Ackerman, Michael J.

Background: Next-Generation Sequencing (NGS) allows for sequencing of a many genes simultaneously, leading genetic testing companies to provide sequencing of all genes associated with a particular disease in large gene test panels. For example, there are over 90 distinct genes associated with premature sudden arrhythmic death. Previous publiclations have shown a background rate of rare non-synonymous single nucleotide variants (nsSNVs) in many of these genes which may be compounded when included in large gene panels. Therefore, we hoped to determine this background rate of nsSNVs in 93 genes that comprise various sudden death genetic heart disease genetic test panels. Methods: The exomes from the 1000 Genomes Project (1kG) and the NHLBI Exome Sequencing Project (ESP) were genotyped in-silico for nsSNVs in 93 genes indentified on clinically available genetic tests available for HCM, DCM, ARVC, LQTS, SQTS, BrS, and CPVT. The number of subjects hosting an exceedingly rare and unique nsSNV was determined for each gene. Results: Strikingly, 40.8% of the 1 kG cohort (12.5% hosted > 2 unique nsSNVs) and 41.9% of the ESP cohort possessed > 1 rare nsSNV within the entire 93 gene panel, while the yield of rare nsSNVs in individual genes only ranged from 0.02% for PLN to 2.3% for DMD. This background rate directly correlated with the number of genes on the panel (r=0.97). When limited to only those genes that account for >10% of the disease (HCM - MYBPC3, MYH7; LQTS - KCNQ1, KCNH2; ARVC - PKP2, DSG2; CPVT - RYR2; BrS - SCN5A), at least 7% of the subjects in both cohorts had at least one nsSNV. Thus, nearly 90% of the background rate is contained within the minor disease-susceptibility genes. Conclusions: Despite not being enriched for the presence of genetic channelopathies and cardiomyopathies, two out of every 5 subjects within these cohorts hosted at least 1 ultra-rare nsSNV within the 93 known channelopathy/cardiomyopathy-susceptibility genes. Despite the anticipated transition from disease-specific genetic testing to whole exome genetic testing, great caution must be exercised to glean the true pathogenic mutation amidst this very daunting burden of background genetic noise. If handled poorly, false positive genetic test results and misguided cascade screens will increase.

Background: Since QT prolongation is a risk factor for sudden death, stroke, and all-cause mortality, we developed an electronic, institution-wide QT alert system to identify patients with significant QT prolongation (i.e. QTc≥500 ms if QRS <120 ms). These patients encountered a significant increased mortality within 1 year, and nearly 40% of patients with ≥4 QTc inciting factors have died. Herein, we examined the patients with ≤1 identifiable QTc inciting factor. Methods: Between November 2010 and June 2011, 53,732 patients underwent ECG-evaluation, 1,145 QT-alerts (2.1%) were triggered, and 470 of the QT-alerted patients had ECG-isolated QT prolongation. Clinical, laboratory, and therapeutic information were collected. Excluding female gender and bradycardia, other potential QT inciting factors (drugs, electrolyte disturbances, and co-morbidities) were synthesized into a pro-QT score and patients with a score of ≤1 were compared to patients with a pro-QT score >1. Results: About one-third (N=150, 59% female) of this QT-alerted cohort had a pro-QT score of ≤1 including 42 patients with an established diagnosis of congenital long QT syndrome (LQTS). Excluding LQTS patients, the remaining 108 patients (60 female, age 59±25 years) were significantly more likely to have a QT prolonging cardiovascular co-morbidity (44%) than those with a QT-score >1 (29%; p=0.006). However, mortality was similar between those with a QT-score ≤1 (19%) and those with a QT-score >1 (27%, p=0.09). Fourteen patients (9 female; age 64±22 years; QTc 507±14 ms) had no discernible QT inciting factors (i.e. pro-QT score=0) including 6 patients with previously documented QT prolongation. Conclusions: Only a minority of patients with ECG-isolated QT-prolongation (~3%) had no discernible QT-inciting factors other than female gender in 9. Careful scrutiny of these patients with seemingly idiopathic QT prolongation may identify novel QT provoking conditions. Alternatively, considering that 14 of 53,732 patients (1 in 3888) activated the alert system without obvious QT inciting factors and that congenital LQTS has an estimated prevalence of 1 in 2000, it is possible that this universal QT alert system has caught patients with yet to be diagnosed LQTS.

