Epistatic Effects of Potassium Channel Variation on Cardiac Repolarization and Atrial Fibrillation Risk

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provided by Elsevier - Publisher Connector Journal of the American College of Cardiology Vol. 59, No. 11, 2012 © 2012 by the American College of Cardiology Foundation ISSN 0735-1097/$36.00 Published by Elsevier Inc. doi:10.1016/j.jacc.2011.11.039

Heart Rhythm Disorders

Stefan A. Mann, PHD,* Robyn Otway, PHD,* Guanglan Guo, PHD,* Magdalena Soka, BSC(HONS),* Lina Karlsdotter, MBIOMEDSCI,* Gunjan Trivedi, BSC(HONS),* Monique Ohanian, BMEDSCI(HONS),* Poonam Zodgekar, MSW, GRADDIPGENCOUNS,* Robert A. Smith, PHD,† Merridee A. Wouters, PHD,‡ Rajesh Subbiah, MBBS, PHD,§ Bruce Walker, MBBS, PHD,§ Dennis Kuchar, MD,§ Prashanthan Sanders, MBBS, PHD,ʈ Lyn Grifﬁths, PHD,† Jamie I. Vandenberg, MBBS, PHD,*¶ Diane Fatkin, MD*§¶ Darlinghurst and Kensington, New South Wales, Gold Coast, Queensland, and Adelaide, South Australia, Australia

Objectives The aim of this study was to evaluate the role of cardiac Kϩ channel gene variants in families with atrial ﬁbrillation (AF).

Background The Kϩ channels play a major role in atrial repolarization but single mutations in cardiac Kϩ channel genes are infrequently present in AF families. The collective effect of background Kϩ channel variants of varying prevalence and effect size on the atrial substrate for AF is largely unexplored.

Methods Genes encoding the major cardiac Kϩ channels were resequenced in 80 AF probands. Nonsynonymous coding sequence variants identiﬁed in AF probands were evaluated in 240 control subjects. Novel variants were characterized using patch-clamp techniques and in silico modeling was performed using the Courtemanche atrial cell model.

Results Nineteen nonsynonymous variants in 9 genes were found, including 11 rare variants. Rare variants were more frequent in AF probands (18.8% vs. 4.2%, p Ͻ 0.001), and the mean number of variants was greater (0.21 vs. 0.04, p Ͻ 0.001). The majority of Kϩ channel variants individually had modest functional effects. Modeling simulations to evaluate combinations of Kϩ channel variants of varying population frequency indicated that simultaneous small perturbations of multiple current densities had nonlinear interactions and could result in substantial (Ͼ30 ms) shortening or lengthening of action potential duration as well as increased dispersion of repolarization.

Conclusions Families with AF show an excess of rare functional Kϩ channel gene variants of varying phenotypic effect size that may contribute to an atrial arrhythmogenic substrate. Atrial cell modeling is a useful tool to assess epistatic interactions between multiple variants. (J Am Coll Cardiol 2012;59:1017–25) © 2012 by the American College of Cardiology Foundation

Genetic variation is increasingly recognized to be a signif- had only modest individual effect size and collectively explain icant determinant of human disease. Over the past decade, relatively little of observed heritability (1). With the advent of genome-wide association studies have sought to identify common genetic variants that affect susceptibility to common See page 1026 complex disorders, but the variants identiﬁed have generally

From the *Molecular Cardiology Division, Victor Chang Cardiac Research Institute, South Wales; the Sylvia and Charles Viertel Charitable Foundation, Melbourne, Darlinghurst, New South Wales, Australia; †Genomics Research Centre, Griffith Victoria; and the St. Vincent’s Clinic Foundation, Sydney, New South Wales, Health Institute, Griffith University, Gold Coast, Queensland, Australia; ‡Structural Australia. Dr. Sanders has served on the advisory board of Bard Electrophysiology, and Computational Biology Division, Victor Chang Cardiac Research Institute, Biosense-Webster, Medtronic, St. Jude Medical, Sanofi-Aventis, and Merck; has Darlinghurst, New South Wales, Australia; §Cardiology Department, St. Vincent’s received lecture fees from Biosense-Webster, St. Jude Medical, and Merck; and has Hospital, Darlinghurst, New South Wales, Australia; ʈCentre for Heart Rhythm received research funding from Biosense-Webster, Boston-Scientific, Biotronik, Disorders, University of Adelaide and Royal Adelaide Hospital, Adelaide, South Medtronic, St. Jude Medical, and Merck. Dr. Vandenberg has received consultancy Australia, Australia; and the ¶Faculty of Medicine, University of New South Wales, fees from Lundbeck Australia Pty. Ltd. All other authors have reported they have no Kensington, New South Wales, Australia. This work was supported by the National relationships relevant to the contents of this paper to disclose. Drs. Mann, Otway, and Health and Medical Research Council of Australia, Canberra, Australian Capital Guo contributed equally to this work. Territory; the National Heart Foundation, Melbourne, Victoria; the Estate of the Manuscript received August 25, 2011; revised manuscript received October 13, Late R.T. Hall, Sydney, New South Wales; the Roth Foundation, Sydney, New 2011, accepted November 1, 2011. 1018 Mann et al. JACC Vol. 59, No. 11, 2012 K؉ Channel Variants in Atrial Fibrillation March 13, 2012:1017–25

