Ackerman, Michael J.

Abstract Background Long-QT syndrome type 1 (LQT1) is a genetic channelopathy characterized by loss-of-function variants in the repolarizing potassium channel KCNQ1/IKs which lead to QT-prolongation and predisposes patients to adverse events such as cardiac arrhythmias. Clincally, during catecholamine infusion or exercise stress test, patients with LQT1 present a paradoxical QT prolongation. Purpose To investigate whether a gene-therapy approach targeting the defective K+-channel, through a KCNQ1 suppression-and-replacement (SupRep) strategy, can restore the healthy phenotype at baseline and under catecholamine infusion in LQT1 transgenic rabbits. Methods Our proprietary dual component suppression-and-replacement (SupRep) gene therapy was created by combining a custom-designed KCNQ1 shRNA and a shRNA-immune (shIMM) KCNQ1 cDNA into AAV9. In vivo KCNQ1-SupRep gene therapy was performed by targeted intra-aortic root injection during balloon occlusion. Two or three weeks post gene therapy, 12-lead ECGs were assessed in adult transgenic LQT1 (KCNQ1-Y315S, loss-of IKs) and wild-type (WT) rabbits to determine the effect of SupRep gene therapy on QT-duration at baseline and under catecholamine infusion (10nM isoproterenol, 5mL/h). Ventricular cardiomyocytes (VCMs) were isolated to investigate the effect of SupRep gene therapy on action potential duration (APD) and calcium transients (Ca2+T) at baseline and under isoproterenol infusion. Results At the cellular level, VCMs isolated from SupRep treated LQT1 rabbits showed a significant shortening of the APD90 as compared to UT-LQT1 (p<0.0001). At the whole animal level, at baseline, the QTi (%) of the UT-LQT1 transgenic rabbits (122±3) was significantly longer compared to the WT rabbits (105±2, p<0.0001). Treatment with AAV9-KCNQ1-SupRep shortened significantly the QTi of the transgenic LQT1 rabbits as compared to the UT-LQT1 rabbits (111±2, p=0.0115). To further investigate the effect of SupRep gene therapy, 12-lead ECGs were obtained in UT-LQT1, sham-treated LQT1, SupRep-treated LQT1, and WT rabbits under ISO infusion. While reaching a 20% increase in heart rate during ISO stimulation, the UT-LQT1 and the sham treated LQT1 rabbits showed a marked QT prolongation, with ΔQTi of 31.4% and 24.5%, respectively, as compared to baseline. In contrast, the SupRep-treated LQT1 rabbit behaved nearly identical to the WT rabbit during ISO infusion, showing a ΔQTi of 16.9% as compared to 16.5% in the WT rabbit. Similarly, in contrast to the UT-LQT1, VCMs isolated from SupRep-treated LQT1 rabbits demonstrated a normalization of the APD and Ca2+T under ISO stimulation, showing a significantly shortened APD90 (p<0.0001) and Ca2+T90 (p=0.0025) which was similar to what was observed in WT VCMs. Conclusion In vivo KCNQ1 suppression-and-replacement gene therapy normalizes the pathologically prolonged APD/QT interval and restores a physiological response to catecholamine infusion in treated LQT1 transgenic rabbits.

AbstractIntroductionAlthough prior studies indicate that a QTc > 500 ms on a single baseline 12‐lead electrocardiogram (ECG) is associated with significantly increased risk of arrhythmic events in long QT syndrome (LQTS), less is known about the risk of persistent QT prolongation. We sought to determine QTc persistence and its prognostic effect on breakthrough cardiac events (BCEs) among pediatric patients treated for LQTS.MethodsWe performed a retrospective analysis of 433 patients with LQTS evaluated, risk‐stratified, and undergoing active guideline‐based LQTS treatment between 1999 and 2019. BCEs were defined as arrhythmogenic syncope/seizure, sudden cardiac arrest (SCA), appropriate VF‐terminating ICD shock, and sudden cardiac death (SCD).ResultsDuring the median follow‐up of 5.5 years (interquartile range [IQR] = 3–9), 32 (7%) patients experienced a total of 129 BCEs. A maximum QTc threshold of 520 ms and median QTc threshold of 490 ms were determined to be strong predictors for BCEs. A landmark analysis controlling for age, sex, genotype, and symptomatic status demonstrated models utilizing both the median QTc and maximum QTc demonstrated the highest discriminatory value (c‐statistic = 0.93–0.95). Patients in the high‐risk group (median QTc > 490 ms and maximum QTc > 520 ms) had a significantly lower BCE free survival (70%–81%) when compared to patients in both medium‐risk (93%–97%) and low‐risk (98%–99%) groups.ConclusionsThe risk of BCE among patients treated for LQTS increases not only based upon their maximum QTc, but also their median QTc (persistence of QTc prolongation). Patients with a maximum QTc > 520 ms and median QTc > 490 ms over serial 12‐lead ECGs are at the highest risk of BCE while on guideline‐directed medical therapy.

