Genotype-phenotype correlations in SCN8A-related disorders reveal prognostic and therapeutic implications

in Brain (2022)

We report detailed functional analyses and genotype-phenotype correlations in 392 individuals carrying disease-causing variants in SCN8A, encoding the voltage-gated Na+ channel NaV1.6, with the aim of ... [more ▼]

We report detailed functional analyses and genotype-phenotype correlations in 392 individuals carrying disease-causing variants in SCN8A, encoding the voltage-gated Na+ channel NaV1.6, with the aim of describing clinical phenotypes related to functional effects. Six different clinical subgroups could be identified: 1) Benign familial infantile epilepsy (BFIE) (n = 15, normal cognition, treatable seizures), 2) intermediate epilepsy (n = 33, mild ID, partially pharmaco-responsive), 3) developmental and epileptic encephalopathy (DEE, n = 177, severe ID, majority pharmaco-resistant), 4) generalized epilepsy (n = 20, mild to moderate ID, frequently with absence seizures), 5) unclassifiable epilepsy (n = 127), and 6) neurodevelopmental disorder without epilepsy (n = 20, mild to moderate ID). Groups 1–3 presented with focal or multifocal seizures (median age of onset: four months) and focal epileptiform discharges, whereas the onset of seizures in group 4 was later (median: 42 months) with generalized epileptiform discharges. We performed functional studies expressing missense variants in ND7/23 neuroblastoma cells and primary neuronal cultures using recombinant tetrodotoxin-insensitive human NaV1.6 channels and whole-cell patch-clamping. Two variants causing DEE showed a strong gain-of-function (GOF, hyperpolarising shift of steady-state activation, strongly increased neuronal firing rate), and one variant causing BFIE or intermediate epilepsy showed a mild GOF (defective fast inactivation, less increased firing). In contrast, all three variants causing generalized epilepsy induced a loss-of-function (LOF, reduced current amplitudes, depolarising shift of steady-state activation, reduced neuronal firing). Including previous studies, functional effects were known for 170 individuals. All 136 individuals carrying a functionally tested GOF variant had either focal (97, groups 1–3), or unclassifiable epilepsy (39), whereas 34 with a LOF variant had either generalized (14), no (11) or unclassifiable (6) epilepsy; only three had DEE. Computational modeling in the GOF group revealed a significant correlation between the severity of the electrophysiological and clinical phenotypes. GOF variant carriers responded significantly better to sodium channel blockers (SCBs) than to other anti-seizure medications, and the same applied for all individuals of groups 1–3.In conclusion, our data reveal clear genotype-phenotype correlations between age at seizure onset, type of epilepsy and gain- or loss-of-function effects of SCN8A variants. Generalized epilepsy with absence seizures is the main epilepsy phenotype of LOF variant carriers and the extent of the electrophysiological dysfunction of the GOF variants is a main determinant of the severity of the clinical phenotype in focal epilepsies. Our pharmacological data indicate that SCBs present a treatment option in SCN8A-related focal epilepsy with onset in the first year of life. [less ▲]

Benchmarking of univariate pleiotropy detection methods applied to epilepsy

in Human Mutation (2022), 43(9), 1314-1332

AbstractPleiotropy is a widespread phenomenon that may increase insight into the etiology of biological and disease traits. Since genome-wide association studies frequently provide information on a single ... [more ▼]

