Bos, J. Martijn

Background Recently, genetic testing for sarcomeric hypertrophic cardiomyopathy (HCM) became a commercially available diagnostic test involving analysis of 8 HCM-associated myofilament genes, four of which encode thin filament proteins - TNNT2- encoded cardiac troponin T, TPM1 -encoded alpha tropomyosin, TNNI3- encoded cardiac troponin I, and ACTC- encoded cardiac actin. Compared to the two most common subtypes of sarcomeric HCM, much less is known about the prevalence and spectrum of thin filament HCM. Methods Comprehensive open reading frame/splice site mutational analysis of these 4 thin filament-encoding genes was performed, using polymerase chain reaction, denaturing high performance liquid chromatography, and direct DNA sequencing, on the largest assembled cohort of unrelated patients (N = 1025, 61% male, 49 ± 18 years at diagnosis, maximal left ventricular wall thickness, LVWT, 22.3 ± 7 mm) with clinically diagnosed HCM that were seen at the Mayo Clinic. Results Nineteen distinct missense mutations, involving conserved residues and absent in 600 reference alleles, were identified in 36 of 1025 patients (3.5%) including TNNT2 (16 patients), TNNI3 (12), TPM1 (7) and ACTC (1). Among these 36 patients with thin filament-HCM, their average age at diagnosis was 33.2 ± 16 years and the average LVWT was 21 ± 8 mm. Eighteen patients (50%) had evidence for familial HCM and 13% had a positive history for sudden cardiac death. Only 13 patients (36%) had a reverse septal curvature by echocardiography. Instead, 12 patients (34%) had either sigmoidal or neutral septal contours and 9 patients (25%) had apical variant-HCM. Conclusion Here, we present the largest cohort analysis of thin filament HCM to date and detail the prevalence and spectrum of HCM-associated mutations in these 4 genes. Compared to the strong association between reverse septal curvature and thick filament HCM, the septal morphology associated with thin filament HCM was quite varied. Contrary to published estimates of the frequency of troponin T-HCM (5 – 10%) and the other subtypes of thin filament HCM, the prevalence of thin filament HCM was very low in this cohort. If confirmed in other cohorts, a revised cascade for HCM genetic testing should be considered.

Coronary microvascular dysfunction (CMD) is an important primary feature of hypertrophic cardiomyopathy (HCM), contributing to myocardial ischemia and ventricular remodelling, and is predictive of adverse outcome. Whether there is any association between presence/severity of CMD and HCM’s pathogenic substrate remains to be determined. To address this issue, we used positron emission tomography (PET) to assess CMD in an HCM cohort that received comprehensive genetic testing for sarcomeric/myofilament-HCM. We measured maximum (intravenous dipyridamole, 0.56 mg/kg) myocardial blood flow (Dip-MBF), using 13 N-labeled ammonia and PET in 46 HCM patients (age 38±14 years, 32 male). Genetic testing was performed by denaturing high performance liquid chromatography and automatic DNA sequencing of nine myofilament-encoding genes including both thick filament proteins (myosin binding protein C, beta-myosin heavy chain, regulatory and essential light chains); and thin filament proteins (troponin-T, troponin-I, troponin-C, alpha-tropomyosin and alpha-actin). Results . Thirty-four mutations were identified in 30/46 patients (myofilament-HCM; 65%), including 29 with thick filament and 6 with thin filament mutations, as well as 4 with complex genotype. Despite similar age and clinical features, patients with myofilament-HCM showed lower Dip-MBF values than the patients with a negative genetic test (1.6±0.7 versus 2.2±0.9 ml/min/g, respectively; p=0.03). Specifically, 13/30 (43%) patients with myofilament-HCM had a Dip-MBF below the lower tertile for the study group (≤1.38 ml/min/g), compared to 2/16 (12%) patients with a negative genetic test (p=0.034). No difference in Dip-MBF was evident with respect to the particular sarcomeric gene involved (ANOVA p=0.63). HCM due to sarcomere gene mutations is characterized by more severe impairment in microvascular function compared to myofilament negative disease, irrespective of the involved contractile protein. These findings suggest that sarcomeric mutations might be implicated in adverse remodelling of the microcirculation in patients with HCM, and account for the greater prevalence of ventricular dysfunction and failure in this subset.

