Clinical features, functional consequences, and rescue pharmacology of missense GRID1 and GRID2 human variants.

GRID1 and GRID2 encode the enigmatic GluD1 and GluD2 proteins, which form tetrameric receptors that play important roles in synapse organization and development of the central nervous system. Variation in these genes has been implicated in neurodevelopmental phenotypes. We evaluated GRID1 and GRID2 human variants from the literature, ClinVar, and clinical laboratories and found that many of these variants reside in intolerant domains, including the amino terminal domain of both GRID1 and GRID2. Other conserved regions, such as the M3 transmembrane domain, show different intolerance between GRID1 and GRID2. We introduced these variants into GluD1 and GluD2 cDNA and performed electrophysiological and biochemical assays to investigate the mechanisms of dysfunction of GRID1/2 variants. One variant in the GRID1 distal amino terminal domain resides at a position predicted to interact with Cbln2/Cbln4, and the variant disrupts complex formation between GluD1 and Cbln2, which could perturb its role in synapse organization. We also discovered that, like the lurcher mutation (GluD2-A654T), other rare variants in the GRID2 M3 domain create constitutively active receptors that share similar pathogenic phenotypes. We also found that the SCHEMA schizophrenia M3 variant GluD1-A650T produced constitutively active receptors. We tested a variety of compounds for their ability to inhibit constitutive currents of GluD receptor variants and found that pentamidine potently inhibited GluD2-T649A constitutive channels (IC50 50 nM). These results identify regions of intolerance to variation in the GRID genes, illustrate the functional consequences of GRID1 and GRID2 variants, and suggest how these receptors function normally and in disease.

Epilepsies of presumed genetic etiology show enrichment of rare variants that occur in the general population.

Previous studies suggested that severe epilepsies, e.g., developmental and epileptic encephalopathies (DEEs), are mainly caused by ultra-rare de novo genetic variants. For milder disease, rare genetic variants could contribute to the phenotype. To determine the importance of rare variants for different epilepsy types, we analyzed a whole-exome sequencing cohort of 9,170 epilepsy-affected individuals and 8,436 control individuals. Here, we separately analyzed three different groups of epilepsies: severe DEEs, genetic generalized epilepsy (GGE), and non-acquired focal epilepsy (NAFE). We required qualifying rare variants (QRVs) to occur in control individuals with an allele count ≥ 1 and a minor allele frequency ≤ 1:1,000, to be predicted as deleterious (CADD ≥ 20), and to have an odds ratio in individuals with epilepsy ≥ 2. We identified genes enriched with QRVs primarily in NAFE (n = 72), followed by GGE (n = 32) and DEE (n = 21). This suggests that rare variants may play a more important role for causality of NAFE than for DEE. Moreover, we found that genes harboring QRVs, e.g., HSGP2, FLNA, or TNC, encode proteins that are involved in structuring the brain extracellular matrix. The present study confirms an involvement of rare variants for NAFE that occur also in the general population, while in DEE and GGE, the contribution of such variants appears more limited.

Classification of missense variants in the N-methyl-d-aspartate receptor GRIN gene family as gain- or loss-of-function.

Advances in sequencing technology have generated a large amount of genetic data from patients with neurological conditions. These data have provided diagnosis of many rare diseases, including a number of pathogenic de novo missense variants in GRIN genes encoding N-methyl-d-aspartate receptors (NMDARs). To understand the ramifications for neurons and brain circuits affected by rare patient variants, functional analysis of the variant receptor is necessary in model systems. For NMDARs, this functional analysis needs to assess multiple properties in order to understand how variants could impact receptor function in neurons. One can then use these data to determine whether the overall actions will increase or decrease NMDAR-mediated charge transfer. Here, we describe an analytical and comprehensive framework by which to categorize GRIN variants as either gain-of-function (GoF) or loss-of-function (LoF) and apply this approach to GRIN2B variants identified in patients and the general population. This framework draws on results from six different assays that assess the impact of the variant on NMDAR sensitivity to agonists and endogenous modulators, trafficking to the plasma membrane, response time course and channel open probability. We propose to integrate data from multiple in vitro assays to arrive at a variant classification, and suggest threshold levels that guide confidence. The data supporting GoF and LoF determination are essential to assessing pathogenicity and patient stratification for clinical trials as personalized pharmacological and genetic agents that can enhance or reduce receptor function are advanced. This approach to functional variant classification can generalize to other disorders associated with missense variants.

