Ion Channel Variants Research Articles

Allele-Specific Editing of a Dominant SCN8A Epilepsy Variant Protects against Seizures and Lethality in a Murine Model.

Developmental and epileptic encephalopathies (DEEs) can result from dominant, gain of function variants of neuronal ion channels. More than 450 de novo missense variants of the sodium channel gene SCN8A have been identified in individuals with DEE. We studied a mouse model carrying the patient Scn8a variant p.Asn1768Asp. An AAV-PHP.eB virus carrying an allele-specific single guide RNA (sgRNA) was administered by intracerebroventricular injection. Cas9 was provided by an inherited transgene. Allele-specific disruption of the reading frame of the pathogenic transcript generated out-of-frame indels in 1/4 to 1/3 of transcripts throughout the brain. This editing efficiency was sufficient to rescue lethality and seizures. Neuronal hyperexcitability was reduced in cells expressing the virus. The data demonstrate efficient allele-specific editing of a dominant missense variant and support the feasibility of allele-specific therapy for DEE epilepsy. ANN NEUROL 2024;96:958-969.

The Need for Speed; Investigating Channelopathy-Associated Epilepsy Using High Throughput Electrophysiological Approaches.

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.

Genetic background of neonatal hypokalemia.

Neonatal hypokalemia (defined as a serum potassium level <3.5 mEq/L) is the most common electrolyte disorder encountered in clinical practice. In addition to common secondary causes, primary genetic etiologies are also closely associated with hypokalemia. Currently, a systematic characterization of these genetic disorders is lacking, making early recognition challenging and clinical management uncertain. This review will aid clinicians by summarizing the genetic background of neonatal hypokalemia from two aspects: (1) increased excretion of K+, whereby genetic factors primarily lead to increased renal Na+ influx, decreased H+ efflux, or reduced Cl- influx, ultimately resulting in increased K+ efflux; and (2) decreased extracellular distribution of K+, whereby genetic factors result in abnormalities in transmembrane ion channels, reducing outward potassium currents or generating inward cation leak currents. We describe over ten genetic diseases associated with neonatal hypokalemia, which involve pathogenic variants in dozens of genes and affect multiple target organs, including the kidneys, intestines, and skeletal muscle. For example, in the renal tubules, pathogenic variants in the SLC12A1 gene encoding the Na+-K+-2Cl- cotransporter lead to renal K+ loss, causing Bartter syndrome type I; in intestinal epithelial cells, pathogenic variants in the SLC26A3 gene result in a defective Cl⁻-HCO₃⁻ exchanger, causing congenital chloride diarrhea; and in skeletal muscle, pathogenic variants in the CACNA1S gene impact membrane calcium ion channels resulting in hypokalemic periodic paralysis. Given the wide variety of organs and genetic alterations that can contribute to neonatal hypokalemia, we believe this review will provide valuable insights for clinical diagnosis and treatment.

Multisite Validation of a Functional Assay to Adjudicate SCN5A Brugada Syndrome-Associated Variants.

Brugada syndrome is an inheritable arrhythmia condition that is associated with rare, loss-of-function variants in SCN5A. Interpreting the pathogenicity of SCN5A missense variants is challenging, and ≈79% of SCN5A missense variants in ClinVar are currently classified as variants of uncertain significance. Automated patch clamp technology enables high-throughput functional studies of ion channel variants and can provide evidence for variant reclassification. An in vitro SCN5A-Brugada syndrome automated patch clamp assay was independently performed at Vanderbilt University Medical Center and Victor Chang Cardiac Research Institute. The assay was calibrated according to ClinGen Sequence Variant Interpretation recommendations using high-confidence variant controls (n=49). Normal and abnormal ranges of function were established based on the distribution of benign variant assay results. Odds of pathogenicity values were derived from the experimental results according to ClinGen Sequence Variant Interpretation recommendations. The calibrated assay was then used to study SCN5A variants of uncertain significance observed in 4 families with Brugada syndrome and other arrhythmia phenotypes associated with SCN5A loss-of-function. Variant channel parameters generated independently at the 2 research sites showed strong correlations, including peak INa density (R2=0.86). The assay accurately distinguished benign controls (24/25 concordant variants) from pathogenic controls (23/24 concordant variants). Odds of pathogenicity values were 0.042 for normal function and 24.0 for abnormal function, corresponding to strong evidence for both American College of Medical Genetics and Genomics/Association for Molecular Pathology benign and pathogenic functional criteria (BS3 and PS3, respectively). Application of the assay to 4 clinical SCN5A variants of uncertain significance revealed loss-of-function for 3/4 variants, enabling reclassification to likely pathogenic. This validated high-throughput assay provides clinical-grade functional evidence to aid the classification of current and future SCN5A-Brugada syndrome variants of uncertain significance.