Abbreviations new sequencing technologies, it is relatives. None of the families studied had other inherited and Acronyms now feasible and affordable to se- cardiac or systemic disorders that would account for AF. quence the entire human exome to Families in which isolated affected members had concurrent atrial fibrillation ؍ AF look for rare gene-coding sequence risk factors for AF, such as hypertension, were not excluded. action potential ؍ AP variants (1–3). Data analysis has be- All subjects provided informed written consent and were -action potential come the major challenge, with evaluated by history and physical examination, electrocardi ؍ APD duration thousands of variants detected in ography, and transthoracic echocardiography. Two Chinese hamster ؍ CHO ovary every individual person. hundred-forty healthy subjects (83 male), 16 to 91 years of Recently, exome sequencing of age (mean 53 years of age), with no history of cardiovascular deoxyribonucleic ؍ DNA acid 237 ion channels in persons with disease comprised a control group. All participants were of idiopathic epilepsy demonstrated Caucasian ethnicity. Protocols were approved by St. Vin- minor allele ؍ MAF frequency that rare deleterious variants in cent’s Hospital human research ethics committee. wild-type known Mendelian disease genes Mutation screening. Protein-coding sequences of the ؍ WT were present not only in affected KCND3, KCNIP2, KCNA5, KCNQ1, KCNH2, KCNE1, cases but also in a majority of KCNE2, KCNE3, KCNE4, KCNE5, KCNJ2, KCNJ4, and age-and race-matched healthy subjects (4). These important KCNJ14, genes were polymerase chain reaction amplified observations clearly demonstrate that single deleterious from genomic deoxyribonucleic acid (DNA) and sequenced variants cannot be assumed to be disease-causing and that using Big Dye terminator (version 3.1, Applied Biosystems, the total variant burden needs to be considered. Variants in Foster City, California) and ABI PRISM 3730 DNA the same gene, or in different genes, can be expected to show Analyzer (Applied Biosystems). Variants identified in AF epistasis, namely, have additive, neutralizing, or synergistic probands were evaluated in control subjects by sequencing actions that are nonintuitive and unpredictable based on or high-resolution melting, using SensiMix HRM (Quan- knowledge of the individual variant effects (5). Conse- tace, London, United Kingdom) or Lightcycler 480 HRM quently, development of in silico methods for modeling the Master (Roche Diagnostics, Mannheim, Germany) master- biological effects of multiple variants is critically required to mix and a Lightcycler 480 Instrument (Roche Diagnostics). derive meaningful information from genomic sequence data. Cellular electrophysiology. Chinese hamster ovary Here we have utilized atrial cell modeling in an analysis of (CHO) cells were transfected with wild-type (WT) or ϩ ϩ variants in multiple cardiac potassium (K ) channel genes in mutant K channel cDNA clones and currents were re- familial atrial fibrillation (AF). Genetic factors have an corded using conventional patch-clamp techniques (see important role in the pathogenesis of AF, but the genes Supplementary Methods). involved and mechanistic links with atrial arrhythmogenesis In silico modeling. Code for the Courtemanche atrial cell are incompletely understood. Given the fundamental im- model (11) was downloaded from the CellML repository ϩ ϩ portance of K currents in atrial repolarization, cardiac K and converted into Matlab M-code using cellular open channel genes have been considered strong candidates, and resource (COR) (12). The model was solved using the mutations in 8 genes have been associated with AF in Matlab ode15s solver with a maximal time-step of 1 ms. families or in sporadic cases (6–9). Several common variants Models were equilibrated for 10 s of simulated time at a that modify susceptibility to AF in the general population pacing rate of 1 Hz, and the duration of the last AP (APD) have also been identified, including an intronic variant in was measured from the time of the peak to the time of 90%

KCNN3, that encodes the calcium-activated small conduc- repolarization (APD90). The sensitivity of the model to ϩ ϩ tance K channel, SK3 (10). We resequenced genes encod- changes in repolarizing K currents was estimated (13). The ϩ ing all the major cardiac K currents in a cohort of persons model was solved for 1,000 consecutive runs. In each run, ϩ with familial AF and coding sequence variants identiﬁed the5K conductances (gK1, gto, gKr, gKs, and gKur) were were evaluated in healthy control subjects. Novel variants individually scaled by a random number drawn from a were characterized using patch-clamp techniques, and an log-normal distribution centered around a mean value of 1