Bi-allelic loss-of-function variants in PPFIBP1 cause a neurodevelopmental disorder with microcephaly, epilepsy, and periventricular calcifications.

PPFIBP1 encodes for the liprin-ß1 protein, which has been shown to play a role in neuronal outgrowth and synapse formation in Drosophila melanogaster. By exome and genome sequencing, we detected nine ultra-rare homozygous loss-of-function variants in 16 individuals from 12 unrelated families. The individuals presented with moderate to profound developmental delay, often refractory early-onset epilepsy, and progressive microcephaly. Further common clinical findings included muscular hyper- and hypotonia, spasticity, failure to thrive and short stature, feeding difficulties, impaired vision, and congenital heart defects. Neuroimaging revealed abnormalities of brain morphology with leukoencephalopathy, ventriculomegaly, cortical abnormalities, and intracranial periventricular calcifications as major features. In a fetus with intracranial calcifications, we identified a rare homozygous missense variant that by structural analysis was predicted to disturb the topology of the SAM domain region that is essential for protein-protein interaction. For further insight into the effects of PPFIBP1 loss of function, we performed automated behavioral phenotyping of a Caenorhabditis elegans PPFIBP1/hlb-1 knockout model, which revealed defects in spontaneous and light-induced behavior and confirmed resistance to the acetylcholinesterase inhibitor aldicarb, suggesting a defect in the neuronal presynaptic zone. In conclusion, we establish bi-allelic loss-of-function variants in PPFIBP1 as a cause of an autosomal recessive severe neurodevelopmental disorder with early-onset epilepsy, microcephaly, and periventricular calcifications.

De novo variants in KCNA3 cause developmental and epileptic encephalopathy.

OBJECTIVE: Variants in several potassium channel genes, including KCNA1 and KCNA2, cause Developmental and Epileptic Encephalopathies (DEEs). We investigated whether variants in KCNA3, another mammalian homologue of the Drosophila shaker family and encoding for Kv1.3 subunits, can cause DEE. METHODS: Genetic analysis of study individuals was performed by routine exome or genome sequencing, usually of parent-offspring trios. Phenotyping was performed via a standard clinical questionnaire. Currents from wild-type and/or mutant Kv1.3 subunits were investigated by whole-cell patch-clamp upon their heterologous expression. RESULTS: Fourteen individuals, each carrying a de novo heterozygous missense variant in KCNA3, were identified. Most (12/14; 86%) had DEE with marked speech delay with or without motor delay, intellectual disability, epilepsy, and autism spectrum disorder. Functional analysis of Kv1.3 channels carrying each variant revealed heterogeneous functional changes, ranging from "pure" loss-of-function (LoF) effects due to faster inactivation kinetics, depolarized voltage-dependence of activation, slower activation kinetics, increased current inactivation, reduced or absent currents with or without dominant-negative effects, to "mixed" loss- and gain-of-function (GoF) effects. Compared to controls, Kv1.3 currents in lymphoblasts from 1 of the proband displayed functional changes similar to those observed upon heterologous expression of channels carrying the same variant. The antidepressant drug fluoxetine inhibited with similar potency the currents from wild-type and 1 of the Kv1.3 GoF variant. INTERPRETATION: We describe a novel association of de novo missense variants in KCNA3 with a human DEE, and provide evidence that fluoxetine might represent a potential targeted treatment for individuals carrying variants with significant GoF effects. ANN NEUROL 2024;95:365-376.

De novo GRIN variants in M3 helix associated with neurological disorders control channel gating of NMDA receptor.