Identification of Polymorphisms in EAAT1 Glutamate Transporter Gene SLC1A3 Associated with Reduced Migraine Risk.

Dysfunction in ion channels or processes involved in maintaining ionic homeostasis is thought to lower the threshold for cortical spreading depression (CSD), and plays a role in susceptibility to associated neurological disorders, including pathogenesis of a migraine. Rare pathogenic variants in specific ion channels have been implicated in monogenic migraine subtypes. In this study, we further examined the channelopathic nature of a migraine through the analysis of common genetic variants in three selected ion channel or transporter genes: SLC4A4, SLC1A3, and CHRNA4. Using the Agena MassARRAY platform, 28 single-nucleotide polymorphisms (SNPs) across the three candidate genes were genotyped in a case-control cohort comprised of 182 migraine cases and 179 matched controls. Initial results identified significant associations between migraine and rs3776578 (p = 0.04) and rs16903247 (p = 0.05) genotypes within the SLC1A3 gene, which encodes the EAAT1 glutamate transporter. These SNPs were subsequently genotyped in an independent cohort of 258 migraine cases and 290 controls using a high-resolution melt assay, and association testing supported the replication of initial findings-rs3776578 (p = 0.0041) and rs16903247 (p = 0.0127). The polymorphisms are in linkage disequilibrium and localise within a putative intronic enhancer region of SLC1A3. The minor alleles of both SNPs show a protective effect on migraine risk, which may be conferred via influencing the expression of SLC1A3.

Unravelling the Genetic Landscape of Hemiplegic Migraine: Exploring Innovative Strategies and Emerging Approaches.

Migraine is a severe, debilitating neurovascular disorder. Hemiplegic migraine (HM) is a rare and debilitating neurological condition with a strong genetic basis. Sequencing technologies have improved the diagnosis and our understanding of the molecular pathophysiology of HM. Linkage analysis and sequencing studies in HM families have identified pathogenic variants in ion channels and related genes, including CACNA1A, ATP1A2, and SCN1A, that cause HM. However, approximately 75% of HM patients are negative for these mutations, indicating there are other genes involved in disease causation. In this review, we explored our current understanding of the genetics of HM. The evidence presented herein summarises the current knowledge of the genetics of HM, which can be expanded further to explain the remaining heritability of this debilitating condition. Innovative bioinformatics and computational strategies to cover the entire genetic spectrum of HM are also discussed in this review.

HiPSC-derived cardiomyocytes as a model to study the role of small-conductance Ca2+-activated K+ (SK) ion channel variants associated with atrial fibrillation.