AbstractPleiotropy is a widespread phenomenon that may increase insight into the etiology of biological and disease traits. Since genome-wide association studies frequently provide information on a single trait only, only univariate pleiotropy detection methods are applicable, with yet unknown comparative performance. Here, we compared five such methods with respect to their ability to detect pleiotropy, including meta-analysis, ASSET, cFDR, CPBayes, and PLACO, by performing extended computer simulations that varied the underlying etiological model for pleiotropy for a pair of traits, including the number of causal variants, degree of traits’ overlap, effect sizes as well as trait prevalence, and varying sample sizes. Our results indicate that ASSET provides the best trade-off between power and protection against false positives. We then applied ASSET to a previously published ILAE consortium dataset on complex epilepsies, comprising genetic generalized epilepsy and focal epilepsy cases and corresponding controls. We identified a novel candidate locus at 17q21.32 and confirmed locus 2q24.3, previously identified to act pleiotropically on both epilepsy subtypes by a mega-analysis. Functional annotation, tissue-specific expression and regulatory function analysis as well as Bayesian co-localization analysis corroborated this result, rendering 17q21.32 a worthwhile candidate for follow-up studies on pleiotropy in epilepsies.This article is protected by copyright. All rights reserved. [less ▲]

Spectrum of Phenotypic, Genetic, and Functional Characteristics in Epilepsy Patients With KCNC2 Pathogenic Variants 10.1212/WNL.0000000000200660

in Neurology (2022)

Background: KCNC2 encodes Kv3.2, a member of the Shaw-related (Kv3) voltage-gated potassium channel subfamily, which is important for sustained high-frequency firing and optimized energy efficiency of ... [more ▼]

Background: KCNC2 encodes Kv3.2, a member of the Shaw-related (Kv3) voltage-gated potassium channel subfamily, which is important for sustained high-frequency firing and optimized energy efficiency of action potentials in the brain. The objective of this study was to analyse the clinical phenotype, genetic background, and biophysical function of disease-associated Kv3.2 variants.Methods: Individuals with KCNC2 variants detected by exome sequencing were selected for clinical, further genetic, and functional analysis. Cases were referred through clinical and research collaborations. Selected de novo variants were examined electrophysiologically in Xenopus laevis oocytes.Results: We identified novel KCNC2 variants in 18 patients with various forms of epilepsy including genetic generalized epilepsy (GGE), developmental and epileptic encephalopathy (DEE) including early-onset absence epilepsy (EOAE), focal epilepsy (FE), and myoclonic-atonic epilepsy (MAE). 10/18 variants were de novo and 8/18 variants were classified as modifying variants. 8 drug responsive cases became seizure-free using valproic acid as monotherapy or in combination including severe DEE cases. Functional analysis of four variants demonstrated gain-of-function in three severely affected DEE cases and loss-of-function in one case with a milder phenotype (GGE) as the underlying pathomechanisms.Conclusion: These findings implicate KCNC2 as a novel causative gene for epilepsy and emphasize the critical role of KV3.2 in the regulation of brain excitability. [less ▲]

Distinct gene-set burden patterns underlie common generalized and focal epilepsies

in EBioMedicine (2021), 72

Background Analyses of few gene-sets in epilepsy showed a potential to unravel key disease associations. We set out to investigate the burden of ultra-rare variants (URVs) in a comprehensive range of ... [more ▼]

Background Analyses of few gene-sets in epilepsy showed a potential to unravel key disease associations. We set out to investigate the burden of ultra-rare variants (URVs) in a comprehensive range of biologically informed gene-sets presumed to be implicated in epileptogenesis. Methods The burden of 12 URV types in 92 gene-sets was compared between cases and controls using whole exome sequencing data from individuals of European descent with developmental and epileptic encephalopathies (DEE, n = 1,003), genetic generalized epilepsy (GGE, n = 3,064), or non-acquired focal epilepsy (NAFE, n = 3,522), collected by the Epi25 Collaborative, compared to 3,962 ancestry-matched controls. Findings Missense URVs in highly constrained regions were enriched in neuron-specific and developmental genes, whereas genes not expressed in brain were not affected. GGE featured a higher burden in gene-sets derived from inhibitory vs. excitatory neurons or associated receptors, whereas the opposite was found for NAFE, and DEE featured a burden in both. Top-ranked susceptibility genes from recent genome-wide association studies (GWAS) and gene-sets derived from generalized vs. focal epilepsies revealed specific enrichment patterns of URVs in GGE vs. NAFE. Interpretation Missense URVs affecting highly constrained sites differentially impact genes expressed in inhibitory vs. excitatory pathways in generalized vs. focal epilepsies. The excess of URVs in top-ranked GWAS risk-genes suggests a convergence of rare deleterious and common risk-variants in the pathogenesis of generalized and focal epilepsies. [less ▲]

