Search

Pathogenic variants in genes encoding ion channels are frequently discovered in monogenic disorders associated with epilepsy and neurodevelopmental disorders. This review covers advances in the use of automated patch clamp recording for determining the functional consequences of epilepsy-associated ion channel variants and the use of induced pluripotent stem cell (iPSC) derived neurons for in-depth investigations of the physiological consequences of such variants. The combination of these advanced technologies was a focus of the recently completed NINDS-funded Channelopathy-associated Epilepsy Research Center without Walls., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2024.)

SCN2A-related disorders secondary to altered function in the voltage-gated sodium channel Nav1.2 are rare, with clinically heterogeneous expressions that include epilepsy, autism and multiple severe to profound impairments and other conditions. To advance understanding of the clinical phenotypes and their relationship to channel function, 81 patients (36 female, 44%, median age 5.4 years) with 69 unique SCN2A variants were systematically phenotyped and their Nav1.2 channel function systematically assessed. Participants were recruited through the FamileSCN2A Foundation. Primary phenotype (epilepsy of neonatal onset, n = 27; infant onset, n = 18; and later onset n = 24; and autism without seizures, n = 12) was strongly correlated with a non-seizure severity index (P = 0.002), which was based on presence of severe impairments in gross motor, fine motor, communication abilities, gastrostomy tube dependence and diagnosis of cortical visual impairment and scoliosis. Non-seizure severity was greatest in the neonatal-onset group and least in the autism group (P = 0.002). Children with the lowest severity indices were still severely impaired, as reflected by an average Vineland Adaptive Behavior composite score of 49.5 (>3 standard deviations below the norm-referenced mean of the test). Epileptic spasms were significantly more common in infant-onset (67%) than in neonatal (22%) or later-onset (29%) epilepsy (P = 0.007). Primary phenotype was also strongly correlated with variant function (P < 0.0001); gain-of-function and mixed function variants predominated in neonatal-onset epilepsy, shifting to moderate loss of function in infant-onset epilepsy and to severe and complete loss of function in later-onset epilepsy and autism groups. Exploratory cluster analysis identified five groups, representing: (i) primarily later-onset epilepsy with moderate loss-of-function variants and low severity indices; (ii) mostly infant-onset epilepsy with moderate loss-of-function variants but higher severity indices; and (iii) late-onset and autism only, with the lowest severity indices (mostly zero) and severe/complete loss-of-function variants. Two exclusively neonatal clusters were distinguished from each other largely on non-seizure severity scores and secondarily on variant function. The relationship between primary phenotype and variant function emphasizes the role of developmental factors in the differential clinical expression of SCN2A variants based on their effects on Nav1.2 channel function. The non-seizure severity of SCN2A disorders depends on a combination of the age at seizure onset (primary phenotype) and variant function. As precision therapies for SCN2A-related disorders advance towards clinical trials, knowledge of the relationship between variant function and clinical disease expression will be valuable for identifying appropriate patients for these trials and in selecting efficient clinical outcomes., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Developmental causes of the most common arrhythmia, atrial fibrillation (AF), are poorly defined, with compensation potentially masking arrhythmic risk. Here, we delete 9 amino acids (Δ9) within a conserved domain of the giant protein titin's A-band in zebrafish and human-induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-aCMs). We find that ttna Δ9/Δ9 zebrafish embryos' cardiac morphology is perturbed and accompanied by reduced functional output, but ventricular function recovers within days. Despite normal ventricular function, ttna Δ9/Δ9 adults exhibit AF and atrial myopathy, which are recapitulated in TTN Δ9/Δ9 -hiPSC-aCMs. Additionally, action potential is shortened and slow delayed rectifier potassium current ( I Ks ) is increased due to aberrant atrial natriuretic peptide (ANP) levels. Strikingly, suppression of I Ks in both models prevents AF and improves atrial contractility. Thus, a small internal deletion in titin causes developmental abnormalities that increase the risk of AF via ion channel remodeling, with implications for patients who harbor disease-causing variants in sarcomeric proteins., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