Background: Mutations in MYBPC3 -encoded myosin binding protein C underlie the most common genotype in hypertrophic cardiomyopathy (HCM). Compared to most HCM-associated mutations which are primarily missense mutations, MYBPC3-HCM is often caused by insertions, deletions, nonsense mutations or mutations involving the canonical splice site. However, the effects of most mutations that prematurely truncate MYBPC3 are unknown. Access to cardiac specific mRNA and tissue has permitted a molecular and cellular elucidation of the consequences of two splice site mutations. Methods: Mutational analysis of MYBPC3 revealed 2 HCM-susceptibility mutations involving the splice donor sites of exons 7 and 30. Reverse transcription was performed on cDNA generated from RNA extracted from cardiac tissue obtained following surgical myectomy. Western blot analysis and immunohistochemistry (IHC) of the myofilaments were performed to further assess the impact of the splice-site mutations. Results: Both splice donor mutations produced abnormal RNA splicing of MYBPC3. The c.821+1 g>a mutation in the splice donor site of exon 7 generated two alternatively-spliced mutant transcripts resulting in two frame-shifted, premature truncations (H257 fs/37 and H257 fs/15). The c.3330+2 t>g mutation in exon 30’s splice donor site produced a single alternatively-spliced mutant transcript that translated into a frame-shifted premature truncation (V1063 fs/37). Expression levels of wild type myosin binding protein C were decreased in the myectomy specimen from the patient with the exon 7 splice donor site mutation. By IHC, the spatial organization of myosin binding protein C was disrupted severely in both patients. Conclusions: This study provides the first molecular and cellular characterization of HCM-causing mutations involving canonical splice-site motifs within the intron. Loss of function of the splice site resulted in exon skipping and generation of frame-shifted and prematurely truncated myosin binding protein C and disruption of the distribution and organization of myosin binding protein C in the heart tissue. This research has received full or partial funding support from the American Heart Association, AHA Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).

Introduction: Vectorcardiography (VCG) has clinical prognostic and diagnostic value in various disease states. Hypertrophic cardiomyopathy is a disease state which can lead to sudden cardiac death, where genotype status has been shown to predict worse outcomes.. Hypothesis: We set out to determine the utility of vectorcardiographic parameters in one of the largest genotyped cohorts of patients with hypertrophic cardiomyopathy (HCM) and to determine whether they can identify genotype status. Methods: Between 1997 and 2007, research based genetic testing was performed on 1053 patients with HCM (60% males; mean age at diagnosis 44.4 years); yield of genetic testing was 34%. Of these, 967 had ECG’s appropriate for computerized derivation of VCG parameters including the spatial mean and peaks QRS-T angles, spatial ventricular gradient and the spatial QRS, QT, and Tpeak-Tend (TpTe) intervals. Results were compared against those from a group of healthy control subjects (N=418).Subsequent genotype positive versus negative comparisons were performed using paired student T-tests or Mann-Whitney U-tests as appropriate. Results: Spatial peaks and spatial mean QRS-T angles were significantly higher in HCM patients (129.3±26.4 and 121.8±38.6 degrees, respectively) than in controls (25.0±15.0 and 43.0±-21.0 degrees, respectively, p<0.001). Sensitivities for HCM, based on cutoff values of 2 standard deviations above the mean values reported for control patients, were 84.5% and 81.5% for spatial peaks and spatial mean QRS-T angles, respectively. VCG predicted genotype status as genotype positive patients had significantly higher spatial peaks (132.4±-29.2 vs. 127.9±24.8 degrees; p = 0.02) and spatial mean (129.7±33.8 vs. 117.6±40.3 degrees; p<0.001) QRS-T angles, spatial TpTe (118.0±40.8 vs. 107.9±48.7 ms; p<0.001) as well as significantly lower spatial ventricular gradients (0.059±0.044 vs. 0.067±0.04; p<0.001). Conclusions: Patients with HCM can be distinguished from normal subjects through the study of the spatial QRS-T angle. Whether incorporation of spatial peaks QRS-T angle to pre-existing genetic test prediction tools will further improve prediction of genotype status remains to be determined.

