVT ranged from 350 ms (171 beats/min) to 220 ms(273 beats/min) and in each patient was associatedwith marked symptoms and varying degrees of he-modynamic instability. In each case of BBRVT, atrio-ventricular dissociation was observed and changes ineither the H-H or right bundle–right bundle potentialswere observed to precede and predict changes in sub-sequent V-V intervals (Figure 4B). All patients weretreated with catheter ablation of the right bundlebranch, which rendered tachycardia noninducible.Fol low-up. Following catheter ablation, there wasno recurrence of VT in any of the cases (mean follow-up: 6.7 � 4.6 years) (Table 1). Patient #1 developedtypical atrial flutter in association with symptomaticsinus bradycardia at 24 years of age, 7 years followingher initial presentation for BBRVT. She was

TABLE 1 Clinical, Electrophysiological, and Genetic Features of BBRVT Study Participants

Case 1 Case 2 Case 3 Case 4 Case 5 Case 6

Clinical features

Age at diagnosis, yrs 17 18 38 37 22 29

Sex F M F M M M

Family history ofarrhythmic disorder

Negative Negative CCD Negative Negative Negative

Presentation Syncope ACA ACA Pre-syncope Syncope Syncope

Structural heart disease Negative Negative BAE BAV BAV Negative

Arrhythmia features

Presenting rhythm BBRVT BBRVT BBRVT AVB AVB BBRVT

Baseline ECG QRS 120 ms Indet axis AF & LBBB AVB AVB iRBBB

Baseline HV, ms 61 79 62 60 78 75

BBRVT morphology RBBB & LBBB LBBB LBBB LBBB RBBB & LBBB LBBB

BBRVT HV, ms 74 91 75 66 86 115

BBRVT rate, beats/min 260, 240 170 250 200 270, 230 220

Follow-up

Years 15 3 8 3 4 7

Other arrhythmias SND, AFl Negative Negative Negative Negative Negative

Clinical status Alive Alive Deceased Alive Alive Alive

Genetic mutation

Gene SCN5A SCN5A LMNA – – –

Nucleotide change C > G C > T C > G – – –

Mutation type Missense Splice Site Missense – – –

Amino acid change Ala1905Gly N/A Leu327Val – – –

In silico analysis

PolyPhen-2 score 0.999 N/A 0.566 – – –

SIFT score 0 N/A 0.05 – – –

Mutation Taster score Disease-causing Disease-causing Disease-causing – – –

ACA ¼ aborted cardiac arrest; AF ¼ atrial fibrillation; AFl ¼ atrial flutter; AVB ¼ third-degree atrioventricular block; BAE ¼ biatrial enlargement; BAV ¼ bicuspid aortic valve;BBRVT ¼ bundle branch re-entrant ventricular tachycardia; CCD ¼ cardiac conduction system disease; ECG ¼ electrocardiogram; F ¼ female; HV ¼ His-ventricular; Indetaxis ¼ indeterminate axis; iRBBB ¼ incomplete right bundle branch block; LBBB ¼ left bundle branch block; M ¼ male; Negative ¼ no family history of an arrhythmic disorder;NIVCD ¼ nonspecific intraventricular conduction delay; PolyPhen-2 ¼ Polymorphism Phenotyping v2; RBBB ¼ right bundle branch block; SIFT ¼ Sorting Intolerant FromTolerant; SND ¼ sinus node dysfunction.

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successfully treated with cavotricuspid isthmusablation and was also noted to have sinus nodedysfunction (corrected sinus node recovery time:2,070 ms) at the time of EPS, leading to permanentpacemaker insertion. Fifteen years following herinitial presentation, she has had no subsequentarrhythmic events. Case 3 (LMNA Leu327Val) devel-oped a dilated cardiomyopathy approximately 6 yearsfollowing her initial presentation in association withproximal muscle weakness. Despite aggressive inter-vention including a left ventricular assist device, shedied from heart failure while awaiting heart trans-plantation. Cases 4 and 5, both of whom presentedwith third-degree atrioventricular block, had recov-ery of atrioventricular conduction during follow-upwith <1% ventricular pacing observed on repeatedannual pacemaker interrogations. Cases 2 and 6 havehad no subsequent arrhythmic episodes during thefollow-up period (Table 1).