KCNQ2 variants in children with neurodevelopmental impairment are difficult to assess due to their heterogeneity and unclear pathogenic mechanisms. We describe a child with neonatal-onset epilepsy, developmental impairment of intermediate severity, and KCNQ2 G256W heterozygosity. Analyzing prior KCNQ2 channel cryoelectron microscopy models revealed G256 as a node of an arch-shaped non-covalent bond network linking S5, the pore turret, and the ion path. Co-expression with G256W dominantly suppressed conduction by wild-type subunits in heterologous cells. Ezogabine partly reversed this suppression. G256W/+ mice have epilepsy leading to premature deaths. Hippocampal CA1 pyramidal cells from G256W/+ brain slices showed hyperexcitability. G256W/+ pyramidal cell KCNQ2 and KCNQ3 immunolabeling was significantly shifted from axon initial segments to neuronal somata. Despite normal mRNA levels, G256W/+ mouse KCNQ2 protein levels were reduced by about 50%. Our findings indicate that G256W pathogenicity results from multiplicative effects, including reductions in intrinsic conduction, subcellular targeting, and protein stability. These studies provide evidence for an unexpected and novel role for the KCNQ2 pore turret and introduce a valid animal model of KCNQ2 encephalopathy. Our results, spanning structure to behavior, may be broadly applicable because the majority of KCNQ2 encephalopathy patients share variants near the selectivity filter.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) recapitulate numerous disease and drug response phenotypes, but cell immaturity may limit their accuracy and fidelity as a model system. Cell culture medium modification is a common method for enhancing maturation, yet prior studies have used complex media with little understanding of individual component contribution, which may compromise long-term hiPSC-CM viability. Here, we developed high-throughput methods to measure hiPSC-CM maturation, determined factors that enhanced viability, and then systematically assessed the contribution of individual maturation medium components. We developed a medium that is compatible with extended culture. We discovered that hiPSC-CM maturation can be sub-specified into electrophysiological/EC coupling, metabolism, and gene expression and that induction of these attributes is largely independent. In this work, we establish a defined baseline for future studies of cardiomyocyte maturation. Furthermore, we provide a selection of medium formulae, optimized for distinct applications and priorities, that promote measurable attributes of maturation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Pathogenic variants in SCN8A, which encodes the voltage-gated sodium (NaV) channel NaV1.6, associate with neurodevelopmental disorders, including developmental and epileptic encephalopathy. Previous approaches to determine SCN8A variant function may be confounded by use of a neonatally expressed, alternatively spliced isoform of NaV1.6 (NaV1.6N) and engineered mutations rendering the channel tetrodotoxin (TTX) resistant. We investigated the impact of SCN8A alternative splicing on variant function by comparing the functional attributes of 15 variants expressed in 2 developmentally regulated splice isoforms (NaV1.6N, NaV1.6A). We employed automated patch clamp recording to enhance throughput, and developed a neuronal cell line (ND7/LoNav) with low levels of endogenous NaV current to obviate the need for TTX-resistance mutations. Expression of NaV1.6N or NaV1.6A in ND7/LoNav cells generated NaV currents with small, but significant, differences in voltage dependence of activation and inactivation. TTX-resistant versions of both isoforms exhibited significant functional differences compared with the corresponding WT channels. We demonstrated that many of the 15 disease-associated variants studied exhibited isoform-dependent functional effects, and that many of the studied SCN8A variants exhibited functional properties that were not easily classified as either gain- or loss-of-function. Our work illustrates the value of considering molecular and cellular context when investigating SCN8A variants.

