Tester, David J.

Background: Long-QT syndrome (LQTS) is characterized by QT prolongation and increased risk for syncope, seizures, and sudden cardiac death. The majority of LQTS stems from pathogenic mutations in KCNQ1 , KCNH2 , or SCN5A . However, ≈10% of patients with LQTS remain genetically elusive. We utilized genome sequencing to identify a novel LQTS genetic substrate in a multigenerational genotype-negative LQTS pedigree. Methods: Genome sequencing was performed on 5 affected family members. Only rare nonsynonymous variants present in all affected family members were considered. The candidate variant was characterized functionally in patient-derived induced pluripotent stem cell and gene-edited, variant corrected, isogenic control induced pluripotent stem cell–derived cardiomyocytes. Results: A missense variant (p.G6S) was identified in ALG10B -encoded α-1,2-glucosyltransferase B protein. ALG10B (alpha-1,2-glucosyltransferase B protein) is a known interacting protein of KCNH2 -encoded K v 11.1 (HERG [human Ether-à-go-go–related gene]). Compared with isogenic control, ALG10B-p.G6S induced pluripotent stem cell–derived cardiomyocytes showed (1) decreased protein expression of ALG10B (p.G6S, 0.7±0.18, n=8 versus control, 1.25±0.16, n=9; P <0.05), (2) significant retention of HERG in the endoplasmic reticulum ( P <0.0005), and (3) a significantly prolonged action potential duration confirmed by both patch clamp (p.G6S, 531.1±38.3 ms, n=15 versus control, 324.1±21.8 ms, n=13; P <0.001) and multielectrode assay ( P <0.0001). Lumacaftor—a compound known to rescue HERG trafficking—shortened the pathologically prolonged action potential duration of ALG10B-p.G6S induced pluripotent stem cell–derived cardiomyocytes by 10.6% (n=31 electrodes; P <0.001). Conclusions: Here, we demonstrate that ALG10B-p.G6S downregulates ALG10B, resulting in defective HERG trafficking and action potential duration prolongation. Therefore, ALG10B is a novel LQTS-susceptibility gene underlying the LQTS phenotype observed in a multigenerational pedigree. ALG10B mutation analysis may be warranted, especially in genotype-negative patients with an LQT2-like phenotype.

Background: Hypertrophic cardiomyopathy (HCM) is characterized by asymmetric left ventricular hypertrophy. Currently, hypertrophy pathways responsible for HCM have not been fully elucidated. Their identification could serve as a nidus for the generation of novel therapeutics aimed at halting disease development or progression. Herein, we performed a comprehensive multi-omic characterization of hypertrophy pathways in HCM. Methods: Flash-frozen cardiac tissues were collected from genotyped HCM patients (n=97) undergoing surgical myectomy and tissue from 23 controls. RNA sequencing and mass spectrometry–enabled deep proteome and phosphoproteomic assessment were performed. Rigorous differential expression, gene set enrichment, and pathway analyses were performed to characterize HCM-mediated alterations with emphasis on hypertrophy pathways. Results: We identified transcriptional dysregulation with 1246 (8%) differentially expressed genes and elucidated downregulation of 10 hypertrophy pathways. Deep proteomic analysis identified 411 proteins (9%) that differed between HCM and controls with strong dysregulation of metabolic pathways. Seven hypertrophy pathways were upregulated with antagonistic upregulation of 5 of 10 hypertrophy pathways shown to be downregulated in the transcriptome. Most upregulated hypertrophy pathways encompassed the rat sarcoma-mitogen-activated protein kinase signaling cascade. Phosphoproteomic analysis demonstrated hyperphosphorylation of the rat sarcoma-mitogen-activated protein kinase system suggesting activation of this signaling cascade. There was a common transcriptomic and proteomic profile regardless of genotype. Conclusions: At time of surgical myectomy, the ventricular proteome, independent of genotype, reveals widespread upregulation and activation of hypertrophy pathways, mainly involving the rat sarcoma-mitogen-activated protein kinase signaling cascade. In addition, there is a counterregulatory transcriptional downregulation of the same pathways. Rat sarcoma-mitogen-activated protein kinase activation may serve a crucial role in hypertrophy observed in HCM.