atrial cell model was used to assess the effects of multiple (SD 22%). In each of 1,000 runs, the APD90 of the 10th AP ϩ simultaneous variations of K channel activation on atrial was determined and saved with the corresponding set of 5 action potential (AP) properties. Our data show that mul- scaling factors. After 1,000 runs, matrices containing ϩ tiple K channel variants are frequently present in families APD90 values and scaling factors were log-transformed, with AF and can contribute to an arrhythmogenic atrial centered on their mean values, and normalized to their substrate. means. These values were used as inputs for the partial least squares function PLSREGRESS in the Matlab Statistics Toolbox. The output of this function is an array of corre- Methods lation coefficients that gives the model sensitivity for each ϩ Subjects. Study subjects comprised 80 persons (56 males), K current. To determine the impact of altered repolariza- 26 to 90 years of age (mean 55 years of age) with a family tion reserve, the sensitivity analysis was repeated while ϩ history of AF, defined by AF in 2 or more first-degree increasing or reducing all K current densities in 10% JACC Vol. 59, No. 11, 2012 Mann et al. 1019 March 13, 2012:1017–25 K؉ Channel Variants in Atrial Fibrillation

increments in the range 140% to 60%. All possible combi- absent from control subjects. The G568V KCNA5 variant ϩ nations of changes in each of the 5 K currents were had a gain-of-function effect on IKur (Online Fig. 1), simulated at 90%, 100%, and 110% of their respective whereas the E444K KCNH2 variant reduced IKr (Online original values (35 ϭ 243 combinations). For each combi- Fig. 2). In genotype-positive subjects in both families, ϩ nation the model was solved 1,000 times for 10 s, and the additional functional K channel variants were present. For

mean (and SD) of APD90 were determined. Mean (and SD) other AF families, there was a maximum of 5 variants values for APD at 60% repolarization (APD60), plateau present (Fig. 1B). potential between 20 ms and 80 ms after AP onset, and Effects of multiple variants on atrial repolarization. The maximum slope during repolarization were also determined. cumulative effects of ion channel variants are difficult to Statistical analysis. The proportions of common, uncom- assess, and direct experimental evaluation of every different mon, and rare variants in AF probands and control subjects combination is impractical. Hence, to get a quantitative ϩ were compared using chi-square analysis. The statistical understanding of how multiple small changes in K cur- significance of differences in the number of alleles between rents could affect atrial repolarization, we used the Courte- these 2 groups was determined using Student’s unpaired t manche atrial cell model (11). We first determined the ϩ test. The robustness of comparisons of the number of sensitivity of APD to changes in the 5 K currents (Ito, IKur, alleles to normality assumptions required for t tests to be IKr, IKs, IK1) (Online Fig. 5). This analysis provides a valid was assessed by also comparing AF probands with distribution of APD90 (Fig. 2A), which for our simulations control subjects using the nonparametric Mann-Whitney was 302.3 Ϯ 39.3 ms. This spread is comparable to U test. All p values Ͻ0.05 were considered statistically variations observed in recordings from isolated atrial myo- significant. cytes (28) that may result from different gene expression levels and/or disruption of normally tight electrotonic interactions between cells. The 2 most important determi- Results nants of APD90 are IK1 and IKr in the Courtemanche model, ؉ K channel gene variants identified. Coding regions of with sensitivity coefficients of 0.59 and 0.46, respectively, ϩ ␣ ␤ 13 K channel genes that comprise the - and -subunits while changes in IKur have almost negligible effect (sensi- of the Ito, IKur, IKr, IKs, and IK1 currents were resequenced in tivity coefficient 0.02) (Fig. 2B). probands from 80 AF families. Nineteen nonsynonymous We extended the sensitivity analysis to investigate small ϩ variants in 9 genes were found (Table 1). Each of these 19 random variations in K currents combined with single or variants was evaluated in 240 healthy control subjects and multiple variants that each have a modest effect. Results for was classified according to the minor allele frequency variations in IKr and IKur (as seen in family IF) are shown in (MAF) in the control population; 4 variants were common Figures 2C to 2E. As predicted by the sensitivity coefficients Ͼ (MAF 10%), 4 were uncommon (MAF 1% to 10%), and in Figure 2B, changes in IKr had significant effects on mean Ͻ 11 were rare (MAF 1%). Nearly all AF probands and APD90 (Fig. 2C) but minimal effect on dispersion of the Ϯ control subjects had at least 1 common variant allele, with APD90. Changing IKur by 20% results in minimal similar numbers of variant alleles in each group (Table 2). changes in mean APD90 (Fig. 2D) but does significantly ϩ The distribution of uncommon variants was also similar in alter APD90 dispersion. In family IF, the K variants are AF probands and controls. Of the 11 rare variants, 6 were predicted to increase IKur (Online Fig. 1) and reduce IKr novel and only seen in persons with AF. Rare variants were (Table 3). We modeled this scenario by increasing IKur by present in a higher proportion of AF probands than control 20% and decreasing IKr by 20%. The resulting APD90 subjects (18.8% vs. 4.2%, p Ͻ 0.001), and the mean number histogram demonstrates that these 2 changes in current of rare variants was greater (0.21 vs. 0.04, p Ͻ 0.001). The densities have an additive effect but not in a simple linear difference in the number of alleles between groups was also manner (Fig. 2E). significant when a nonparametric method was used. We also calculated sensitivity coefficients for each of the ϩ Functional effects of single variants. Novel variants were K currents in the context of a single variant that has a characterized by whole-cell voltage clamp assays (Online modest effect. This enables predictions about a “second hit,”