Background: Many cardiomyopathy-associated FLNC pathogenic variants are heterozygous truncations, and FLNC pathogenic variants are associated with arrhythmias. Arrhythmia triggers in filaminopathy are incompletely understood., Methods and Results: We describe an individual with biallelic FLNC pathogenic variants, p.Arg650X and c.970-4A>G, with peripartum cardiomyopathy and ventricular arrhythmias. We also describe clinical findings in probands with FLNC variants including Val2715fs87X, Glu2458Serfs71X, Phe106Leu, and c.970-4A>G with hypertrophic and dilated cardiomyopathy, atrial fibrillation, and ventricular tachycardia. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated. The FLNC truncation, Arg650X/c.970-4A>G, showed a marked reduction in filamin C protein consistent with biallelic loss of function mutations. To assess loss of filamin C, gene editing of a healthy control iPSC line was used to generate a homozygous FLNC disruption in the actin binding domain. Because filamin C has been linked to protein quality control, we assessed the necessity of filamin C in iPSC-CMs for response to the proteasome inhibitor bortezomib. After exposure to low-dose bortezomib, FLNC- null iPSC-CMs showed an increase in the chaperone proteins BAG3, HSP70 (heat shock protein 70), and HSPB8 (small heat shock protein B8) and in the autophagy marker LC3I/II. FLNC null iPSC-CMs had prolonged electric field potential, which was further prolonged in the presence of low-dose bortezomib. FLNC null engineered heart tissues had impaired function after low-dose bortezomib., Conclusions: FLNC pathogenic variants associate with a predisposition to arrhythmias, which can be modeled in iPSC-CMs. Reduction of filamin C prolonged field potential, a surrogate for action potential, and with bortezomib-induced proteasome inhibition, reduced filamin C led to greater arrhythmia potential and impaired function.

Myocarditis is clinically characterized by chest pain, arrhythmias, and heart failure, and treatment is often supportive. Mutations in DSP, a gene encoding the desmosomal protein desmoplakin, have been increasingly implicated in myocarditis. To model DSP-associated myocarditis and assess the role of innate immunity, we generated engineered heart tissues (EHTs) using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients with heterozygous DSP truncating variants (DSPtvs) and a gene-edited homozygous deletion cell line (DSP-/-). At baseline, DSP-/- EHTs displayed a transcriptomic signature of innate immune activation, which was mirrored by cytokine release. Importantly, DSP-/- EHTs were hypersensitive to Toll-like receptor (TLR) stimulation, demonstrating more contractile dysfunction compared with isogenic controls. Relative to DSP-/- EHTs, heterozygous DSPtv EHTs had less functional impairment. DSPtv EHTs displayed heightened sensitivity to TLR stimulation, and when subjected to strain, DSPtv EHTs developed functional deficits, indicating reduced contractile reserve compared with healthy controls. Colchicine or NF-κB inhibitors improved strain-induced force deficits in DSPtv EHTs. Genomic correction of DSP p.R1951X using adenine base editing reduced inflammatory biomarker release from EHTs. Thus, EHTs replicate electrical and contractile phenotypes seen in human myocarditis, implicating cytokine release as a key part of the myogenic susceptibility to inflammation. The heightened innate immune activation and sensitivity are targets for clinical intervention.

Pathogenic variants in KCNB1 are associated with a neurodevelopmental disorder spectrum that includes global developmental delays, cognitive impairment, abnormal electroencephalogram (EEG) patterns, and epilepsy with variable age of onset and severity. Additionally, there are prominent behavioral disturbances, including hyperactivity, aggression, and features of autism spectrum disorder. The most frequently identified recurrent variant is KCNB1-p.R306C, a missense variant located within the S4 voltage-sensing transmembrane domain. Individuals with the R306C variant exhibit mild to severe developmental delays, behavioral disorders, and a diverse spectrum of seizures. Previous in vitro characterization of R306C described altered sensitivity and cooperativity of the voltage sensor and impaired capacity for repetitive firing of neurons. Existing Kcnb1 mouse models include dominant negative missense variants, as well as knockout and frameshifts alleles. While all models recapitulate key features of KCNB1 encephalopathy, mice with dominant negative alleles were more severely affected. In contrast to existing loss-of-function and dominant-negative variants, KCNB1-p.R306C does not affect channel expression, but rather affects voltage-sensing. Thus, modeling R306C in mice provides a novel opportunity to explore impacts of a voltage-sensing mutation in Kcnb1. Using CRISPR/Cas9 genome editing, we generated the Kcnb1 R306C mouse model and characterized the molecular and phenotypic effects. Consistent with the in vitro studies, neurons from Kcnb1 R306C mice showed altered excitability. Heterozygous and homozygous R306C mice exhibited hyperactivity, altered susceptibility to chemoconvulsant-induced seizures, and frequent, long runs of slow spike wave discharges on EEG, reminiscent of the slow spike and wave activity characteristic of Lennox Gastaut syndrome. This novel model of channel dysfunction in Kcnb1 provides an additional, valuable tool to study KCNB1 encephalopathies. Furthermore, this allelic series of Kcnb1 mouse models will provide a unique platform to evaluate targeted therapies., Competing Interests: Declaration of competing interest The authors declare no competing interests related to this study., (Copyright © 2023. Published by Elsevier Inc.)