Assessing the role of rare genetic variants in drug-resistant, non-lesional focal epilepsy

in Annals of Clinical and Translational Neurology (2021), n/a(n/a),

Abstract Objective Resistance to antiseizure medications (ASMs) is one of the major concerns in the treatment of epilepsy. Despite the increasing number of ASMs available, the proportion of individuals ... [more ▼]

Abstract Objective Resistance to antiseizure medications (ASMs) is one of the major concerns in the treatment of epilepsy. Despite the increasing number of ASMs available, the proportion of individuals with drug-resistant epilepsy remains unchanged. In this study, we aimed to investigate the role of rare genetic variants in ASM resistance. Methods We performed exome sequencing of 1,128 individuals with non-familial non-acquired focal epilepsy (NAFE) (762 non-responders, 366 responders) and were provided with 1,734 healthy controls. We undertook replication in a cohort of 350 individuals with NAFE (165 non-responders, 185 responders). We performed gene-based and gene-set-based kernel association tests to investigate potential enrichment of rare variants in relation to drug response status and to risk for NAFE. Results We found no gene or gene set that reached genome-wide significance. Yet, we identified several prospective candidate genes – among them DEPDC5, which showed a potential association with resistance to ASMs. We found some evidence for an enrichment of truncating variants in dominant familial NAFE genes in our cohort of non-familial NAFE and in association with drug-resistant NAFE. Interpretation Our study identifies potential candidate genes for ASM resistance. Our results corroborate the role of rare variants for non-familial NAFE and imply their involvement in drug-resistant epilepsy. Future large-scale genetic research studies are needed to substantiate these findings. [less ▲]

Sub-genic intolerance, ClinVar, and the epilepsies: A whole-exome sequencing study of 29,165 individuals

in The American Journal of Human Genetics (2021)

Summary Both mild and severe epilepsies are influenced by variants in the same genes, yet an explanation for the resulting phenotypic variation is unknown. As part of the ongoing Epi25 Collaboration, we ... [more ▼]

Summary Both mild and severe epilepsies are influenced by variants in the same genes, yet an explanation for the resulting phenotypic variation is unknown. As part of the ongoing Epi25 Collaboration, we performed a whole-exome sequencing analysis of 13,487 epilepsy-affected individuals and 15,678 control individuals. While prior Epi25 studies focused on gene-based collapsing analyses, we asked how the pattern of variation within genes differs by epilepsy type. Specifically, we compared the genetic architectures of severe developmental and epileptic encephalopathies (DEEs) and two generally less severe epilepsies, genetic generalized epilepsy and non-acquired focal epilepsy (NAFE). Our gene-based rare variant collapsing analysis used geographic ancestry-based clustering that included broader ancestries than previously possible and revealed novel associations. Using the missense intolerance ratio (MTR), we found that variants in DEE-affected individuals are in significantly more intolerant genic sub-regions than those in NAFE-affected individuals. Only previously reported pathogenic variants absent in available genomic datasets showed a significant burden in epilepsy-affected individuals compared with control individuals, and the ultra-rare pathogenic variants associated with DEE were located in more intolerant genic sub-regions than variants associated with non-DEE epilepsies. MTR filtering improved the yield of ultra-rare pathogenic variants in affected individuals compared with control individuals. Finally, analysis of variants in genes without a disease association revealed a significant burden of loss-of-function variants in the genes most intolerant to such variation, indicating additional epilepsy-risk genes yet to be discovered. Taken together, our study suggests that genic and sub-genic intolerance are critical characteristics for interpreting the effects of variation in genes that influence epilepsy. [less ▲]