GENETIC ANALYSIS. Case 1. Next-generation sequ-encing of the 12 pre-specified genes identified a novel

SCN5A Ala1905Glymissensemutation (Table 1) locatedon the C-terminus of the ion channel (Figure 5). Theamino acid was highly conserved among mammalianspecies (Online Figure 2A) and the mutation was pre-dicted to be pathogenic on in silico analysis (Table 1).Cascade screening of first-degree family members hasbeen offered, but to date has been declined.Case 2 . Genetic testing was performed using acommercially available arrhythmia panel (AmbryGenetics) comprised of 29 different genes (OnlineAppendix). The patient was found to carry anSCN5A c.4719C>T mutation (Table 1) located in exon27 predicted by 2 in silico splicing tools (Splice SitePrediction by Neural Network, Human SplicingFinder) to generate a cryptic donor splice site locatedwithin exon 27 that does not involve the canonical 5’dinucleotide GT donor site. The splice site mutationis predicted to lead to deletion of 32 amino acidsfrom Domain IV of the ion channel (Figure 5). Insilico analysis with Mutation Taster predicted thevariant to be “disease causing.” The nucleotideposition is highly conserved in mammalian species

FIGURE 2 QRS Complex on Surface Electrocardiograms in the Absence of Bundle Branch Re-Entrant Ventricular Tachycardia in

Cases 1, 2, 3, and 6

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(Online Figure 2B), has previously been reported asthe genetic culprit in 3 separate cases of Brugadasyndrome (BrS) (18,19), and is absent from the ExomeAggregation Consortium database. Another splice site

mutation that disrupts the canonical 5’ donor splicesite of exon 27 resulting in activation of the crypticsplice site at c.4917C has previously been identifiedin a case of BrS and presence of the aberrant

FIGURE 3 Atrioventricular Block Associated With an Infranodal Escape

Atrioventricular block associated with an infranodal escape observed on presentation in Cases 4 (A) and 5 (B).

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transcript containing the identical 96 base pairdeletion predicted to occur secondary to our splicesite mutation was demonstrated (20). Electrophysio-logical studies with patch clamping on the

corresponding protein product containing an in-frame 32 amino acid deletion (the identical proteinproduct predicted in our case) revealed a completeloss-of-function (20).

FIGURE 4 Intracardiac Electrograms

(A) Long-short initiation of BBRVT via pacing from the right ventricle. (B) Variations in

RB-RB potential intervals preceding and predicting variations in V-V activation intervals.

RB ¼ right bundle; other abbreviations as in Figure 1.

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Following identification of the SCN5A mutation,modified ECG with precordial leads moved up 2intercostal spaces revealed no evidence of a Brugadapattern. Procainamide challenge was not pursuedbecause of the presence of right bundle branch blockfollowing ablation. First-degree relatives havedeclined subsequent attempts at clinical and geneticcascade screening.Case 3. Genetic screening identified a novel LMNALeu327Val mutation (Table 1, Online Figure 3). Theamino acid was highly conserved among mammalianspecies (Online Figure 2C) and in silico analysis sug-gested that the mutation was possibly pathogenic(Table 1). Family history revealed that the brother ofthe proband had a pacemaker inserted at 52 years ofage for sick sinus syndrome and had mild PR-intervalprolongation (210 ms) on surface ECG. To date, ge-netic and cascade screening of family members hasbeen declined.Cases 4 , 5 , and 6. Next-generation sequencing ofthe 12 pre-specified genes revealed no rare variants inthe remaining cases.

ELECTROPHYSIOLOGICAL ANALYSIS OF SCN5A Ala1905Gly.

The peak current densities of homozygous and het-erozygous Ala1905Gly Nav1.5 channels in mammaliantsA201 cells were significantly reduced relative to WT(p ¼ 0.018 and p ¼ 0.044, respectively) (Table 2,Figures 6A to 6C). Analysis of gating properties over awide range of membrane voltages revealed thatsteady-state activation of the homozygous and het-erozygous Ala1905Gly Nav1.5 currents was shiftedsignificantly to more depolarized membrane poten-tials relative to WT (Table 2, Figure 6D). Half-maximalactivation voltages (V1/2) for homozygous and het-erozygous Ala1905Gly mutant Nav1.5 channelsexhibited statistically significant positive shifts rela-tive to WT (p ¼ 0.042 and p ¼ 0.020, respectively)(Table 2). Evaluation of the inactivation kinetics ofthe Nav1.5 currents revealed that the half-maximalinactivation voltage of the heterozygous and homo-zygous Ala1905Gly Nav1.5 channels differed signifi-cantly relative to WT (p ¼ 0.002 and p ¼ 0.024,respectively) (Figure 6D, Table 2). The activation slopefactor (k) was significantly increased in heterozygousAla1905Gly Nav1.5 channels relative to WT (p < 0.01),whereas no differences were observed in inactivationslope factors between mutant and WT Nav1.5 currents(Table 2).