Figs. 1 to 4, Online Table 1). The functional effects of novel e.g., from an ion channel-blocking drug. When levels of IK1 and previously identified variants (14–27) are summarized or IKur were systematically varied, there were significant in Table 3. Most variants had demonstrable electrophysio- changes to the sensitivity coefficients for other currents logical effects, with 9 of the 19 variants also having exper- compared to baseline (Online Figs. 6A and 6B). Conversely, imentally validated or predicted effects on protein phos- when IKr or IKs was systematically varied, the sensitivity phorylation or protein-protein interactions. coefficients for other currents were not significantly altered ؉ Multiple K channel gene variants present in AF fami- (Online Figs. 6D and 6E). lies. Sixty of the 80 AF probands (75%) had 2 or more Finally, we examined the functional consequences of ϩ variants. In families IF and HF, the novel variants G568V multiple variations in baseline K current levels. This is the KCNA5 and E444K KCNH2 showed good segregation with putative situation in families HF, HW, and BN where a disease amongst family members tested (Fig. 1A) and were number of variants were present, including some in acces- 1020 Mann et al. JACC Vol. 59, No. 11, 2012 K؉ Channel Variants in Atrial Fibrillation March 13, 2012:1017–25

؉ CodingTable Sequence1 Coding Variants Sequence Identiﬁed Variants in IdentiﬁedK Channel in GenesK؉ Channel Genes

(240 ؍ Controls (n (80 ؍ AF Probands (n Nucleotide Amino Acid Gene Change Change Genotype Allele Frequency Genotype Allele Frequency Reference KCND3 5CϾA A2E CC 79 (98.8%) C 0.99 CC 239 (99.6%) C 0.998 Novel CA 1 (1.2%) A 0.01 CA 1 (0.4%) A 0.002 AA 0 (0%) AA 0 (0%) 641AϾG K214R AA 79 (98.8%) A 0.99 AA 240 (100%) A 1.0 Novel AG 1 (1.2%) G 0.01 AG 0 (0%) G 0.0 GG 0 (0%) GG 0 (0%) KCNA5 751GϾA A251T GG 77 (96.3%) G 0.98 GG 234 (97.5%) G 0.99 rs12720442 GA 2 (2.5%) A 0.02 GA 6 (2.5%) A 0.01 AA 1 (1.2%) AA 0 (0%) 919CϾT P307S CC 78 (97.5%) C 0.99 CC 238 (99.2%) C 0.996 rs17215409 CT 2 (2.5%) T 0.01 CT 2 (0.8%) T 0.004 TT 0 (0%) TT 0 (0%) 929CϾT P310L CC 78 (97.5%) C 0.99 CC 238 (99.2%) C 0.996 rs17215402 CT 2 (2.5%) T 0.01 CT 2 (0.8%) T 0.004 TT 0 (0%) TT 0 (0%) 1703GϾT G568V GG 79 (98.8%) G 0.99 GG 240 (100%) G 1.0 Novel GT 1 (1.2%) T 0.01 GT 0 (0%) T 0.0 TT 0 (0%) TT 0 (0%) 1733GϾA R578K GG 78 (97.5%) G 0.99 GG 237 (98.8%) G 0.99 rs12720445 GA 2 (2.5%) A 0.01 GA 3 (1.2%) A 0.01 AA 0 (0%) AA 0 (0%) KCNH2 526CϾT R176W CC 79 (98.8%) C 0.99 CC 240 (100%) C 1.0 rs36210422 CT 1 (1.2%) T 0.01 CT 0 (0%) T 0.0 TT 0 (0%) TT 0 (0%) 1330GϾA E444K GG 79 (98.8%) G 0.99 GG 240 (100%) G 1.0 Novel GA 1 (1.2%) A 0.01 GA 0 (0%) A 0.0 AA 0 (0%) AA 0 (0%) 2690AϾC K897T AA 48 (60.0%) A 0.77 AA 144 (60.0%) A 0.76 rs1805123 AC 27 (33.8%) C 0.23 AC 79 (32.9%) C 0.24 CC 5 (6.2%) CC 17 (7.1%) 3140GϾT R1047L GG 77 (96.2%) G 0.98 GG 228 (95.0%) G 0.98 rs36210421 GT 3 (3.8%) T 0.02 GT 12 (5.0%) T 0.02 TT 0 (0%) TT 0 (0%) KCNQ1 40CϾT R14C CC 79 (98.8%) C 0.99 CC 240 (100%) C 1.0 Novel CT 1 (1.2%) T 0.01 CT 0 (0%) T 0.0 TT 0 (0%) TT 0 (0%) KCNJ14 1292CϾT A431V CC 78 (97.5%) C 0.99 CC 225 (93.8%) C 0.97 Novel CT 2 (2.5%) T 0.01 CT 15 (6.2%) T 0.03 TT 0 (0%) TT 0 (0%) KCNE1 112AϾG S38G AA 9 (11.2%) A 0.37 AA 23 (9.6%) A 0.33 rs17846179 AG 41 (51.3%) G 0.63 AG 111 (46.3%) G 0.67 GG 30 (37.5%) GG 106 (44.1%) 253GϾA D85N GG 77 (96.3%) G 0.98 GG 230 (95.8%) G 0.98 rs1805128 GA 3 (3.7%) A 0.02 GA 10 (4.2%) A 0.02 AA 0 (0%) AA 0 (0%) KCNE3 248GϾA R83H GG 76 (95.0%) G 0.98 GG 238 (99.2%) G 0.996 rs17215437 GA 4 (5.0%) A 0.02 GA 2 (0.8%) A 0.004 AA 0 (0%) AA 0 (0%) KCNE4 422AϾC E141A AA 79 (98.8%) A 0.99 AA 240 (100%) A 1.0 Novel AC 1 (1.2%) C 0.01 AC 0 (0%) C 0.0 CC 0 (0%) CC 0 (0%) 435GϾT E145D GG 45 (56.3%) G 0.76 GG 135 (56.3%) G 0.76 rs12621643 GT 32 (40.0%) T 0.24 GT 94 (39.2%) T 0.24 TT 3 (3.7%) TT 11 (4.6%) KCNE5* 97CϾT P33S CC 68 (85.0%) C 0.93 CC 181 (75.4%) C 0.88 rs17003955 CT 12 (15.0%) T 0.07 CT 59 (24.6%) T 0.12 TT 0 (0%) TT 0 (0%)