Ultra-rare constrained missense variants in the epilepsies: Shared and specific enrichment patterns in neuronal gene-sets 2021.04.18.440264

E-print/Working paper (2021)

Background: Burden analysis in epilepsy has shown an excess of deleterious ultra-rare variants (URVs) in few gene-sets, such as known epilepsy genes, constrained genes, ion channel or GABAA receptor genes ... [more ▼]

Background: Burden analysis in epilepsy has shown an excess of deleterious ultra-rare variants (URVs) in few gene-sets, such as known epilepsy genes, constrained genes, ion channel or GABAA receptor genes. We set out to investigate the burden of URVs in a comprehensive range of gene-sets presumed to be implicated in epileptogenesis. Methods: We investigated several constraint and conservation-based strategies to study whole exome sequencing data from European individuals with developmental and epileptic encephalopathies (DEE, n = 1,003), genetic generalized epilepsy (GGE, n = 3,064), and non-acquired focal epilepsy (NAFE, n = 3,522), collected by the Epi25 Collaborative, compared to 3,962 ancestry-matched controls. The burden of 12 URVs types in 92 gene-sets was compared between epilepsy cases (DDE, GGE, NAFE) and controls using logistic regression analysis. Results: Burden analysis of brain-expressed genes revealed an excess of different URVs types in all three epilepsy categories which was largest for constrained missense variants. The URVs burden was prominent in neuron-specific, synaptic and developmental genes as well as genes encoding ion channels and receptors, and it was generally higher for DEE and GGE compared to NAFE. The patterns of URVs burden in gene-sets expressed in inhibitory vs. excitatory neurons or receptors suggested a high burden in both in DEE but a differential involvement of inhibitory genes in GGE, while excitatory genes were predominantly affected in NAFE. Top ranking susceptibility genes from a recent genome-wide association study (GWAS) of generalized and focal epilepsies displayed a higher URVs burden in constrained coding regions in GGE and NAFE, respectively. Conclusions: Using exome-based gene-set burden analysis, we demonstrate that missense URVs affecting mainly constrained sites are enriched in neuronal genes in both common and rare severe epilepsy syndromes. Our results indicate a differential impact of these URVs in genes expressed in inhibitory vs. excitatory neurons and receptors in generalized vs. focal epilepsies. The excess of URVs in top-ranking GWAS risk-genes suggests a convergence of rare deleterious and common risk-variants in the pathogenesis of generalized and focal epilepsies. [less ▲]

Predicting functional effects of missense variants in voltage-gated sodium and calcium channels

in Science Translational Medicine (2020), 12(556), 6848

Malfunctions of voltage-gated sodium and calcium channels (encoded by SCNxA and CACNA1x family genes, respectively) have been associated with severe neurologic, psychiatric, cardiac, and other diseases ... [more ▼]

Malfunctions of voltage-gated sodium and calcium channels (encoded by SCNxA and CACNA1x family genes, respectively) have been associated with severe neurologic, psychiatric, cardiac, and other diseases. Altered channel activity is frequently grouped into gain or loss of ion channel function (GOF or LOF, respectively) that often corresponds not only to clinical disease manifestations but also to differences in drug response. Experimental studies of channel function are therefore important, but laborious and usually focus only on a few variants at a time. On the basis of known gene-disease mechanisms of 19 different diseases, we inferred LOF (n = 518) and GOF (n = 309) likely pathogenic variants from the disease phenotypes of variant carriers. By training a machine learning model on sequence- and structure-based features, we predicted LOF or GOF effects [area under the receiver operating characteristics curve (ROC) = 0.85] of likely pathogenic missense variants. Our LOF versus GOF prediction corresponded to molecular LOF versus GOF effects for 87 functionally tested variants in SCN1/2/8A and CACNA1I (ROC = 0.73) and was validated in exome-wide data from 21,703 cases and 128,957 controls. We showed respective regional clustering of inferred LOF and GOF nucleotide variants across the alignment of the entire gene family, suggesting shared pathomechanisms in the SCNxA/CACNA1x family genes. [less ▲]