Abstract Atrial fibrillation (AF) is a globally prevalent cardiac arrhythmia with significant genetic underpinnings, as highlighted by recent large-scale genetic studies. A prominent clinical and genetic overlap exists between AF, heritable ventricular cardiomyopathies, and arrhythmia syndromes, underlining the potential of AF as an early indicator of severe ventricular disease in younger individuals. Indeed, several recent studies have demonstrated meaningful yields of rare pathogenic variants among early-onset AF patients (∼4%–11%), most notably for cardiomyopathy genes in which rare variants are considered clinically actionable. Genetic testing thus presents a promising opportunity to identify monogenetic defects linked to AF and inherited cardiac conditions, such as cardiomyopathy, and may contribute to prognosis and management in early-onset AF patients. A first step towards recognizing this monogenic contribution was taken with the Class IIb recommendation for genetic testing in AF patients aged 45 years or younger by the 2023 American College of Cardiology/American Heart Association guidelines for AF. By identifying pathogenic genetic variants known to underlie inherited cardiomyopathies and arrhythmia syndromes, a personalized care pathway can be developed, encompassing more tailored screening, cascade testing, and potentially genotype-informed prognosis and preventive measures. However, this can only be ensured by frameworks that are developed and supported by all stakeholders. Ambiguity in test results such as variants of uncertain significance remain a major challenge and as many as ∼60% of people with early-onset AF might carry such variants. Patient education (including pretest counselling), training of genetic teams, selection of high-confidence genes, and careful reporting are strategies to mitigate this. Further challenges to implementation include financial barriers, insurability issues, workforce limitations, and the need for standardized definitions in a fast-moving field. Moreover, the prevailing genetic evidence largely rests on European descent populations, underscoring the need for diverse research cohorts and international collaboration. Embracing these challenges and the potential of genetic testing may improve AF care. However, further research—mechanistic, translational, and clinical—is urgently needed.

Introduction: Most of long QT syndrome (LQTS) is explained by pathogenic variants in KCNQ1 , KCNH2 , or SCN5A . However, ~10-20% of LQTS index cases remain genetically elusive following commercial genetic testing. Here, we identified and functionally characterized a novel LQTS genetic substrate in a multi-generational, “genotype negative” LQTS pedigree. Methods: The index case was a 40-year-old female with a history of syncope, seizures, ventricular fibrillation, and a family history of LQTS and sudden death. Although anticipated to have KCNH2- mediated LQT2, commercial genetic testing of all LQTS-causative genes was negative. Genome sequencing and variant analysis was performed on 6 affected family members. Patient-specific and CRISPR/Cas9 “gene-corrected” isogenic control induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated. RT-PCR was used to determine the effects of the identified variant on splicing. Action potential duration (APD) and field potential duration (FPD) were measured using voltage-sensing dye (Fluovolt) and microelectrode array, respectively. Results: No ultra-rare, nonsynonymous heterozygous variants co-segregated among the 6 LQTS phenotype-positive individuals. Instead, a deep intronic KCNH2 variant (c.3331-316 G>T) was present in all affected individuals. RT-PCR analysis of patient-specific iPSC-CM-derived RNA revealed that c.3331-316 G>T creates a novel 89 base-pair exon residing between canonical exons 14 and 15 of KCNH2 that results in a frame-shift variant annotated as p.S1112Pfs*171. The APD 90 was significantly longer in p.S1112Pfs*171-iPSC-CMs (647 ± 12 ms, n=50) compared to isogenic control iPSC-CMs (412 ± 10 ms, n=50, p < 0.0001). Further, the FPD was significantly longer in p.S1112Pfs*171-iPSC-CMs (349 ± 8 ms, n=48) compared to isogenic control iPSC-CMs (209 ± 10 ms, n=20, p < 0.0001). Conclusions: A novel deep intronic KCNH2 variant was identified in a multi-generational genetically elusive LQTS pedigree. The patient-derived and isogenic control iPSC-CMs establish that the p.S1112Pfs*171-KCNH2 variant is the monogenetic cause for this family’s LQTS. As suggested by the phenotype, this family indeed has KCNH2 -mediated LQT2 albeit stemming from a deep intronic variant .

