original article - homepage | circulation: arrhythmia and...

1122

Each year, >400 000 cases (>1000 per day) of sudden car-diac death (SCD) are reported in the United States.1 Many

of these deaths can be attributed to coronary artery disease; however, a small portion are caused by inherited structural heart diseases, such as hypertrophic cardiomyopathy (HCM) or structurally normal heart inherited arrhythmia syndromes, such as long-QT syndrome (LQTS).2,3 Although both HCM and LQTS can cause sudden death caused by cardiac arrhyth-mias, the pathophysiology, disease progression, and underly-ing molecular/genetic substrates are thought to be distinct.

Largely considered a disorder of the cardiac sarcomere, HCM is defined by cardiac hypertrophy, most commonly of the left ventricle, in the absence of a clinically identifiable cause, and often presents with fibrosis, myocyte disarray, ventricular septal dysmorphology, coronary artery microvas-cular disease, and left ventricular outflow tract obstruction.4,5 Conversely, LQTS is a disorder of delayed ventricular myo-cardial repolarization that often manifests as prolongation of the QT interval on an ECG in the setting of a structurally nor-mal heart.6 Although >25 HCM-susceptibility genes (mostly

© 2015 American Heart Association, Inc.

Circ Arrhythm Electrophysiol is available at http://circep.ahajournals.org DOI: 10.1161/CIRCEP.115.002745

Original Article

Background—A portion of sudden cardiac deaths can be attributed to structural heart diseases, such as hypertrophic cardiomyopathy (HCM) or cardiac channelopathies such as long-QT syndrome (LQTS); however, the underlying molecular mechanisms are distinct. Here, we identify a novel CACNA1C missense mutation with mixed loss-of-function/gain-of-function responsible for a complex phenotype of LQTS, HCM, sudden cardiac death, and congenital heart defects.

Methods and Results—Whole exome sequencing in combination with Ingenuity variant analysis was completed on 3 affected individuals and 1 unaffected individual from a large pedigree with concomitant LQTS, HCM, and congenital heart defects and identified a novel CACNA1C mutation, p.Arg518Cys, as the most likely candidate mutation. Mutational analysis of exon 12 of CACNA1C was completed on 5 additional patients with a similar phenotype of LQTS plus a personal or family history of HCM-like phenotypes and identified 2 additional pedigrees with mutations at the same position, p.Arg518Cys/His. Whole cell patch clamp technique was used to assess the electrophysiological effects of the identified mutations in Ca

V1.2 and revealed a complex phenotype, including loss of current density and inactivation in

combination with increased window and late current.Conclusions—Through whole exome sequencing and expanded cohort screening, we identified a novel genetic substrate

p.Arg518Cys/His-CACNA1C, in patients with a complex phenotype including LQTS, HCM, and congenital heart defects annotated as cardiac-only Timothy syndrome. Our electrophysiological studies, identification of mutations at the same amino acid position in multiple pedigrees, and cosegregation with disease in these pedigrees provide evidence that p.Arg518Cys/His is the pathogenic substrate for the observed phenotype. (Circ Arrhythm Electrophysiol. 2015;8:1122-1132. DOI: 10.1161/CIRCEP.115.002745.)

Key Words: calcium channels, L-type ◼ cardiomyopathy, hypertrophic ◼ death, sudden, cardiac ◼ genetics ◼ long QT syndrome ◼ Timothy syndrome

Received January 16, 2015; accepted July 23, 2015.From the Department of Molecular Pharmacology and Experimental Therapeutics, Windland Smith Rice Sudden Death Genomics Laboratory (N.J.B.,

D.Y., D.J.T., J.M.B., M.J.A.), Division of Immunology and Neurology (F.J., A.H., A.J.J.), and Departments of Medicine (Division of Cardiovascular Diseases) and Pediatrics (Division of Pediatric Cardiology) (M.J.A.), Mayo Clinic, Rochester, MN; Division of Cardiology, Nicklaus Children's Hospital, Miami, FL (R.K.).

The Data Supplement is available at http://circep.ahajournals.org/lookup/suppl/doi:10.1161/CIRCEP.115.002745/-/DC1.Correspondence to Michael J. Ackerman, MD, PhD, Windland Smith Rice Sudden Death Genomics Laboratory, Mayo Clinic, Guggenheim 501,

Rochester, MN 55905. E-mail [email protected]

Identification and Functional Characterization of a Novel CACNA1C-Mediated Cardiac Disorder Characterized by

Prolonged QT Intervals With Hypertrophic Cardiomyopathy, Congenital Heart Defects, and Sudden Cardiac Death

Nicole J. Boczek, PhD; Dan Ye, MD; Fang Jin, MD; David J. Tester, BS; April Huseby, BS; J. Martijn Bos, MD, PhD; Aaron J. Johnson, PhD; Ronald Kanter, MD;

Michael J. Ackerman, MD, PhD

by guest on May 19, 2018

http://circep.ahajournals.org/D

ownloaded from

by guest on M

ay 19, 2018http://circep.ahajournals.org/

Dow

nloaded from



ownloaded from

by guest on M


Dow

nloaded from



ownloaded from

by guest on M


Dow

nloaded from



ownloaded from

http://circep.ahajournals.org/lookup/suppl/doi:10.1161/CIRCEP.115.002745/-/DC1

mailto:[email protected]

http://circep.ahajournals.org/







Boczek et al Novel CACNA1C Mutation in New Disorder COTS 1123

encoding for structural proteins)7 and 17 LQTS-susceptibility genes (encoding for ion channels or channel interacting pro-teins)8 have been discovered, a significant number of patients expressing these potentially lethal phenotypes remains geneti-cally elusive.

Here, using next-generation pedigree-based whole exome sequencing (WES) on a multigenerational pedigree and a sub-sequent analysis of additional unrelated patients/pedigrees presenting with a similar cardiac phenotype, we have iden-tified a novel perturbation in the CACNA1C-encoded Ca

V1.2

calcium channel that confers susceptibility for the concomi-tant phenotypes of LQTS, HCM, congenital heart defects (CHDs), and SCD that are present in these families.

MethodsStudy SubjectsAn 11-member (5 affected) multigenerational pedigree present-ed with a multitude of cardiac signs and symptoms, including marked QT prolongation, HCM, SCD, and CHDs. However, none of the family members had syndactyly, cognitive impairments, facial dysmorphisms, or any other noncardiac clinical character-istics suggestive of Timothy syndrome (TS). The proband, who was genotype negative by commercially available LQTS genetic testing, was referred to the Mayo Clinic Windland Smith Rice Sudden Death Genomics Laboratory for further genetic test-ing. After written consent was obtained for this institutional re-view board–approved study, peripheral blood lymphocytes were

obtained from 6 family members. Genomic DNA was obtained using the Puregene DNA Isolation Kit (Qiagen Inc, Valencia, CA). The symptomatic index case, unaffected mother, affected sister, and affected nephew were selected for WES (Figure 1A, cases III.1, II.2, III.4, and IV.1).

Whole Exome SequencingExpanded Methods on the whole exome sequencing and subsequent bioinformatic analysis are available in the Data Supplement.

CACNA1C Mutational AnalysisNext, we examined our cohort of 37 unrelated patients with clini-cally robust but genetically elusive LQTS for coexisting echocardio-graphic evidence of HCM or family history of HCM-like phenotypes; we found 5 cases with this phenotype (Table 1). These cases were mutation negative after LQTS mutational analysis (by denaturing high-performance liquid chromatography and DNA sequencing) of the 3 major LQTS genes, KCNQ1, KCHN2, and SCN5A, and 8 mi-nor LQTS genes, AKAP9, ANKB, CAV3, KCNE1, KCNE2, KCNJ2, SCN4B, and SNTA1.

For these 5 unrelated cases with LQTS and concomitant person-al or familial HCM, genetic analysis was performed on exon 12 of CACNA1C (NM_000719) using polymerase chain reaction and DNA sequencing (ABI Prism 377; Applied Biosystems Inc, Foster City, CA). Primer sequences and polymerase chain reaction conditions are available on request.

WHAT IS KNOWN

•LQTS is a disorder of delayed ventricular myocar-dial repolarization that often manifests as prolonged QT intervals on a resting ECG in the setting of a structurally normal heart, and to date, 17 LQTS-susceptibility genes, encoding ion channel or ion channel interacting proteins, have been described.

•Hypertrophic cardiomyopathy (HCM) is defined by cardiac hypertrophy and to date, 25 HCM-susceptibility genes, primarily encoding for structur-al proteins, have been described; and although some cases of HCM have concurrent QT prolongation, a genetic etiology has never been identified for patients presenting with LQTS and HCM-like phenotypes.