*KCNE5 variants are X-linked. Values shown are for males and females combined. Sex-specific allele frequencies were as follows: males, atrial fibrillation (n ϭ 56), C 0.94, T 0.06; controls (n ϭ 83), C 0.90, T 0.10; females, atrial fibrillation (n ϭ 24), C 0.90, T 0.10; controls (n ϭ 157), C 0.86, T 0.14. JACC Vol. 59, No. 11, 2012 Mann et al. 1021 March 13, 2012:1017–25 K؉ Channel Variants in Atrial Fibrillation

DistributionTable 2 ofDistribution Variants in of AF Variants Probands in AF and Probands Control Subjectsand Control Subjects

No. of Individuals No. of Alleles

Controls AF Probands Controls AF Probands †p Value (160 ؍ n) (480 ؍ p Value (n (80 ؍ n) (240 ؍ Variants* (n Common variants 235 (97.9%) 77 (96.3%) 0.35 2.5 Ϯ 1.1 2.4 Ϯ 1.2 0.19 Uncommon variants 42 (17.5%) 10 (12.5%) 0.29 0.18 Ϯ 0.40 0.15 Ϯ 0.45 0.58 Rare variants 10 (4.2%) 15 (18.8%) Ͻ0.001 0.04 Ϯ 0.20 0.21 Ϯ 0.47 Ͻ0.001

*Variant groups deﬁned by minor allele frequency in control group (see Table 1). †The p values for Student’s unpaired t test are shown; p values for the nonparametric Mann-Whitney U test are (common) 0.23, (uncommon) 0.52, (rare) 0.049. AF ϭ atrial ﬁbrillation.

␤ sory -subunits that can inﬂuence the properties of several in Ito and IKur at the less variable end that these 2 currents channels (29). We simulated all possible combinations of are the main determinants of APD90 variability, as de- ϩ changes in K channel currents at 90%, 100%, and 110% of scribed by the SD of the APD90 histograms. Notably, in the baseline (i.e., 35 ϭ 243 possible combinations). In Figure 3A, middle regions of both Figs. 3A and 3B, there is no the clustering of white and black IK1 and IKr segments apparent clustering, indicating that a large number of apparent at the “short” and “long” ends of the circular different combinations can give a “normal” value. Further- diagram, respectively, clearly shows that these current com- more, Ͼ54% of the 243 combinations give mean APD Ϯ ponents are the main determinants for APD90 in the values that are within 10 ms of the normal APD90 (302 Ͼ Courtemanche model (Fig. 2B). A 10% reduction in all ms), whereas only 4% give APD90 values 25 ms different repolarizing currents did not increase APD90 to the greatest from the normal APD90. This perturbation of APD90 is extent. Rather, a 10% reduction in IK1, IKr, and IKs and 10% comparable to that observed for single variants with very increase in IKur and Ito was the combination that gave the large effect (e.g., 50% loss of function or 100% gain of longest APD90. This superﬁcially counterintuitive result is a function) (Online Fig. 5). ϩ consequence of highly nonlinear relationships among the The effects of multiple K channel variations on other ϩ effects of variations in each of the K currents on APD90.In AP parameters, including APD60, the plateau potential, and Figure 3B, where these variations have been sorted accord- the maximum slope of repolarization, were also evaluated. It ing to their effects on dispersion (i.e., SD of the APD90 was found that Ito and IKur are the major determinants of histograms), it is apparent from clustering of black segments the plateau potential (Online Fig. 7C). Interestingly,