Introduction: Long QT syndrome (LQTS) is an autosomal dominant disorder characterized by a prolonged QT interval on a 12-lead electrocardiogram (ECG). Cascade genetic testing for LQTS is standard of care, including in infants, but takes 2-4 weeks for return of results. For newborns of parent with confirmed LQTS, the ECG is obtained after birth as an initial assessment, but QTc values during the immediate newborn period may be misleading. Thus, our objective was to determine the ECG’s diagnostic accuracy in the first 48 hours of life for neonates born to a parent with LQTS. Methods: We performed a retrospective review of the electronic medical records of neonates born to a parent with ≥ 1 pathogenic variant in a LQTS-causative gene for which at least 1 ECG in the first 48 hours of life was obtained. Clinical data was collected on all neonates with focus on the 12-lead ECG, family history, and genetic testing results. The sensitivity and specificity of the diagnostic ECG were calculated using QTc thresholds of 440, 460, and 470 ms. Results: Overall, 70 infants (35 [50%] females) met inclusion criteria and the mean QTc on the first ECG was 484 ± 61 ms. Following genetic testing, a LQTS-causing variant was identified in 46 infants (66%), including 22 (48%) with LQT1 ( KCNQ1 ), 12 (26%) with LQT2 ( KCNH2 ), and 8 (17%) with LQT3 ( SCN5A ). Mean QTc in the first 48 hours of life for neonates that ultimately were genotype-positive was 502 ± 59 ms compared to 451 ± 50 ms (p < 0.001) for genotype-negative neonates. When using a recommended threshold QTc of ≥ 440 ms, 6/46 genotype-positive neonates (13%) were missed (underdiagnosed) and 13/24 genotype-negative neonates (54%) were overdiagnosed (sensitivity: 87%, specificity: 45%). At a QTc threshold ≥ 460 ms, 15 (21%) were classified erroneously (sensitivity: 85%, specificity: 67%) while at a threshold of ≥ 470 ms, 18 (26%) were misclassified (sensitivity: 76%, specificity: 71%). Conclusions: Although a baseline ECG should still be obtained before a newborn’s dismissal, reliance solely on the QTc for diagnosing LQTS and initiating therapy will result in an unacceptable number of misclassifications. Thus, genetic testing must be initiated prior to the baby’s discharge and proper anticipatory guidance is vital while awaiting genetic test results.

Introduction: Hypertrophic cardiomyopathy (HCM) is a major cause of sudden cardiac death in the young and especially in young athletes. The treadmill exercise test (TEST) has an important role in the functional assessment of patients with HCM. Herein, TEST-derived peak oxygen uptake (VO2 max) is a proxy for cardiopulmonary fitness. However, the correlation between symptoms and VO2 - especially in active young patients with HCM - is unclear. Objective: To explore the relationship between TEST-derived VO2 and symptomology in athletes and non-athletes with HCM. Methods: Overall, 91 patients (40% female) with HCM and a TEST with VO 2 data were included in this case-control study. Data were abstracted from the electronic health record using the first TEST performed at Mayo Clinic. Patients were defined as an athlete if they participated in an organized, competitive sport. Results: Overall, 60 HCM patients (33% female) were categorized as an athlete. HCM athletes were younger (17 vs. 27 years; p<0.0001) and less likely to be symptomatic at the time of their TEST (OR=0.24; p=0.01). Additionally, athletes had a higher VO2 max (86% vs. 68% of predicted; p<0.0001) and reached higher exercise times (93% vs 76% of predicted; p<0.0001) compared to the non-athletes with HCM. When comparing symptomatic (n = 18) to asymptomatic patients (n = 73), symptomatic patients had i) a reduced exercise time (68% vs. 92% of predicted; p=0.002) and ii) lower VO2 max (62% vs 84% of predicted; p<0.001). Finally, among the 27 patients (30%) with a VO 2 max < 70% predicted (n=27), they i) more likely had a history of cardiac arrest (4 vs 0; p=0.006) and ii) were more likely to be symptomatic (OR=7.7; p< 0.0001) than those with VO2 ≥ 70% of predicted (n=64). Importantly, however, 15/27 (56%) patients (8 athletes/7 non-athletes) with a VO2 max < 70% were asymptomatic. Conclusions: Reduced exercise capacity is common in athletes with HCM and does not correlate with symptom status. However, if the TEST-derived VO2 max is < 70%, treatment intensification may be necessary as it may portend an adverse outcome both arrhythmically and mechanically.