WHAT THE STUDY ADDS

• This study identifies a novel disorder, cardiac-only Timothy syndrome (COTS), characterized by QT pro-longation, hypertrophic cardiomyopathy, congenital heart defects, and sudden cardiac death.

•Using whole exome sequencing and expanded cohort screening, novel genetic substrates p.Arg518Cys/His, within the CACNA1C-encoded L-type calcium channel, were attributed to the COTS phenotype.

•The p.Arg518Cys/His variants were characterized functionally and found to lead to a complex electro-physiological phenotype including loss of current density, increased window and late current, and de-celerating voltage-dependent inactivation.

A

B

Figure 1. Pedigree and whole exome sequencing (WES) filtering strategy. A, Pedigree with long-QT syndrome (LQTS), sudden cardiac death, cardiomyopathy, and congenital heart defects (see key to right) that was used for WES. Individuals who under-went WES are highlighted with gray, red, yellow, and blue circles. B, The filtering strategy used on this pedigree, showing the num-ber of variants eliminated at each step.



ownloaded from


1124 Circ Arrhythm Electrophysiol October 2015

We expanded the pedigrees to include additional blood samples from family members of 2 of 5 of these patients to examine exon 12 of CACNA1C. All patients and family members signed written con-sent for this institutional review board–approved study.

CACNA1C Mammalian Expression Vectors and Site-Directed MutagenesisThe human wild-type (WT) CACNA1C cDNA with an N-terminal enhanced yellow fluorescence protein (EYFP) tag ([EYFP] Nα1c,77) in the pcDNA vector and the cDNA of the CACNA2D1 gene cloned in pcDNA3.1 vector, and the CACNB2 cDNA were gifts from Dr. Charles Antzelevitch, Masonic Medical Research Laboratory, Utica, NY. The cDNA of CACNB2b was subcloned into the bicis-tronic pIRES2-dsRED2 vector (Clontech, Mountain View, CA). The p.Arg518Cys-CACNA1C and p.Arg518His-CACNA1C missense mutations were engineered into pcDNA3-CACNA1C-WT–EYFP vector using primers containing the missense mutations (available on request) in combination with the Quikchange II XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). The integrity of all con-structs was verified by DNA sequencing.

HEK293 Cell Culture and TransfectionExpanded Methods on the HEK293 cell culture and transfection, as well as subsequent electrophysiological measurements, data analysis, and con-focal examination of CACNA1C, are available in the Data Supplement.

Statistical AnalysisAll data points are shown as the mean value, and error bars represent the standard error of the mean (SEM). Statistics were completed used SigmaPlot 12.0 (San Jose, CA) and GraphPad Prism (La Jolla, CA). Normality was tested by Shapiro–Wilk, and constant variance was analyzed for each comparison, and if normality failed, nonparamet-ric methods were used. Student t test or Mann–Whitney rank sum test was performed to determine statistical significance between two groups, and a P<0.05 was considered significant. One-way ANOVA or Kruskal–Wallis in combination with Dunn method for multiple comparisons was performed to determine statistical significance among our variants versus controls. After correction for multiple comparisons, a P<0.05 was considered to be significant.

ResultsMultigenerational Pedigree With LQTS, HCM, SCD, and CHDsThe index case is a 33-year-old woman (Figure 1A, III.1; Table 2, III.1) who presented at 25 years of age with QT prolongation during pregnancy. Subsequent medical history revealed a ventric-ular septal defect and a family history of SCD. Her ECG exhib-ited a QTc of 500 ms, first-degree AV block, and her medical therapy included the placement of an implantable cardioverter

defibrillator. She later experienced peripartum cardiomyopathy after giving birth to triplets. Several years later, she had an abnor-mal echocardiogram with ventricular septal hypertrophy with a maximum septal wall thickness of 15 mm.

After reviewing her family history (Table 2), it was identi-fied that her father (II.1) died at the age of 36 years because of a cardiac arrhythmia secondary to HCM with cardiac hyper-trophy and fibrosis noted at autopsy. Her brother (III.2) died at the age of 24 years, when first responders found him in ventricular fibrillation and were unable to resuscitate him. His autopsy also revealed underlying HCM with cardiomegaly and interstitial fibrosis. An ECG performed 2 years before his death revealed QT prolongation with a QTc of 490 ms. Her deceased brother’s son (IV.1) has marked QT prolonga-tion (QTc, 522 ms) and has been treated with an implantable cardioverter defibrillator. Her living brother (III.3) has normal QTc values. Her sister’s (III.4) ECGs showed QT prolonga-tion with a QTc of 491 ms.

Overall, the family history is unique and atypical, with overt QT prolongation presenting in some cases and cardio-myopathies including HCM, with or without CHDs, in an autosomal dominant inheritance pattern. However, none of the patients have extracardiac phenotypes, such as those observed in TS. Taken altogether, the phenotypic sequela is representa-tive of an autosomal dominant condition, with features consis-tent with cardiac-only TS (COTS).

WES for Novel Pathogenic Substrate Identification in a Large Multigenerational PedigreeUsing WES, we genetically interrogated our index case (III.1), her affected sister (III.4), affected nephew (IV.1), and unaf-fected mother (II.2). Overall, 146 020 variants were identified (Figure 1B). Using Ingenuity variant analysis, we were able to filter our total number of plausible candidate variants to 29 (Figure 1B). Using genes that have been associated previously with LQTS, cardiomyopathy, SCD, and diseases consistent with these phenotypes, we identified 1 novel, ultrarare vari-ant, c.1552C>T in exon 12 of the CACNA1C-encoded Ca

V1.2

L-type calcium channel (LTCC), which leads to an arginine to cysteine change at amino acid position 518 (p.Arg518Cys) in the I–II cytoplasmic linker of Ca

V1.2 (Figure 2).

Follow-Up Cohort AnalysisOn the basis of the unique and novel phenotype identified within this pedigree, we examined our cohort of 37 unrelated

Table 1. Patients With LQTS With Personal or Family History of HCM-Like Phenotype

Patient No. Sex Age at Dx, y QTc, ms Symptomatic HCM-Like Phenotype

1* M 10 480 No Father with HCM

2 M 13 493 No SCD in 2 cousins with reported increased left ventricular wall thickness on autopsy

3* F 25 558 Yes: torsades de pointes during surgery Sister, aunt, and cousin with HCM

4 F 28 513 Yes: syncope Cardiomyopathy with septal wall motion abnormalities

5 F 46 493 Yes: cardiac arrest Postpartum cardiomyopathy

Dx indicates diagnosis; F, female; HCM, hypertrophic cardiomyopathy; LQTS, long-QT syndrome; M, male; and SCD, sudden cardiac death.*p.Arg518Cys/His mutation positive.



ownloaded from



patients with clinically robust but genetically elusive LQTS for cases with a personal or family history of phenotypes sug-gestive of HCM, and a total of 5 patients met those criteria (Table 1). Using polymerase chain reaction amplification and Sanger-based DNA sequencing, we interrogated exon 12 of CACNA1C. Interestingly, 2 of 5 (40%) had a variant in exon 12, both at the 518th amino acid, first encoding for arginine to cysteine (p.Arg518Cys), like our original pedigree, and the second encoding for arginine to histidine (p.Arg518His).

The index case (II.1) in pedigree 2 was diagnosed with LQTS with a QTc of 480 ms after a screening ECG was performed because of his father’s (I.2) diagnosis of HCM. (Table 2, pedigree 2; Figure 3, pedigree 2). The index case (III.1) in pedigree 3 was diagnosed with LQTS with a QTc of 500 ms. She was born with a cleft mitral valve and had surgical repair of an atrial septal defect at 19 months. During an unrelated hernia repair, she had intraoperative torsades des pointes for 5 to 10 beats and was implanted subsequently with an implantable cardioverter defibrillator. Her sister (III.2) was diagnosed with obstructive HCM at a young age, and she had 2 myectomies before the age of 5 years. She also

had QT prolongation which at the time was attributed to her HCM. She died suddenly at 30 years of age, and her autopsy reported symmetrical hypertrophy and fibrosis; cause of death was likely because of arrhythmia. Her mother (II.1) has an enlarged septum and QT prolongation, and she has a pace-maker. Her maternal aunt (II.2) and maternal cousin (III.3) have been diagnosed with HCM. Family member III.3 had subaortic stenosis, had a left ventricular septal myectomy, has a history of supraventricular tachycardia and first-degree AV block, and has been treated with verapamil. Her maternal grandfather (I.1) died suddenly in the hospital at the age of 64 years (Table 2, pedigree 3; Figure 3, pedigree 3). Genetic inter-rogation using polymerase chain reaction and Sanger-based DNA sequencing of exon 12 of CACNA1C in family members in all 3 pedigrees revealed that p.Arg518Cys/His cosegregated with the abnormal cardiac phenotypes (Figure 3).