KCNQ1 encodes the voltage-gated α-subunit of the I Ks channel complex (Kv7.1), and KCNQ1 mutations are associated with multiple arrhythmia syndromes. Over the past 12 years, hundreds of different KCNQ1 mutations have been linked to congenital long QT syndrome but only three KCNQ1 variants, each identified in only one family, are linked to familial early-onset AF (<50 years of age). The scant genetic linkage between KCNQ1 and early-onset AF raises the question as to whether these KCNQ1 variants actually cause familial early-onset AF or simply unmask a latent genetic predisposition. We now report that a single KCNQ1 variant, pArg231His or R231H, is identified in five unrelated families with a high penetrance of familial early-onset AF. Whole-cell patch clamp of cells heterologously expressing the obligatory I Ks β-subunit KCNE1 with wild-type KCNQ1 (WT) or WT and R231H (to mimic the patients’ genotypes) showed that cells co-expressing R231H increased current at negative and resting membrane potentials. Additionally, currents recorded from cells using a human atrial action potential (AP) waveform at physiological temperature showed that WT is minimally activated during the atrial AP and co-expression of R231H increases current measured during the atrial AP waveform by greater than 20-fold (WT, n = 10; WT & R231H, n = 12, p<0.0001). Simulations using a computational model of a human atrial AP also predicted that I Ks is normally minimally activated in the atria and R231H would dramatically increase I Ks and shorten the atrial AP. We conclude that KCNQ1 mutations are capable of causing familial early-onset AF by increasing I Ks during the atrial AP and shortening atrial refractoriness.

Background: Hypertrophic cardiomyopathy (HCM) is the most common cause of sudden cardiac death in young people. Recently, a patient with HCM with paroxysmal atrial fibrillation (AF) and sinoatrial block was found to have a missense mutation, E169K, in the junctophilin 2 (JPH2) protein. Loss of JPH2 has been shown to affect intracellular Ca2+ release in the heart. To date, no study has evaluated the effect of E169K mutation on Ca2+ handling and arrhythmogenesis. Hypothesis: The JPH2 E169K mutation promotes sarcoplasmic reticulum Ca2+ leak causing abnormal atrial conduction and AF. Methods and Results: We evaluated our hypothesis by generating 3 mice that overexpressed 1) JPH2 with E169K mutation (EK-Tg), 2) WT JPH2 (WT-Tg) and 3) JPH2 with inactive P584S mutation (PS-Tg). On Western Blot, JPH2 expression was increased by 2-3 fold in all the mouse groups compared to non-transgenic controls. Echocardiography at 6 months of age revealed normal fractional shortening in all mice. At baseline, EK-Tg mice had a shorter RR interval (104±1 ms, n=10) vs. WT-Tg (123±5 ms, n=8, p=0.001) and PS-Tg mice (121±5 ms, n=5, p<0.001). EK-Tg mice also had a shorter PR interval (36±1 ms, n=10) vs. WT-Tg (41±1 ms, n=8, p=0.01) and PS-Tg mice (39±1 ms, n=5, p=0.15). On intracardiac electrophysiology studies, EK-Tg mice had shorter AVERP (39±2 ms, n=10) vs. WT-Tg (51±2 ms, n=8, p=0.005) and PS-Tg mice (48±3 ms, n=5, p=0.05). On overdrive pacing of the atria in the presence of epinephrine and caffeine, EK-Tg mice had a 57% incidence of AF (n=10) vs. 21% in WT-Tg (n=8, p<0.05) and 33% in PS-Tg mice (n=5, p=0.2). Isolated cardiomyocytes from EK-Tg mice had a higher Ca2+ spark frequency (6.7±0.8 events/100 um/s, n=20) vs. cardiomyocytes from WT-Tg (1.8±0.5 events/100 um/s, n=10, p<0.001) and PS-Tg mice (3.3±1.0 events/100 um/s, n=16, p<0.05). Lastly, JPH2 levels were lower in RyR2 channels immunoprecipitated from EK-Tg mice vs. RyR2 from WT-Tg mice (65% vs. 100%, p<0.05). Conclusion: The JPH2 mutation, E169K, leads to reduced binding of JPH2 with RyR2, increased SR Ca2+ leak, abnormal atrial conduction and AF. The data suggest that JPH2 may serve as a critical stabilizing molecule for RyR2; further approaches to stabilize this interaction may lead to improved control of AF in susceptible populations.