N-methyl-D-aspartate receptors (NMDARs) are members of the glutamate receptor family and participate in excitatory postsynaptic transmission throughout the central nervous system. Genetic variants in GRIN genes encoding NMDAR subunits are associated with a spectrum of neurological disorders. The M3 transmembrane helices of the NMDAR couple directly to the agonist-binding domains and form a helical bundle crossing in the closed receptors that occludes the pore. The M3 functions as a transduction element whose conformational change couples ligand binding to opening of an ion conducting pore. In this study, we report the functional consequences of 48 de novo missense variants in GRIN1, GRIN2A, and GRIN2B that alter residues in the M3 transmembrane helix. These de novo variants were identified in children with neurological and neuropsychiatric disorders including epilepsy, developmental delay, intellectual disability, hypotonia and attention deficit hyperactivity disorder. All 48 variants in M3 for which comprehensive testing was completed produce a gain-of-function (28/48) compared to loss-of-function (9/48); 11 variants had an indeterminant phenotype. This supports the idea that a key structural feature of the M3 gate exists to stabilize the closed state so that agonist binding can drive channel opening. Given that most M3 variants enhance channel gating, we assessed the potency of FDA-approved NMDAR channel blockers on these variant receptors. These data provide new insight into the structure-function relationship of the NMDAR gate, and suggest that variants within the M3 transmembrane helix produce a gain-of-function.

De novo missense variants in FBXO11 alter its protein expression and subcellular localization.

Recently, others and we identified de novo FBXO11 (F-Box only protein 11) variants as causative for a variable neurodevelopmental disorder (NDD). We now assembled clinical and mutational information on 23 additional individuals. The phenotypic spectrum remains highly variable, with developmental delay and/or intellectual disability as the core feature and behavioral anomalies, hypotonia and various facial dysmorphism as frequent aspects. The mutational spectrum includes intragenic deletions, likely gene disrupting and missense variants distributed across the protein. To further characterize the functional consequences of FBXO11 missense variants, we analyzed their effects on protein expression and localization by overexpression of 17 different mutant constructs in HEK293 and HeLa cells. We found that the majority of missense variants resulted in subcellular mislocalization and/or reduced FBXO11 protein expression levels. For instance, variants located in the nuclear localization signal and the N-terminal F-Box domain lead to altered subcellular localization with exclusion from the nucleus or the formation of cytoplasmic aggregates and to reduced protein levels in western blot. In contrast, variants localized in the C-terminal Zn-finger UBR domain lead to an accumulation in the cytoplasm without alteration of protein levels. Together with the mutational data, our functional results suggest that most missense variants likely lead to a loss of the original FBXO11 function and thereby highlight haploinsufficiency as the most likely disease mechanism for FBXO11-associated NDDs.

CNV-ClinViewer: enhancing the clinical interpretation of large copy-number variants online.

MOTIVATION: Pathogenic copy-number variants (CNVs) can cause a heterogeneous spectrum of rare and severe disorders. However, most CNVs are benign and are part of natural variation in human genomes. CNV pathogenicity classification, genotype-phenotype analyses, and therapeutic target identification are challenging and time-consuming tasks that require the integration and analysis of information from multiple scattered sources by experts. RESULTS: Here, we introduce the CNV-ClinViewer, an open-source web application for clinical evaluation and visual exploration of CNVs. The application enables real-time interactive exploration of large CNV datasets in a user-friendly designed interface and facilitates semi-automated clinical CNV interpretation following the ACMG guidelines by integrating the ClassifCNV tool. In combination with clinical judgment, the application enables clinicians and researchers to formulate novel hypotheses and guide their decision-making process. Subsequently, the CNV-ClinViewer enhances for clinical investigators' patient care and for basic scientists' translational genomic research. AVAILABILITY AND IMPLEMENTATION: The web application is freely available at https://cnv-ClinViewer.broadinstitute.org and the open-source code can be found at https://github.com/LalResearchGroup/CNV-clinviewer.