In Vitro Functional Analysis of p.Arg518Cys/ His-CACNA1CTo better understand the effect that p.Arg518Cys/His has on the Ca

V1.2 channel, we used whole cell patch clamp

Table 2. Summary of Pedigrees 1, 2, and 3

Pedigree Patient Symptoms QTc, ms R518 Status

1 I.1 Died unexpectedly at the age of 67 y: heart attack NA NA

1 I.2 Died unexpectedly at the age of 66 y: heart attack NA NA

1 I.3 Died at the age of 52 y during heart surgery NA NA

1 II.1 SCD at 36 y caused by cardiac arrhythmia secondary to primary cardiomyopathy and hypertrophy

NA Obligate positive

1* II.2 Asymptomatic NA Negative

1*,† III.1 LQTS, VSD, peripartum cardiomyopathy, HCM, first-degree AV block

500 Positive

1 III.2 SCD at 24 y, prolonged QT, autopsy-revealed HCM-like phenotype of cardiomegaly and interstitial fibrosis

490 Obligate positive

1 III.3 Asymptomatic 405 Negative

1* III.4 LQTS 491 Positive

1* IV.1 LQTS: syncope 522 Positive

1 IV.2 Asymptomatic NA Negative

2 I.1 Asymptomatic NA Negative

2 I.2 HCM NA Positive

2 I.3 Died at the age of 23 y during single motor vehicle accident NA NA

2† II.1 LQTS, sinoatrial node dysfunction 480 Positive

2 II.2 Asymptomatic NA NA

3 I.1 SCD at the age of 64 y while in the hospital NA NA

3 I.2 Asymptomatic NA NA

3 II.1 Septal hypertrophy and QT prolongation NA Obligate positive

3 II.2 HCM NA Obligate positive

3† III.1 LQTS, cleft mitral valve, ASD, torsades de pointes during surgery, and premature atrial complexes

500 Positive

3 III.2 SCD at the age of 30 y, previous history of HCM and QT prolongation

NA Positive

3 III.3 HCM, subaortic stenosis, and first-degree AV block 444 Positive

ASD indicates atrial septal defect; HCM, hypertrophic cardiomyopathy; LQTS, long-QT syndrome; NA, not available; SCD, sudden cardiac death; and VSD, ventricular septal defect.

*Whole exome sequenced.†Index cases.



ownloaded from



technique to determine whether there are electrophysi-ological differences between these mutants and WT-Ca

V1.2

channels in HEK293 cells. Typical ICa

tracings of voltage-dependent activation from WT and p.Arg518Cys/His are shown in Figure 4A. Analysis of the current–voltage rela-tionship shows that p.Arg518Cys/His mutants dramatically decrease I

Ca current density from −10 to +70 mV (P<0.05 ver-

sus WT; Figure 4B). The peak current density was reduced by 55.6% and 63.2%, respectively, from −69.3±7.0 pA/pF (WT, n=15) to −30.8±5.2 pA/pF (p.Arg518Cys, n=14; P<0.0001) and to −25.5±5.8 pA/pF (p.Arg518His, n=15; P<0.0001; Figure 4C).

Because of the reduction of current density observed with the p.Arg518Cys/His variants, we used the EYFP-tag

on CACNA1C to examine the localization of mutant and WT-Ca

V1.2 proteins. Visual comparison highlights a cen-

tralized punctate pattern of fluorescence with p.Arg518Cys, which is not observed in WT cells (Figure 4D). Using the confocal microscopy images, we compared the periph-eral (membrane):central (cytosol) ratio between WT and p.Arg518Cys. We found that WT had a ratio of 0.97±0.05 (n=10), whereas p.Arg518Cys had a ratio of 0.81±0.04 (n=10; P=0.02; Figure 4E), suggesting that less mutant channels are localized to the membrane and that p.Arg518Cys may affect trafficking.

Typical tracings of steady-state inactivation for WT and p.Arg518Cys/His are shown in Figure 5A. Steady-state inac-tivation was assessed by a standard 2-pulse voltage-clamp

Figure 2. Topology of CACNA1C. Shown is the topology of CACNA1C-encoded CaV1.2 in the membrane. The location of the p.Arg518Cys/His mutations, associated with cardiac-only Timothy syndrome (COTS), as identified in the pedigrees, are highlighted in red circles. In addition, other published CACNA1C mutations in Brugada syndrome (BrS)/Brugada syndrome+short-QT syndrome (BrS+SQTS; black diamonds),9,10 long-QT syndrome (LQTS; black circles),11–15 and Timothy syndrome (TS; white circles),13,14,16–19 are high-lighted. Shown in green is the α-interaction domain (AID; amino acids 428–445), the domain in which the β-subunit is known to bind. *Variants represent those that have been functionally characterized.

Figure 3. Pedigrees harboring p.Arg518Cys/His. The original whole exome sequencing (WES) pedigree (pedigree 1), in addition to the pedigrees identified through the cohort analysis (pedigrees 2 and 3), shows the phenotypic status of each patient (key on right). In addition, each individual with a red circle is p.Arg518Cys/His positive, dotted red circles represent obligate positive p.Arg518Cys/His individuals, those who have been tested and are negative for p.Arg518Cys/His are demarcated with neg. LQTS indicates long-QT syndrome; NA, samples that were not available.



ownloaded from



protocol (Figure 5A, inset). A plot of the inactivation curves shows that p.Arg518Cys and p.Arg518His shifted V

1/2 of

inactivation to more depolarized potentials by +6.8 and +7.0 mV, respectively, from −20.9±0.4 mV (WT, n=15) to −14.1±0.5 (Arg518Cys, n=14; P<0.001) and to −13.9±0.7 mV (p.Arg518His, n=15; P<0.001; Figure 5B). The respec-tive k (slope factor) showed a significant difference between the mutants and the WT from 5.6±0.2 (WT, n=15) to 8.2±0.4 (p.Arg518Cys, n=14; P<0.001) and to 8.6±0.6 (p.Arg518His, n=15; P<0.001). A plot of the activation curves shows that the p.Arg518His mutation shifted V

1/2 of activation to a more

depolarized potential by +4.5 mV from +13.4±1.1 mV (WT, n=15) to +17.9±0.8 mV (p.Arg518His, n=15; P=0.004; Figure 5B). However, p.Arg518Cys did not significantly shift activation. In addition, the respective k (slope factor) showed no significant difference between the groups: 6.9±0.4 (WT, n=15), 7.7±0.4 (p.Arg518Cys, n=14), 8.0±0.4 (p.Arg518His, n=15). When voltage-dependent activation and steady-state inactivation are plotted together, large increases in the window current can be observed (Figure 5B).

ICa

decay after 90% of peak was best fit by 2 exponen-tials with 2 τ values representing fast and slow inactivation.

CACNA1C-WT CACNA1C-Arg518Cys

-90 mV

+70 mV500 ms

-50 mV

CACNA1C-Arg518His

50 ms50 pA

-60 -40 -20 0 20 40 60 80

-80

-60

-40

-20

0

Arg518His (n=15)

I-V Curves

WT (n=15)

Voltage (mV)

Cur

rent

Den

sity

(pA/

pF)

Arg518Cys (n=14)

*

**

**

0

-20

-40

-60

-80

Cur

rent

Den

sity

(pA/

pF)

**

WTn=15

Arg518Cysn=14

Arg518Hisn=15

Peak Current Density

DAPI DAPIEYFP-N-terminally tagged CACNA1C

EYFP-N-terminally tagged CACNA1C

MergeWT Arg518Cys Pe

riphe

ral:C

entr

alR

elat

ive

Fluo

resc

ence

1.0

0.5

0.0WT

n=10Arg518Cys

n=10

*

* * *

** ****

****

**

A

BC

D E

Figure 4. p.Arg518Cys/His reduced ICaL current density in heterologous cells. A, Whole cell ICaL current representative tracings from HEK293 cells expressing wild-type (WT) or mutant p.Arg518Cys/His determined from a holding potential of −90 mV to testing potential of +70 mV in 10-mV increments with 500-ms duration. B, Current–voltage relationship for WT (n=15), and mutant p.Arg518Cys (n=14) and p.Arg518His (n=15) missense mutations. C, Bar graph representing peak current density for WT (n=15), p.Arg518Cys (n=14), and p.Arg518His (n=15). All values represent mean±SEM. *P<0.05 vs CACNA1C-WT. D, Confocal studies examining WT and mutant expres-sion of N-terminally tagged CACNA1C with enhanced yellow fluorescence protein (EYFP). The top left corner of each image represents the DAPI nuclear stain, the top right corner represents EYFP-CACNA1C, and the bottom left corner shows the merged image. E, Bar graph representing the peripheral:central fluorescence of WT (n=10) and p.Arg518Cys (n=10) cells. Bars represent mean±SEM. *P<0.05 vs CACNA1C-WT for Student t tests; *P<0.05 after ANOVA/Kruskal–Wallis with Dunn correction for multiple comparisons.



ownloaded from



From +40 to +50, p.Arg518Cys/His revealed slower inactiva-tion τ in the fast component of decay time (corrected P<0.05; Figure 5C). From +20 to +50 mV, p.Arg518Cys/His revealed a slower inactivation τ in the slow component of the decay time (corrected P<0.05; Figure 5D).

