Gao, Xiaozhi

BACKGROUND: Genetic testing for cardiac channelopathies is the standard of care. However, many rare genetic variants remain classified as variants of uncertain significance (VUS) due to lack of epidemiological and functional data. Whether deep protein language models may aid in VUS resolution remains unknown. Here, we set out to compare how 2 deep protein language models perform at VUS resolution in the 3 most common long-QT syndrome–causative genes compared with the gold-standard patch clamp. METHODS: A total of 72 rare nonsynonymous VUS (9 KCNQ1, 19 KCNH2 , and 50 SCN5A ) were engineered by site-directed mutagenesis and expressed in either HEK293 cells or TSA201 cells. Whole-cell patch-clamp technique was used to functionally characterize these variants. The protein language models, evolutionary scale modeling, version 1b and AlphaMissense, were used to predict the variant effect of missense variants and compared with patch clamp. RESULTS: Considering variants in all 3 genes, the evolutionary scale modeling, version 1b model had a receiver operating characteristic curve–area under the curve of 0.75 ( P =0.0003). It had a sensitivity of 88% and a specificity of 50%. AlphaMissense performed well compared with patch-clamp with an receiver operating characteristic curve-area under the curve of 0.85 ( P <0.0001), sensitivity of 80%, and specificity of 76%. CONCLUSIONS: Deep protein language models aid in VUS resolution with high sensitivity but lower specificity. Thus, these tools cannot fully replace functional characterization but can aid in reducing the number of variants that may require functional analysis.

Introduction: 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. Hypothesis: To investigate the electrophysiology phenotype of MEPPC in a novel, patient-specific, re-engineered cardiomyocyte model. Methods: Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated from a 27-year-old male with a history of frequent, multifocal PVCs and atrial tachycardia, positive for a putative MEPPC-causative SCN5A variant (p.Q419P). A CRISPR-Cas9 Q419P variant-corrected isogenic control (IC) was created to establish the normalized electrophysiology recordings for that MEPPC model. The standard whole-cell patch clamp technique was used to measure Q419P-SCN5A- and IC-derived NaV1.5 sodium currents using voltage clamp configuration. In addition, action potential duration (APD) of the iPSC-CMs were measured using FluoVolt optical voltage-sensing dye. Results: Analysis of the current-voltage relationship revealed that neither peak sodium current densities nor late inward sodium current were impacted by Q419P-SCN5A. However, Q419P-SCN5A significantly right-shifted the V 1/2 in inactivation by 8.3 mV from -81.1±1.7 mV (IC, n=5) to -72.8±0.2 mV (Q419P-SCN5A, n=6, p<0.05 vs. IC) and left-shifted the V 1/2 in activation by -4.1 mV from -39.5±0.3 mV (IC, n=5) to -43.6±0.9 mV (Q419P-SCN5A, n=6, p<0.05 vs. IC) leading to a larger hyperpolarized window current. Optical APD90 measurement revealed no difference between the Q419P-containing (476 ± 23 ms) and the IC (474 ± 46 ms, p=0.867) cardiomyocytes. Conclusions: This is the first in-vitro disease model of MEPPC using patient-specific, re-engineered heart cells. Consistent with the findings in the non-cardiac HEK cell system, this MEPPC-causative variant contributes to increased window current in cardiomyocytes without affecting the cardiac APD. This novel cardiac cell model for MEPPC may facilitate new insights into the pathobiology of MEPPC and enable the testing of novel therapies for it.