Rationale: Few studies of the effect of the dynamic physiologic changes during pregnancy on plasma concentrations of fluoxetine (FLX) have been published., Objectives: We determined the change in concentration to dose (C/D) ratios of R- and S-FLX and R- and S-norfluoxetine monthly during pregnancy and postpartum, assessed their relationships to cytochrome P450 (CYP) 2D6 and CYP2C9 metabolizer phenotypes, and evaluated the course of their depressive and anxiety symptoms., Methods: In this observational study, 10 FLX-treated pregnant individuals provided blood samples at steady state every 4 weeks during pregnancy and once postpartum for measurement of plasma FLX and norfluoxetine enantiomer concentrations. Participants were genotyped for variants in CYP2C9 and CYP2D6 using commercial assays with Taqman probes. At each assessment, depressive and anxiety symptoms were quantified., Results: The C/D ratios of all FLX and norfluoxetine enantiomers, and the active moiety, decreased steadily through pregnancy and rose after birth. In the final trimester, the mean C/D ratio of the active moiety was 24.9% lower compared with the mean nonpregnant, 12-week postpartum C/D ratio. One individual with CYP2D6 ultrarapid metabolizer status was prescribed the highest FLX dose among participants. In these treated individuals, the mean depressive and anxiety symptoms remained in the mild range across the perinatal period., Conclusions: These data do not support a recommendation for routine plasma concentration monitoring or CYP2D6 pharmacogenetic testing for pregnant people treated with FLX; however, monitoring for symptom relapse is recommended because of declining plasma drug concentrations., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Alternating hemiplegia of childhood (AHC) is a rare neurodevelopment disorder that is typically characterized by debilitating episodic attacks of hemiplegia, seizures, and intellectual disability. Over 85% of individuals with AHC have a de novo missense variant in ATP1A3 encoding the catalytic α3 subunit of neuronal Na +/ K + ATPases. The remainder of the patients are genetically unexplained. Here, we used next-generation sequencing to search for the genetic cause of 26 ATP1A3-negative index patients with a clinical presentation of AHC or an AHC-like phenotype. Three patients had affected siblings. Using targeted sequencing of exonic, intronic, and flanking regions of ATP1A3 in 22 of the 26 index patients, we found no ultra-rare variants. Using exome sequencing, we identified the likely genetic diagnosis in 9 probands (35%) in five genes, including RHOBTB2 (n = 3), ATP1A2 (n = 3), ANK3 (n = 1), SCN2A (n = 1), and CHD2 (n = 1). In follow-up investigations, two additional ATP1A3-negative individuals were found to have rare missense SCN2A variants, including one de novo likely pathogenic variant and one likely pathogenic variant for which inheritance could not be determined. Functional evaluation of the variants identified in SCN2A and ATP1A2 supports the pathogenicity of the identified variants. Our data show that genetic variants in various neurodevelopmental genes, including SCN2A, lead to AHC or AHC-like presentation. Still, the majority of ATP1A3-negative AHC or AHC-like patients remain unexplained, suggesting that other mutational mechanisms may account for the phenotype or that cases may be explained by oligo- or polygenic risk factors., (© 2023. The Author(s), under exclusive licence to European Society of Human Genetics.)