Finally, we examined late current of ICaL

; the typical traces can be seen in Figure 5E. In addition, we normalized late cur-rent to the peak current, and compared the percent of each group, and the mutations conferred an increased late cur-rent by 7.0- and 6.6-fold, respectively, from 2.6±0.5% (WT, n=15) to 20.9±2.5% (p.Arg518Cys, n=14; P<0.001) and to 19.7±1.9% (p.Arg518His, n=15; P<0.001; Figure 5F).

In Vitro Functional Analysis of p.Arg518Cys-CACNA1C With or Without BaCl2 PerfusionI

Ca CACNA1C-WT and p.Arg518Cys reached peak at +30 mV,

whereas IBa

CACNA1C-WT and p.Arg518Cys reached peak at +10 mV. Both WT and p.Arg518Cys mutant displayed robust I

Ba currents at +10 mV when compared with I

Ca currents. The

p.Arg518Cys mutation exhibited slower inactivation kinet-ics than WT under both CaCl

2 and BaCl

2 perfusion. Typical

CaV1.2 CACNA1C-WT and p.Arg518Cys I

Ca and I

Ba tracings

of voltage-dependent activation at +10 and +30 mV are shown in Figure 6A. Voltage-dependent inactivation (VDI) was pre-sented as fraction of current remaining after 500-ms depolariza-tion normalized to peak current (r

500) across various voltages.

The extent of Ca2+-dependent inactivation was calculated as f500

=(r500/Ba

−r500/Ca

)/r500/Ba.

The p.Arg518Cys mutation signifi-cantly decelerated VDI from +10 to +30 mV under both BaCl

2

and CaCl2 perfusion (Figure 6B). However, p.Arg518Cys did

not change Ca2+-dependent inactivation of CaV1.2 channel at

+30 mV significantly with f500

=0.62±0.07 (p.Arg518Cys, n = 5) when compared with f

500=0.55±0.09 (WT, n=5).

DiscussionFamilial WES is a powerful tool for identifying the underlying genetic substrate in novel disorders. In this study, we identified a pedigree with multiple cardiac abnormalities, including LQTS,

-90 mV

+20 mV+ 30 mV

500 ms

-100 mV

10 s

CACNA1C-WTA

CACNA1C-Arg518Cys

100 ms100 pA

CACNA1C-Arg518His

F

0 100 200 300 400 600

-600

-400

-200

0

200

400

Cur

rent

Am

plitu

de a

t +30

mV

Time (ms)

WTArg518CysArg518His

Calcium Late Current Traces

500

E

**

WTn=15

Arg518Cysn=14

Arg518Hisn=15

0

5

10

15

20

25

Late

Cur

rent

/Pea

k C

urre

nt (%

)

Calcium Late Current

-60 -40 -20 0 20 40 60

0.0

0.2

0.4

0.6

0.8

1.0

WT (n=15)

I/Im

ax

Arg518Cys (n=14)

Activation-Inactivation Curves

Arg518His (n=15)

Voltage (mV)

B

0.0

0.2

0.4

0.6

0.8

1.0

G/G

max

10 20 30 40 50

20

30

40

50

60

70

80

Inac

tivat

ion

Tau

Fast

(ms)

WT (n=11)

Fast Decay Time

Arg518Cys (n=10)Arg518His (n=6)

*

Voltage (mV)

C

**

**

10 20 30 40 5075

125

175

225

275

325

375

Inac

tivat

ion

Tau

Slow

(ms)

WT (n=11)

Slow Decay Time

Voltage (mV)

Arg518Cys (n=10)Arg518His (n=6)

D

** ****

**

Figure 5. CACNA1C-Arg518Cys/His negatively shifted ICaL V1/2 inactivation. A, Representative tracings of steady-state ICaL inactivation representing wild-type (WT), p.Arg518Cys, and p.Arg518His determined from a holding potential of −90 mV to prepulse of 20 mV in 10-mV increments with 10-s duration followed by a test pulse of 30 mV with 500-ms duration. B, Inactivation curves of ICaL WT (n=15), p.Arg518Cys (n=14), and p.Arg518His (n=15). I/Imax represents normalized calcium current fitted with a Boltzmann function. Activation curves of ICaL WT (n=15), p.Arg518Cys (n=14), and p.Arg518His (n=15). G/Gmax represents normalized conductance fitted with a Boltzmann function. C, Inactivation time constants (τ) for the fast phase of ICaL decay time of WT (n=11), p.Arg518Cys (n=10), and p.Arg518His (n=6) as a function of voltage. Time constants for each voltage step were determined by fitting a biexponential function to current decay. D, Inactivation time constants (τ) for the slow phase of ICaL decay time of WT (n=11), p.Arg518Cys (n=10), and p.Arg518His (n=6) as a function of voltage. E, Representative tracings of late current representing WT, p.Arg518Cys, and p.Arg518His measured at the end of 500-ms-long depolarization. F, Normalized late current to peak current shown as percentages in a bar graph for WT, p.Arg518Cys, and p.Arg518His. All values represent mean±SEM. *P<0.05 vs CACNA1C-WT for Student t tests; *P<0.05 after ANOVA/Kruskal–Wallis with Dunn correction for multiple comparisons.



ownloaded from



HCM, CHDs, and SCD. WES of 3 affected and 1 unaffected family member in combination with Ingenuity variant analysis suggested a novel mutation p.Arg518Cys-CACNA1C as the probable pathogenic substrate for the COTS phenotype observed in this pedigree. Follow-up cohort analysis revealed 2 additional pedigrees, with similar phenotypes, having mutations at the exact same amino acid position (p.Arg518Cys and p.Arg518His).

CACNA1C encodes for the α-subunit of the CaV1.2 LTCC,

which is critical for the plateau phase of the cardiac action potential, cellular excitability, excitation–contraction cou-pling, and regulation of gene expression.20,21 Perturbations of CACNA1C have been associated with several different car-diac arrhythmia disorders, which can be differentiated into 2 groups: loss-of-function CACNA1C-mediated disease lead-ing to Brugada syndrome9 and gain-of-function CACNA1C-mediated disease leading to either TS16 or LQTS.11

Of the gain-of-function CACNA1C-mediated diseases, TS, is an extremely rare, sporadic disorder, characterized by a myriad of multisystem abnormalities, including a cardiac

phenotype of QT prolongation, HCM, CHDs, premature SCD and an extracardiac phenotype of syndactyly, facial dysmor-phisms, and neurological symptoms including autism and intellectual disability.22 Although it was speculated initially that gain-of-function mutations within CACAN1C would lead to this complex multisystem phenotype of TS, CACNA1C mutations were described recently in cases of pure con-genital LQTS devoid of other cardiac or other organ system abnormalities.11 Conversely, concomitant QT prolongation is common in patients with HCM although the exact pathophysi-ology of this electrocardiographic finding remains unclear.23 Interestingly, the pedigrees identified within this study seem to have a phenotype that lies between TS and LQTS, repre-senting a COTS phenotype of LQTS, HCM, CHDs, and SCD. Akin to patients with CACNA1C-mediated LQTS, none of the individuals within the 3 pedigrees have any of the extracardiac phenotypes associated with TS.