Atrial fibrillation (AF), the most common arrhythmia, has been associated with different electrophysiological, molecular, and structural alterations in atrial cardiomyocytes. Therefore, more studies are required to elucidate the genetic and molecular basis of AF. Various genome-wide association studies (GWAS) have strongly associated different single nucleotide polymorphisms (SNPs) with AF. One of these GWAS identified the rs13376333 risk SNP as the most significant one from the 1q21 chromosomal region. The rs13376333 risk SNP is intronic to the KCNN3 gene that encodes for small conductance calcium-activated potassium channels type 3 (SK3). However, the functional electrophysiological effects of this variant are not known. SK channels represent a unique family of K+ channels, primarily regulated by cytosolic Ca2+ concentration, and different studies support their critical role in the regulation of atrial excitability and consequently in the development of arrhythmias like AF. Since different studies have shown that both upregulation and downregulation of SK3 channels can lead to arrhythmias by different mechanisms, an important goal is to elucidate whether the rs13376333 risk SNP is a gain-of-function (GoF) or a loss-of-function (LoF) variant. A better understanding of the functional consequences associated with these SNPs could influence clinical practice guidelines by improving genotype-based risk stratification and personalized treatment. Although research using native human atrial cardiomyocytes and animal models has provided useful insights, each model has its limitations. Therefore, there is a critical need to develop a human-derived model that represents human physiology more accurately than existing animal models. In this context, research with human induced pluripotent stem cells (hiPSC) and subsequent generation of cardiomyocytes derived from hiPSC (hiPSC-CMs) has revealed the underlying causes of various cardiovascular diseases and identified treatment opportunities that were not possible using in vitro or in vivo studies with animal models. Thus, the ability to generate atrial cardiomyocytes and atrial tissue derived from hiPSCs from human/patients with specific genetic diseases, incorporating novel genetic editing tools to generate isogenic controls and organelle-specific reporters, and 3D bioprinting of atrial tissue could be essential to study AF pathophysiological mechanisms. In this review, we will first give an overview of SK-channel function, its role in atrial fibrillation and outline pathophysiological mechanisms of KCNN3 risk SNPs. We will then highlight the advantages of using the hiPSC-CM model to investigate SNPs associated with AF, while addressing limitations and best practices for rigorous hiPSC studies.

Human pain channelopathies.

There has been significant progress in our understanding of the molecular basis by which nociceptors transduce and transmit noxious (tissue damaging) stimuli. This is dependent on ion channels, many of which are selectively expressed in nociceptors. Mutations in such proteins have recently been linked to inherited pain disorders in humans. An exemplar is the voltage-gated sodium channel (VGSC) NaV1.7. Loss of function mutations in NaV1.7 result in congenital inability to experience pain while gain-of-function mutations can cause a number of distinct neuropathic pain disorders, including erythromelalgia, paroxysmal extreme pain disorder, and small-fiber neuropathy. Furthermore, variants in the VGSCs 1.8 and 1.9 have also been linked to human pain disorders. There is a correlation between the impact of mutations on the biophysical properties of the ion channel and the severity of the clinical phenotype. Pain channelopathies are not restricted to VGSCs: a mutation in the ligand-gated ion channel TRPA1, (which responds to environmental irritants) causes a familial episodic pain disorder. Ion channel variants have also been linked to more common neuropathic pain disorders such as painful diabetic neuropathy. Not only do these ion channels present targets for novel analgesics, but stratification based on genotype may improve treatment selection of existing analgesics.

Development of automated patch clamp assays to overcome the burden of variants of uncertain significance in inheritable arrhythmia syndromes.

Advances in next-generation sequencing have been exceptionally valuable for identifying variants in medically actionable genes. However, for most missense variants there is insufficient evidence to permit definitive classification of variants as benign or pathogenic. To overcome the deluge of Variants of Uncertain Significance, there is an urgent need for high throughput functional assays to assist with the classification of variants. Advances in parallel planar patch clamp technologies has enabled the development of automated high throughput platforms capable of increasing throughput 10- to 100-fold compared to manual patch clamp methods. Automated patch clamp electrophysiology is poised to revolutionize the field of functional genomics for inheritable cardiac ion channelopathies. In this review, we outline i) the evolution of patch clamping, ii) the development of high-throughput automated patch clamp assays to assess cardiac ion channel variants, iii) clinical application of these assays and iv) where the field is heading.

A novel de novo HCN2 loss-of-function variant causing developmental and epileptic encephalopathy treated with ketogenic diet.