؉ EffectsTable of3 K EffectsGene Variants of K؉ Gene on In Variants Vitro Cellular on In Vitro Electrophysiology Cellular Electrophysiology and Post-Translational and Post-Translational Modiﬁcation Modiﬁcation

Variant Location Cellular Electrophysiology Post-Translational Modiﬁcation* Common variants (MAF Ͼ10%) ϩ ϩ KCNE1 S38G N-terminus (E) Loss-of-function IKs (16) Loss of PKA cdc2 kinase Erk D-domain sites† ϩ KCNH2 K897T C-terminus (I) Variable effects on IKr (17,18) Creates PKA Akt kinase sites (19)

KCNE4 E145D C-terminus (I) Gain-of-function IKs (20) None predicted KCNE5 P33S N-terminus (E) Unknown None predicted Uncommon variants (MAF 1%–10%) KCNJ14 A431V C-terminus (I) No change (Online Fig. 4) Loss of Erk1 kinase site

KCNH2 R1047L C-terminus (I) Variable effects on IKr (18,21) Creates Erk D-domain site

KCNE1 D85N C-terminus (I) Loss-of-function IKs (22) Loss of CKII site KCNA5 A251T S1 domain No change (23,24) None predicted Rare variants (MAF Ͻ1%)

KCNA5 R578K C-terminus (I) Loss-of-function IKur (23) None predicted

KCNA5 P307S S1-S2 linker (E) Variable effects on IKur (23,24) Loss of Nck SH3 site† KCNA5 P310L S1-S2 linker (E) No change (23) Loss of PLC␥ ϩ Nck SH3 site†

KCNE3 R83H C-terminus (I) Loss-of-function IKs, gain-of-function IKr (25) Creates PKA site KCND3 A2E N-terminus (I) No change (Online Fig. 3) None predicted KCND3 K214R S1-S2 linker (E) No change (Online Fig. 3) None predicted

KCNA5 G568V C-terminus (I) Gain-of-function IKur (Online Fig. 1) None predicted

KCNH2 R176W N-terminus (I) Loss-of-function IKr (26) Loss of PKC site

KCNH2 E444K S1-S2 linker (E) Loss-of-function IKr (Online Fig. 2) None predicted

KCNQ1 R14C N-terminus (I) Gain-of-function IKs (stretch-induced) (27) None predicted

KCNE4 E141A C-terminus (I) Possible change in IKs, IKr, Ito None predicted

*Effects of amino acid substitutions on post-translational protein modiﬁcation were predicted by Netphos (14) or Scansite (14) programs, or experimental data as indicated. †Functional signiﬁcance of predicted changes in extracellular regions uncertain. CKII ϭ casein kinase II; E ϭ extracellular; I ϭ intracellular; MAF ϭ minor allele frequency in control subjects; PKA ϭ protein kinase A; PKC ϭ protein kinase C; PLC ϭ phospholipase C. 1022 Mann et al. JACC Vol. 59, No. 11, 2012 K؉ Channel Variants in Atrial Fibrillation March 13, 2012:1017–25

A Family IF Family HF

12 I 12 I 12 II 12 + / – + / – + / – – / – II KCNH2 E444K + / – – / – 1234KCNE1 S38G + / + + / – III KCNE4 E145D + / – + / – – / – + / – – / – – / – 1234 + / – + / – + / – – / – III

1 2 3 45 6 7 89 IV

KCNA5 G568V + / – – / – + / – – / – – / – – / – KCNH2 R1047L + / + – / – + / – + / – – / – – / –

B Family HW Family BN

12 12 I I

12 3 4 1 23456 789 10 II II

KCNH2 R176W + / – + / – + / – – / – KCNH2 K897T + / – – / – KCNH2 K897T – / – + / – + / – – / – KCNE1 S38G + / – + / – KCNE1 S38G + / – + / – + / – – / – KCNE1 D85N + / – – / – KCNE3 R83H – / – + / – + / – – / – KCNE4 E145D + / – – / – KCNE5 P33S* + / 0 – / – KCNE5 P33S* – / 0 + / – + / – + / –

Figure 1 K؉ Channel Variants in AF Families

(A) Genotype-phenotype distribution of novel rare variants, G568V KCNA5 (family IF), and E444K KCNH2 (family HF). Additional Kϩ channel variants were present in both families. (B) Persons with AF in families HW and BN have multiple common and rare Kϩ variants. Phenotypes are denoted as follows: AF (dark blue symbols),noAF(open symbols), possible AF (symptomatic but AF not documented [pale blue symbols]), unknown (pink symbols). For family HW, solid symbols -denote AF (left half) or conduction defects (right half). The presence (؉) or absence (؊) of variant alleles is shown. *Gene located on X-chromosome with sin gle allele present in males.