Background: Type 2 long QT syndrome (LQT2) is an inherited arrhythmia syndrome caused by pathogenic variants in the KCNH2 gene which result in QT prolongation and confers susceptibility to arrhythmic syncope/seizures and sudden cardiac arrest/death. Oral progestin-based contraceptives increase the risk of cardiac events in women with LQT2 and we previously reported the case of a LQT2 woman with recurrent cardiac events attributed to the use of the progestin-based contraceptive, Medroxyprogesterone Acetate (“Depo-Provera”, Depo). Objective: To evaluate the arrhythmic-risk of Depo in a LQT2-specific, induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model. Methods: An iPSC-CM line was generated from the previously reported woman (KCNH2-G1006A*fs) as well as a control line. Voltage-sensing dye was used to assess the optical action potential duration (APD) with 10 μM Depo. 3-D erratic beating patterns were then assessed using multiple electrode array (MEA) with Depo and isoproterenol (ISO) to mimic an adrenergic surge. Results: Addition of Depo to the G1006A*fs iPSC-CMs decreased the optical APD from baseline 438±14 ms (n=6) to 363±35 ms (n=12; p=0.0001). Despite the APD shortening at baseline, in the setting of ISO, Depo administration induced erratic beating patterns in MEA, characterized as alternating spike amplitudes, alternans, or early after depolarization-like phenomena. Combined Depo and ISO treatment increased the percent of electrodes displaying erratic beating in G1006A*fs [baseline 8%±13 vs. Depo + ISO 38%±40 (p=0.0097)] but not in controls [baseline 2%±5 vs. Depo + ISO 7%±11 (p=0.14)]. Neither Depo nor ISO alone changed beating patterns in G1006A*fs [baseline 8%±13, Depo 30%±41 (p=0.08), ISO 21%±30, (p=0.17)] and controls [baseline 2%±5, Depo 3%±7 (p=0.61), ISO 0%±0 (p=0.4)]. Conclusion: This patient-specific, re-engineered heart cell study provides a potential mechanism for the patient’s clinically documented, Depo-associated episodes of recurrent ventricular fibrillation. This in-vitro data should prompt a large-scale clinical assessment of Depo’s potential pro-arrhythmic effect in women with LQT2.

Introduction: Gain-of-function mutations in SCN5A- encoded cardiac sodium channel (Na V 1.5) cause type 3 long QT syndrome (LQT3). Patients with SCN5A variants localizing to the local anesthetic binding domain might not be sensitive to lidocaine or mexiletine treatment. Hypothesis: Phenytoin, a sodium channel blocker used to treat epilepsy, may be a novel LQT3-directed therapy for a variant involving a critical residue of the Nav1.5’s local anesthetic binding domain. Methods: SCN5A-F1760C was identified in a now deceased infant with severe LQT3 (QTc of 680-1200 ms) who was refractory to left cardiac sympathetic denervation and pharmacological treatments with either lidocaine or mexiletine. SCN5A-F1760C was engineered by site directed mutagenesis and expressed in TSA201 cells. Patient-specific pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from the SCN5A-F1760C-positive infant and gene-edited/variant-corrected isogenic control (IC) iPSC-CMs were generated. Na V 1.5 currents were recorded in TSA201 cells transfected with SCN5A-WT/ F1760C and action potentials (APs) from iPSC-CMs were recorded before and after treatment with 5μM Phenytoin using the whole cell patch-clamp technique. Results: SCN5A-F1760C did not change the NaV1.5 current density. However, the V1/2 of inactivation was right-shifted significantly by +14.1 mV from -86.3±0.9 mV (WT) to -72.2±0.7 mV (F1760C, p<0.0001) resulting in a marked increase in window current. F1760C increased sodium late current 2-fold from 0.18±0.04% in WT to 0.49±0.07% in F1760C (p=0.0005). Baseline APD to 90% repolarization (APD90) was prolonged significantly in F1760C-derived iPSC-CMs (641±40 ms) compared to IC iPSC-CMs (412±22 ms, p<0.0001). Treatment with 5μM phenytoin normalized the APD90 in F1760C-derived iPSC-CMs to 423+34 ms (p<0.0001), values approximating the ‘gene/variant-edited’ cure. Conclusions: Na V 1.5 gain-of-function caused by a novel SCN5A-F1760C variant results in marked APD90 prolongation which were rescued by phenytoin indicating the antiepileptic drug, phenytoin, may be a mutation-specific, LQT3-directed therapy for those LQT3-causative variants that disrupt the local anesthetic/lidocaine-binding domain.

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.