KCNT2-Related Disorders: Phenotypes, Functional, and Pharmacological Properties.

OBJECTIVE: Pathogenic variants in KCNT2 are rare causes of developmental epileptic encephalopathy (DEE). We herein describe the phenotypic and genetic features of patients with KCNT2-related DEE, and the in vitro functional and pharmacological properties of KCNT2 channels carrying 14 novel or previously untested variants. METHODS: Twenty-five patients harboring KCNT2 variants were investigated: 12 were identified through an international collaborative network, 13 were retrieved from the literature. Clinical data were collected and included in a standardized phenotyping sheet. Novel variants were detected using exome sequencing and classified using ACMG criteria. Functional and pharmacological studies were performed by whole-cell electrophysiology in HEK-293 and SH-SY5Y cells. RESULTS: The phenotypic spectrum encompassed: (a) intellectual disability/developmental delay (21/22 individuals with available information), ranging from mild to severe/profound; (b) epilepsy (15/25); (c) neurological impairment, with altered muscle tone (14/22); (d) dysmorphisms (13/20). Nineteen pathogenic KCNT2 variants were found (9 new, 10 reported previously): 16 missense, 1 in-frame deletion of a single amino acid, 1 nonsense, and 1 frameshift. Among tested variants, 8 showed gain-of-function (GoF), and 6 loss-of-function (LoF) features when expressed heterologously in vitro. Quinidine and fluoxetine blocked all GoF variants, whereas loxapine and riluzole activated some LoF variants while blocking others. INTERPRETATION: We expanded the phenotypic and genotypic spectrum of KCNT2-related disorders, highlighting novel genotype-phenotype associations. Pathogenic KCNT2 variants cause GoF or LoF in vitro phenotypes, and each shows a unique pharmacological profile, suggesting the need for in vitro functional and pharmacological investigation to enable targeted therapies based on the molecular phenotype. ANN NEUROL 2023;94:332-349.

Clinical features and genotype-phenotype correlations in epilepsy patients with de novo DYNC1H1 variants.

OBJECTIVE: DYNC1H1 variants are involved on a disease spectrum from neuromuscular disorders to neurodevelopmental disorders. DYNC1H1-related epilepsy has been reported in small cohorts. We dissect the electroclinical features of 34 patients harboring de novo DYNC1H1 pathogenic variants, identify subphenotypes on the DYNC1H1-related epilepsy spectrum, and compare the genotype-phenotype correlations observed in our cohort with the literature. METHODS: Patients harboring de novo DYNC1H1 pathogenic variants were recruited through international collaborations. Clinical data were retrospectively collected. Latent class analysis was performed to identify subphenotypes. Multivariable binary logistic regression analysis was applied to investigate the association with DYNC1H1 protein domains. RESULTS: DYNC1H1-related epilepsy presented with infantile epileptic spasms syndrome (IESS) in 17 subjects (50%), and in 25% of these individuals the epileptic phenotype evolved into Lennox-Gastaut syndrome (LGS). In 12 patients (35%), focal onset epilepsy was defined. In two patients, the epileptic phenotype consisted of generalized myoclonic epilepsy, with a progressive phenotype in one individual harboring a frameshift variant. In approximately 60% of our cohort, seizures were drug-resistant. Malformations of cortical development were noticed in 79% of our patients, mostly on the lissencephaly-pachygyria spectrum, particularly with posterior predominance in a half of them. Midline and infratentorial abnormalities were additionally reported in 45% and 27% of subjects. We have identified three main classes of subphenotypes on the DYNC1H1-related epilepsy spectrum. SIGNIFICANCE: We propose a classification in which pathogenic de novo DYNC1H1 variants feature drug-resistant IESS in half of cases with potential evolution to LGS (Class 1), developmental and epileptic encephalopathy other than IESS and LGS (Class 2), or less severe focal or genetic generalized epilepsy including a progressive phenotype (Class 3). We observed an association between stalk domain variants and Class 1 phenotypes. The variants p.Arg309His and p.Arg1962His were common and associated with Class 1 subphenotype in our cohort. These findings may aid genetic counseling of patients with DYNC1H1-related epilepsy.

