Variants In Ion Channel Genes Research Articles

Long-term effectiveness and tolerability of ketogenic diet therapy in patients with genetic developmental and epileptic encephalopathy onset within the first 6 months of life.

To investigate the effectiveness and tolerability of ketogenic diet therapy (KDT) in patients with developmental and epileptic encephalopathy (DEE) associated with genetic etiology which onset within the first 6 months of life, and to explore the association between response to KDT and genotype/clinical parameters. We retrospectively reviewed data from patients with genetic DEE who started KDT at Beijing Children's Hospital between January 1, 2016, and December 31, 2021. A total of 32 patients were included, involving 14 pathogenic or likely pathogenic single genes, and 16 (50.0%) patients had sodium/potassium channel gene variants. The median age at onset of epilepsy was 1.0 (IQR: 0.1, 3.0) months. The median age at initiation of KDT was 10.0 (IQR: 5.3, 13.8) months and the median duration of maintenance was 14.0 (IQR: 7.0, 26.5) months, with a mean blood β-hydroxybutyrate of 2.49 ± 0.62 mmol/L. During the maintenance period of KDT, 26 (81.3%) patients had a ≥50% reduction of seizure frequency, of which 12 (37.5%) patients achieved seizure freedom. Better responses were observed in patients with STXBP1 variants, with four out of five patients achieving seizure freedom. There were no statistically differences in the age of onset, duration of epilepsy before KDT, blood ketone values, or the presence of ion channel gene variants between the seizure-free patients and the others. The most common adverse effects were gastrointestinal side effects, which occurred in 21 patients (65.6%), but all were mild and easily corrected. Only one patient discontinued KDT due to nephrolithiasis. KDT is effective in treating early onset genetic DEE, and no statistically significant relationship has been found between genotype and effectiveness in this study. KDT is well tolerated in most young patients, with mild and reversible gastrointestinal side effects being the most common, but usually not the reason to discontinue KDT. This study evaluated the response and side effects of ketogenic diet therapy (KDT) in patients who had seizures within the first 6 months of life, and were diagnosed with genetic developmental and epileptic encephalopathy (DEE), a type of severe epilepsy with developmental delay caused by gene variants. Thirty-two patients involving 14 gene variants who started KDT at Beijing Children's Hospital between were included. KDT was effective in treating early onset genetic DEE in this cohort, and patients with STXBP1 variants responded better; however, no statistically significant relationship was found between gene variant and response. Most young patients tolerated KDT well, with mild and reversible gastrointestinal side effects being the most common.

Mechanosensitive ion channel gene survey suggests potential roles in primary open angle glaucoma

Although glaucoma is a disease modulated by eye pressure, the mechanisms of pressure sensing in the eye are not well understood. Here, we investigated associations between mechanosensitive ion channel gene variants and primary open-angle glaucoma (POAG). Common (minor allele frequency > 5%) single nucleotide polymorphisms located within the genomic regions of 20 mechanosensitive ion channel genes in the K2P, TMEM63, PIEZO and TRP channel families were assessed using genotype data from the NEIGHBORHOOD consortium of 3853 cases and 33,480 controls. Rare (minor allele frequency < 1%) coding variants were assessed using exome array genotyping data for 2606 cases and 2606 controls. Association with POAG was analyzed using logistic regression adjusting for age and sex. Two rare PIEZO1 coding variants with protective effects were identified in the NEIGHBOR dataset: R1527H, (OR 0.17, P = 0.0018) and a variant that alters a canonical splice donor site, g.16-88737727-C-G Hg38 (OR 0.38, P = 0.02). Both variants showed similar effects in the UK Biobank and the R1527H also in the FinnGen database. Several common variants also reached study-specific thresholds for association in the NEIGHBORHOOD dataset. These results identify novel variants in several mechanosensitive channel genes that show associations with POAG, suggesting that these channels may be potential therapeutic targets.

Prophecy or Empiricy? Clinical value of predicting vs determining genetic variant functions.

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.

Peripheral Ion Channel Gene Screening in Painful- and Painless-Diabetic Neuropathy.