To confirm the disease-causing nature of the p.Arg518Cys/His mutations and to attempt to better understand the resultant

WT Ca2+

WT Ba2+

R518C Ca2+

R518C Ba2+

WT Ca2+

WT Ba2+

R518C Ca2+

R518C Ba2+

50 ms

100 pA

Current Traces at +10 mV

Current Traces at +30 mV

A

B

0

0

-60 -40 -20 0 20 40

0.0

0.2

0.4

0.6

0.8

1.0

0.0

0.2

0.4

0.6

0.8

1.0

r 500

Voltage (mV)

WT Ca2+ (n=5)WT Ba2+ (n=5)R518C Ca2+ (n=5)R518C Ba2+ (n=5)

WT Ca2+ Arg518Cys Ca2+

WT Ba2+ Arg518Cys Ba2+

WT Ba2+

WT Ca2+ Arg518Cys Ca2+

Arg518Cys Ba2+

WT Ca2+ (n=5)WT Ba2+ (n=5)Arg518Cys Ca2+ (n=5)Arg518Cys Ba2+ (n=5)

**

**

* ***

*

*

Figure 6. Deceleration of voltage-dependent inactivation by p.Arg518Cys-CACNA1C. A, Whole cell ICa and IBa current representative tracings from HEK293 cells expressing wild-type (WT) or mutant p.Arg518Cys determined from a holding potential of −90 mV to testing potential of +10 mV and +30 mV with 500-ms duration. B, Voltage-dependent inactivation of ICa and IBa currents for WT and p.Arg518Cys channels (n=5 for each group, red *P<0.05 vs WT IBa, black *P<0.05 vs WT ICa; statistics completed for each of the 10 voltages). r500 repre-sented fraction of current remaining after 500-ms depolarization normalized to peak current (r500).



ownloaded from



COTS phenotype, heterologous expression of the CaV1.2 was

used to elucidate the functional perturbation of p.Arg518Cys/His. Interestingly, unlike the previously reported LQTS-associated mutations, which produced an overall gain of cur-rent density,11,12 p.Arg518Cys/His led to an overall loss of current density by ≈59%. Confocal imaging provided evidence that there was a higher proportion of CACNA1C mutant chan-nels in the center versus the periphery of the cell, highlight-ing the possibility of a trafficking defect, which may explain the overall reduction in current density. The p.Arg518Cys/His variant lies within the I-II linker of CACNA1C, and within this linker is the α-interaction domain, in which the β-subunit binds (Figure 2). Although p.Arg518Cys/His does not fall within the α-interaction domain, the β-subunit is known to aid in α-subunit trafficking and regulation of channel kinet-ics24,25; therefore one could hypothesize that the p.Arg518Cys/His may disrupt folding of the I–II linker, altering the nor-mal interaction between the α-interaction domain and the β-subunit, leading to significant changes in trafficking and channel kinetics.

In addition, p.Arg518Cys/His also caused a gain-of-func-tion shift in the inactivation curves to more depolarized poten-tials, leading to a significant increase in the window current. Increases in window current, because of gain-of-function shifts in activation were identified recently in p.Ile1166Thr-CAC-NA1C, a mutation attributed to TS.17 Finally, p.Arg518Cys/His elicited a ≈7-fold increase in the overall late current because of slower inactivation of Ca

V1.2 mutant channels and led to decel-

erating VDI. Similarly, the original TS-mutations p.Gly406Arg and p.Gly402Ser (also in the I–II linker) lead to almost com-plete loss of inactivation of Ca

V1.2 and alterations in VDI.16,18,26

Although a mixed phenotype including loss of current den-sity in combination with an increased window current and loss of inactivation was shown, we think that the p.Arg518Cys/His mutations are capable of producing prolonged QT inter-vals. Modeling studies of the TS-associated mutations, p.Gly406Arg16 and p.Ile1166Thr,17 with the electrophysiolog-ical phenotypes including loss of inactivation and increased window current with loss of current density, respectively, have shown action potential prolongation and spontaneous delayed afterdepolarizations (p.Gly406Arg) or early afterdepolariza-tions (p.Ilell66Thr). Both delayed afterdepolarizations and early afterdepolarizations are capable of leading to ventricular fibrillation and sudden death. The modeling studies completed on p.Gly406Arg were further validated in induced pluripotent stem cell models, in which prolonged action potentials were observed.27 The similarities in the electrophysiological pheno-types of our identified variants and those previously described in TS provide evidence that the p.Arg518Cys/His mutations can cause action potential prolongation at the myocyte level and QT prolongation clinically.

However, it is less well understood why these mutations would lead to a HCM phenotype or CHDs, as observed in our pedigrees. Interestingly, TS has clinical manifestations includ-ing HCM and CHDs. In fact, as many as 61% of TS cases have some form of CHDs including HCM22; therefore, it may not be that surprising that the patients within our identified pedi-grees similarly manifest with cardiac hypertrophy and CHDs including ventricular septal defect and atrial septal defect.

The exact pathophysiology of HCM and CHDs in TS is yet to be established. However, it has been suggested previously that protein expression in pathways regulating Ca2+ within car-diac tissue may be perturbed in patients with CHDs and may result in hypertrophy.28 Those studies, in combination with what has been observed in our pedigrees and TS, suggest that proper regulation of Ca2+ and potentially the LTCC are crucial for cardiac development, and perturbation of Ca2+ handling could lead to developmental abnormalities leading to CHDs.

Abnormal calcium handling has also been described in var-ious models of cardiac hypertrophy. What has been unclear in the past is whether this Ca2+ irregularity is secondary to hyper-trophy or related to the primary pathogenesis of disease.29 Recently, Lan et al30 examined Ca2+ handling in an induced pluripotent stem cell model of HCM and found irregular Ca2+ transients and elevated diastolic [Ca2+]

i before overt pheno-

typic expression of cardiac hypertrophy as seen in HCM. This finding, in combination with their finding that LTCC blockers, such as verapamil, mitigated cellular hypertrophy, suggest that dysregulation of Ca2+ may be a central mechanism for disease development.30 The phenotypes observed within our pedigrees also support the findings of Lan et al,30 suggesting that Ca2+ handling could be a primary cause for hypertrophy. As shown in our electrophysiological studies, the p.Arg518Cys/His leads to Ca2+ mishandling, through constitutively active LTCCs, and over time, many of the patients within the pedigrees who are mutation positive acquire left ventricular hypertrophy.

CACNA1C-Mediated Clinical PhenotypesA large paradox emerges in the context of CACNA1C muta-tions in the human heart as to why some gain-of-function mutations cause a multiple organ system phenotype like TS, some an LQTS-only phenotype, and some a COTS phenotype such as the one described within this study. Initially, analyzing electrophysiological phenotypes of each of these diseases as described above, it was easy to distinguish CACNA1C-medi-ated LQTS. On the basis of our original findings, we found that the major electrophysiological phenotype of CACNA1C-associated LQTS was a marked gain in Ca

V1.2 current den-

sity,11 whereas TS16,20 and p.Arg518Cys/His altered the kinetic properties of the channel. It also seemed as if TS-associated mutations were localized to specific channel regions, at the S6/interdomain linker boundaries,17 whereas the COTS- and LQTS-associated mutations were primarily localized to intra-cellular linker loops and the N and C terminus (Figure 2). Therefore, it could have been hypothesized that the mecha-nism for pure LQTS is largely because of the location and the electrophysiological perturbation caused by the mutation.

However, since the initial discovery of CACNA1C-mediated LQTS, the spectrum and prevalence of mutations within CACNA1C have expanded greatly. Two novel case reports identified the previously TS-associated p.Gly402Ser mutation in patients without the neurological phenotypes associated with TS, rather these patients presented primarily with QT prolongation and ventricular fibrillation. In addi-tion, these case reports suggest that the neurological pheno-type observed with p.Gly402Ser may stem from the cardiac insults that had taken place early in development, rather than observed with the p.Gly402Ser mutation itself.13,14 In a similar



ownloaded from



fashion, Wemhöner et al15 found an individual with phenotypes consistent with TS with a p.Ile1166Thr mutation, matching previous reports.17 However, they found a second mutation, p.Ile1166Val, in a patient exhibiting a LQTS-only phenotype. Although the electrophysiological characteristics between p.Ile1166Thr and p.Ile1166Val were distinct, this finding, along with the novel case reports surrounding p.Gly402Ser, begins to highlight that mutation location may not be the final determinant of disease manifestation.

After the identification of more CACNA1C mutations in LQTS and TS, a more comprehensive comparison of the electrophysiological characteristics could be made. Taken altogether, there are LQTS-associated mutations that pres-ent with only increases in peak current density (p.Pro857Arg and p.Ilell66Val), there are mutations that affect only channel kinetics (p.Arg860Gly, p.Ile1475Met, and p.Glu1496Lys), and there are mutations that affect both (p.Ala28Thr, p.Ala582Asp, and p.Arg858His). In addition to our CACNA1C-mediated LQTS, we have TS mutations (p.Gly406Arg exon 8/8A, p.Gly402Ser exon 8, and p.Ile1166Thr) that affect kinetics and in some cases current density. And finally, we have the COTS-associated mutations (p.Arg518Cys/His) that affect both current density and channel kinetics. Therefore, these variants cannot easily be separated by the electrophysiologi-cal characteristics.