The shifts in steady-state activation and inactiva-tion between mutant andWT Nav1.5 channels resultedin a reduced overlap of the activation and inactivationcurves resulting in a reduced “window current.” In

addition to the fast-recovered inactivation compo-nent of steady-state inactivation, the slow recoveredinactivation component, also referred to as the inter-mediate inactivation, was also investigated in WT andhomozygous Ala1905Gly Nav1.5 channels using a2-pulse voltage protocol. The first voltage pulselength was varied from 3 to 3,000 ms and was sepa-rated from the second voltage pulse by 20 ms of hy-perpolarization to -100 mV to permit recovery of thefast inactivation component. The intermediate inac-tivation of homozygous Ala1905Gly Nav1.5 channels(0.43 � 0.03) was increased compared with the WTNav1.5 channels (0.34 � 0.03; p ¼ 0.035) (Table 2,Online Figure 4). Inactivation recovery and the late,persistent inward sodium current did not differsignificantly in WT and homozygous Ala1905GlyNav1.5 channels (Table 2, Online Figure 5).

FIGURE 5 SCN5A c.4719 C>T Mutation Results in Aberrant Splicing and a 32 Amino Acid In-Frame Deletion in Domain IV

of the Ion Channel

SCN5A Ala1905Gly resides within the C-terminus of the ion channel.

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DISCUSSION

Our investigation into cases of apparent idiopathicBBRVT has identified the first genetic culprits in thislife-threatening condition. Pathogenic mutations in 2separate genes were identified in 3 of 6 individuals,providing evidence to support the use of clinical ge-netic testing in cases of idiopathic BBRVT. In additionto establishing idiopathic BBRVT as a genetic condi-tion, our study sheds additional insight into its un-derlying pathogenesis and emphasizes thatconduction system disease in isolation provides asufficient substrate for arrhythmia development. Ourfindings also highlight BBRVT as a novel genetic eti-ology of unexplained sudden cardiac death amongindividuals with structurally normal hearts. The latterfurther emphasizes the important role for EPS in the

evaluation and management of cases of aborted car-diac arrest, a particularly important concept giventhat BBRVT can be cured with catheter ablation.

The findings from our study implicate both SCN5Aand LMNA as genetic culprits of BBRVT. Case 1possessed a novel SCN5A mutation in a highlyconserved residue (Ala1905Gly) located on theC-terminus of the ion channel (Figure 5). Findingsfrom in vitro electrophysiological analyses revealedthat, relative to WT Nav1.5 current, homozygous andheterozygous Ala1905Gly mutants had reduced peakcurrent densities and steady-state activation andhalf-maximal activation voltages that were signifi-cantly shifted to more depolarized membrane po-tential. Collectively, these findings were consistentwith the SCN5A Ala1905Gly missense variant being apathogenic loss-of-function mutation that was

TABLE 2 Peak Current and Kinetics of Activation and

Inactivation in Wild-Type and Mutant Ala1905Gly Nav1.5 Currents

Nav1.5

Wt Ala1905Gly wt/Ala1905Gly

Ipeak, pA/pF -263.3 � 40.5 -144.3 � 21.4* -164.8 � 21.4*

Activation kinetics

V1/2, mV -45.0 � 1.8 -39.9 � 1.6* -39.7 � 1.2*

Slope factor, k 2.3 � 0.4 3.2 � 0.4 4.3 � 0.3†

Inactivation kinetics

V1/2, mV -72.0 � 1.1 -76.2 � 1.3* -80.0 � 1.1*

Slope factor, k -8.8 � 1.8 -6.6 � 0.5 -5.8 � 0.9

sf, ms 13.2 � 1.4 14.2 � 1.2 –

ss, ms 157.4 � 38.3 119.4 � 24.8 –

Iintermediate inact 0.34 � 0.03 0.43 � 0.03* –

Isus 0.12 � 0.04 0.12 � 0.06 –

Values are mean � SD. *p < 0.05. †p < 0.01 relative to the wild-type.