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia treatable with antiarrhythmic drugs; however, patient responses remain highly variable. Human induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs) are useful for discovering precision therapeutics, but current platforms yield phenotypically immature cells and are not easily scalable for high-throughput screening. Here, primary adult atrial, but not ventricular, fibroblasts induced greater functional iPSC-aCM maturation, partly through connexin-40 and ephrin-B1 signaling. We developed a protein patterning process within multiwell plates to engineer patterned iPSC-aCM and atrial fibroblast coculture (PC) that significantly enhanced iPSC-aCM structural, electrical, contractile, and metabolic maturation for 6+ weeks compared to conventional mono-/coculture. PC displayed greater sensitivity for detecting drug efficacy than monoculture and enabled the modeling and pharmacological or gene editing treatment of an AF-like electrophysiological phenotype due to a mutated sodium channel. Overall, PC is useful for elucidating cell signaling in the atria, drug screening, and modeling AF.

Background: Sudden unexpected death in children is a tragic event. Understanding the genetics of sudden death in the young (SDY) enables family counseling and cascade screening. The objective of this study was to characterize genetic variation in an SDY cohort using whole genome sequencing., Methods: The SDY Case Registry is a National Institutes of Health/Centers for Disease Control and Prevention surveillance effort to discern the prevalence, causes, and risk factors for SDY. The SDY Case Registry prospectively collected clinical data and DNA biospecimens from SDY cases < 20 years of age. SDY cases were collected from medical examiner and coroner offices spanning 13 US jurisdictions from 2015 to 2019. The cohort included 211 children (median age 0.33 year; range 0-20 years), determined to have died suddenly and unexpectedly and from whom DNA biospecimens for DNA extractions and next-of-kin consent were ascertained. A control cohort consisted of 211 randomly sampled, sex- and ancestry-matched individuals from the 1000 Genomes Project. Genetic variation was evaluated in epilepsy, cardiomyopathy, and arrhythmia genes in the SDY and control cohorts. American College of Medical Genetics/Genomics guidelines were used to classify variants as pathogenic or likely pathogenic. Additionally, pathogenic and likely pathogenic genetic variation was identified using a Bayesian-based artificial intelligence (AI) tool., Results: The SDY cohort was 43% European, 29% African, 3% Asian, 16% Hispanic, and 9% with mixed ancestries and 39% female. Six percent of the cohort was found to harbor a pathogenic or likely pathogenic genetic variant in an epilepsy, cardiomyopathy, or arrhythmia gene. The genomes of SDY cases, but not controls, were enriched for rare, potentially damaging variants in epilepsy, cardiomyopathy, and arrhythmia-related genes. A greater number of rare epilepsy genetic variants correlated with younger age at death., Conclusions: While damaging cardiomyopathy and arrhythmia genes are recognized contributors to SDY, we also observed an enrichment in epilepsy-related genes in the SDY cohort and a correlation between rare epilepsy variation and younger age at death. These findings emphasize the importance of considering epilepsy genes when evaluating SDY., (© 2024. The Author(s).)

The recent explosion of epilepsy genetic testing has created challenges for interpretation of gene variants. Assessments of the functional consequences of genetic variants either by predictive or experimental strategies can contribute to estimating pathogenicity, but there is no consensus on which approach is best. The Special Interest Group on Epilepsy Genetics hosted a session during the Annual American Epilepsy Society Meeting in December 2022 to discuss this topic. The session featured a debate of the relative advantages and limitations of predicting (prophecy) versus experimentally determining (empiricism) variant function using ion channel gene variants as examples. This commentary summarizes these discussions., (© 2023 The Authors. Epilepsia published by Wiley Periodicals LLC on behalf of International League Against Epilepsy.)