Collectively, it seems as if the disease phenotypes observed with mutations in the calcium channel are more complex than the location of the mutation within the channel and the underlying electrophysiological perturbations that the partic-ular mutation causes. There are several different unexplored explanations that may determine the disease pathogenesis. It may be possible that the amino acid change itself, for instance p.Ile1166Thr, causes TS, whereas p.Ile1166Val causes LQTS and confers disease phenotype. It is also possible that the bind-ing of partner proteins leading to complex signaling cascades may also be important to disease manifestation. For example, Krey et al31 found that the location of the p.Gly406Arg muta-tion may be critical for the neurological phenotypes seen in TS. The p.Gly406Arg mutation leads to more dendritic retrac-tion than WT cells regardless of the presence of pore mutations eliminating Ca2+ influx, highlighting the independence of this function from proper Ca

V1.2 electrophysiology. They identi-

fied that p.Gly406Arg mutation disrupted binding between Gem and Ca

V1.2, altering signaling cascades regulated by Gem

and RhoA, providing evidence that this interaction is essential to prevent dendritic retraction, and highlighting that binding partners and signaling cascades may be critical for disease presentation.31 Finally, it is also may be possible that there are other genetic or epigenetic factors may play a role in the dis-ease pathogenesis causing different CACNA1C mutations to lead to different phenotypic manifestations in each patient.

Only 1 conclusion can be made for certain; there definitely are regions within CACNA1C that seem to resemble genetic hotspots, which lead to cardiac disease. The S6/interdomain linker loop regions are capable of leading to severe LQTS or TS phenotypes, p.Arg518Cys/His lead to a COTS phe-notype, the II–III linker has a multitude of LQTS-associated CACNA1C mutations. Future studies will be necessary to unravel the differences between these mutations, and why,

despite their location and electrophysiological similarities, these regions are capable of producing distinct clinical pheno-types ranging the spectrum of TS, COTS, and LQTS.

ConclusionsThrough WES and expanded cohort screening, we identified a novel genetic substrate, p.Arg518Cys/His-CACNA1C, in patients with a complex phenotype including LQTS, HCM, CHDs, and SCD here referred to as COTS. Our electrophysi-ological studies identified a complex phenotype, including loss of current density in combination with increased window current and late current, and decelerating VDI. On the basis of functional studies, the identification of mutations at the same amino acid position, and cosegregation with disease in 3 different pedigrees, these 2 novel, ultrarare missense muta-tions—p.Arg518Cys and p.Arg518His—cause COTS.

Sources of FundingThis work was supported by the Windland Smith Rice Comprehensive Sudden Cardiac Death Program, the Dr. Scholl Foundation, Hannah Wernke Memorial Foundation, the Sheikh Zayed Saif Mohammed Al Nahyan Fund in Pediatric Cardiology Research (Dr Ackerman). Dr Boczek was supported by CTSA Grant (ULI TR000135) from the National Center for Advancing Translational Science, a component of the National Institutes of Health, as well as was a member of the Clinical and Translational Science program and was supported by the Mayo Clinic Graduate School.

DisclosuresMayo Clinic and Dr Ackerman received sales-based royalties from Transgenomic’s FAMILION-long-QT syndrome and FAMILION-CPVT (catecholaminergic polymorphic ventricular tachycardia) ge-netic tests

References 1. Go AS, Mozaffarian D, Roger VL, Benjamin EJ, Berry JD, Blaha MJ, Dai

S, Ford ES, Fox CS, Franco S, Fullerton HJ, Gillespie C, Hailpern SM, Heit JA, Howard VJ, Huffman MD, Judd SE, Kissela BM, Kittner SJ, Lackland DT, Lichtman JH, Lisabeth LD, Mackey RH, Magid DJ, Marcus GM, Marelli A, Matchar DB, McGuire DK, Mohler ER III, Moy CS, Mussolino ME, Neumar RW, Nichol G, Pandey DK, Paynter NP, Reeves MJ, Sorlie PD, Stein J, Towfighi A, Turan TN, Virani SS, Wong ND, Woo D, Turner MB; American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Executive summary: heart disease and stroke statistics–2014 update: a report from the American Heart Association. Circulation. 2014;129:399–410. doi: 10.1161/01.cir.0000442015.53336.12.

2. Chugh SS, Reinier K, Teodorescu C, Evanado A, Kehr E, Al Samara M, Mariani R, Gunson K, Jui J. Epidemiology of sudden cardiac death: clini-cal and research implications. Prog Cardiovasc Dis. 2008;51:213–228. doi: 10.1016/j.pcad.2008.06.003.

3. Boczek NJ, Tester DJ, Ackerman MJ. The molecular autopsy: an indispens-able step following sudden cardiac death in the young? Herzschrittmacherther Elektrophysiol. 2012;23:167–173. doi: 10.1007/s00399-012-0222-x.

4. Maron BJ. Hypertrophic cardiomyopathy: a systematic review. JAMA. 2002;287:1308–1320.

5. Maron BJ, Towbin JA, Thiene G, Antzelevitch C, Corrado D, Arnett D, Moss AJ, Seidman CE, Young JB; American Heart Association; Council on Clinical Cardiology, Heart Failure and Transplantation Committee; Quality of Care and Outcomes Research and Functional Genomics and Translational Biology Interdisciplinary Working Groups; Council on Epidemiology and Prevention. Contemporary definitions and classifica-tion of the cardiomyopathies: an American Heart Association Scientific Statement from the Council on Clinical Cardiology, Heart Failure and Transplantation Committee; Quality of Care and Outcomes Research and Functional Genomics and Translational Biology Interdisciplinary Working Groups; and Council on Epidemiology and Prevention. Circulation. 2006;113:1807–1816. doi: 10.1161/CIRCULATIONAHA.106.174287.



ownloaded from



6. Schwartz PJ, Ackerman MJ. The long QT syndrome: a transatlantic clini-cal approach to diagnosis and therapy. Eur Heart J. 2013;34:3109–3116. doi: 10.1093/eurheartj/eht089.

7. Roma-Rodrigues C, Fernandes AR. Genetics of hypertrophic cardiomy-opathy: advances and pitfalls in molecular diagnosis and therapy. Appl Clin Genet. 2014;7:195–208. doi: 10.2147/TACG.S49126.

8. Giudicessi JR, Ackerman MJ. Genotype- and phenotype-guided manage-ment of congenital long QT syndrome. Curr Probl Cardiol. 2013;38:417–455. doi: 10.1016/j.cpcardiol.2013.08.001.

9. Burashnikov E, Pfeiffer R, Barajas-Martinez H, Delpón E, Hu D, Desai M, Borggrefe M, Häissaguerre M, Kanter R, Pollevick GD, Guerchicoff A, Laiño R, Marieb M, Nademanee K, Nam GB, Robles R, Schimpf R, Stapleton DD, Viskin S, Winters S, Wolpert C, Zimmern S, Veltmann C, Antzelevitch C. Mutations in the cardiac L-type calcium channel associ-ated with inherited J-wave syndromes and sudden cardiac death. Heart Rhythm. 2010;7:1872–1882. doi: 10.1016/j.hrthm.2010.08.026.

10. Antzelevitch C, Pollevick GD, Cordeiro JM, Casis O, Sanguinetti MC, Aizawa Y, Guerchicoff A, Pfeiffer R, Oliva A, Wollnik B, Gelber P, Bonaros EP Jr, Burashnikov E, Wu Y, Sargent JD, Schickel S, Oberheiden R, Bhatia A, Hsu LF, Haïssaguerre M, Schimpf R, Borggrefe M, Wolpert C. Loss-of-function mutations in the cardiac calcium channel underlie a new clinical entity characterized by ST-segment elevation, short QT in-tervals, and sudden cardiac death. Circulation. 2007;115:442–449. doi: 10.1161/CIRCULATIONAHA.106.668392.

11. Boczek NJ, Best JM, Tester DJ, Giudicessi JR, Middha S, Evans JM, Kamp TJ, Ackerman MJ. Exome sequencing and systems biology converge to identify novel mutations in the L-type calcium channel, CACNA1C, linked to autosomal dominant long QT syndrome. Circ Cardiovasc Genet. 2013;6:279–289. doi: 10.1161/CIRCGENETICS.113.000138.

12. Fukuyama M, Wang Q, Kato K, Ohno S, Ding WG, Toyoda F, Itoh H, Kimura H, Makiyama T, Ito M, Matsuura H, Horie M. Long QT syndrome type 8: novel CACNA1C mutations causing QT prolongation and vari-ant phenotypes. Europace. 2014;16:1828–1837. doi: 10.1093/europace/euu063.

13. Hiippala A, Tallila J, Myllykangas S, Koskenvuo JW, Alastalo TP. Expanding the phenotype of Timothy syndrome type 2: an adolescent with ventricular fibrillation but normal development. Am J Med. Genet A. 2015;167A:629–634. doi: 10.1002/ajmg.a.36924.