Iintermediate inact ¼ intermediate inactivation; Ipeak ¼ peak current density;Isus ¼ sustained current (also referred to as late, persistent inward sodium current);pA/pF ¼ picoamperes per picofarad; sf ¼ time constant of fast inactivationrecovery; ss ¼ time constant of slow inactivation recovery; V1/2 ¼ half-maximalvoltage of activation or inactivation.

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causative for the constellation of clinical arrhythmiasin Case 1, including BBRVT, cardiac conduction sys-tem disease, atrial flutter, and sinus nodedysfunction.

Case 2 possessed an SCN5A c.4719C>T mutationlocated in exon 27 predicted to result in activation ofa cryptic 5’ donor splice site leading to loss of 96nucleotides from the mRNA product. This nucleotideis highly conserved among mammalian species(Online Figure 1B) and has been reported as thegenetic culprit in 3 cases of BrS (18,19). The impact ofthe aberrant splicing is a 32 amino acid in-framedeletion involving the S2/S3 segments and inter-vening cytoplasmic loop of Domain IV (Figure 5).Previous functional work on another splice-site mu-tation disrupting the 5’ donor site at the distal end ofexon 27 and resulting in the identical 32 amino acidin-frame deletion revealed a complete loss-of-function, consistent with it being pathogenic (20).

Notably, Case 2 did not have ECG findings of BrSwith surface leads in the standard and modified highpositions, although a procainamide challenge was notperformed because of the presence of right bundlebranch block following ablation (21). Identification ofan identical mutation causing 2 different phenotypes,termed genetic pleiotropy, is a common finding withSCN5A (22,23). In reference to BrS, it should also benoted that affected patients often have conductionsystem disease, as evidenced by prolongedHV-intervals at the time of EPS (24). Although ven-tricular arrhythmias in BrS are almost exclusivelypolymorphic, in rare instances when monomorphicVT is observed, consideration should be given to

BBRVT as a potential underlying etiology, as high-lighted by a recent study (25,26).

The LMNA gene encodes both the lamin A and Cproteins, generated through alternative splicing,which are constituents of nuclear lamina that resideimmediately inside the inner nuclear membrane(Online Figure 3) (27). In the context of cardiac disease,LMNA mutations most often cause an autosomal-dominant form of dilated cardiomyopathy associatedwith conduction system disease (28). A select group ofother genetic dilated cardiomyopathies are also linkedto conduction system disease, including those associ-ated with the PRKAG2, TBX5, and NKX2-5 genes, andboth phenotypic features most often developconcomitantly (29–31). Our patient presented withunderlying conduction system disease and BBRVTbefore subsequently developing dilated cardiomyop-athy. The LMNA Leu327Val mutation is novel and thephenotype of the proband, which included conductionsystem disease, atrial fibrillation, subsequent devel-opment of cardiomyopathy (following onset ofBBRVT), and proximalmuscle weakness, is classic for alaminopathy, lending support for the identified mu-tation being the genetic culprit.

The dramatic presentations of our cases,including syncope and cardiac arrests requiringresuscitation secondary to heart rates frequentlyexceeding 200 beats/min, also emphasize the ma-lignant potential of BBRVT. Our findings highlightthat BBRVT should be considered as a potentialculprit in cases of unexplained sudden cardiacdeath, a concept that is generally not incorporatedin diagnostic algorithms for this patient population(32–34). The need to screen for BBRVT highlightsthe critical importance of invasive EPS, particularlywhen there is evidence of underlying conductionsystem disease on surface ECG. Because of thedependence of BBRVT on the specialized conduc-tion system, these induction protocols should al-ways include long-short extra-stimuli. In addition tofacilitating accurate diagnosis, catheter ablation canalso serve as a curative therapy for this life-threatening arrhythmia, particularly notable giventhat none of the other causes of unexplained abor-ted cardiac arrest in this patient population can becured. That being said, implantation of animplantable cardioverter-defibrillator is still likelyreasonable given that the genetic mutation maypotentially lead to additional cardiac abnormalitiesthat continue to place patients at risk of suddencardiac death. Both patients in this study that suf-fered aborted cardiac arrests were offered animplantable cardioverter-defibrillator, although Case2 declined.