Neuropathic pain is common in diabetic peripheral neuropathy (DN), probably caused by pathogenic ion channel gene variants. Therefore, we performed molecular inversion probes-next generation sequencing of 5 transient receptor potential cation channels, 8 potassium channels and 2 calcium-activated chloride channel genes in 222 painful- and 304 painless-DN patients. Twelve painful-DN (5.4%) patients showed potentially pathogenic variants (five nonsense/frameshift, seven missense, one out-of-frame deletion) in ANO3 (n = 3), HCN1 (n = 1), KCNK18 (n = 2), TRPA1 (n = 3), TRPM8 (n = 3) and TRPV4 (n = 1) and fourteen painless-DN patients (4.6%—three nonsense/frameshift, nine missense, one out-of-frame deletion) in ANO1 (n = 1), KCNK18 (n = 3), KCNQ3 (n = 1), TRPA1 (n = 2), TRPM8 (n = 1), TRPV1 (n = 3) and TRPV4 (n = 3). Missense variants were present in both conditions, presumably with loss- or gain-of-functions. KCNK18 nonsense/frameshift variants were found in painless/painful-DN, making a causal role in pain less likely. Surprisingly, premature stop-codons with likely nonsense-mediated RNA-decay were more frequent in painful-DN. Although limited in number, painful-DN patients with ion channel gene variants reported higher maximal pain during the night and day. Moreover, painful-DN patients with TRP variants had abnormal thermal thresholds and more severe pain during the night and day. Our results suggest a role of ion channel gene variants in neuropathic pain, but functional validation is required.

A calibrated functional patch-clamp assay to enhance clinical variant interpretation in KCNH2-related long QT syndrome

Modern sequencing technologies have revolutionized our detection of gene variants. However, in most genes, including KCNH2, the majority of missense variants are currently classified as variants of uncertain significance (VUSs). The aim of this study was to investigate the utility of an automated patch-clamp assay for aiding clinical variant classification in KCNH2. The assay was designed according to recommendations proposed by the Clinical Genome Sequence Variant Interpretation Working Group. Thirty-one variants (17 pathogenic/likely pathogenic, 14 benign/likely benign) were classified internally as variant controls. They were heterozygously expressed in Flp-In HEK293 cells for assessing the effects of variants on current density and channel gating in order to determine the sensitivity and specificity of the assay. All 17 pathogenic variant controls had reduced current density, and 13 of 14 benign variant controls had normal current density, which enabled determination of normal and abnormal ranges for applying evidence of moderate or supporting strength for VUS reclassification. Inclusion of functional assay evidence enabled us to reclassify 6 out of 44 KCNH2 VUSs as likely pathogenic. The high-throughput patch-clamp assay can provide moderate-strength evidence for clinical interpretation of clinical KCNH2 variants and demonstrates the value of developing automated patch-clamp assays for functional characterization of ion channel gene variants.

The Role of Ion Channel-Related Genes in Autism Spectrum Disorder: A Study Using Next-Generation Sequencing.

The clinical heterogeneity of autism spectrum disorder (ASD) is closely associated with the diversity of genes related to ASD pathogenesis. With their low effect size, it has been hard to define the role of common variants of genes in ASD phenotype. In this study, we reviewed genetic results and clinical scores widely used for ASD diagnosis to investigate the role of genes in ASD phenotype considering their functions in molecular pathways. Genetic data from next-generation sequencing (NGS) were collected from 94 participants with ASD. We analyzed enrichment of cellular processes and gene ontology using the Database for Annotation, Visualization, and Integrated Discovery (DAVID). We compared clinical characteristics according to genetic functional characteristics. We found 266 genes containing nonsense, frame shift, missense, and splice site mutations. Results from DAVID revealed significant enrichment for “ion channel” with an enrichment score of 8.84. Moreover, ASD participants carrying mutations in ion channel-related genes showed higher total IQ (p = 0.013) and lower repetitive, restricted behavior (RRB)-related scores (p = 0.003) and mannerism subscale of social responsiveness scale scores, compared to other participants. Individuals with variants in ion channel genes showed lower RRB scores, suggesting that ion channel genes might be relatively less associated with RRB pathogenesis. These results contribute to understanding of the role of common variants in ASD and could be important in the development of precision medicine of ASD.