14. Fröhler S, Kieslich M, Langnick C, Feldkamp M, Opgen-Rhein B, Berger F, Will JC, Chen W. Exome sequencing helped the fine diagnosis of two siblings afflicted with atypical Timothy syndrome (TS2). BMC Med Genet. 2014;15:48. doi: 10.1186/1471-2350-15-48.

15. Wemhöner K, Friedrich C, Stallmeyer B, Coffey AJ, Grace A, Zumhagen S, Seebohm G, Ortiz-Bonnin B, Rinné S, Sachse FB, Schulze-Bahr E, Decher N. Gain-of-function mutations in the calcium channel CACNA1C (Cav1.2) cause non-syndromic long-QT but not Timothy syndrome. J Mol Cell Cardiol. 2015;80:186–195. doi: 10.1016/j.yjmcc.2015.01.002.

16. Splawski I, Timothy KW, Sharpe LM, Decher N, Kumar P, Bloise R, Napolitano C, Schwartz PJ, Joseph RM, Condouris K, Tager-Flusberg H, Priori SG, Sanguinetti MC, Keating MT. Ca(V)1.2 calcium channel dys-function causes a multisystem disorder including arrhythmia and autism. Cell. 2004;119:19–31. doi: 10.1016/j.cell.2004.09.011.

17. Boczek NJ, Miller EM, Ye D, Nesterenko VV, Tester DJ, Antzelevitch C, Czosek RJ, Ackerman MJ, Ware SM. Novel Timothy syndrome muta-tion leading to increase in CACNA1C window current. Heart Rhythm. 2015;12:211–219. doi: 10.1016/j.hrthm.2014.09.051.

18. Splawski I, Timothy KW, Decher N, Kumar P, Sachse FB, Beggs AH, Sanguinetti MC, Keating MT. Severe arrhythmia disorder caused by cardiac L-type calcium channel mutations. Proc Natl Acad Sci U S A. 2005;102:8089–96; discussion 8086. doi: 10.1073/pnas.0502506102.

19. Gillis J, Burashnikov E, Antzelevitch C, Blaser S, Gross G, Turner L, Babul-Hirji R, Chitayat D. Long QT, syndactyly, joint contractures, stroke and novel CACNA1C mutation: expanding the spectrum of Timothy syndrome. Am J Med. Genet A. 2012;158A:182–187. doi: 10.1002/ajmg.a.34355.

20. Benitah JP, Alvarez JL, Gómez AM. L-type Ca(2+) current in ventricu-lar cardiomyocytes. J Mol Cell Cardiol. 2010;48:26–36. doi: 10.1016/j.yjmcc.2009.07.026.

21. Dai S, Hall DD, Hell JW. Supramolecular assemblies and localized regu-lation of voltage-gated ion channels. Physiol Rev. 2009;89:411–452. doi: 10.1152/physrev.00029.2007.

22. NCBI: Books: GeneReviews. Timothy Syndrome. National Center for Biotechnology Information web site. www.ncbi.nlm.nih.gov/books/NBK1403. Accessed December 15, 2014.

23. Johnson JN, Grifoni C, Bos JM, Saber-Ayad M, Ommen SR, Nistri S, Cecchi F, Olivotto I, Ackerman MJ. Prevalence and clinical correlates of QT prolongation in patients with hypertrophic cardiomyopathy. Eur Heart J. 2011;32:1114–1120. doi: 10.1093/eurheartj/ehr021.

24. Van Petegem F, Clark KA, Chatelain FC, Minor DL Jr. Structure of a com-plex between a voltage-gated calcium channel beta-subunit and an alpha-subunit domain. Nature. 2004;429:671–675. doi: 10.1038/nature02588.

25. Almagor L, Chomsky-Hecht O, Ben-Mocha A, Hendin-Barak D, Dascal N, Hirsch JA. The role of a voltage-dependent Ca2+ channel intracellular linker: a structure-function analysis. J Neurosci. 2012;32:7602–7613. doi: 10.1523/JNEUROSCI.5727-11.2012.

26. Almagor L, Chomsky-Hecht O, Ben-Mocha A, Hendin-Barak D, Dascal N, Hirsch JA. Ca(V)1.2 I-II linker structure and Timothy syndrome. Channels (Austin). 2012;6:468–472. doi: 10.4161/chan.22078.

27. Yazawa M, Hsueh B, Jia X, Pasca AM, Bernstein JA, Hallmayer J, Dolmetsch RE. Using induced pluripotent stem cells to investigate car-diac phenotypes in Timothy syndrome. Nature. 2011;471:230–234. doi: 10.1038/nature09855.

28. Wu Y, Feng W, Zhang H, Li S, Wang D, Pan X, Hu S. Ca²+-regulatory pro-teins in cardiomyocytes from the right ventricle in children with congenital heart disease. J Transl Med. 2012;10:67. doi: 10.1186/1479-5876-10-67.

29. Han L, Li Y, Tchao J, Kaplan AD, Lin B, Li Y, Mich-Basso J, Lis A, Hassan N, London B, Bett GC, Tobita K, Rasmusson RL, Yang L. Study familial hypertrophic cardiomyopathy using patient-specific induced plu-ripotent stem cells. Cardiovasc Res. 2014;104:258–269. doi: 10.1093/cvr/cvu205.

30. Lan F, Lee AS, Liang P, Sanchez-Freire V, Nguyen PK, Wang L, Han L, Yen M, Wang Y, Sun N, Abilez OJ, Hu S, Ebert AD, Navarrete EG, Simmons CS, Wheeler M, Pruitt B, Lewis R, Yamaguchi Y, Ashley EA, Bers DM, Robbins RC, Longaker MT, Wu JC. Abnormal calcium han-dling properties underlie familial hypertrophic cardiomyopathy pathol-ogy in patient-specific induced pluripotent stem cells. Cell Stem Cell. 2013;12:101–113. doi: 10.1016/j.stem.2012.10.010.

31. Krey JF, Paşca SP, Shcheglovitov A, Yazawa M, Schwemberger R, Rasmusson R, Dolmetsch RE. Timothy syndrome is associated with ac-tivity-dependent dendritic retraction in rodent and human neurons. Nat Neurosci. 2013;16:201–209. doi: 10.1038/nn.3307.



ownloaded from


Johnson, Ronald Kanter and Michael J. AckermanNicole J. Boczek, Dan Ye, Fang Jin, David J. Tester, April Huseby, J. Martijn Bos, Aaron J.

Congenital Heart Defects, and Sudden Cardiac DeathDisorder Characterized by Prolonged QT Intervals With Hypertrophic Cardiomyopathy,

-Mediated CardiacCACNA1CIdentification and Functional Characterization of a Novel

Print ISSN: 1941-3149. Online ISSN: 1941-3084 Copyright © 2015 American Heart Association, Inc. All rights reserved.

Avenue, Dallas, TX 75231is published by the American Heart Association, 7272 GreenvilleCirculation: Arrhythmia and Electrophysiology

doi: 10.1161/CIRCEP.115.0027452015;8:1122-1132; originally published online August 7, 2015;Circ Arrhythm Electrophysiol.

http://circep.ahajournals.org/content/8/5/1122World Wide Web at:

The online version of this article, along with updated information and services, is located on the

http://circep.ahajournals.org/content/suppl/2015/08/07/CIRCEP.115.002745.DC1Data Supplement (unedited) at:

http://circep.ahajournals.org//subscriptions/

is online at: Circulation: Arrhythmia and Electrophysiology Information about subscribing to Subscriptions:

http://www.lww.com/reprints Information about reprints can be found online at: Reprints:

document. Answer

Permissions and Rights Question andunder Services. Further information about this process is available in thepermission is being requested is located, click Request Permissions in the middle column of the Web pageClearance Center, not the Editorial Office. Once the online version of the published article for which

can be obtained via RightsLink, a service of the CopyrightCirculation: Arrhythmia and Electrophysiologyin Requests for permissions to reproduce figures, tables, or portions of articles originally publishedPermissions:



ownloaded from

http://circep.ahajournals.org/content/8/5/1122

http://circep.ahajournals.org/content/suppl/2015/08/07/CIRCEP.115.002745.DC1

http://www.ahajournals.org/site/rights/

http://www.ahajournals.org/site/rights/

http://www.lww.com/reprints

http://circep.ahajournals.org//subscriptions/


1

SUPPLEMENTAL MATERIAL

Supplemental Methods

Whole Exome Sequencing

Paired-end libraries were prepared following the manufacturer’s protocol (Illumina, San Diego,

CA and Agilent, Santa Clara, CA) using the Bravo liquid handler from Agilent. Whole exon

capture was carried out using the protocol for Agilent’s SureSelect Human All Exon v5 + UTRs

75 MB kit. Exome libraries were loaded onto TruSeq Rapid run paired end flow cells at

concentrations of 9 pM to generate cluster densities of 600,000-800,000/mm2 following

Illumina’s standard protocol using the Illumina cBot and TruSeq Rapid Paired end cluster kit

version 1. The flow cells were sequenced as 100 X 2 paired end reads on an Illumina HiSeq 2500

using TruSeq Rapid SBS kit version 1 and HiSeq data collection version 2.0.12.0 software. Base-

calling was performed using Illumina’s RTA version 1.17.21.3.