Background: KCNH2 -mediated arrhythmia syndromes are caused by loss-of-function (type 2 long QT syndrome [LQT2]) or gain-of-function (type 1 short QT syndrome [SQT1]) pathogenic variants in the KCNH2 -encoded K v 11.1 potassium channel, which is essential for the cardiac action potential. Methods: A dual-component “suppression-and-replacement” (SupRep) KCNH2 gene therapy was created by cloning into a single construct a custom-designed KCNH2 short hairpin RNA with ~80% knockdown (suppression) and a “short hairpin RNA-immune” KCNH2 cDNA (replacement). Induced pluripotent stem cell-derived cardiomyocytes and their CRISPR-Cas9 variant-corrected isogenic control (IC) induced pluripotent stem cell-derived cardiomyocytes were made for 2 LQT2- (G604S, N633S) and 1 SQT1- (N588K) causative variants. All variant lines were treated with KCNH2-SupRep or non-targeting control short hairpin RNA (shCT). The action potential duration (APD) at 90% repolarization (APD 90 ) was measured using FluoVolt voltage dye. Results: KCNH2-SupRep achieved variant-independent rescue of both pathologic phenotypes. For LQT2-causative variants, treatment with KCNH2-SupRep resulted in shortening of the pathologically prolonged APD 90 to near curative (IC-like) APD 90 levels (G604S IC, 471±25 ms; N633S IC, 405±55 ms) compared with treatment with shCT (G604S: SupRep-treated, 452±76 ms versus shCT-treated, 550±41 ms; P <0.0001; N633S: SupRep-treated, 399±105 ms versus shCT-treated, 577±39 ms, P <0.0001). Conversely, for the SQT1-causative variant, N588K, treatment with KCNH2-SupRep resulted in therapeutic prolongation of the pathologically shortened APD 90 (IC: 429±16 ms; SupRep-treated: 396±61 ms; shCT-treated: 274±12 ms). Conclusions: We provide the first proof-of-principle gene therapy for correction of both LQT2 and SQT1. KCNH2-SupRep gene therapy successfully normalized the pathologic APD 90 , thereby eliminating the pathognomonic feature of both LQT2 and SQT1.

Background: The transient outward current (Ito) that mediates early (phase 1) repolarization is conducted by the KCND3 -encoded Kv4.3 pore-forming α-subunit. KCND3 gain-of-function mutations have been reported previously as a pathogenic substrate for J wave syndromes (JWS), including the Brugada syndrome and early repolarization syndrome, as well as autopsy-negative sudden unexplained death (SUD). Acacetin, a natural flavone, is a potent Ito current blocker. Acacetin may be a novel therapeutic for KCND3 -mediated J wave syndrome. Methods: KCND3-V392I was identified in an 18-year-old male with J wave syndrome/early repolarization syndrome, and a history of cardiac arrest including ventricular tachycardia/ventricular fibrillation and atrial fibrillation/atrial flutter. Pathogenic KCND3 mutation was engineered by site-directed mutagenesis and co-expressed with wild-type KChIP2 in TSA201 cells. Gene-edited/variant-corrected isogenic control and patient-specific pluripotent stem cell–derived cardiomyocytes (iPSC-CMs) from the p. Val392Ile-KCND3-positive patient were generated. I to currents and action potentials were recorded before and after treatment with Acacetin using the whole cell patch-clamp and multielectrode array technique. Western blot and immunocytochemistry were performed to investigate KCND3 expression. Results: KCND3-V392I demonstrated a marked gain-of-function phenotype, increasing peak I to current density by 92.2% ( P <0.05 versus KCND3-WT). KCND3 expression was significantly increased in KCND3-V392I-derived iPSC-CMs ( P <0.05 versus isogenic control). While KCND3-WT revealed an IC50 of 7.2±1.0 µmol/L for acacetin effect, 30 µmol/L acacetin dramatically inhibited KCND3-V392I peak Ito current density by 96.2% ( P <0.05 versus before Acacetin). Ito was also increased by 60.9% in Kv4.3-V392I iPSC-CM ( P <0.05 versus isogenic control iPSC-CM). Ten micromoles per liter acacetin, a concentration approaching its IC50 value, inhibited Ito by ≈50% in patient-derived iPSC-CMs and reduced the accentuated action potential notch displayed in KCND3-V392I-derived iPSC-CMs. Conclusions: This preclinical study provides pharmacological and functional evidence to suggest that Acacetin may be a novel therapeutic for patients with KCND3 gain-of-function-associated J wave syndrome by inhibiting I to and abolishing the accentuated action potential notch in patient-derived iPSC-CMs.