Spectrum of Cardiac Arrhythmias in children with left ventricular non-compaction

Abstract Left ventricular non-compaction (LVNC) represents a genetically heterogeneous primary cardiomyopathy which occurs in both children and adults, can be isolated or combined with other congenital heart diseases and reveals substantial clinical polymorphism. Isolated LVNC is a rare phenomenon more commonly being a part other cardiovascular phenotypes such as hypertrophic cardiomyopathy, restrictive cardiomyopathy or congenital heart disorders. Clinical features on LVNC include main triade: heart failure, ventricular arrhythmias (VA) and thromboembolism. Purpose The aim of this study was to analyze the clinical characteristics of arrhythmias in children with LVNC. Methods We enrolled 15 patients diagnosed with LVNC. For all patients enrolled the clinical examination included ECG, echocardiography, Holter monitoring, family history and neurological examination. The echocardiographic diagnosis for LVNC was based on the criteria of Stollberger and Jenni. Several patients additionally received MRI. Target sequencing and WES were performed using a targeted panel of 108 cardiomyopathy-associated genes. All patients had a written parental consent form for including in this study. Results The mean age oat presentation was 7.46±4.4 year (from 0.5 to 14 year old). 4 patients had ventricular arrhythmias, which manifested as isolated premature ventricular contractions (PVCs) in two patients; ventricular tachycardia (VT) in two patients, 3 – supraventricular arrhythmias, 2 – WPW syndrome/phenomenon, 5 - atrioventricular block (1 and 2 degrees), atrial fibrillation – 1. Antiarrhythmic therapy received 7 patients (3 with VA; 3 with SVA). In total 21 variants of interest were identified in all 15 patients in 18 different genes. All four patients with variants in ion channel genes had documented supraventricular and ventricular arrhythmias. In 3 out of 5 patients with AV block variants in contractile sarcomeric and related genes were detected. Four patients reached endpoints such as cardiovascular death (2 patients) and heart transplantation (2 patients). Conclusions Arrhythmic manifestations in children with left ventricular non-compaction can vary from conduction disease to SVT and ventricular arrhythmias and these arrhythmias have been associated with prognosis and outcome. Funding Acknowledgement Type of funding source: None

Increased Burden of Ion Channel Gene Variants Is Related to Distinct Phenotypes in Pediatric Patients With Left Ventricular Noncompaction.

Left ventricular noncompaction (LVNC) is a hereditary type of cardiomyopathy. Although it is associated with high morbidity and mortality, the related ion channel gene variants in children have not been fully investigated. This study aimed to elucidate the ion channel genetic landscape of LVNC and identify genotype-phenotype correlations in a large Japanese cohort. We enrolled 206 children with LVNC from 2002 to 2017 in Japan. LVNC was classified as follows: LVNC with congenital heart defects, arrhythmia, dilated phenotype, or normal function. In the enrolled patients, 182 genes associated with cardiomyopathy were screened using next-generation sequencing. We identified 99 pathogenic variants in 40 genes in 87 patients. Of the pathogenic variants, 8.8% were in genes associated with channelopathies, 27% were in sarcomere genes, and 11.5% were in mitochondrial genes. Ion channel gene variants were mostly associated with the arrhythmia classification, whereas sarcomere and mitochondrial gene variants were associated with the dilated phenotype. Echocardiography revealed that the group with ion channel gene variants had almost normal LV ejection fraction and LV diastolic diameter Z scores. Fragmented QRS, old age, and an arrhythmia phenotype were the most significant risk factors for ventricular tachycardia (P=0.165, 0.0428, and 0.0074, respectively). Moreover, the group with ion channel variants exhibited a greater risk of a higher prevalence of arrhythmias such as ventricular tachycardia, rather than congestive heart failure. This is the first study that focused on genotype-phenotype correlations in a large pediatric LVNC patient cohort with ion channel gene variants that were determined using next-generation sequencing. Ion channel gene variants were strongly correlated with arrhythmia phenotypes. Genetic testing and phenotype specification allow for appropriate medical management of specific LVNC targets.

Deep Mutational Scan of an SCN5A Voltage Sensor.

Variants in ion channel genes have classically been studied in low throughput by patch clamping. Deep mutational scanning is a complementary approach that can simultaneously assess function of thousands of variants. We have developed and validated a method to perform a deep mutational scan of variants in SCN5A, which encodes the major voltage-gated sodium channel in the heart. We created a library of nearly all possible variants in a 36 base region of SCN5A in the S4 voltage sensor of domain IV and stably integrated the library into HEK293T cells. In preliminary experiments, challenge with 3 drugs (veratridine, brevetoxin, and ouabain) could discriminate wild-type channels from gain- and loss-of-function pathogenic variants. High-throughput sequencing of the pre- and postdrug challenge pools was used to count the prevalence of each variant and identify variants with abnormal function. The deep mutational scan scores identified 40 putative gain-of-function and 33 putative loss-of-function variants. For 8 of 9 variants, patch clamping data were consistent with the scores. These experiments demonstrate the accuracy of a high-throughput in vitro scan of SCN5A variant function, which can be used to identify deleterious variants in SCN5A and other ion channel genes.

The Clinical and Genetic Features of Co-occurring Epilepsy and Autism Spectrum Disorder in Chinese Children.