Missense variants of HCN ion channels cause variable phenotypes, ranging from mild generalized epilepsy to developmental and epileptic encephalopathy (DEE). While variants of HCN1 are an established cause of DEE, those of HCN2 have been reported in generalized epilepsies. Here, we describe the first case of DEE caused by the novel de novo heterozygous missense variant c.1379G>A (p.G460D) of HCN2. Functional characterization in transfected HEK293 cells and neonatal rat cortical neurons revealed that HCN2 p.G460D currents were strongly reduced compared to wild-type, consistent with a dominant negative loss-of-function effect. Immunofluorescence staining showed that mutant channels are retained within the cell and do not reach the membrane. Moreover, mutant HCN2 also affect HCN1 channels, by reducing the Ih current expressed by the HCN1-HCN2 heteromers. Due to the persistence of frequent seizures despite pharmacological polytherapy, the patient was treated with ketogenic diet, reporting a significant and long-lasting reduction of seizures. In vitro experiments conducted in a ketogenic environment demonstrated that the clinical improvement observed with this dietary regimen was not mediated by a direct action on HCN2 activity. These results expand the clinical spectrum related to HCN2 channelopathies, further broadening our understanding of the pathogenesis of DEE.

Predicting functional effects of ion channel variants using new phenotypic machine learning methods.

Missense variants in genes encoding ion channels are associated with a spectrum of severe diseases. Variant effects on biophysical function correlate with clinical features and can be categorized as gain- or loss-of-function. This information enables a timely diagnosis, facilitates precision therapy, and guides prognosis. Functional characterization presents a bottleneck in translational medicine. Machine learning models may be able to rapidly generate supporting evidence by predicting variant functional effects. Here, we describe a multi-task multi-kernel learning framework capable of harmonizing functional results and structural information with clinical phenotypes. This novel approach extends the human phenotype ontology towards kernel-based supervised machine learning. Our gain- or loss-of-function classifier achieves high performance (mean accuracy 0.853 SD 0.016, mean AU-ROC 0.912 SD 0.025), outperforming both conventional baseline and state-of-the-art methods. Performance is robust across different phenotypic similarity measures and largely insensitive to phenotypic noise or sparsity. Localized multi-kernel learning offered biological insight and interpretability by highlighting channels with implicit genotype-phenotype correlations or latent task similarity for downstream analysis.

Investigating genotype-phenotype relationship of extreme neuropathic pain disorders in a UK national cohort.

The aims of our study were to use whole genome sequencing in a cross-sectional cohort of patients to identify new variants in genes implicated in neuropathic pain, to determine the prevalence of known pathogenic variants and to understand the relationship between pathogenic variants and clinical presentation. Patients with extreme neuropathic pain phenotypes (both sensory loss and gain) were recruited from secondary care clinics in the UK and underwent whole genome sequencing as part of the National Institute for Health and Care Research Bioresource Rare Diseases project. A multidisciplinary team assessed the pathogenicity of rare variants in genes previously known to cause neuropathic pain disorders and exploratory analysis of research candidate genes was completed. Association testing for genes carrying rare variants was completed using the gene-wise approach of the combined burden and variance-component test SKAT-O. Patch clamp analysis was performed on transfected HEK293T cells for research candidate variants of genes encoding ion channels. The results include the following: (i) Medically actionable variants were found in 12% of study participants (205 recruited), including known pathogenic variants: SCN9A(ENST00000409672.1): c.2544T>C, p.Ile848Thr that causes inherited erythromelalgia, and SPTLC1(ENST00000262554.2):c.340T>G, p.Cys133Tr variant that causes hereditary sensory neuropathy type-1. (ii) Clinically relevant variants were most common in voltage-gated sodium channels (Nav). (iii) SCN9A(ENST00000409672.1):c.554G>A, pArg185His variant was more common in non-freezing cold injury participants than controls and causes a gain of function of NaV1.7 after cooling (the environmental trigger for non-freezing cold injury). (iv) Rare variant association testing showed a significant difference in distribution for genes NGF, KIF1A, SCN8A, TRPM8, KIF1A, TRPA1 and the regulatory regions of genes SCN11A, FLVCR1, KIF1A and SCN9A between European participants with neuropathic pain and controls. (v) The TRPA1(ENST00000262209.4):c.515C>T, p.Ala172Val variant identified in participants with episodic somatic pain disorder demonstrated gain-of-channel function to agonist stimulation. Whole genome sequencing identified clinically relevant variants in over 10% of participants with extreme neuropathic pain phenotypes. The majority of these variants were found in ion channels. Combining genetic analysis with functional validation can lead to a better understanding as to how rare variants in ion channels lead to sensory neuron hyper-excitability, and how cold, as an environmental trigger, interacts with the gain-of-function NaV1.7 p.Arg185His variant. Our findings highlight the role of ion channel variants in the pathogenesis of extreme neuropathic pain disorders, likely mediated through changes in sensory neuron excitability and interaction with environmental triggers.