FIGURE 6 Biophysical Properties of Wild-Type and Mutant Ala1905Gly Nav1.5 Currents Expressed in tsA201 Cells

(A) Whole cell currents of wild-type, homozygous, and heterozygous Ala1905Gly Nav1.5 channels. (B) Peak currents of mutant Nav1.5 channels were significantly

reduced relative to wild-type. (C) Current-voltage relationships revealed significant reductions in current densities of homozygous and heterozygous Ala1905Gly Nav1.5

channels relative to wild-type at multiple different membrane voltages. (D) Steady-state voltage-dependent properties of activation and inactivation for wild-type,

homozygous, and heterozygous Ala1905Gly Nav1.5 channels. wt ¼ wild-type; *p < 0.05; **p < 0.01.

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Among the remaining cases of BBRVT in ourcohort, the inability to identify an underlying geneticetiology may be secondary to the presence of undis-covered genetic culprits or a nongenetic mechanismfor their underlying conduction system disease.Although we excluded cases of known myocarditis, itis notable that Cases 4 and 5 seemed to recoveratrioventricular conduction during follow-up, as evi-denced by their requiring <1% ventricular pacing af-ter 1 year of follow-up. It is conceivable that theirconduction system disease may have been secondaryto a focal myocarditis that subsequently resolved.The third-degree atrioventricular block and distalconduction system disease observed in Cases 4 and 5could also have been secondary to Lev disease thatmay have developed in association with their

bicuspid aortic valves, particularly for Case 5, whoseaortic valve was heavily calcified (35). Althoughconceivable, Lev disease is generally progressive,whereas the atrioventricular block in both of thesepatients resolved during follow-up. Analogous toheart block among young individuals (36), it is prob-able that BBRVT in the setting of structurally normalhearts may have multiple etiologies.

STUDY LIMITATIONS. Although our study examiningBBRVT in the absence of structural heart diseaseinvolves the largest case series to date, being drawnfrom 6 North American centers, our study size of 6patients is modest. The primary goal of this inves-tigation was to identify novel genetic culprits and, ashighlighted by our novel findings, the cohort was

PERSPECTIVES

COMPETENCY IN MEDICAL KNOWLEDGE: Bundle branch

re-entrant ventricular tachycardiamay have an underlying genetic

origin and is a novel genetic etiology of unexplained sudden car-

diac death. Genetic testing of the SCN5A and LMNA genes should

be considered among patients with bundle branch re-entrant

ventricular tachycardia in the setting of a structurally normal

heart. Survivors of unexplained sudden cardiac death should be

considered for an invasive electrophysiology study that includes

long-short extra-stimuli during the induction protocol to screen

for bundle branch re-entrant ventricular tachycardia.

TRANSLATIONAL OUTLOOK: The role of genetics in bundle

branch re-entrant ventricular tachycardia associated with struc-

tural heart disease is unknown and should be investigated in

future studies. The prevalence of bundle branch re-entrant

ventricular tachycardia among survivors of unexplained sudden

cardiac death should be evaluated to further clarify the role of

invasive electrophysiology study among this patient population.

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sufficient to accomplish this goal. A larger samplesize is necessary to more definitively establish theprevalence of genetic mutations in this patientpopulation. The families of each proband found tocarry a presumed pathogenic mutation havedeclined cascade screening, which has precludedevaluation for genotype-phenotype segregation.Although this may be viewed as a limitation fordefinitively concluding that the identified mutationswere the genetic culprits for the BBRVT phenotype,for both SCN5A mutations, we provided multiplelines of in silico and in vitro evidence consistentwith their being pathogenic. Support for the novelLMNA mutation being pathogenic was provided bythe clinical phenotype being classic for alaminopathy.

CONCLUSIONS

Our investigation into BBRVT in the setting of normalbiventricular size and systolic function has identifiedthe first genetic culprits for this life-threateningventricular arrhythmia. Identification of culprit mu-tations within SCN5A and LMNA provides furtherinsight into the pathophysiology underlying thecondition and emphasizes a potential role for routineclinical genetic testing for idiopathic BBRVT. Ourfindings also highlight BBRVT as a novel genetic eti-ology of unexplained sudden cardiac death that canbe cured with catheter ablation.

ADDRESS FOR CORRESPONDENCE: Dr. Jason D.Roberts, Sectionof CardiacElectrophysiology,DivisionofCardiology, Department of Medicine, Western Univer-sity, 339 Windermere Road, B6-129B, London, OntarioN6A 5A5, Canada. E-mail: jason.roberts@lhsc.on.ca.

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KEY WORDS conduction system disease,genetics, sudden cardiac death, ventriculartachycardia

APPENDIX For a supplemental Methodssection and figures, please see the onlineversion of this article.