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.

Widespread genomic influences on phenotype in Dravet syndrome, a 'monogenic' condition.

Dravet syndrome is an archetypal rare severe epilepsy, considered 'monogenic', typically caused by loss-of-function SCN1A variants. Despite a recognizable core phenotype, its marked phenotypic heterogeneity is incompletely explained by differences in the causal SCN1A variant or clinical factors. In 34 adults with SCN1A-related Dravet syndrome, we show additional genomic variation beyond SCN1A contributes to phenotype and its diversity, with an excess of rare variants in epilepsy-related genes as a set and examples of blended phenotypes, including one individual with an ultra-rare DEPDC5 variant and focal cortical dysplasia. The polygenic risk score for intelligence was lower, and for longevity, higher, in Dravet syndrome than in epilepsy controls. The causal, major-effect, SCN1A variant may need to act against a broadly compromised genomic background to generate the full Dravet syndrome phenotype, whilst genomic resilience may help to ameliorate the risk of premature mortality in adult Dravet syndrome survivors.

SLC6A1 variant pathogenicity, molecular function and phenotype: a genetic and clinical analysis.

Genetic variants in the SLC6A1 gene can cause a broad phenotypic disease spectrum by altering the protein function. Thus, systematically curated clinically relevant genotype-phenotype associations are needed to understand the disease mechanism and improve therapeutic decision-making. We aggregated genetic and clinical data from 172 individuals with likely pathogenic/pathogenic (lp/p) SLC6A1 variants and functional data for 184 variants (14.1% lp/p). Clinical and functional data were available for a subset of 126 individuals. We explored the potential associations of variant positions on the GAT1 3D structure with variant pathogenicity, altered molecular function and phenotype severity using bioinformatic approaches. The GAT1 transmembrane domains 1, 6 and extracellular loop 4 (EL4) were enriched for patient over population variants. Across functionally tested missense variants (n = 156), the spatial proximity from the ligand was associated with loss-of-function in the GAT1 transporter activity. For variants with complete loss of in vitro GABA uptake, we found a 4.6-fold enrichment in patients having severe disease versus non-severe disease (P = 2.9 × 10-3, 95% confidence interval: 1.5-15.3). In summary, we delineated associations between the 3D structure and variant pathogenicity, variant function and phenotype in SLC6A1-related disorders. This knowledge supports biology-informed variant interpretation and research on GAT1 function. All our data can be interactively explored in the SLC6A1 portal (https://slc6a1-portal.broadinstitute.org/).

Clinical and functional consequences of GRIA variants in patients with neurological diseases.

AMPA receptors are members of the glutamate receptor family and mediate a fast component of excitatory synaptic transmission at virtually all central synapses. Thus, their functional characteristics are a critical determinant of brain function. We evaluate intolerance of each GRIA gene to genetic variation using 3DMTR and report here the functional consequences of 52 missense variants in GRIA1-4 identified in patients with various neurological disorders. These variants produce changes in agonist EC50, response time course, desensitization, and/or receptor surface expression. We predict that these functional and localization changes will have important consequences for circuit function, and therefore likely contribute to the patients' clinical phenotype. We evaluated the sensitivity of variant receptors to AMPAR-selective modulators including FDA-approved drugs to explore potential targeted therapeutic options.

KCND2 variants associated with global developmental delay differentially impair Kv4.2 channel gating.