Antisense oligonucleotide therapy for KCNT1 encephalopathy.

Developmental and epileptic encephalopathies (DEEs) are characterized by pharmaco-resistant seizures with concomitant intellectual disability. Epilepsy of infancy with migrating focal seizures (EIMFS) is one of the most severe of these syndromes. De novo variants in ion channels, including gain-of-function variants in KCNT1, which encodes for sodium activated potassium channel protein KNa1.1, have been found to play a major role in the etiology of EIMFS. Here, we test a potential precision therapeutic approach in KCNT1-associated DEE using a gene-silencing antisense oligonucleotide (ASO) approach. We generated a mouse model carrying the KCNT1 p.P924L pathogenic variant; only the homozygous animals presented with the frequent, debilitating seizures and developmental compromise that are seen in patients. After a single intracerebroventricular bolus injection of a Kcnt1 gapmer ASO in symptomatic mice at postnatal day 40, seizure frequency was significantly reduced, behavioral abnormalities improved, and overall survival was extended compared with mice treated with a control ASO (nonhybridizing sequence). ASO administration at neonatal age was also well tolerated and effective in controlling seizures and extending the life span of treated animals. The data presented here provide proof of concept for ASO-based gene silencing as a promising therapeutic approach in KCNT1-associated epilepsies.

Peripheral Ion Channel Genes Screening in Painful Small Fiber Neuropathy.

Neuropathic pain is a characteristic feature of small fiber neuropathy (SFN), which in 18% of the cases is caused by genetic variants in voltage-gated sodium ion channels. In this study, we assessed the role of fifteen other ion channels in neuropathic pain. Patients with SFN (n = 414) were analyzed for ANO1, ANO3, HCN1, KCNA2, KCNA4, KCNK18, KCNN1, KCNQ3, KCNQ5, KCNS1, TRPA1, TRPM8, TRPV1, TRPV3 and TRPV4 variants by single-molecule molecular inversion probes-next-generation sequencing. These patients did not have genetic variants in SCN3A, SCN7A-SCN11A and SCN1B-SCN4B. In twenty patients (20/414, 4.8%), a potentially pathogenic heterozygous variant was identified in an ion-channel gene (ICG). Variants were present in seven genes, for two patients (0.5%) in ANO3, one (0.2%) in KCNK18, two (0.5%) in KCNQ3, seven (1.7%) in TRPA1, three (0.7%) in TRPM8, three (0.7%) in TRPV1 and two (0.5%) in TRPV3. Variants in the TRP genes were the most frequent (n = 15, 3.6%), partly in patients with high mean maximal pain scores VAS = 9.65 ± 0.7 (n = 4). Patients with ICG variants reported more severe pain compared to patients without such variants (VAS = 9.36 ± 0.72 vs. VAS = 7.47 ± 2.37). This cohort study identified ICG variants in neuropathic pain in SFN, complementing previous findings of ICG variants in diabetic neuropathy. These data show that ICG variants are central in neuropathic pain of different etiologies and provides promising gene candidates for future research.

Evolutionary coupling analysis guides identification of mistrafficking-sensitive variants in cardiac K+ channels: Validation with hERG.

