In some crosses, the hybrid progeny perform better than their parental inbred lines in terms of beneficial traits (e.g., greater biomass, faster development), a phenomenon known as hybrid vigor or heterosis. While classical genetic paradigms explain much of its basis, how these interactions translate into specific molecular frameworks remains a significant challenge. Mounting evidence indicates that epigenetic regulation—encompassing chromatin states, histone modifications, DNA methylation, and small RNAs—reshapes the hybrid transcriptional landscape. While many studies provide compelling correlations between epigenetic remodeling and heterotic phenotypes, a definitive mechanistic synthesis remains elusive due to the complexity of distinguishing cause from consequence. This review integrates current knowledge of these multilayered regulatory systems, emphasizing the redundant and hierarchical nature of epigenetic interactions that sustain the heterotic state. We highlight the transition from descriptive profiling to functional validation, and discuss how emerging tools such as targeted epigenome editing for recreating optimal hybrid methylotypes may finally bridge the gap between epigenetic variation and hybrid vigor.

BACKGROUND AND AIMS:Patients with catecholaminergic polymorphic ventricular tachycardia (CPVT) are at risk for potentially life-threatening arrhythmic events (AEs) even while treated with β-blockers. The aim was to develop a model for individualized prediction of AEs in patients with RYR2-mediated CPVT on β-blocker monotherapy. METHODS:The derivation and independent validation cohorts included 743 and 129 patients, respectively. AEs were defined as arrhythmic syncope, appropriate implantable cardioverter-defibrillator shock, sudden cardiac arrest (SCA), and sudden cardiac death. Near-fatal or fatal AEs (nf/fAEs) included all AEs except for arrhythmic syncope. Prediction models using Cox regression were developed and internally and externally validated. RESULTS:A total of 102 (13.7%) patients in the derivation cohort and 24 (18.6%) patients in the validation cohort experienced ≥1 AE over a median follow-up of 5.1 [interquartile range (IQR), 7.7] and 2.4 (IQR, 4.4) years, respectively. Predictors of AE were arrhythmic syncope or SCA prior to diagnosis and age at β-blocker initiation. In the derivation and validation cohorts, the optimism-corrected C-indices of the models for AE were 0.67 [95% confidence interval (CI) 0.62-0.72] and 0.59 (95% CI 0.48-0.71), respectively. For nf/fAEs, ventricular arrhythmia severity before β-blocker initiation was a fourth independent predictor, and C-indices of the models in the derivation and validation cohorts were 0.74 (95% CI 0.68-0.80) and 0.60 (95% CI 0.47-0.72), respectively. In the derivation cohort, calibration slopes were 1.00 (95% CI 0.59-1.41) for AE and 1.00 (95% CI 0.69-1.32) for nf/fAE. CONCLUSIONS:These externally validated risk prediction models using clinical parameters accurately distinguished CPVT patients on β-blocker monotherapy at low and high risk for future AEs while treated with β-blockers. These models provide guidance for implementation of clinical management therapies to prevent AEs in patients with CPVT.

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have emerged as a pivotal model for research. Specialized devices can generate Extracellular Field Potential (EFP) measurements from these cells, analogous to the ventricular complex of the electrocardiogram. However, electrophysiological analysis can be complex and requires specialized expertise, posing a barrier to broader adoption in non-specialized labs. We present the EFP-Analyzer (EFPA), a semi-automized analyzer for EFP traces, which identifies and averages beats, identifies landmarks, and calculates intervals. We demonstrate an analysis of 358 EFP traces from 22 patient-derived lines. We analyzed spontaneously beating iPSC-CMs and optically paced iPSC-CMs through channelrhodopsin. We developed stringent quality criteria and measured EFP intervals, including Field Potential Duration (FPD). We further analyzed the usability and data replicability of EFPA through an inter-intra observer analysis. Correlation coefficient for inter-reader tangent and threshold measurements for these FPD ranged between r: 0.93-1.00. Bland-Altman plots comparing inter observer results for spontaneously beating and paced iPSC-CMs showed 95% limits of agreement (- 13.6 to 19.4 ms and - 13.2 to 15.3 ms, respectively). EFPA could accurately detect FPD prolongation due to drug (moxifloxacin) or pathogenic loss of function mutations (CACNA1C N639T). This program and instructions are available for download at https://github.com/kroncke-lab/EFPA .