Background. Na + /Ca 2+ exchanger (NCX) is essential in tuning intracellular Ca 2+ and the development of heart failure (HF). We reported that loss of Junctophilin 2 (JPH2) in adult mouse hearts leads to reduced NCX activity in addition to hyperactive RyR2s and more arrhythmogenic Ca 2+ waves. NCX has been found to co-localize with RyR2 recently. However, what is the physiological significance of junctional NCX and whether the reduced junctional NCX activity following JPH2 knockdown contributes to the arrhythmogenesis is still unknown. Hypothesis. Dysfunction of Junctional NCX in response to JPH2 knockdown alters spatial properties of Ca 2+ sparks and contributes to arrhythmogenesis. Methods. Tamoxifen-treated MCM and MCM-shJPH2 (KD) mice were studied. Contractile function was measured by echocardiography. Open probability of single RyR2 was evaluated by lipid bilayer. Ca 2+ spark were assessed with confocal microscopy. NCX activity was measured by exponential fitting of the declining phase of caffeine-induced Ca 2+ transient. Results. JPH2 knockdown increased spark size (MCM: 2.55 ± 0.14; KD: 4.25 ± 0.16, P < 0.001), suggesting an increased occurrence of proarrhythmic macrosparks. Cardiac Contractile function (EF% ) is negatively correlated with Ca 2+ spark size (n = 8, P < 0.05). Increased Open probability of RyR2 (MCM: 0.038 ± 0.018 KD: 0.274 ± 0.142, P < 0.01) is unlikely the reason for the ~70% increase in spark size, since it is not observed in 3 other mouse models with hyperactive RyR2s but normal JPH2 levels (RyR2:S2814D, mdx and FKBP12.6 -/- ). Loss of JPH2 also slows Ca 2+ efflux via NCX by ~50% (MCM: 2.70 ± 0.23 vs. KD: 5.11 ± 0.61 s, P < 0.01) without altering NCX expression level. Spark size and NCX activity are negatively correlated (n = 8 mice, P < 0.05), and partial inhibition (~30%) of NCX with 1 mM Cd 2+ significantly increased spark size by 14.0 ± 2.8% (n = 8, P <0.05) in wt mice. Conclusion. For the first time we showed that reduced NCX activity increases spatial properties of Ca 2+ sparks and thus arrhythmogenesis. Since reduced JPH2 is associated with HF and NCX plays more important role in human than in murine, reduced junctional NCX activity following loss of JPH2 could be a novel mechanism underlying the development of HF and associated ventricular arrhythmias.