combinations of high IKur/low Ito or low IKur/high Ito give Discussion intermediate plateau levels, but Ito mainly determines the plateau level variability (Online Fig. 7D). The maximal There has been intense recent interest in rare variants as a cause of “missing” heritability in complex disorders (1–3). slope of repolarization appears to be mostly set by IKur, Although individual rare variants might occur in relatively and to a lesser extend by IKr and IKs levels (Online Fig. 7E), with the SD of the repolarization slope showing no few cases, it has been suggested that different rare variants obvious patterns (Online Fig. 7F). might collectively account for a substantial proportion of Application of the partial least squares sensitivity analysis phenotypic traits in populations. For example, rare variants to the scenario where multiple conductances are varied in genes related to lipid metabolism are more frequent in enables estimation of the phenotypic variation one could persons with extremes of cholesterol levels (3). expect if 1 or more of the 5 variants were absent or an At least 70% of rare variants in gene coding sequences are additional hit, such as drug-block, was added. For example, estimated to have functional effects, with ϳ20% being in the case of the group of 5 variants that gave the longest strongly deleterious and ϳ50% mildly deleterious (2). In

APD90 (decreased IK1, IKr, IKs, increased Ito, IKur) the order disorders with Mendelian patterns of inheritance, single rare of sensitivity (ranked from most sensitive to least sensitive) variants of major effect size are regarded as sufﬁcient to was IK1, IKr, Ito, IKs, and IKur. cause disease. The role of less deleterious rare variants in JACC Vol. 59, No. 11, 2012 Mann et al. 1023 March 13, 2012:1017–25 K؉ Channel Variants in Atrial Fibrillation

earlier analysis of our first 50 AF probands (27), was clearly pathogenic due to its close correlation with affection status in the family, absence from a control population, and substantial functional effects. Two novel variants reported here, G568V KCNA5 and E444K KCNH2, had family segregation data that were consistent with pathogenicity (albeit limited in family HF) and were not detected in ethnically matched controls. For both variants, the in vitro electrophysiological effects appeared relatively modest, suggesting that the rare variants might not be the sole cause of AF in these families. The limitations of cellular assays need to be taken into account, however, as demonstrated recently in a mouse model of the D1275N SCN5A mutation in which there were minimal effects in vitro, but striking abnormalities of cardiac rhythm and contraction in vivo (31). In this study, we found that AF probands and controls subjects had similar prevalence of common and low frequency ϩ variants in the 13 K channel genes evaluated, but that there was an excess of rare variants in the AF probands. ϩ We hypothesized that rare K channel variants, in combination with common variants, cumulatively affect atrial repolarization properties and propensity for AF. Combinations of variants may have additive, opposing, or synergistic actions, and the net effects of multiple variants are difficult to predict or demonstrate experimen- tally. Recent advances in in silico cardiomyocyte modeling have provided a powerful tool for evaluating simultaneous perturbations of multiple ion channels. For example, the Courtemanche atrial cell model has been used previously to simulate effects of single gene mutations (27), electrolyte changes (32), and ion channel ؉ changes associated with atrial remodeling (33) on atrial Figure 2 Effects of K Current Variants on Atrial Cell APD90 AP characteristics. We used this model to systematically (A) Distribution of time of the peak action potential duration (APD) to the time analyze the consequences of all possible combinations of ϩ ϩ of 90% repolarization (APD90) values after 1,000 simulations, where all 5 K small variations in the 5 K channels, I , I , I , I , channel conductances were independently altered using a log-normal distribu- to Kur Kr Ks tion with SD of 22% (inset). The resulting mean APD is 302 ms and SD and IK1. Our modeling method enabled us to predict the 90 ϩ (pink shaded area) is 39 ms. (B) Sensitivity coefficients for influence of each nonlinear interactions between different K channel ϩ K current on APD90 values derived from partial least squares regression anal- variants at baseline and with variants of small effect. ysis of 1,000 randomized simulations. The larger the sensitivity coefficient, the more pronounced the effect current changes have on APD . Distribution of Importantly, our data indicate that uniform decreases or 90 ϩ APD90 values after 1,000 simulations where (C) IKr or (D) IKur was changed to increases in K current densities do not produce the most 80% (red squares), 100% (black circles), or 120% (blue triangles) of base- dramatic effects on repolarization. Moreover, small vari- line, and then all currents allowed to vary randomly over the same range as in ϩ