Background:KCNQ1 loss of function variants are thought to cause type 1 long QT syndrome by reducing I Ks. However, we have recently reported that pharmacologic block of I Ks in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) produced minimal increases in action potential duration at 90% repolarization (APD90), while genetic loss of KCNQ1 markedly prolonged APD90. We sought here to define mechanisms underlying APD prolongation by genetic loss of KCNQ1. Methods:We studied iPSC-CMs from population controls, an isogenic KCNQ1 knock out (KO) line created by a homozygous edit for the R518X loss of function variant, and 2 unrelated patients with the Jervell and Lange-Nielsen syndrome (JLN) due to compound heterozygosity for loss of function KCNQ1 variants. Results:In both JLN and the KCNQ1-KO lines, I Ks was absent, APD90 was markedly prolonged, and L-type Ca channel (LTCC) current (I Ca-L) was significantly increased, 2-3-fold, compared to the control cells with no change in kinetics or gating. RNA-sequencing identified 298 and 584 genes that were up- and down-regulated, respectively, by KCNQ1-KO compared to the isogenic control cells. Gene ontology analysis identified down-regulation of 6 Ca2+ channel negative regulatory genes (p=0.0002, FDR=0.02), and in knockdown experiments in wild-type iPSC-CMs, three of these, CBARP, FKBP1B, and RRAD, increased I Ca-L, and RRAD increased APD90. A therapeutic low concentration (1 μM) of the Ca channel antagonist diltiazem significantly shortened APD90 in the two JLN cell lines and in KCNQ1-KO cells. A single low dose of intravenous diltiazem in one of the JLN patients shortened QTc. Conclusions:These data further support the concept that delayed repolarization in JLN cannot be explained solely by loss of I Ks. Our findings demonstrate that KCNQ1 mutations lead to down-regulation of Ca2+ channel inhibitory genes, with resultant increased I Ca-L that underlies delayed repolarization in JLN. We further propose that diltiazem can be repurposed for treatment of patients with JLN.

Rationale:Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are an emerging model for determining drug effects and modeling disease. Specialized devices can generate Extracellular Field Potential (EFP) measurements from these cells, analogous to the ventricular complex of the electrocardiogram. Objective:The objective of this study was to develop an easy-to-use, easy-to-teach, reproducible software tool to measure EFPs. Methods-Results:We present the EFP-Analyzer (EFPA), a semi-automized analyzer for EFP traces, which identifies and averages beats, identifies landmarks, and calculates intervals. We evaluated the tool in an analysis of 358 EFP traces from 22 patient-derived lines. We analyzed spontaneously beating iPSC-CMs, as well as optically paced iPSC-CMs through channelrhodopsin. We developed stringent quality criteria and measured EFP intervals, including Field Potential Duration (FPD). FPD from optically paced iPSC-CMs were shorter than those of spontaneously beating iPSC-CMs (283.7.0±54.2 vs. 293.0±47.5, p: 0.32, respectively). We further analyzed the usability and data replicability of EFPA through an inter-intra observer analysis. Correlation coefficient for inter-reader tangent and threshold measurements for these FPD ranged between r: 0.93-1.00. Bland-Altman plots comparing inter observer results for spontaneously beating and paced iPSC-CMs showed 95% limits of agreement (-13.6 to 19.4ms and -13.2 to 15.3ms, respectively). The EFP-analyzer could accurately detect FPD prolongation due to drug (moxifloxacin) or pathogenic loss of function mutations ( CACNA1C N639T). This program is available for download at https://github.com/kroncke-lab/EFPA . The instructions will be available at the same listed website under the README section of the Github main page. Conclusions:The EFP-Analyzer tool is a useful tool that enables the efficient use of iPSC-CMs as a model to study drug effects and disease.