Introduction: Hypertrophic cardiomyopathy (HCM) is one of the most common genetic heart diseases characterized by asymmetric left ventricular hypertrophy (LVH) and vast clinical and genetic heterogeneity. Several transcriptional studies in HCM heart tissues have shown severe alterations in gene expression. However, these earlier studies relied on microarray technology or were limited by small sample size or lack of genotype-specific analyses. Methods: We obtained flash-frozen cardiac tissue from patients with HCM undergoing surgical myectomy/septal reduction therapy for relief of left ventricular outflow tract obstruction. RNA-sequencing (RNAseq) was performed using paired-end index reading on 97 myectomy tissues (44 female [45%]; mean age at surgery of 47 ± 18 years) and 23 control tissues (10 female [43%]; mean age at death of 43 ± 12 years) to identify differentially expressed genes (DEGs) using edgeR and pathways in HCM using Ingenuity Pathway Analysis. Additionally, genome sequencing (GS) was performed and HCM patients were divided into genotype-based subgroups to compare the transcriptional profile across different genotypes. Results: Overall, 43% had a pathogenic, HCM-associated variant including 23% with MYBPC3-HCM and 15% with MYH7-HCM. Among the 15,634 genes expressed in the cardiac tissue,1,246 genes (8%) were upregulated and 738 genes (3%) were downregulated significantly in HCM vs. controls. Pathway analysis showed alterations in 126 pathways with downregulation of 44 pathways and upregulation of 1 pathway. There were notable transcriptional alterations of most canonical, pro-hypertrophic pathways, including statistically and biologically significant downregulation of five pathways [-log (BH p-value)≥ 1.3; Z-score≤ -2]: ERK/MAPK, p38 MAPK, ERK5, IL6, and NF-κB signaling. Far less DEGs were observed between genotypic subgroups, with only 37 DEGSs identified between sarcomere positive- and genotype negative-HCM. Conclusions: At the time of surgical myectomy, the ventricular myocardium’s transcriptome, independent of genotype, reveals widespread downregulation of pro-hypertrophic pathways. Whether these counter regulatory measures can be targeted therapeutically warrants further investigation.

Introduction: Loss-of-function variants in the RYR2-encoded cardiac calcium release channel underlie a small number of unexplained sudden cardiac arrest and death (SCA/SCD) cases. Objective: To determine if the yield of ultra-rare RYR2 variants in unexplained SCA patients with a default diagnosis of idiopathic ventricular fibrillation (IVF) depends on pre-defined IVF endophenotype and whether RYR2 variant-positive IVF cases have a discernible electrocardiographic phenotype. Methods: All SCA cases referred to Mayo Clinic’s Windland Smith Rice Genetic Heart Rhythm Clinic between 01/2000 and 04/2021 were reviewed. After exclusion of 116 cases where a probable SCA root cause was identified and/or RYR2 genetic testing was not pursued, a total of 64 unexplained SCA cases (36% female; 26 ± 11 years at the time of SCA) were included. Clinical data was extracted from the medical record and patients classified as having i) IVF (no diagnostic clues), N=38 (59%) ii) PVC-triggered ventricular fibrillation (PTVF; documented short coupled PVC-triggered ventricular arrhythmias), N=19 (30%), or iii) early repolarization syndrome (ERS; J-point elevation ≥0.1 mV in ≥2 contiguous leads, excluding leads V 1 -V 3 ), N=7 (11%). The yield of ultra-rare, non-synonymous RYR2 variants (MAF<0.0001 in the Genome Aggregation Database) and electrocardiographic profiles were then compared. Results: Overall, a qualifying, ultra-rare RYR2 non-synonymous variant was identified in 5 out of 64 (7.8%) unexplained SCA patients. Of note, the yield of ultra-rare RYR2 missense variants was significantly higher in individuals with an PTVF endophenotype than those without [4/19 (21%) versus 1/45 (2.2%), p=0.02]. Interestingly, the majority of ultra-rare RYR2 variant-positive unexplained SCA survivors [3/5 (60%)] displayed an exercise stress test pattern characterized by the onset of ventricular ectopy at ~120 bpm with suppression at heart rates ≥140 bpm. Conclusions: The burden of ultra-rare RYR2 variants is higher in unexplained SCA patients with documented PTVF. Collectively, this data suggests that i) a subset of PTVF may have an underlying genetic basis and ii) some IVF- and PTVF-associated RYR2 variants may result in an identifiable electrocardiographic profile.