A. Systematic modest changes in IKr level have significant effects on mean ations in multiple K conductances can produce signifi- APD90 as predicted by the large sensitivity coefficient for IKr, shown in B, cant prolongation or shortening of atrial repolarization, whereas systematic modest changes in IKur have minimal effect on mean increased dispersion of repolarization, or changes in the APD90 but a pronounced effect on the dispersion of APD90. (E) A combination of a 20% increase in IKur and 20% decrease in IKr (blue squares) increased AP shape that could facilitate AF. Ϯ APD90 to 322 44 ms. Black circles indicate 100%. Study limitation. A limitation of this study is that only ϩ cardiac K channel variants were evaluated. Although we ϩ modeled 243 possible combinations of K current human diseases has been very little investigated. With the changes, the specific combinations of greatest clinical exponential increase in exome sequencing for human genet- relevance remain to be determined. ics studies, investigators are being confronted increasingly by this problem. ϩ Conclusions A surprisingly low prevalence of single K channel gene mutations has been found in cohorts of AF families As clearly demonstrated in exome sequencing data, func- (27,30). Only 1 variant, R14C KCNQ1, described in an tionally deleterious variants in numerous sodium and cal- 1024 Mann et al. JACC Vol. 59, No. 11, 2012 K؉ Channel Variants in Atrial Fibrillation March 13, 2012:1017–25

؉ Figure 3 Interactions Between Multiple K Channel Variants Have Nonlinear Effects on Atrial Cell APD90

The action potential (AP) simulations were performed starting with all possible 243 combinations of changes in the 5 Kϩ currents to 90% (black), 100% (gray), and 110% (white) of baseline (inner concentric circles), then all currents were varied randomly with the same distribution as in Figure 2A. Results are sorted

according to (A) mean time of the peak action potential duration (APD) to the time of 90% repolarization (APD90) (inner colored band) and (B) SD of APD90 values (outer colored band). The positions where all currents are 90%, 100%, or 110% are highlighted. The APs are shown for the 2 extremes of the wheel. The

dashed line indicates the level of 90% repolarization. In A, clustering of black sections in the inner 2 wheel segments, corresponding to IK1 and IKr, toward long APD90 indicates the pronounced effects that these currents have on mean APD90. Other current densities appear to be randomly distributed. In B, there is apparent clustering of segments in the 2 outer segments representing Ito and IKur. Apart from clustering toward the extreme ends, the segments appear to be distributed randomly. cium ion channels as well as other cardiomyocyte compo- cus, David Ross, Jonathan Silberberg, Suresh Singarayar, nents are present in every person, and it is the net effect of Charles Thorburn, John Uther, Paul West, and Thomas all these variants together with acquired “environmental” Yeoh. factors that contribute to the electrophysiological properties of the atrial wall. Interpretation of human genome sequence Reprint requests and correspondence: Dr. Diane Fatkin, Victor data requires global perspectives that look beyond the single Chang Cardiac Research Institute, Lowy Packer Building, 405 variant. Accordingly, integration of comprehensive genetic Liverpool Street, P.O. Box 699, Darlinghurst, NSW 2010, Aus- screening, cellular electrophysiological data, atrial cell mod- tralia. E-mail: [email protected]. eling, and systems biology approaches will be instrumental in achieving these goals. Further delineation of genetic signatures for AF is an important step toward personalized REFERENCES approaches to risk stratification and therapeutic decision making. 1. Manolio TA, Collins FS, Cox NJ, et al. Finding the missing heritability of complex diseases. Nature 2009;461:747–53. 2. Kryukov GV, Pennacchio LA, Sunyaev SR. Most rare missense alleles Acknowledgments are deleterious in humans: implications for complex disease and The authors thank Olivia Baddeley, Haley Crotty, Jessica association studies. Am J Hum Genet 2007;80:727–39. Hansen, Louise Lynagh, Zara Richmond, Nan Yang, and 3. Cohen JC, Kiss RS, Pertsemlidis A, Marcel YL, McPherson R, Hobbs HH. Multiple rare alleles contribute to low plasma levels of HDL Kathryn North for assistance with clinical data and blood cholesterol. Science 2004;305:869–72. sample collection; Matthew Law for statistical advice; 4. Klassen T, Davis C, Goldman A, et al. Exome sequencing of ion and the following physicians for referral of study pro- channel genes reveals complex profiles confounding personal risk assessment in epilepsy. Cell 2011:145:1036–48. bands: Ruth Arnold, Tim Carruthers, Suchitra Chandar, 5. Moore JH. The ubiquitous nature of epistasis in determining suscep- Richard Cranswick, Michael Feneley, Peter French, Da- tibility to common human diseases. Hum Hered 2003;56:73–82. vid Gray, Dean Guy, Deborah Hayes, Peter Hayes, 6. Fatkin D, Otway R, Vandenberg JI. Genes and atrial fibrillation: a new Christopher Hayward, William Heddle, Pramesh Ko- look at an old problem. Circulation 2007;116:782–92. 7. Lundby A, Ravn LS, Svendsen JH, Hauns S, Olesen SP, Schmitt N. voor, Anne Keogh, Jo-Dee Lattimore, Jim Leitch, Alex KCNE3 mutation V17M identified in a patient with lone atrial Levendel, Harry Lowe, Peter Macdonald, Matthew Pin- fibrillation. Cell Physiol Biochem 2008;21:47–54. JACC Vol. 59, No. 11, 2012 Mann et al. 1025 March 13, 2012:1017–25 K؉ Channel Variants in Atrial Fibrillation

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