Background We identified a novel SCN5A variant, E171Q, in a neonate with very frequent ectopy and reduced ejection fraction which normalized after arrhythmia suppression by flecainide. This clinical picture is consistent with multifocal ectopic Purkinje-related premature contractions (MEPPC). Most previous reports of MEPPC have implicated SCN5A variants such as R222Q that neutralize positive charges in the S4 voltage sensor helix of the channel protein Na V 1.5 and generate a gating pore current. Methods and Results E171 is a highly conserved negatively-charged residue located in the S2 transmembrane helix of Na V 1.5 domain I. E171 is a key component of the Gating Charge Transfer Center, a region thought to be critical for normal movement of the S4 voltage sensor helix. We used heterologous expression, CRISPR-edited induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), and molecular dynamics simulations to demonstrate that E171Q generates a gating pore current, which was suppressed by a low concentration of flecainide (IC50 = 0.71±0.07 µM). R222Q shifts voltage dependence of activation and inactivation in a negative direction but we observed positive shifts with E171Q. E171Q iPSC-CMs demonstrated abnormal spontaneous activity and prolonged action potentials. Molecular dynamics simulations revealed that both R222Q and E171Q proteins generate a water-filled permeation pathway that underlies generation of the gating pore current. Conclusion Previously identified MEPPC-associated variants that create gating pore currents are located in positively-charged residues in the S4 voltage sensor and generate negative shifts in the voltage dependence of activation and inactivation. We demonstrate that neutralizing a negatively charged S2 helix residue in the Gating Charge Transfer Center generates positive shifts but also create a gating pore pathway. These findings implicate the gating pore pathway as the primary functional and structural determinant of MEPPC and widen the spectrum of variants that are associated with gating pore-related disease in voltage-gated ion channels.

BACKGROUND Variants in the KCNQ1 gene, encoding the a- subunit of the slow component of delayed recti fi er K + channel Kv7.1, cause long QT syndrome (LQTS) type 1. The location of variants may be one of the factors in determining prognosis. However, detailed genotype-phenotype relationships associated with C-terminus variants remain unelucidated. OBJECTIVE We investigated the clinical characteristics and variant-speci fi c arrhythmic risks in patients with LQTS carrying Kv7.1 C-terminus variants. METHODS The study comprises 202 consecutive patients with LQTS (98 probands and 104 family members) who carry a rare heterozygous variant in the Kv7.1 C-terminus. Their clinical characteristics and arrhythmic events were investigated. RESULTS We identi fi ed 36 unique C-terminus variants (25 missense and 11 non-missense). The p.R366W variant was identi fi ed in 8 families, and p.T587M was identi fi ed in 21 families in large numbers from northwestern Japan. As for the location of the variant, we found that the variants in highly conserved regions and nonhelical domains were associated with longer QTc intervals compared with the variants in other regions. Both p.R366W and p.T587M variants are located in the highly conserved and functionally pivotal regions close to helices A and D, which are associated with calmodulin binding and channel assembly (tetramerization), respectively. The probands carrying p.T587M and p.R366W variants had worse arrhythmia outcomes compared with those with other C-terminus variants. The haplotype analysis of p.T587M families was suggestive of a founder effect. CONCLUSION The arrhythmic risk of C-terminus variants in Kv7.1 in LQTS is not homogeneous, and locations of variants can be a determining factor for prognosis.

Background As gene-specific therapy for inherited retinal dystrophy (IRD) advances, unified variant interpretation across institutes is becoming increasingly important. This study aims to update the genetic findings of 86 retinitis pigmentosa (RP)-related genes in a large number of Japanese patients with RP by applying the standardised variant interpretation guidelines for Japanese patients with IRD (J-IRD-VI guidelines) built upon the American College of Medical Genetics and Genomics and the Association for Molecular Pathology rules, and assess the contribution of these genes in RP-allied diseases. Methods We assessed 2325 probands with RP (n=2155, including n=1204 sequenced previously with the same sequencing panel) and allied diseases (n=170, newly analysed), including Usher syndrome, Leber congenital amaurosis and cone-rod dystrophy (CRD). Target sequencing using a panel of 86 genes was performed. The variants were interpreted according to the J-IRD-VI guidelines. Results A total of 3564 variants were detected, of which 524 variants were interpreted as pathogenic or likely pathogenic. Among these 524 variants, 280 (53.4%) had been either undetected or interpreted as variants of unknown significance or benign variants in our earlier study of 1204 patients with RP. This led to a genetic diagnostic rate in 38.6% of patients with RP, with EYS accounting for 46.7% of the genetically solved patients, showing a 9% increase in diagnostic rate from our earlier study. The genetic diagnostic rate for patients with CRD was 28.2%, with RP-related genes significantly contributing over other allied diseases. Conclusion A large-scale genetic analysis using the J-IRD-VI guidelines highlighted the population-specific genetic findings for Japanese patients with IRD; these findings serve as a foundation for the clinical application of gene-specific therapies.