Background: Multifocal ectopic Purkinje-related premature contractions (MEPPC), the most recent addition to the spectrum of SCN5A -mediated cardiac channelopathies, is characterized by frequent premature ventricular complexes (PVCs) originating from the Purkinje system, atrial arrhythmias, and a predilection for PVC-mediated dilated cardiomyopathy. MEPPC-causative variants tend to cluster within the voltage sensing domain (VSD) of Na v 1.5. Objective: To describe the clinical and cellular electrophysiological phenotype associated with a novel, putative MEPPC-causative SCN5A variant (p.Gln419Pro-SCN5A) that resides outside the VSD of Na v 1.5. Methods: p.Gln419Pro-SCN5A was identified in a 21-year-old male with a history of frequent, multifocal PVCs and atrial tachycardia. Subsequently, three p.Gln419Pro-SCN5A-positive relatives, each with early onset atrial arrhythmias and/or frequent, multifocal PVCs, were identified. All individuals had structurally normal hearts by echocardiography and/or cardiac magnetic resonance imaging. p.Gln419Pro-SCN5A was engineered by site directed mutagenesis and heterologously expressed in TSA201 cells. The standard whole-cell patch clamp technique was used to measure SCN5A wild-type (WT) and p.Gln419Pro-SCN5A sodium currents. Results: Analysis of the current-voltage relationship revealed that neither peak sodium current densities nor late inward sodium current were impacted by p.Gln419Pro-SCN5A. However, p.Gln419Pro-SCN5A significantly shifted V 1/2 in inactivation by -4.6 mV from -84.5±0.7 mV (WT, n=10) to -89.1±0.4 mV (Gln419Pro, n=12, p<0.05 vs. WT) and V 1/2 in activation by -11.6 mV from -30.9±1.3 mV (WT, n=10) to -42.5±1.0 mV (Gln419Pro, n=12, p<0.05 vs. WT) leading to a larger hyperpolarized window current. Flecainide dramatically reduced PVC burden in p.Gln419Pro-SCN5A-positive patients. Conclusions: p.Gln419Pro-SCN5A represents only the fifth MEPPC-causative variant described to date and the first associated with a classic MEPPC phenotype localizing outside Na v 1.5’s VSD. As class I antiarrhythmic drugs are highly efficacious in MEPPC, this clinical entity merits consideration in all young patients found to have a high burden of Purkinje-related PVCs.