Introduction: Type 2 long QT syndrome (LQT2) is caused by loss-of-function variants in the KCNH2 -encoded K v 11.1 potassium channel which is essential for the rapid delayed rectifier channel (I Kr ) that governs phase 3 of cardiac action potential (AP). No current therapies target the molecular cause of LQT2. Methods: A dual-component “suppression-and-replacement” (SupRep) KCNH2 gene therapy was created by cloning into a single construct a custom-designed KCNH2 shRNA that produces ~80% knockdown (suppression) and a “shRNA-immune” (shIMM) KCNH2 cDNA modified by the introduction of synonymous single nucleotide substitutions within the shRNA target site (replacement). The ability of KCNH2-SupRep gene therapy to suppress and replace LQT2-causative variants in KCNH2 was evaluated via heterologous expression in TSA201 cells. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated from two patients with LQT2 (KCNH2-G604S and -N633S). CRISPR-Cas9 corrected isogenic control iPSC-CMs were made. Both lines were treated with KCNH2-SupRep or non-targeting control shRNA (shCT). FluoVolt voltage dye was used to measure the APD. Results: KCNH2-SupRep achieved mutation-independent suppression of wild-type KCNH2 and both LQT2-causative variants with simultaneous replacement of KCNH2-shIMM as measured by allele-specific qRT-PCR. Treatment with KCNH2-SupRep resulted in shortening of the pathologically prolonged APD at 90% repolarization (APD 90 ) to near isogenic control APD 90 levels (G604S: isogenic controls, 424 + 39 ms and N633S: isogenic controls, 433 + 16 ms) compared to treatment with non-targeting shCT (G604S: SupRep treated, 399±105 ms vs. shCT treated, 577±39 ms, p<0.0001; N633S: SupRep treated, 432±85 ms vs. shCT treated, 483±65 ms, p<0.005). Conclusions: We provide the first proof-of-principle gene therapy for correction of LQT2. Like our sentinel discovery of SupRep gene therapy for LQT1, KCNH2-SupRep successfully suppressed and replaced KCNH2 to normal wild-type levels. In TSA201 cells, co-transfection of LQT2 variants and KCNH2-SupRep caused mutation-independent suppression-and-replacement of KCNH2 . In LQT2 iPSC-CMs, KCNH2-SupRep shortened the APD, thereby eliminating the pathognomonic feature of LQT2.

Introduction: Loss-of-function (LOF) and gain-of-function (GOF) pathogenic variants in the KCNH2 -encoded Kv11.1 potassium channel cause type 2 long QT syndrome(LQT2) and type 1 short QT syndrome (SQT1), respectively. Given the prevalence of KCNH2- mediated heart disease (1 in 8000 for LQT2,1 in 100,000 for SQT1) and the reduced penetrance/variable expressivity seen in LQTS and SQTS, large databases such as the Genome Aggregation Database (gnomAD, n=141,352 individuals) might harbor subjects with unidentified LQT2/SQT1-causative mutations. Objective: To determine which “ultra-rare” KCNH2 missense variants (MVs) in gnomAD are most likely to be LQT2- or SQT1-causative. Methods: A list of gnomAD-derived MVs in KCNH2 was compiled. The frequency of each MV in gnomAD was assessed. Each MV was analyzed by 8 in silico pathogenicity prediction tools. MVs that were seen once in gnomAD and predicted damaging by all 8 tools were considered possible LQT2/SQT1-causative MVs worthy of functional validation via whole-cell patch clamp. Results: Overall, 233/474 (49%) of gnomAD KCNH2 MVs were seen in just 1 of >140,000 subjects. Of these, 2 (0.4%) MVs (S320W, S379Y) were predicted damaging by 8 in silico tools. Both S320W and S379Y localize to the N-terminus of the Kv11.1 potassium channel. There was no significant change in peak or tail current density between heterozygously expressed KCNH2-S320W (n=13, p=NS) and KCNH2-WT (n=13). However, heterozygous KCNH2-S379Y (n=12) produced a marked increase in peak current density across the range -30 mV to +20 mV and shifted the V 1/2 of activation by -17 mV (p < 0.001). Also, tail current density of S379Y channels significantly increased across the range -20 mV to +20 mV and the V 1/2 of deactivation shifted by -26 mV (p < 0.001). Conclusion: With this stringent requirement of ultra-rarity, universal prediction of damaging impact, and functionally abnormal electrophysiological properties, one SQT1-causative pathogenic variant in gnomAD was identified consistent with the predicted frequency of SQTS. The search continues among the remaining 32 ultra-rare MVs in KCNH2 where at least 75% of the in silico tools predicted deleterious impact of the MV to find the 15-20 subjects in gnomAD’s population who ought to have an LQT2-causative MV.