Bi-allelic GAD1 variants cause a neonatal onset syndromic developmental and epileptic encephalopathy

in Brain: a Journal of Neurology (2020)

Developmental and Epileptic Encephalopathies are a heterogeneous group of early-onset epilepsy syndromes dramatically impairing neurodevelopment. Modern genomic technologies have revealed a number of ... [more ▼]

Developmental and Epileptic Encephalopathies are a heterogeneous group of early-onset epilepsy syndromes dramatically impairing neurodevelopment. Modern genomic technologies have revealed a number of monogenic origins and opened the door to therapeutic hopes. Here we describe a new syndromic developmental and epileptic encephalopathies caused by bi-allelic loss of function variants in GAD1, as presented by eleven patients from 6 independent consanguineous families. Seizure onset occurred in the two first months of life in all patients. All 10 patients from whom early disease history was available, presented seizure onset in the first month of life, mainly consisting of epileptic spasms or myoclonic seizures. Early electroencephalography showed suppression-burst or pattern of burst attenuation or hypsarrhythmia if only recorded in the post-neonatal period. Eight patients had joint contractures and/or pes equinovarus. Seven patients presented a cleft palate and two also had an omphalocele, reproducing the phenotype of the knockout Gad1-/- mouse model. Four patients died before four years of age. GAD1 encodes the glutamate decarboxylase enzyme GAD67, a critical actor of the γ-aminobutyric acid (GABA) metabolism as it catalyzes the decarboxylation of glutamic acid to form GABA. Our findings evoke a novel syndrome related to GAD67 deficiency, characterized by the unique association of developmental and epileptic encephalopathies, cleft palate, joint contractures and/or omphalocele. [less ▲]

Ultra-Rare Genetic Variation in the Epilepsies: A Whole-Exome Sequencing Study of 17,606 Individuals

in American Journal of Human Genetics (2019)

Sequencing-based studies have identified novel risk genes associated with severe epilepsies and revealed an excess of rare deleterious variation in less-severe forms of epilepsy. To identify the shared ... [more ▼]

Sequencing-based studies have identified novel risk genes associated with severe epilepsies and revealed an excess of rare deleterious variation in less-severe forms of epilepsy. To identify the shared and distinct ultra-rare genetic risk factors for different types of epilepsies, we performed a whole-exome sequencing (WES) analysis of 9,170 epilepsy-affected individuals and 8,436 controls of European ancestry. We focused on three phenotypic groups: severe developmental and epileptic encephalopathies (DEEs), genetic generalized epilepsy (GGE), and non-acquired focal epilepsy (NAFE). We observed that compared to controls, individuals with any type of epilepsy carried an excess of ultra-rare, deleterious variants in constrained genes and in genes previously associated with epilepsy; we saw the strongest enrichment in individuals with DEEs and the least strong in individuals with NAFE. Moreover, we found that inhibitory GABAA receptor genes were enriched for missense variants across all three classes of epilepsy, whereas no enrichment was seen in excitatory receptor genes. The larger gene groups for the GABAergic pathway or cation channels also showed a significant mutational burden in DEEs and GGE. Although no single gene surpassed exome-wide significance among individuals with GGE or NAFE, highly constrained genes and genes encoding ion channels were among the lead associations; such genes included CACNA1G, EEF1A2, and GABRG2 for GGE and LGI1, TRIM3, and GABRG2 for NAFE. Our study, the largest epilepsy WES study to date, confirms a convergence in the genetics of severe and less-severe epilepsies associated with ultra-rare coding variation, and it highlights a ubiquitous role for GABAergic inhibition in epilepsy etiology. [less ▲]