Technologies capable of programmable translation activation offer strategies to develop therapeutics for diseases caused by insufficient gene expression. Here, we present "translation-activating RNAs" (taRNAs), a bifunctional RNA-based molecular technology that binds to a specific mRNA of interest and directly upregulates its translation. taRNAs are constructed from a variety of viral or mammalian RNA internal ribosome entry sites (IRESs) and upregulate translation for a suite of target mRNAs. We minimize the taRNA scaffold to 94 nucleotides, identify two translation initiation factor proteins responsible for taRNA activity, and validate the technology by amplifying SYNGAP1 expression, a haploinsufficiency disease target, in patient-derived cells. Finally, taRNAs are suitable for delivery as RNA molecules by lipid nanoparticles (LNPs) to cell lines, primary neurons, and mouse liver in vivo. taRNAs provide a general and compact nucleic acid-based technology to upregulate protein production from endogenous mRNAs, and may open up possibilities for therapeutic RNA research., (© 2023. The Author(s).)

Background: Pathogenic variants in genes encoding CaM (calmodulin) are associated with a life-threatening ventricular arrhythmia syndrome (calmodulinopathy). The in vivo consequences of CaM variants have not been studied extensively and there is incomplete understanding of the genotype-phenotype relationship for recurrent variants. We investigated effects of different factors on calmodulinopathy phenotypes using 2 mouse models with a recurrent pathogenic variant (N98S) in Calm1 or Calm2 ., Methods: Genetically engineered mice with heterozygous N98S pathogenic variants in Calm1 or Calm2 were generated. Differences between the sexes and affected genes were assessed using multiple physiological assays at the cellular and whole animal levels. Statistical significance among groups was evaluated using 1-way ANOVA or the Kruskal-Wallis test when data were not normally distributed., Results: Calm1 N98S/+ ( Calm1 S/+ ) or Calm2 N98S/+ ( Calm2 S/+ ) mice exhibited sinus bradycardia and were more susceptible to arrhythmias after exposure to epinephrine and caffeine. Male Calm1 S/+ mice had the most severe arrhythmia phenotype with evidence of early embryonic lethality, greater susceptibility for arrhythmic events, frequent premature beats, corrected QT prolongation, and more heart rate variability after epinephrine and caffeine than females with the same genotype. Calm2 S/+ mice exhibited a less severe phenotype, with female Calm2 S/+ mice having the least severe arrhythmia susceptibility. Flecainide was not effective in preventing arrhythmias in heterozygous CaM-N98S mice. Intracellular Ca 2+ transients observed in isolated ventricular cardiomyocytes from male heterozygous CaM-N98S mice had lower peak amplitudes and slower sarcoplasmic reticulum Ca 2+ release following in vitro exposure to epinephrine and caffeine, which were not observed in cardiomyocytes from heterozygous female CaM-N98S mice., Conclusions: We report heterogeneity in arrhythmia susceptibility and cardiomyocyte Ca 2+ dynamics among male and female mice heterozygous for a recurrent pathogenic variant in Calm1 or Calm2 , illustrating a complex calmodulinopathy phenotype in vivo. Further investigation of sex and genetic differences may help identify the molecular basis for this heterogeneity., Competing Interests: Disclosures None.