Background: Heart rate–corrected QT interval (QTc) prolongation, whether secondary to drugs, genetics including congenital long QT syndrome, and/or systemic diseases including SARS-CoV-2–mediated coronavirus disease 2019 (COVID-19), can predispose to ventricular arrhythmias and sudden cardiac death. Currently, QTc assessment and monitoring relies largely on 12-lead electrocardiography. As such, we sought to train and validate an artificial intelligence (AI)–enabled 12-lead ECG algorithm to determine the QTc, and then prospectively test this algorithm on tracings acquired from a mobile ECG (mECG) device in a population enriched for repolarization abnormalities. Methods: Using >1.6 million 12-lead ECGs from 538 200 patients, a deep neural network (DNN) was derived (patients for training, n = 250 767; patients for testing, n = 107 920) and validated (n = 179 513 patients) to predict the QTc using cardiologist-overread QTc values as the “gold standard”. The ability of this DNN to detect clinically-relevant QTc prolongation (eg, QTc ≥500 ms) was then tested prospectively on 686 patients with genetic heart disease (50% with long QT syndrome) with QTc values obtained from both a 12-lead ECG and a prototype mECG device equivalent to the commercially-available AliveCor KardiaMobile 6L. Results: In the validation sample, strong agreement was observed between human over-read and DNN-predicted QTc values (−1.76±23.14 ms). Similarly, within the prospective, genetic heart disease–enriched dataset, the difference between DNN-predicted QTc values derived from mECG tracings and those annotated from 12-lead ECGs by a QT expert (−0.45±24.73 ms) and a commercial core ECG laboratory [10.52±25.64 ms] was nominal. When applied to mECG tracings, the DNN’s ability to detect a QTc value ≥500 ms yielded an area under the curve, sensitivity, and specificity of 0.97, 80.0%, and 94.4%, respectively. Conclusions: Using smartphone-enabled electrodes, an AI DNN can predict accurately the QTc of a standard 12-lead ECG. QTc estimation from an AI-enabled mECG device may provide a cost-effective means of screening for both acquired and congenital long QT syndrome in a variety of clinical settings where standard 12-lead electrocardiography is not accessible or cost-effective.

AbstractBackgroundUnfortunately, some patients with long QT syndrome (LQTS) experience breakthrough cardiac events (BCEs) despite maximal therapy. Small studies have shown that refractory LQTS cases may benefit from intentional permanent atrial pacing (IPAP). As such, we sought to determine the genotype‐specific utilization and efficacy of IPAP in a single‐center LQTS registry.Methods and ResultsIn this retrospective study, electronic medical records from 1065 patients diagnosed with LQTS were used to identify individuals that received IPAP. Pre‐ and post‐IPAP heart rate, heart rate‐corrected QT (QTc) values, annual BCE rate, and IPAP‐related complications were compared between genotypes. BCEs were defined as LQTS‐associated syncope/seizures, sustained ventricular arrhythmia (VA)‐terminating ICD therapies, and sudden cardiac arrest/death.Overall, 52 out of 1065 LQTS patients received adjunctive IPAP therapy (77% female; median age 18.5 [interquartile range, 1–35.5] years; 73% with prior VA). Over an average IPAP follow‐up of 121 ± 82 months, the average heart rate increased from 65.8 ± 20.4 bpm to 78.9 ± 17.1 bpm; (p < .01) and the average QTc decreased from 533.4 ± 66.6 to 488.3 ± 52.4 ms; (p < .01). The mean BCE rate dropped from 0.88 to 0.19 per patient‐year (p = .01), driven by a marked decrease in the LQT2 cohort (1.01 BCE/year to 0.02 BCE/year; p = .003). No serious IPAP‐related complications were observed.ConclusionIn high‐risk LQTS patients, namely those with recalcitrant LQT2, IPAP appears to be a safe and efficacious adjunct therapy. The beneficial effects of IPAP may stem from attenuating the QTc and circumventing a pause‐dependent trigger. Whether IPAP might obviate the need for an ICD in some instances warrants further study.

Background: Type 1 long QT syndrome (LQT1) is caused by loss-of-function variants in the KCNQ1 -encoded K v 7.1 potassium channel α-subunit that is essential for cardiac repolarization, providing the slow delayed rectifier current. No current therapies target the molecular cause of LQT1. Methods: A dual-component suppression-and-replacement (SupRep) KCNQ1 gene therapy was created by cloning a KCNQ1 short hairpin RNA and a short hairpin RNA-immune KCNQ1 cDNA modified with synonymous variants in the short hairpin RNA target site, into a single construct. The ability of KCNQ1-SupRep gene therapy to suppress and replace LQT1-causative variants in KCNQ1 was evaluated by means of heterologous expression in TSA201 cells. For a human in vitro cardiac model, induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs) were generated from 4 patients with LQT1 (KCNQ1-Y171X, -V254M, -I567S, and -A344A/spl) and an unrelated healthy control. CRISPR-Cas9 corrected isogenic control iPSC-CMs were made for 2 LQT1 lines (correction of KCNQ1-V254M and KCNQ1-A344A/spl). FluoVolt voltage dye was used to measure the cardiac action potential duration (APD) in iPSC-CMs treated with KCNQ1-SupRep. Results: In TSA201 cells, KCNQ1-SupRep achieved mutation-independent suppression of wild-type KCNQ1 and 3 LQT1-causative variants (KCNQ1-Y171X, -V254M, and -I567S) with simultaneous replacement of short hairpin RNA-immune KCNQ1 as measured by allele-specific quantitative reverse transcription polymerase chain reaction and Western blot. Using FluoVolt voltage dye to measure the cardiac APD in the 4 LQT1 patient-derived iPSC-CMs, treatment with KCNQ1-SupRep resulted in shortening of the pathologically prolonged APD at both 90% and 50% repolarization, resulting in APD values similar to those of the 2 isogenic controls. Conclusions: This study provides the first proof-of-principle gene therapy for complete correction of long QT syndrome. As a dual-component gene therapy vector, KCNQ1-SupRep successfully suppressed and replaced KCNQ1 to normal wild-type levels. In TSA201 cells, cotransfection of LQT1-causative variants and KCNQ1-SupRep caused mutation-independent suppression and replacement of KCNQ1 . In LQT1 iPSC-CMs, KCNQ1-SupRep gene therapy shortened the APD, thereby eliminating the pathognomonic feature of LQT1.