Loss of function (LOF) mutations of voltage sensitive K+ channel proteins hERG (Kv11.1) and KCNQ1 (Kv7.1) account for the majority of instances of congenital Long QT Syndrome (cLQTS) with the dominant molecular phenotype being a mistrafficking one resulting from protein misfolding. We explored the use of Evolutionary Coupling (EC) analysis, which identifies evolutionarily conserved pairwise amino acid interactions that may contribute to protein structural stability, to identify regions of the channels susceptible to misfolding mutations. Comparison with published experimental trafficking data for hERG and KCNQ1 showed that the method strongly predicts "scaffolding" regions of the channel membrane domains and has useful predictive power for trafficking phenotypes of individual variants. We identified a region in and around the cytoplasmic S2-S3 loop of the hERG Voltage Sensor Domain (VSD) as susceptible to destabilising mutation, and this was confirmed using a quantitative LI-COR ® based trafficking assay that showed severely attenuated trafficking in eight out of 10 natural hERG VSD variants selected using EC analysis. Our analysis highlights an equivalence in the scaffolding structures of the hERG and KCNQ1 membrane domains. Pathogenic variants of ion channels with an underlying mistrafficking phenotype are likely to be located within similar scaffolding structures that are identifiable by EC analysis.

A TRPM7 mutation linked to familial trigeminal neuralgia: Omega current and hyperexcitability of trigeminal ganglion neurons

Trigeminal neuralgia (TN) is a unique pain disorder characterized by intense paroxysmal facial pain within areas innervated by the trigeminal nerve. Although most cases of TN are sporadic, familial clusters of TN suggest that genetic factors may contribute to this disorder. Whole-exome sequencing in patients with TN reporting positive family history demonstrated a spectrum of variants of ion channels including TRP channels. Here, we used patch-clamp analysis and Ca2+ and Na+ imaging to assess a rare variant in the TRPM7 channel, p.Ala931Thr, within transmembrane domain 3, identified in a man suffering from unilateral TN. We showed that A931T produced an abnormal inward current carried by Na+ and insensitive to the pore blocker Gd3+. Hypothesizing that replacement of the hydrophobic alanine at position 931 with the more polar threonine destabilizes a hydrophobic ring, near the voltage sensor domain, we performed alanine substitutions of F971 and W972 and obtained results suggesting a role of A931-W972 hydrophobic interaction in S3-S4 hydrophobic cleft stability. Finally, we transfected trigeminal ganglion neurons with A931T channels and observed that expression of this TRPM7 variant lowers current threshold and resting membrane potential, and increases evoked firing activity in TG neurons. Our results support the notion that the TRPM7-A931T mutation located in the S3 segment at the interface with the transmembrane region S4, generates an omega current that carries Na+ influx in physiological conditions. A931T produces hyperexcitability and a sustained Na+ influx in trigeminal ganglion neurons that may underlie pain in this kindred with trigeminal neuralgia.

Conserved patterns across ion channels correlate with variant pathogenicity and clinical phenotypes.

Clinically identified genetic variants in ion channels can be benign or cause disease by increasing or decreasing the protein function. As a consequence, therapeutic decision-making is challenging without molecular testing of each variant. Our biophysical knowledge of ion-channel structures and function is just emerging, and it is currently not well understood which amino acid residues cause disease when mutated. We sought to systematically identify biological properties associated with variant pathogenicity across all major voltage and ligand-gated ion-channel families. We collected and curated 3049 pathogenic variants from hundreds of neurodevelopmental and other disorders and 12 546 population variants for 30 ion channel or channel subunits for which a high-quality protein structure was available. Using a wide range of bioinformatics approaches, we computed 163 structural features and tested them for pathogenic variant enrichment. We developed a novel 3D spatial distance scoring approach that enables comparisons of pathogenic and population variant distribution across protein structures. We discovered and independently replicated that several pore residue properties and proximity to the pore axis were most significantly enriched for pathogenic variants compared to population variants. Using our 3D scoring approach, we showed that the strongest pathogenic variant enrichment was observed for pore-lining residues and alpha-helix residues within 5Å distance from the pore axis centre and not involved in gating. Within the subset of residues located at the pore, the hydrophobicity of the pore was the feature most strongly associated with variant pathogenicity. We also found an association between the identified properties and both clinical phenotypes and functional in vitro assays for voltage-gated sodium channels (SCN1A, SCN2A, SCN8A) and N-methyl-D-aspartate receptor (GRIN1, GRIN2A, GRIN2B) encoding genes. In an independent expert-curated dataset of 1422 neurodevelopmental disorder pathogenic patient variants and 679 electrophysiological experiments, we show that pore axis distance is associated with seizure age of onset and cognitive performance as well as differential gain versus loss-of-channel function. In summary, we identified biological properties associated with ion-channel malfunction and show that these are correlated with in vitro functional readouts and clinical phenotypes in patients with neurodevelopmental disorders. Our results suggest that clinical decision support algorithms that predict variant pathogenicity and function are feasible in the future.