Abstract Background Pathogenic variants in the lamin A/C gene (LMNA) can lead to a wide range of phenotypes from dilated and arrhythmogenic cardiomyopathies and conduction abnormalities to partial lipodystrophies. This case highlights a coincidental pathogenic LMNA variant identified in a patient with sudden cardiac arrest (SCA). We demonstrate the need for careful interpretation of pathogenic variants identified in cardiomyopathy genes by highlighting a case in which a coincidental pathogenic LMNA variant was found in a patient with premature ventricular complex (PVC)-induced ventricular fibrillation (VF). Case summary We present the case of a 16-year-old male with SCA secondary to VF. Genetic testing identified a maternally inherited pathogenic variant in LMNA annotated c.1961dup; p.T655Nfs*49. The patient received an implantable cardiac defibrillator and was discharged on nadolol. The patient’s two brothers were also variant-positive. However, the patient and both brothers had normal chamber dimensions on echocardiogram and no late gadolinium enhancement on cardiac magnetic resonance imaging. The family members with the variant were recommended to have prophylactic implantable cardiac defibrillators and thus sought a second opinion. The patient received an appropriate shock and device interrogation identified PVCs. Electrophysiology study identified PVC-induced VF which was ablated with no recurrent ventricular arrhythmias/implantable cardioverter defibrillator therapies over 8 months of follow-up. Although the variant in LMNA could lead to cardiac arrest, the clinical phenotype was consistent with a non-genetic aetiology. The family members were told to have periodic cardiac evaluation. Discussion This case demonstrates the identification of a coincidental pathogenic variant in a cardiomyopathy gene in a patient with cardiac arrest. Although this variant could lead to cardiomyopathy, it appears the cardiac arrest was not due to the pathogenic variant. This highlights the need to consider the clinical phenotype when interpreting genetic test results for cardiomyopathies even in the presence of a positive genetic test result.

Background: Sudden cardiac arrest (SCA) and sudden unexplained death (SUD) are feared sequelae of many genetic heart diseases. In rare circumstances, pathogenic variants in cardiomyopathy-susceptibility genes may result in electrical instability leading to SCA/SUD before any structural manifestations of underlying cardiomyopathy are evident. Methods: Collectively, 38 unexplained SCA survivors (21 males; mean age at SCA 26.4±13.1 years), 68 autopsy-inconclusive SUD cases (46 males; mean age at death 20.4±9.0 years) without disease-causative variants in the channelopathy genes, and 973 ostensibly healthy controls were included. Following exome sequencing, ultrarare (minor allele frequency ≤0.00005 in any ethnic group within Genome Aggregation Database [gnomAD, N=141 456 individuals]) nonsynonymous variants identified in 24 Clinical Genome Resource adjudicated definitive/strong evidence cardiomyopathy-susceptibility genes were analyzed. Eligible variants were adjudicated as pathogenic, likely pathogenic, or variant of uncertain significance in accordance with current American College of Medical Genetics and Genomics guidelines. Results: Overall, 7 out of 38 (18.4%) SCA survivors and 14 out of 68 (20.5%) autopsy-inconclusive, channelopathic-negative SUD cases had at least one pathogenic/likely pathogenic or a variant of uncertain significance nonsynonymous variant within a strong evidence, cardiomyopathy-susceptibility gene. Following American College of Medical Genetics and Genomics criterion variant adjudication, a pathogenic or likely pathogenic variant was identified in 3 out of 38 (7.9%; P =0.05) SCA survivors and 8 out of 68 (11.8%; P =0.0002) autopsy-inconclusive SUD cases compared to 20 out of 973 (2.1%) European controls. Interestingly, the yield of pathogenic/likely pathogenic variants was significantly greater in autopsy-inconclusive SUD cases with documented interstitial fibrosis (4/11, 36%) compared with only 4 out of 57 (7%, P <0.02) SUD cases without ventricular fibrosis. Conclusions: Our data further supports the inclusion of strong evidence cardiomyopathy-susceptibility genes on the genetic testing panels used to evaluate unexplained SCA survivors and autopsy-inconclusive/negative SUD decedents. However, to avoid diagnostic miscues, the careful interpretation of genetic test results in patients without overt phenotypes is vital.

Significance Approximately 400 United States children 1 y of age and older die suddenly from unexplained causes annually. We studied whole-exome sequence data from 124 “trios” (decedent child and living parents) to identify genetic risk factors. Nonsynonymous mutations, mostly de novo (present in child but absent in both biological parents), were highly enriched in genes associated with cardiac and seizure disorders relative to controls, and contributed to 9% of deaths. We found significant overtransmission of loss-of-function or pathogenic missense variants in cardiac and seizure disorder genes. Most pathogenic variants were de novo in origin, highlighting the importance of trio studies. Many of these pathogenic de novo mutations altered a protein network regulating calcium-related excitability at submembrane junctions in cardiomyocytes and neurons.