It is challenging to apply traditional mutational scanning to voltage-gated sodium channels (Na V s) and functionally annotate the large number of coding variants in these genes. Using a cytosine base editor and a pooled viability assay, we screen a library of 368 guide RNAs (gRNAs) tiling Na V 1.2 to identify more than 100 gRNAs that change Na V 1.2 function. We sequence base edits made by a subset of these gRNAs to confirm specific variants that drive changes in channel function. Electrophysiological characterization of these channel variants validates the screen results and provides functional mechanisms of channel perturbation. Most of the changes caused by these gRNAs are classifiable as loss of function along with two missense mutations that lead to gain of function in Na V 1.2 channels. This two-tiered strategy to functionally characterize ion channel protein variants at scale identifies a large set of loss-of-function mutations in Na V 1.2., Competing Interests: Declaration of interests A.L.G. consults for and receives sponsored research funding from Praxis Precision Medicines and Neurocrine Biosciences for unrelated work., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Neuropsychiatric disorders (NPDs) are frequently co-morbid with epilepsy, but the biological basis of shared risk remains poorly understood. The 16p11.2 duplication is a copy number variant that confers risk for diverse NPDs including autism spectrum disorder, schizophrenia, intellectual disability and epilepsy. We used a mouse model of the 16p11.2 duplication (16p11.2 dup/+ ) to uncover molecular and circuit properties associated with this broad phenotypic spectrum, and examined genes within the locus capable of phenotype reversal. Quantitative proteomics revealed alterations to synaptic networks and products of NPD risk genes. We identified an epilepsy-associated subnetwork that was dysregulated in 16p11.2 dup/+ mice and altered in brain tissue from individuals with NPDs. Cortical circuits from 16p11.2 dup/+ mice exhibited hypersynchronous activity and enhanced network glutamate release, which increased susceptibility to seizures. Using gene co-expression and interactome analysis, we show that PRRT2 is a major hub in the epilepsy subnetwork. Remarkably, correcting Prrt2 copy number rescued aberrant circuit properties, seizure susceptibility and social deficits in 16p11.2 dup/+ mice. We show that proteomics and network biology can identify important disease hubs in multigenic disorders, and reveal mechanisms relevant to the complex symptomatology of 16p11.2 duplication carriers., (© 2023. The Author(s).)

Protein-protein interactions are essential for normal cellular processes and signaling events. Defining these interaction networks is therefore crucial for understanding complex cellular functions and interpretation of disease-associated gene variants. We need to build a comprehensive picture of the interactions, their affinities and interdependencies in the specific organ to decipher hitherto poorly understood signaling mechanisms through ion channels. Here we report the experimental identification of the ensemble of protein interactors for 13 types of ion channels in murine cardiac tissue. Of these, we validated the functional importance of ten interactors on cardiac electrophysiology through genetic knockouts in zebrafish, gene silencing in mice, super-resolution microscopy and patch clamp experiments. Furthermore, we establish a computational framework to reconstruct human cardiomyocyte ion channel networks from deep proteome mapping of human heart tissue and human heart single-cell gene expression data. Finally, we integrate the ion channel interactome with human population genetics data to identify proteins that influence the electrocardiogram (ECG). We demonstrate that the combined channel network is enriched for proteins influencing the ECG, with 44% of the network proteins significantly associated with an ECG phenotype. Altogether, we define interactomes of 13 major cardiac ion channels, contextualize their relevance to human electrophysiology and validate functional roles of ten interactors, including two regulators of the sodium current (epsin-2 and gelsolin). Overall, our data provide a roadmap for our understanding of the molecular machinery that regulates cardiac electrophysiology., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2023.)

Major depressive disorder (MDD) is a common disorder in pregnancy. Although sertraline is the most frequently prescribed antidepressant for pregnant people in the United States, limited information about its pharmacokinetics in pregnancy is available. Our objectives were to characterize plasma sertraline concentration to dose (C/D) ratios across pregnancy and postpartum and investigate the effect of pharmacogenetic variability on sertraline elimination. We performed a prospective observational cohort study in people with a singleton pregnancy ≤ 18 weeks gestation and a lifetime diagnosis of MDD at the 3 Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)-funded Obstetrical-Fetal Pharmacology Research Center sites. Subjects (N = 47) were receiving maintenance sertraline therapy and chose to continue it during pregnancy. Blood samples were obtained 24-hours postdose every 4 weeks across pregnancy and twice postpartum for measurement of plasma concentrations of sertraline and desmethylsertraline. Overall mean sertraline C/D ratios were decreased at study onset and remained consistently low until after delivery. During the last 4 weeks of pregnancy the mean sertraline C/D ratio (95% confidence interval (CI)), 0.25 (95% CI, 0.19, 0.3) ng/mL/dose (mg/day), was smaller than the mean ratio at ≥ 8 weeks after delivery, 0.32 (95% CI, 0.27, 0.37) ng/mL/dose (mg/day), a 22% difference. Mean sertraline/desmethylsertraline ratios were highest after birth, which confirmed increased sertraline elimination during pregnancy. Sertraline C/D ratios in participants with functional CYP2C19 activity did not change significantly during pregnancy, whereas ratios in participants with poor or intermediate CYP2C19 activity decreased by 51%. Exploratory pharmacogenomic analysis indicated that pregnant people with poor or intermediate CYP2C19 activity are at risk for subtherapeutic sertraline concentrations during pregnancy., (© 2022 The Authors. Clinical Pharmacology & Therapeutics © 2022 American Society for Clinical Pharmacology and Therapeutics.)