Here, we report on six unrelated individuals, all presenting with early-onset global developmental delay, associated with impaired motor, speech and cognitive development, partly with developmental epileptic encephalopathy and physical dysmorphisms. All individuals carry heterozygous missense variants of KCND2, which encodes the voltage-gated potassium (Kv) channel α-subunit Kv4.2. The amino acid substitutions associated with the variants, p.(Glu323Lys) (E323K), p.(Pro403Ala) (P403A), p.(Val404Leu) (V404L) and p.(Val404Met) (V404M), affect sites known to be critical for channel gating. To unravel their likely pathogenicity, recombinant mutant channels were studied in the absence and presence of auxiliary ß-subunits under two-electrode voltage clamp in Xenopus oocytes. All channel mutants exhibited slowed and incomplete macroscopic inactivation, and the P403A variant in addition slowed activation. Co-expression of KChIP2 or DPP6 augmented the functional expression of both wild-type and mutant channels; however, the auxiliary ß-subunit-mediated gating modifications differed from wild type and among mutants. To simulate the putative setting in the affected individuals, heteromeric Kv4.2 channels (wild type + mutant) were studied as ternary complexes (containing both KChIP2 and DPP6). In the heteromeric ternary configuration, the E323K variant exhibited only marginal functional alterations compared to homomeric wild-type ternary, compatible with mild loss-of-function. By contrast, the P403A, V404L and V404M variants displayed strong gating impairment in the heteromeric ternary configuration, compatible with loss-of-function or gain-of-function. Our results support the etiological involvement of Kv4.2 channel gating impairment in early-onset monogenic global developmental delay. In addition, they suggest that gain-of-function mechanisms associated with a substitution of V404 increase epileptic seizure susceptibility.

Biallelic variants in PIGN cause Fryns syndrome, multiple congenital anomalies-hypotonia-seizures syndrome, and neurologic phenotypes: A genotype-phenotype correlation study.

PURPOSE: Biallelic PIGN variants have been described in Fryns syndrome, multiple congenital anomalies-hypotonia-seizure syndrome (MCAHS), and neurologic phenotypes. The full spectrum of clinical manifestations in relation to the genotypes is yet to be reported. METHODS: Genotype and phenotype data were collated and analyzed for 61 biallelic PIGN cases: 21 new and 40 previously published cases. Functional analysis was performed for 2 recurrent variants (c.2679C>G p.Ser893Arg and c.932T>G p.Leu311Trp). RESULTS: Biallelic-truncating variants were detected in 16 patients-10 with Fryns syndrome, 1 with MCAHS1, 2 with Fryns syndrome/MCAHS1, and 3 with neurologic phenotype. There was an increased risk of prenatal or neonatal death within this group (6 deaths were in utero or within 2 months of life; 6 pregnancies were terminated). Incidence of polyhydramnios, congenital anomalies (eg, diaphragmatic hernia), and dysmorphism was significantly increased. Biallelic missense or mixed genotype were reported in the remaining 45 cases-32 showed a neurologic phenotype and 12 had MCAHS1. No cases of diaphragmatic hernia or abdominal wall defects were seen in this group except patient 1 in which we found the missense variant p.Ser893Arg to result in functionally null alleles, suggesting the possibility of an undescribed functionally important region in the final exon. For all genotypes, there was complete penetrance for developmental delay and near-complete penetrance for seizures and hypotonia in patients surviving the neonatal period. CONCLUSION: We have expanded the described spectrum of phenotypes and natural history associated with biallelic PIGN variants. Our study shows that biallelic-truncating variants usually result in the more severe Fryns syndrome phenotype, but neurologic problems, such as developmental delay, seizures, and hypotonia, present across all genotypes. Functional analysis should be considered when the genotypes do not correlate with the predicted phenotype because there may be other functionally important regions in PIGN that are yet to be discovered.

The constitutional gain-of-function variant p.Glu1099Lys in NSD2 is associated with a novel syndrome.