BACKGROUND: Long QT syndrome is a lethal arrhythmia syndrome, frequently caused by rare loss-of-function variants in the potassium channel encoded by KCNH2. Variant classification is difficult, often because of lack of functional data. Moreover, variant-based risk stratification is also complicated by heterogenous clinical data and incomplete penetrance. Here we sought to test whether variant-specific information, primarily from high-throughput functional assays, could improve both classification and cardiac event risk stratification in a large, harmonized cohort of KCNH2 missense variant heterozygotes. METHODS: We quantified cell-surface trafficking of 18 796 variants in KCNH2 using a multiplexed assay of variant effect (MAVE). We recorded KCNH2 current density for 533 variants by automated patch clamping. We calibrated the strength of evidence of MAVE data according to ClinGen guidelines. We deeply phenotyped 1458 patients with KCNH2 missense variants, including QTc, cardiac event history, and mortality. We correlated variant functional data and Bayesian long QT syndrome penetrance estimates with cohort phenotypes and assessed hazard ratios for cardiac events. RESULTS: Variant MAVE trafficking scores and automated patch clamping peak tail currents were highly correlated (Spearman rank-order rho=0.69; n=433). The MAVE data were found to provide up to pathogenic very strong evidence for severe loss-of-function variants. In the cohort, both functional assays and Bayesian long QT syndrome penetrance estimates were significantly predictive of cardiac events when independently modeled with patient sex and corrected QT interval (QTc); however, MAVE data became nonsignificant when peak tail current and penetrance estimates were also available. The area under the receiver operator characteristic curve for 20-year event outcomes based on patient-specific sex and QTc (area under the curve, 0.80 [0.76-0.83]) was improved with prospectively available penetrance scores conditioned on MAVE (area under the curve, 0.86 [0.83-0.89]) or attainable automated patch clamping peak tail current data (area under the curve, 0.84 [0.81-0.88]). CONCLUSIONS: High-throughput KCNH2 variant MAVE data meaningfully contribute to variant classification at scale, whereas long QT syndrome penetrance estimates and automated patch clamping peak tail current measurements meaningfully contribute to risk stratification of cardiac events in patients with heterozygous KCNH2 missense variants.

Aims While variants in KCNQ1 are the commonest cause of the congenital long QT syndrome, we and others find only a small IKs in cardiomyocytes from human-induced pluripotent stem cells (iPSC-CMs) or human ventricular myocytes. Methods and results We studied population control iPSC-CMs and iPSC-CMs from a patient with Jervell and Lange-Nielsen (JLN) syndrome due to compound heterozygous loss-of-function (LOF) KCNQ1 variants. We compared the effects of pharmacologic I-Ks block to those of genetic KCNQ1 ablation, using JLN cells, cells homozygous for the KCNQ1 LOF allele G643S, or siRNAs reducing KCNQ1 expression. We also studied the effects of two blockers of I-Kr, the other major cardiac repolarizing current, in the setting of pharmacologic or genetic ablation of KCNQ1: moxifloxacin, associated with a very low risk of drug-induced long QT, and dofetilide, a high-risk drug. In control cells, a small I-Ks was readily recorded but the pharmacologic I-Ks block produced no change in action potential duration at 90% repolarization (APD(90)). In contrast, in cells with genetic ablation of KCNQ1 (JLN), baseline APD(90) was markedly prolonged compared with control cells (469 +/- 20 vs. 310 +/- 16 ms). JLN cells displayed increased sensitivity to acute I-Kr block: the concentration (mu M) of moxifloxacin required to prolong APD(90) 100 msec was 237.4 [median, interquartile range (IQR) 100.6-391.6, n = 7] in population cells vs. 23.7 (17.3-28.7, n = 11) in JLN cells. In control cells, chronic moxifloxacin exposure (300 mu M) mildly prolonged APD(90) (10%) and increased I-Ks, while chronic exposure to dofetilide (5 nM) produced greater prolongation (67%) and no increase in I-Ks. However, in the siRNA-treated cells, moxifloxacin did not increase I-Ks and markedly prolonged APD(90). Conclusion Our data strongly suggest that KCNQ1 expression modulates baseline cardiac repolarization, and the response to IKr block, through mechanisms beyond simply generating IKs. [GRAPHICS] .