Background: Potentially lethal syndromes such as long QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT) may account for a significant number of autopsy-negative sudden unexplained deaths (SUD). Here, the spectrum and prevalence of putative LQTS-/CPVT-causative mutations in autopsy- negative SUD was determined following a cardiac channel molecular autopsy. Methods: From September 1998 to October 2010, 173 cases of SUD (106 males, 18 ± 13 years, 89% white) were referred by Medical Examiners/Coroners for a cardiac channel molecular autopsy. Using PCR, DHPLC, and DNA sequencing, a comprehensive mutational analysis of the LQTS-susceptibility genes ( KCNQ1 , KCNH2 , SCN5A, KCNE1, and KCNE2 ) and a targeted analysis of the CPVT1-associated gene ( RYR2 ) was conducted. Results: Overall, 45 putative pathogenic mutations (20 RYR2 , 11 KCNQ1 , 6 KCNH2 , 6 SCN5A , and 2 KCNE2 , absent in 400 - 700 controls, were identified in 45 (26%) autopsy negative SUD cases. The yield was greatest for those with an exercise-induced death (16/46, 35%) or a positive personal/family history of a syncope (13/25, 52%), or sudden death (9/20, 45%). Females had a higher yield (26/67, 39%) than males (19/106, 18%, p < 0.005) especially if the SUD was during adolescence (12/25, 48% females vs. 9/50, 18% males). Females were more likely to have LQTS-associated mutations (65%) while males were more likely to have CPVT1-associated mutations (58%). Among SUD victims with exercise-induced death, the yield was much higher among the 1 - 10 year olds compared to the 11 -20 year olds (females, 71% vs 14%, p<0.05; males, 60% vs 15%, p = 0.07). However for those with death during a period of sleep, the 11 - 20 year olds had a much higher yield than the 1-10 year old group (females, 75% vs 0%, p<0.01; males 18% vs 6%). Conclusions: Over one-fourth of autopsy-negative SUD may stem from LQTS and CPVT. A cardiac channel molecular autopsy should be considered as a standard part of the evaluation of autopsy-negative SUD especially among children with exercise-induced SUD, adolescent females, and those with a positive personal/family history of cardiac events. A two-tiered approach starting with the LQTS-susceptibility genes in females and the CPVT-susceptibility genes in males may be prudent.

Introduction: Long QT Syndrome (LQTS) is a potentially lethal, but highly treatable cardiac channelopathy. Current treatment options include pharmacotherapy (mostly beta blockers), device therapy (mostly implantable cardioverter defibrillators (ICDs)), and/or left cardiac sympathetic denervation (LCSD) with the majority of patients requiring only beta blocker therapy. However, beta blockers are not without side effects and beta blocker intolerance can have a profoundly negative effect on a patient's quality of life. Heretofore, a prophylactic ICD has been utilized for the patient with LQTS who can not tolerate high dose beta blocker therapy. Here we examined the role of a prophylactic LCSD rather than an ICD for the LQTS patient with beta blocker intolerance. Methods: In this IRB-approved study, we retrospectively reviewed the electronic medical record for 58 patients (28 male (48%), mean age at diagnosis 9.6 ± 9 years (yrs), mean baseline QTc 524 ± 73 ms, 93% genotype positive) with LQTS who underwent LCSD at our institution from November 2005 through May 2011 (mean age at LCSD 14.0 ± 9.8 yrs). Results: Out of 58, 19 patients (33%; 15 female (79%) had LCSD as primary prevention because of beta blocker intolerance and were older at LQTS diagnosis (15.1 ± 8 vs 7.0 ± 9 yrs;.p=0.001), older at LCSD (20.5 ± 8 vs 10.9 ± 9 yrs; p<0.001), and had a shorter baseline QTc (493 ± 32 vs 539 ± 82 ms; p = 0.004) compared to the other 38 patients. Post-LCSD, 5/19 (26 %) had QTc-shortening of >30ms. With an average follow-up of 1.3 ± 0.8 yrs, none of these patients have experienced a post-LCSD cardiac event even though 7/19 (37%) had at least 1 LQTS-associated cardiac event. Qualitatively, there has been a dramatic improvement in quality of life following LCSD with subsequent decrease in beta blocker dose in 13 or discontinuation of beta blockers altogether in 5. Conclusions: Prophylactic LCSD may provide a beta blocker-reducing or even sparing strategy without increasing risk in carefully selected patients. However, LCSD should not be viewed as curative or an ICD-alternative for high-risk patients. Among appropriately selected patients who are failing to tolerate beta blocker therapy, LCSD may be an acceptable, quality of life improving alternative to an ICD and its associated comorbidities.