Objectives: We sought to describe the prevalence of midazolam treatment failure in children with refractory status epilepticus (RSE) and define a threshold dose associated with diminishing frequency of seizure cessation., Design: Single center retrospective cohort study., Setting: Single-center, quaternary-care PICU., Patients: Children younger than 18 years old admitted to the PICU from 2009 to 2018 who had RSE requiring a continuous midazolam infusion., Interventions: None., Measurements and Main Results: We identified individuals with RSE through a data analytics inquiry. Receiver operating characteristic (ROC) curve analysis and Youden's index were used to assess the midazolam dose threshold associated with the highest sensitivity and specificity in identifying seizure cessation. A logistic regression model was used to determine if there was an association between maximum midazolam dose and seizure cessation. Of the 45 patients who met inclusion criteria for this study, 27 (60%) had seizure cessation with a midazolam infusion, whereas 18 (40%) required an additional pentobarbital infusion for seizure cessation. There was an association between maximum midazolam dose and seizure cessation, with patients more likely to fail treatment when midazolam was administered at higher doses. The maximum midazolam dose displayed high area under the ROC curve value for seizure cessation, and the Youden's J index cut-off point was 525 μg/kg/hr. Treatment above this dose was associated with diminishing frequency of seizure cessation. The median time spent titrating midazolam above 500 μg/kg/hr for those patients who required pentobarbital for seizure cessation was 3.83 hours (interquartile range, 2.28-5.58 hr)., Conclusions: In pediatric patients with RSE requiring high dose midazolam, considerable time is spent titrating doses in a range (above 500 µg/kg/hr) that is associated with diminishing frequency of seizure cessation., Competing Interests: Dr. George’s institution received funding from Tevard Biosciences, Praxis Precision Medicines, and Neurocrine Biosciences; he received support for article research from the National Institutes of Health; and he received research grants from Tevard Biosciences, Praxis Precision Medicines, and Neurocrine Biosciences. The remaining authors have disclosed that they do not have any potential conflicts of interest., (Copyright © 2022 by the Society of Critical Care Medicine and the World Federation of Pediatric Intensive and Critical Care Societies.)

The genetic basis of many epilepsies is increasingly understood, giving rise to the possibility of precision treatments tailored to specific genetic etiologies. Despite this, current medical therapy for most epilepsies remains imprecise, aimed primarily at empirical seizure reduction rather than targeting specific disease processes. Intellectual and technological leaps in diagnosis over the past 10 years have not yet translated to routine changes in clinical practice. However, the epilepsy community is poised to make impressive gains in precision therapy, with continued innovation in gene discovery, diagnostic ability, and bioinformatics; increased access to genetic testing and counseling; fuller understanding of natural histories; agility and rigor in preclinical research, including strategic use of emerging model systems; and engagement of an evolving group of stakeholders (including patient advocates, governmental resources, and clinicians and scientists in academia and industry). In each of these areas, we highlight notable examples of recent progress, new or persistent challenges, and future directions. The future of precision medicine for genetic epilepsy looks bright if key opportunities on the horizon can be pursued with strategic and coordinated effort., (© 2022 International League Against Epilepsy.)