NSD2 dimethylates histone H3 at lysine 36 (H3K36me2) and is located in the Wolf-Hirschhorn syndrome (WHS) critical region. Recent descriptions have delineated loss-of-function (LoF) variants in NSD2 with a distinct disorder. The oncogenic missense variant p.Glu1099Lys occurs somatically in leukemia and has a gain-of-function (GoF) effect. We describe two individuals carrying p.Glu1099Lys as heterozygous de novo germline variant identified by exome sequencing (ES) of blood DNA and subsequently confirmed in two ectodermal tissues. Clinically, these individuals are characterized by intellectual disability, coarse/ square facial gestalt, abnormalities of the hands, and organomegaly. Public cell lines with NSD2 GoF variants had increased K36me2, DNA promoter methylation, and dysregulated RNA expression. NSD2 GoF caused by p.Glu1099Lys is associated with a novel phenotype different from WHS and Rauch-Steindl syndrome (RAUST).

De Novo Missense Variants in SLC32A1 Cause a Developmental and Epileptic Encephalopathy Due to Impaired GABAergic Neurotransmission.

OBJECTIVE: Rare inherited missense variants in SLC32A1, the gene that encodes the vesicular gamma-aminobutyric acid (GABA) transporter, have recently been shown to cause genetic epilepsy with febrile seizures plus. We aimed to clarify if de novo missense variants in SLC32A1 can also cause epilepsy with impaired neurodevelopment. METHODS: Using exome sequencing, we identified four individuals with a developmental and epileptic encephalopathy and de novo missense variants in SLC32A1. To assess causality, we performed functional evaluation of the identified variants in a murine neuronal cell culture model. RESULTS: The main phenotype comprises moderate-to-severe intellectual disability, infantile-onset epilepsy within the first 18 months of life, and a choreiform, dystonic, or dyskinetic movement disorder. In silico modeling and functional analyses reveal that three of these variants, which are located in helices that line the putative GABA transport pathway, result in reduced quantal size, consistent with impaired filling of synaptic vesicles with GABA. The fourth variant, located in the vesicular gamma-aminobutyric acid N-terminus, does not affect quantal size, but increases presynaptic release probability, leading to more severe synaptic depression during high-frequency stimulation. Thus, variants in vesicular gamma-aminobutyric acid can impair GABAergic neurotransmission through at least two mechanisms, by affecting synaptic vesicle filling and by altering synaptic short-term plasticity. INTERPRETATION: This work establishes de novo missense variants in SLC32A1 as a novel cause of a developmental and epileptic encephalopathy. SUMMARY FOR SOCIAL MEDIA IF PUBLISHED: @platzer_k @lemke_johannes @RamiJamra @Nirgalito @GeneDx The SLC family 32 Member 1 (SLC32A1) is the only protein identified to date, that loads gamma-aminobutyric acid (GABA) and glycine into synaptic vesicles, and is therefore also known as the vesicular GABA transporter (VGAT) or vesicular inhibitory amino acid transporter (VIAAT). Rare inherited missense variants in SLC32A1, the gene that encodes VGAT/vesicular inhibitory amino acid transporter, have recently been shown to cause genetic epilepsy with febrile seizures plus. We aimed to clarify if de novo missense variants in SLC32A1 can also cause epilepsy with impaired neurodevelopment. We report on four individuals with de novo missense variants in SLC32A1 and a developmental and epileptic encephalopathy with infantile onset epilepsy. We establish causality of the variants via in silico modeling and their functional evaluation in a murine neuronal cell culture model. SLC32A1 variants represent a novel genetic etiology in neurodevelopmental disorders with epilepsy and a new GABA-related disease mechanism. ANN NEUROL 2022;92:958-973.

Gain-of-function variants in GABRD reveal a novel pathway for neurodevelopmental disorders and epilepsy.