Personalized structural biology reveals the molecular mechanisms underlying heterogeneous epileptic phenotypes caused by de novo KCNC2 variants.

SummaryWhole-exome sequencing (WES) in the clinic has identified several rare monogenic developmental and epileptic encephalopathies (DEE) caused by ion channel variants. However, WES often fails to provide actionable insight for rare diseases, such as DEEs, due to the challenges of interpreting variants of unknown significance (VUS). Here, we describe a “personalized structural biology” (PSB) approach that leverages recent innovations in the analysis of protein 3D structures to address this challenge. We illustrate this approach in an Undiagnosed Diseases Network (UDN) individual with DEE symptoms and a de novo VUS in KCNC2 (p.V469L), the Kv3.2 voltage-gated potassium channel. A nearby KCNC2 variant (p.V471L) was recently suggested to cause DEE-like phenotypes. Computational structural modeling suggests that both affect protein function. However, despite their proximity, the p.V469L variant is likely to sterically block the channel pore, while the p.V471L variant is likely to stabilize the open state. Biochemical and electrophysiological analyses demonstrate heterogeneous loss-of-function and gain-of-function effects, as well as differential response to 4-aminopyridine treatment. Molecular dynamics simulations illustrate that the pore of the p.V469L variant is more constricted, increasing the energetic barrier for K+ permeation, whereas the p.V471L variant stabilizes the open conformation. Our results implicate variants in KCNC2 as causative for DEE and guide the interpretation of a UDN individual. They further delineate the molecular basis for the heterogeneous clinical phenotypes resulting from two proximal pathogenic variants. This demonstrates how the PSB approach can provide an analytical framework for individualized hypothesis-driven interpretation of protein-coding VUS.

PO-714-04 HIGH PREVALENCE OF NEURODEVELOPMENTAL DISORDERS IN PEDIATRIC LONG QT SYNDROME PATIENTS

Long QT Syndrome (LQTS) is a familial ion channelopathy associated with a structurally normal heart and risk of sudden cardiac death. Most cases are associated with variants in KCNQ1 and KCNH2 genes. Ion channel variants have been implicated in neurodevelopmental disorders (NDDs), yet the link between LQTS and NDDs in children has not been well characterized.

The effect of alterations of schizophrenia-associated genes on gamma band oscillations

Abnormalities in the synchronized oscillatory activity of neurons in general and, specifically in the gamma band, might play a crucial role in the pathophysiology of schizophrenia. While these changes in oscillatory activity have traditionally been linked to alterations at the synaptic level, we demonstrate here, using computational modeling, that common genetic variants of ion channels can contribute strongly to this effect. Our model of primary auditory cortex highlights multiple schizophrenia-associated genetic variants that reduce gamma power in an auditory steady-state response task. Furthermore, we show that combinations of several of these schizophrenia-associated variants can produce similar effects as the more traditionally considered synaptic changes. Overall, our study provides a mechanistic link between schizophrenia-associated common genetic variants, as identified by genome-wide association studies, and one of the most robust neurophysiological endophenotypes of schizophrenia.