Copy-number variants (CNVs) play a substantial role in the molecular pathogenesis of hereditary disease and cancer, as well as in normal human interindividual variation. However, they are still rather difficult to identify in mainstream sequencing projects, especially involving exome sequencing, because they often occur in DNA regions that are not targeted for analysis. To overcome this problem, we developed OFF-PEAK, a user-friendly CNV detection tool that builds on a denoising approach and the use of "off-target" DNA reads, which are usually discarded by sequencing pipelines. We benchmarked OFF-PEAK on data from targeted sequencing of 96 cancer samples, as well as 130 exomes of individuals with inherited retinal disease from three different populations. For both sets of data, OFF-PEAK demonstrated excellent performance (>95% sensitivity and >80% specificity vs. experimental validation) in detecting CNVs from in silico data alone, indicating its immediate applicability to molecular diagnosis and genetic research.

Long QT syndrome (LQTS) is a lethal arrhythmia syndrome, frequently caused by rare loss-of-function variants in the potassium channel encoded by KCNH2. Variant-based risk stratification is complicated by heterogenous clinical data, incomplete penetrance, and low-throughput functional data. While variant-specific functional data can assist with variant classification, whether functional data can assist with prediction of outcomes is unknown.

The presence of multiple pathogenic variants in desmosomal genes ( DSC2 , DSG2 , DSP , JUP , and PKP2 ) in patients with arrhythmogenic right ventricular cardiomyopathy (ARVC) has been linked to a severe phenotype. However, the pathogenicity of variants is reclassified frequently, which may result in a changed clinical risk prediction. Here, we present the collection, reclassification, and clinical outcome correlation for the largest series of ARVC patients carrying multiple desmosomal pathogenic variants to date ( n = 331). After reclassification, only 29% of patients remained carriers of two (likely) pathogenic variants. They reached the composite endpoint (ventricular arrhythmias, heart failure, and death) significantly earlier than patients with one or no remaining reclassified variant (hazard ratios of 1.9 and 1.8, respectively). Periodic reclassification of variants contributes to more accurate risk stratification and subsequent clinical management strategy.

AIMS More than one-third of type 2 long QT syndrome (LQT2) patients carry KCNH2 non-missense variants that can result in haploinsufficiency (HI), leading to mechanistic loss-of-function. However, their clinical phenotypes have not been fully investigated. The remaining two-thirds of patients harbour missense variants, and past studies uncovered that most of these variants cause trafficking deficiency, resulting in different functional changes: either HI or dominant-negative (DN) effects. In this study, we examined the impact of altered molecular mechanisms on clinical outcomes in LQT2 patients. METHODS AND RESULTS We included 429 LQT2 patients (234 probands) carrying a rare KCNH2 variant from our patient cohort undergoing genetic testing. Non-missense variants showed shorter corrected QT (QTc) and less arrhythmic events (AEs) than missense variants. We found that 40% of missense variants in this study were previously reported as HI or DN. Non-missense and HI-groups had similar phenotypes, while both exhibited shorter QTc and less AEs than the DN-group. Based on previous work, we predicted the functional change of the unreported variants-whether they cause HI or DN via altered functional domains-and stratified them as predicted HI (pHI)- or pDN-group. The pHI-group including non-missense variants exhibited milder phenotypes compared to the pDN-group. Multivariable Cox model showed that the functional change was an independent risk of AEs (P = 0.005). CONCLUSION Stratification based on molecular biological studies enables us to better predict clinical outcomes in the patients with LQT2.

Pre-zygotic interspecies incompatibility in angiosperms is an important mechanism to prevent unfavourable hybrids between species. Here we report our identification of STIGMATIC PRIVACY 2 (SPRI2), a transcription factor that has a zinc-finger domain and regulates interspecies barriers in Arabidopsis thaliana, via genome-wide association study. Knockout analysis of SPRI2/SRS7 and its paralogue SPRI2-like/SRS5 demonstrated their necessity in rejecting male pollen from other species within female pistils. Additionally, they govern mRNA transcription of xylan O-acetyltransferases (TBL45 and TBL40) related to cell wall modification, alongside SPRI1, a pivotal transmembrane protein for interspecific pollen rejection. SPRI2/SRS7 is localized as condensed structures in the nucleus formed via liquid–liquid phase separation (LLPS), and a prion-like sequence in its amino-terminal region was found to be responsible for the formation of the condensates. The LLPS-regulated SPRI2/SRS7 discovered in this study may contribute to the establishment of interspecific reproductive barriers through the transcriptional regulation of cell wall modification genes and SPRI1. Pre-zygotic reproductive barriers are a vital defence against interspecific pollen transfers in plants. This study shows that SPRI2/SRS7, a transcription factor, regulates the formation of interspecific reproductive barriers in Arabidopsis.