There is still no comprehensive description of the general population regarding clinical features and genetic etiology for co-occurring epilepsy and autism spectrum disorder (ASD) in Chinese children. This study was a retrospective study of children diagnosed with epilepsy and ASD from January 1st, 2015, to May 1st, 2018, at the Children's Hospital of Fudan University. A total of 117 patients met the inclusion criteria, and 103 subjects were eligible. Among them, 88 underwent genetic testing, and 47 children (53.4%) were identified as having pathogenic or likely pathogenic variants: 39 had single gene mutations (83.0%, 39/47), and eight had copy number variants (17.0%, 8/47), with SCN1A (14.9%, 7/47) and MECP2 (10.6%, 5/47) gene mutations being the most common. Mutations in other genes encoding voltage-gated ion channels including SCN2A, CACNA1A, CACNA1H, CACNA1D, and KCNQ2 were also common, but the number of individual cases for each gene was small. Epilepsy syndrome and epilepsy-associated syndrome were more common (P = 0.014), and higher rates of poly-therapy (P = 0.01) were used in the positive genetic test group than in the negative group. There were no statistically significant differences in drug-refractory epilepsy, ASD severity, or intellectual disability between the positive genetic test group and the negative genetic group. These data strongly indicate the need for ASD screening in children with epilepsy with voltage-gated ion channel gene variants for better diagnosis and early intervention.

Novel and de novo mutations in pediatric refractory epilepsy

Pediatric refractory epilepsy is a broad phenotypic spectrum with great genetic heterogeneity. Next-generation sequencing (NGS) combined with Sanger sequencing could help to understand the genetic diversity and underlying disease mechanisms in pediatric epilepsy. Here, we report sequencing results from a cohort of 172 refractory epilepsy patients aged 0–14 years. The pathogenicity of identified variants was evaluated in accordance with the American College of Medical Genetics and Genomics (ACMG) criteria. We identified 43 pathogenic or likely pathogenic variants in 40 patients (23.3%). Among these variants, 74.4% mutations (32/43) were de novo and 60.5% mutations (26/43) were novel. Patients with onset age of seizures ≤12 months had higher yields of deleterious variants compared to those with onset age of seizures > 12 months (P = 0.006). Variants in ion channel genes accounted for the greatest functional gene category (55.8%), with SCN1A coming first (16/43). 81.25% (13/16) of SCN1A mutations were de novo and 68.8% (11/16) were novel in Dravet syndrome. Pathogenic or likely pathogenic variants were found in the KCNQ2, STXBP1, SCN2A genes in Ohtahara syndrome. Novel deleterious variants were also found in West syndrome, Doose syndrome and glucose transporter type 1 deficiency syndrome patients. One de novoMECP2 mutation were found in a Rett syndrome patient. TSC1/TSC2 variants were found in 60% patients with tuberous sclerosis complex patients. Other novel mutations detected in unclassified epilepsy patients involve the SCN8A, CACNA1A, GABRB3, GABRA1, IQSEC2, TSC1, VRK2, ATP1A2, PCDH19, SLC9A6 and CHD2 genes. Our study provides novel insights into the genetic origins of pediatric epilepsy and represents a starting-point for further investigations into the molecular pathophysiology of pediatric epilepsy that could eventually lead to better treatments.

Using Clinical Datasets to Optimize Models of Human Ventricular Electrophysiology: Implications for In Silico Drug Screening

Recent advances in Computational Cardiology mean we can now examine the causes, mechanisms and impact of cardiac dysfunction in silico, particularly in regards to risk stratification and treatment of heart rhythm disturbances. As a result, computational cardiology stands at the threshold of clinical utility. For example, in-silico models of human cardiac electrophysiology are being considered by the FDA for prediction of proarrhythmic cardiotoxicity as a core component of the preclinical assessment phase of all new drugs. However, one issue that exists with current models is that they each respond differently to insults such as drug block of ion channels or mutation of cardiac ion channel genes. Clearly this poses a problem in relation to the utility of these models in making quantitative predictions that are physiologically or clinically meaningful. To examine this in detail we tested the ability of three models of the human ventricular action potential, the O’hara-Rudy the Grandi-Bers and the Ten Tusscher models, to reproduce the clinical phenotype of different subtypes of the long QT syndrome. All models, in their original form, produce markedly different and unrealistic predictions of QT prolongation. To address this, we used a global optimization approach to constrain existing in silico models to clinical datasets. After optimization, all models have similar current densities during the action potential, despite differences in kinetic properties of the channels in the different models, and closely reproduced the prolongation of repolarization seen in clinical data. We suggest that models optimized using this approach can be utilized with more confidence in clinical and preclinical applications such as prediction of proarrhythmic risk as part of in silico drug screening, examining pathogenic interactions of electrical dysfunction and structural alteration in the myocardium and assessing the impact of genetic variants in ion channel genes in contributing to heart rhythm disturbances.