Introduction: The incidence and etiologies underlying sudden cardiac arrest (SCA) in the young are derived from population- and referral-based cohorts of young SCA survivors. The extent to which the use of these cohorts contributes to discrepancies in our understanding of SCA in the young is unknown. As such, we sought to determine and compare the spectrum of SCA etiologies observed in young SCA survivors evaluated at a single tertiary center on both a primary and referral basis. Methods: Retrospective review of 940 young SCA survivors evaluated between 1990 and 2017 was used to identify all individuals ≤ 50 years of age that experienced a SCA. SCA root cause was determined by review of the medical records and diagnoses classified as ischemic, primary electrical, structural, or unexplained. SCA survivors that initially presented to our institution were classified as primary, while those who presented for secondary evaluation received a referral classification. Results: Overall, 566 SCA survivors were identified (age at event 33 ± 14 years; 39% females). In comparison to primary SCA survivors, referral SCA survivors were younger at the time of SCA (39 ± 12 vs. 30 ± 14 years; p<0.0001) and less likely to experience pre-SCA symptoms (45% vs. 27%; p<0.0001). Among all SCA survivors, the most common etiologies were idiopathic ventricular fibrillation (27%), acute coronary syndrome (21%), long QT syndrome (10%), and hypertrophic (7%) and dilated (6%) cardiomyopathies. Ischemic etiologies were less common (45% vs. 25%; p<0.0001) and primary electrical disorders more common (6% vs. 23%; p<0.0001) in referral SCA survivors. Conversely, genetic heart diseases (hypertrophic cardiomyopathy, long QT syndrome, etc.) were over-represented in referral cases (8% vs. 29%; p<0.0001), whereas no difference was observed in the number of primary and referral SCAs that remained unexplained. Conclusion: This study demonstrates, for the first time, that differences in SCA root cause exist between young SCA survivors evaluated at the same tertiary medical center on a primary vs. referral basis. Ongoing comparison of SCA etiologies between primary and referral SCA survivors may help resolve discrepancies regarding the contribution of specific disorders to SCA in the young.

Introduction: Loss-of-function (LOF) variants in the KCNH2 -encoded Kv11.1 potassium channel cause type 2 long QT syndrome (LQT2). To date, hundreds of KCNH2 missense variants (MVs) have been published as “disease-causative”. However, it has been suggested that 10% of rare LQTS variants in the literature may be "false positives”. Objective: To determine previously published KCNH2 variants that are likely false positives and warrant demotion to at least likely benign status. Methods: A list of all case-derived LQT2-associated MVs from six large published compendia was compiled. The frequency of each MV within the Genome Aggregation Database (gnomAD, n = 141,352 individuals) was recorded. Eight in silico variant assessment tools were used to grade each MV. Variants (n=8) absent in gnomAD but predicted “benign” by all 8 tools were considered potential false positives, despite being ultra-rare, and were characterized functionally using whole-cell patch clamp. Results: Overall, 339 KCNH2 MVs were identified among LQTS cases. Of these, 13 (4%) were seen at a minor allele frequency (MAF) > 0.0004 in gnomAD. This MAF cut-off is derived from the frequency of R176W-KCNH2, a well-established albeit weakly penetrant (i.e. < 20%) LQT2-causative MV. Variants seen at MAF > R176W-KCNH2 are unlikely to be LQT2-contributing. However, 253 MVs (75%) were absent in gnomAD. Of these, 8 (4%) MVs (I96V, G187S, A203T, P241L, H254Q, G314S, P935S, and P963T) were predicted by all 8 in silico tools to be benign and had never been characterized functionally. Patch clamp studies did not demonstrate any LOF perturbation for these 8 MVs. Conclusion: This study offers compelling evidence for the demotion from LQT2-causative status to likely benign status for 21 (6%) of the 339 previously published LQT2 MVs based either on i) gnomAD MAF unacceptably high for a 1:8000 disorder (LQT2) for 13 MVs or ii) ultra-rarity (never seen in gnomAD) but in silico variant assessment tools predicting benign impact of the amino acid substitution and in vitro functional validation studies devoid of LOF for 8 additional MVs. This meticulous “pruning” exercise must be conducted for all published variants previously implicated as the monogenic cause for LQTS in particular and all genetic heart diseases in general.