A potential link between GABRD encoding the δ subunit of extrasynaptic GABAA receptors and neurodevelopmental disorders has largely been disregarded due to conflicting conclusions from early studies. However, we identified seven heterozygous missense GABRD variants in 10 patients with neurodevelopmental disorders and generalized epilepsy. One variant occurred in two sibs of healthy parents with presumed somatic mosaicism, another segregated with the disease in three affected family members, and the remaining five occurred de novo in sporadic patients. Electrophysiological measurements were used to determine the functional consequence of the seven missense δ subunit variants in receptor combinations of α1ß3δ and α4ß2δ GABAA receptors. This was accompanied by analysis of electroclinical phenotypes of the affected individuals. We determined that five of the seven variants caused altered function of the resulting α1ß3δ and α4ß2δ GABAA receptors. Surprisingly, four of the five variants led to gain-of-function effects, whereas one led to a loss-of-function effect. The stark differences between the gain-of-function and loss-of function effects were mirrored by the clinical phenotypes. Six patients with gain-of-function variants shared common phenotypes: neurodevelopmental disorders with behavioural issues, various degrees of intellectual disability, generalized epilepsy with atypical absences and generalized myoclonic and/or bilateral tonic-clonic seizures. The EEG showed qualitative analogies among the different gain-of-function variant carriers consisting of focal slowing in the occipital regions often preceding irregular generalized epileptiform discharges, with frontal predominance. In contrast, the one patient carrying a loss-of-function variant had normal intelligence and no seizure history, but has a diagnosis of autism spectrum disorder and suffers from elevated internalizing psychiatric symptoms. We hypothesize that increase in tonic GABA-evoked current levels mediated by δ-containing extrasynaptic GABAA receptors lead to abnormal neurotransmission, which represent a novel mechanism for severe neurodevelopmental disorders. In support of this, the electroclinical findings for the gain-of-function GABRD variants resemble the phenotypic spectrum reported in patients with missense SLC6A1 (GABA uptake transporter) variants. This also indicates that the phenomenon of extrasynaptic receptor overactivity is observed in a broader range of patients with neurodevelopmental disorders, because SLC6A1 loss-of-function variants also lead to overactive extrasynaptic δ-containing GABAA receptors. These findings have implications when selecting potential treatment options, as a substantial portion of available antiseizure medication act by enhancing GABAergic function either directly or indirectly, which could exacerbate symptoms in patients with gain-of-function GABRD variants.

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

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 were identified: Group 1, benign familial infantile epilepsy (n = 15, normal cognition, treatable seizures); Group 2, intermediate epilepsy (n = 33, mild intellectual disability, partially pharmaco-responsive); Group 3, developmental and epileptic encephalopathy (n = 177, severe intellectual disability, majority pharmaco-resistant); Group 4, generalized epilepsy (n = 20, mild to moderate intellectual disability, frequently with absence seizures); Group 5, unclassifiable epilepsy (n = 127); and Group 6, neurodevelopmental disorder without epilepsy (n = 20, mild to moderate intellectual disability). Those in Groups 1-3 presented with focal or multifocal seizures (median age of onset: 4 months) and focal epileptiform discharges, whereas the onset of seizures in patients with generalized epilepsy 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 developmental and epileptic encephalopathy showed a strong gain-of-function (hyperpolarizing shift of steady-state activation, strongly increased neuronal firing rate) and one variant causing benign familial infantile epilepsy or intermediate epilepsy showed a mild gain-of-function (defective fast inactivation, less increased firing). In contrast, all three variants causing generalized epilepsy induced a loss-of-function (reduced current amplitudes, depolarizing shift of steady-state activation, reduced neuronal firing). Functional effects were known for 170 individuals. All 136 individuals carrying a functionally tested gain-of-function variant had either focal (n = 97, Groups 1-3) or unclassifiable (n = 39) epilepsy, whereas 34 individuals with a loss-of-function variant had either generalized (n = 14), no (n = 11) or unclassifiable (n = 6) epilepsy; only three had developmental and epileptic encephalopathy. Computational modelling in the gain-of-function group revealed a significant correlation between the severity of the electrophysiological and clinical phenotypes. Gain-of-function variant carriers responded significantly better to sodium channel blockers than to other anti-seizure medications, and the same applied for all individuals in 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 loss-of-function variant carriers and the extent of the electrophysiological dysfunction of the gain-of-function variants is a main determinant of the severity of the clinical phenotype in focal epilepsies. Our pharmacological data indicate that sodium channel blockers present a treatment option in SCN8A-related focal epilepsy with onset in the first year of life.