Bioinformatics

The Illumina paired end reads were aligned to the hg19 reference genome using Novoalign 2.08

(http://novocraft.com) followed by the sorting and marking of duplicate reads using Picard

(http://picard.sourceforge.net). Local realignment of INDELs and base quality score recalibration

were then performed using the Genome Analysis Toolkit 2.7-4 (GATK).1 Single nucleotide

variants (SNVs) and insertions/deletions (INDELs) were called across all of the samples

simultaneously using GATK's Unified Genotyper with variant quality score recalibration.2

Ingenuity Variant Analysis (Qiagen, Valencia, CA) was utilized to complete our filtering

strategy. Variants with a call quality <20, read depth <10, found in the top 1% of genes with

high variability; with a frequency of >0.01% in National Heart Lung and Blood Institute Exome

Sequencing Project (NHLBI ESP), 1000 Genomes Project, or public Complete Genomics

Genomes; and synonymous and noncoding variants with the exception of potential splice site

2

variants were excluded. We then excluded any variants present in the unaffected mother (II.2),

and kept only variants shared by the index case (III.1), affected sister (III.4), and affected

nephew (IV.1; Figure 1A). Finally, we looked for mutations in genes related to LQTS,

cardiomyopathy, SCD, and other diseases consistent with these phenotypes (Figure 1B).

HEK293 Cell Culture and Transfection

HEK293 cells were cultured in minimum essential medium supplemented with 1% nonessential

amino acid solution, 10% horse serum, 1% sodium pyruvate solution, and 1.4%

penicillin/streptomycin solution in a 5% CO2 incubator at 37oC. Heterologous expression of

Cav1.2 was accomplished by co-transfecting 1 μg CACNA1C wild type (WT) or mutant cDNA

with 1 μg CACNB2b, 1 μg CACNA2D1 and 0.25 μg Green Fluorescence Protein (GFP) cDNA

(kindly provided by Dr. Gianrico Farrugia, Mayo Clinic, Rochester, MN) with the use of 9 μl

Lipofectamine 2000 (Invitrogen, Carlsbad, CA). The media was replaced with OPTI-MEM

(Gibco, Carlsbad, CA) after 4-6 hours. Transfected HEK293 cells were cultured in OPTI-MEM

and incubated for 48 hours. Cells exhibiting yellow (CACNA1C), green (transfection control),

and red (CACNB2b) fluorescence were selected for electrophysiological experiments.

Electrophysiological Measurements and Data Analysis

Standard whole-cell patch clamp technique was used to measure CACNA1C wild type and

mutant calcium currents at room temperature (22-24oC) with the use of an Axopatch 200B

amplifier, Digidata 1440A and pclamp version 10.4 software (Axon Instruments, Sunnyvale,

CA). The extracellular (bath) solution contained (mmol/L): 130 NMDG, 5 KCl, 15 CaCl2, 1

MgCl2, 5 mM TEA-Cl and 10 HEPES, pH adjusted to 7.35 with HCl. The pipette solution

contained (mmol/L): 120 CsCl, 2 MgCl2, 10 EGTA, 2 MgATP, 5 CaCl2 and 10 HEPES, pH

adjusted to 7.25 with CsOH.3 Microelectrodes were pulled on a P-97 puller (Sutter Instruments,

3

Novato, CA) and fire polished to a final resistance of 2-3 MΏ. Series resistance was

compensated by 80-85%. Currents were filtered at 1 kHz and digitized at 5 kHz with an eight-

pole Bessel filter. The voltage dependence of activation and inactivation was determined using

voltage-clamp protocols described in the relevant figures. Data were analyzed using Clampfit

(Axon Instruments, Sunnyvale, CA), Excel (Microsoft, Redmond, WA), and fitted with Origin 8

(OriginLab Corporation, Northampton, MA) software.

The voltage-dependence of activation curve was fitted with a Boltzmann function:

GCa/GCa max={1+exp [(V-V1/2)/k]}-1, where V1/2 and k are the half-maximal voltage of activation

and the slope factor respectively. The steady-state inactivation curve was fitted with a Boltzmann

function: ICa/ICa max={1+exp [(V-V1/2)/k]}-1, where V1/2 and k (slope factor) are the half-

maximal voltage of inactivation and the slope factor respectively. ICa decay was fitted with a

two-exponential function: y= y0+{1-[Af exp(-t/τf)]+[As exp(-t/τs)]}, where Af and As represent

the amplitudes of the fast and the slow inactivating components respectively and τf and τs

represent the fast and slow time constants of inactivation respectively. Late ICa current was

measured at the end of 500 ms long depolarization of +30 mV.

In order to explore the BaCl2 effects on CaV1.2 wild type (WT) and mutant channels and

explain the mechanism by which mutants caused slower inactivation, the standard whole-cell

patch clamp technique was used to measure CaV1.2-WT or p.Arg518Cys calcium currents at

room temperature (22-24oC) under both 15 mM CaCl2 and 15 mM BaCl2 extracellular (bath)

solution in the same cells. Fraction of current remaining after 500 ms depolarization normalized

to peak current (r500) across various voltages was measured. The extent of Ca2+-dependent

inactivation (CDI) was calculated as f500= (r500/Ba-r500/Ca)/r500/Ba.4-6

4

Confocal Examination of CACNA1C

48-hours after transfection, cells grown on 10-micro well slides were examined using Olympus

Leica DM 2500 confocal microscope and images were acquired using LAS AF 6000 acquisition

software. The Tiff images (WT n=10 vs p.Arg518Cys n=10) were analyzed with Olympus

Fluoview Ver3.1a Software. After software conversion, we scaled a region of interest

measurement confined to within 1.163 μm of the membrane. The average intensity within this

region was defined as peripheral staining. The average intensity of the inside of the cells was

defined as central staining. We calculated the ratio of the peripheral to central fluorescence.7

Supplemental References

1. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA. The genome analysis toolkit: A mapreduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010;20:1297-1303. 2. DePristo MA, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C, Philippakis AA, del Angel G, Rivas MA, Hanna M, McKenna A, Fennell TJ, Kernytsky AM, Sivachenko AY, Cibulskis K, Gabriel SB, Altshuler D, Daly MJ. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet. 2011;43:491-498. 3. Cordeiro J, Marieb M, Pfeiffer R, Calloe K, Burashnikov E, Antzelevitch C. Accelerated inactivation of the l-type calcium current due to a mutation in CACNB2b underlies Brugada syndrome. J Mol Cell Cardiol. 2009;46:695-703. 4. Almagor L, Chomsky-Hecht O, Ben-Mocha A, Hendin-Barak D, Dascal N, Hirsch J. The role of a voltage-dependent Ca2+ channel intracellular linker: A structure-function analysis. J Neurosci. 2012;32:7602-7613. 5. Almagor L, Chomsky-Hecht O, Ben-Mocha A, Hendin-Barak D, Dascal N, Hirsch JA. Cav1.2 I-II linker structure and timothy syndrome. Channels. 2012;6:468-472. 6. Limpitikul WB, Dick IE, Joshi-Mukherjee R, Overgaard MT, George AL, Jr., Yue DT. Calmodulin mutations associated with long QT syndrome prevent inactivation of cardiac L-type Ca(2+) currents and promote proarrhythmic behavior in ventricular myocytes. J Mol Cell Cardiol. 2014;74:115-124. 7. Antzelevitch C, Pollevick GD, Cordeiro JM, Casis O, Sanguinetti MC, Aizawa Y, Guerchicoff A, Pfeiffer R, Oliva A, Wollnik B, Gelber P, Bonaros EP, Jr., Burashnikov E, Wu

5

Y, Sargent JD, Schickel S, Oberheiden R, Bhatia A, Hsu LF, Haissaguerre M, Schimpf R, Borggrefe M, Wolpert C. Loss-of-function mutations in the cardiac calcium channel underlie a new clinical entity characterized by ST-segment elevation, short QT intervals, and sudden cardiac death. Circulation. 2007;115:442-449.

original article - homepage | circulation: arrhythmia and...

Documents