WNK4 Polymorphisms and Essential Hypertension in the Uyghur Population

Subtle genetic variants in ion-channel genes might be at the origin of less rare forms of hypertension in the general population. To test this hypothesis, we observed the role of several important polymorphisms (T1155547C at exon7, G1155942T at exon8, G1156666A at intron10, and C1163527T at intron14) of WNK4 (with-no-kinase) gene on the prevalence of essential hypertension in a Chinese minority ethnic group—the Uyghur population. We did not find a significantly different distribution of genotype and allele frequency of T1155547C, G1155942T, and G1156666A between hypertensives and controls, but we observed a significantly higher frequency of T allele in hypertensive subjects than controls (8.4% vs. 5.7%) (P = 0.02) of C1163527T polymorphism at intron14. Compared with the individuals with two C allele, individuals with at least one T allele show 55% of excess risk in developing hypertension (OR = 1.56, 95% CI:1.06–2.28). In a further analysis, we did not observe a significantly higher or lower odds ratio of haplotype frequency in the hypertensives than in the controls across haplotype encompassing G1155942T, G1156666A, and C1163527T. Thus, a positive association of C1163527T at intron 14 reveals that the WNK4 gene might be involved in the prevalence of essential hypertension in the Uyghur population. Further study should be conducted to observe the role of this gene on hypertension in other populations.

Effect of common KCNE1 and SCN5A ion channel gene variants on T-wave alternans, a marker of cardiac repolarization, during clinical exercise stress test: the Finnish Cardiovascular Study

T-wave alternans (TWA) in electrocardiography (ECG) is a marker of cardiac repolarization, the molecular regulation of which is incompletely understood. High TWA and prolonged QT intervals are both associated with ventricular arrhythmias and sudden death. Therefore, we tested the hypothesis of whether the same mutations that influence the QT interval also affect TWA variation. We examined the effect of 3 ion channel gene single nucleotide polymorphisms (SNPs), rs1805127, rs727957 KCNE1, and rs1805124 SCN5A, on TWA during a clinical exercise test. A total of 2008 subjects from the Finnish Cardiovascular Study underwent an exercise test with online ECG recording. TWA was measured by using the time-domain, modified moving average method. Maximum values at rest, during maximal exercise, and during recovery were used as outcome measures in statistical analysis. Moreover, 4-year survival data were collected and ion channel SNPs were determined. TWA was lowest in subjects with the TT genotype of rs1805127 during all phases of the exercise test (RANOVA main effect for genotype, P = 0.018). The result remained significant after adjustment for age, existing coronary heart disease, and beta-blocker medication status (RANCOVA, P = 0.035). Of the polymorphisms studied, only rs1805127 had a significant association with mortality (P = 0.047). The most common G-C haplotype, formed by rs727957 and rs1805127, was associated with TWA (RANOVA, P = 0.007) but not with mortality. The rs1805124 polymorphism was not associated with TWA. The common KCNE1 gene variant rs1805127 is associated with TWA during an exercise test in a Finnish population, which provides additional evidence that KCNE1 genetics may influence cardiac repolarization and cardiovascular mortality.

Cardiac Sodium Channel ( SCN5A ) Variants Associated with Atrial Fibrillation

Genetic studies have identified ion channel gene variants in families segregating atrial fibrillation (AF), the most common arrhythmia in clinical practice. Here, we tested the hypothesis that vulnerability to AF is associated with variation in SCN5A, the gene encoding the cardiac sodium channel. We resequenced the entire SCN5A coding region in 375 subjects with either lone AF (n=118) or AF associated with heart disease (n=257). Controls (n=360) from the same population were then genotyped for the presence of mutations or rare variants identified in the AF cases. In 10 probands (2.7%), 8 novel variants not found in the control population (0%; P=0.001) were identified. All variants affect highly conserved residues in the SCN5A protein. In 6 families with >1 affected member, the novel variant cosegregated with AF. We also identified 11 rare missense variants in 12 probands (3.2%) that have previously been associated with inherited arrhythmia syndromes (eg, congenital long-QT syndrome and Brugada syndrome). Mutations or rare variants in SCN5A may predispose patients with or without underlying heart disease to AF. The findings of the present study expand the clinical spectrum of disorders of the cardiac sodium channel to include AF and represent important progress toward molecular phenotyping and directed rather than empirical therapy for this common arrhythmia.

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