Abstract Aims Recently, deep learning artificial intelligence (AI) models have been trained to detect cardiovascular conditions, including hypertrophic cardiomyopathy (HCM), from the 12-lead electrocardiogram (ECG). In this external validation study, we sought to assess the performance of an AI-ECG algorithm for detecting HCM in diverse international cohorts. Methods and results A convolutional neural network-based AI-ECG algorithm was developed previously in a single-centre North American HCM cohort (Mayo Clinic). This algorithm was applied to the raw 12-lead ECG data of patients with HCM and non-HCM controls from three external cohorts (Bern, Switzerland; Oxford, UK; and Seoul, South Korea). The algorithm’s ability to distinguish HCM vs. non-HCM status from the ECG alone was examined. A total of 773 patients with HCM and 3867 non-HCM controls were included across three sites in the merged external validation cohort. The HCM study sample comprised 54.6% East Asian, 43.2% White, and 2.2% Black patients. Median AI-ECG probabilities of HCM were 85% for patients with HCM and 0.3% for controls (P &lt; 0.001). Overall, the AI-ECG algorithm had an area under the receiver operating characteristic curve (AUC) of 0.922 [95% confidence interval (CI) 0.910–0.934], with diagnostic accuracy 86.9%, sensitivity 82.8%, and specificity 87.7% for HCM detection. In age- and sex-matched analysis (case–control ratio 1:2), the AUC was 0.921 (95% CI 0.909–0.934) with accuracy 88.5%, sensitivity 82.8%, and specificity 90.4%. Conclusion The AI-ECG algorithm determined HCM status from the 12-lead ECG with high accuracy in diverse international cohorts, providing evidence for external validity. The value of this algorithm in improving HCM detection in clinical practice and screening settings requires prospective evaluation.

Abstract Aims In patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), a rare inherited arrhythmia syndrome, arrhythmic events can be prevented by medication and lifestyle recommendations. In patients who experience breakthrough arrhythmic events, non-adherence plays an essential role. We aimed to investigate the incidence and potential reasons for non-adherence to medication and lifestyle recommendations in a large, international cohort of patients with CPVT. Methods and results An online multilingual survey was shared with CPVT patients worldwide by their cardiologists, through peer-recruitment, and on social media from November 2022 until July 2023. Self-reported non-adherence was measured using the validated Medication Adherence Rating Scale (MARS) and a newly developed questionnaire about lifestyle. Additionally, validated questionnaires were used to assess potential reasons for medication non-adherence. Two-hundred-and-eighteen patients completed the survey, of whom 200 (92%) were prescribed medication [122 (61%) female; median age 33.5 years (interquartile range: 22–50)]. One-hundred-and-three (52%) were prescribed beta-blocker and flecainide, 85 (43%) beta-blocker, and 11 (6%) flecainide. Thirty-four (17%) patients experienced a syncope, aborted cardiac arrest or appropriate implantable cardioverter defibrillator shock after diagnosis. Nineteen (13.4%) patients were exercising more than recommended. Thirty (15%) patients were non-adherent to medication. Female sex [odds ratio (OR) 3.7, 95% confidence interval (CI) 1.3–12.0, P = 0.019], flecainide monotherapy compared to combination therapy (OR 6.8, 95% CI 1.6–31.0, P = 0.010), and a higher agreement with statements regarding concerns about CPVT medication (OR 1.2, 95% CI 1.1–1.3, P &lt; 0.001) were independently associated with non-adherence. Conclusion The significant rate of non-adherence associated with concerns regarding CPVT-related medication, emphasizes the potential for improving therapy adherence by targeted patient education.