AbstractBackgroundA subset of patients with hypertrophic cardiomyopathy (HCM) is at high risk of sudden cardiac death (SCD). Practice guidelines endorse use of a risk calculator, which requires entry of left atrial (LA) diameter. However, American Society of Echocardiography (ASE) guidelines recommend the use of LA volume index (LAVI) for routine quantification of LA size. The aims of this study were to (a) develop a model to estimate LA diameter from LAVI and (b) evaluate whether substitution of measured LA diameter by estimated LA diameter derived from LAVI reclassifies HCM‐SCD risk.MethodsThe study cohort was comprised of 500 randomly selected HCM patients who underwent transthoracic echocardiography (TTE). LA diameter and LAVI were measured offline using digital clips from TTE. Linear regression models were developed to estimate LA diameter from LAVI. A European Society of Cardiology endorsed equation estimated SCD risk, which was measured using LA diameter and estimated LA diameter derived from LAVI.ResultsThe mean LAVI was 48.5 ± 18.8 mL/m2. The derived LA diameter was 45.1 mm (SD: 5.5 mm), similar to the measured LA diameter (45.1 mm, SD: 7.1 mm). Median SCD risk at 5 years estimated by measured LA diameter was 2.22% (interquartile range (IQR): 1.39, 3.56), while median risk calculated by estimated LA diameter was 2.18% (IQR: 1.44, 3.52). 476/500 (95%) patients maintained the same risk classification regardless of whether the measured or estimated LA diameter was used.ConclusionsSubstitution of measured LA diameter by estimated LA diameter in the HCM‐SCD calculator did not reclassify risk.

Abstract Background Left cardiac sympathetic denervation (LCSD) is an effective anti-fibrillatory, minimally invasive therapy for patients with either LQTS or CPVT. As a pre-ganglionic fiber resection, re-growth or re-innervation is not possible. However, if a suboptimal CSD was performed, chances for post-LCSD breakthrough cardiac events (BCEs) are increased. Purpose To examine the prevalence and outcomes of patients requiring a re-do LCSD. Methods We performed a retrospective review of the 251 LQTS and CPVT patients who underwent CSD (mostly LCSD) at our institution to identify the subset referred for additional CSD because of recurrent BCEs after their primary LCSD that was performed elsewhere. Clinical data on symptomatic status prior to diagnosis and BCEs after first surgery as well as the surgical reports were reviewed to determine the reasons for the repeat procedure. Results Among the 15 patients (6% overall, 6 female; 13 LQTS, 2 CPVT) referred for additional CSD, 3 patients (20%) had a re-do LCSD performed instead of a sequential RCSD because of incomplete resection with the primary LCSD. Patient 1 is a male with Jervell-and-Lange Nielsen Syndrome (JLNS; KCNQ1-K421fs9X; KCNQ1-N365H), who first experienced syncope with torsades at age 3 after which a primary bilateral CSD was performed elsewhere. He had 2 BCEs and at age 12 underwent re-do LCSD, where his left-sided T2-T4 sympathetic chain with remnants of T1 were found to be intact. Patient 2 is a male who had 3 cardiac events prior to diagnosis (genotype negative LQTS) and LCSD at age 3. Following 3 BCEs, he underwent re-do LCSD at age 5 where scarring of T3 and intact stellate ganglion were found. Patient 3 is an adult female with CPVT (RYR2-deletion exon 3) who had multiple syncopal events, out-of-hospital arrest (leading to ICD) and 2 ICD-storms before primary bilateral CSD. Re-do LCSD was performed after identifying an intact left stellate ganglion and T2. Conclusions Before performing a primary bilateral CSD, proceeding to a RCSD after post-LCSD BCEs, or doing a videoscopic LCSD in the first place, it must be recognized that LCSD's success hinges on fully resecting the lower half of the stellate ganglion and the sympathetic chain through T4. Anything less is a partial denervation and likely ineffective procedure. Funding Acknowledgement Type of funding source: None