Identification of a Novel Homozygous GLS Gene Variant Associated with Developmental and Epileptic Encephalopathy (DEE) Type 71.

Developmental and epileptic encephalopathy (DEEs) (OMIM#618,328) is characterized by seizures, hypotonia, and brain abnormalities, often arising from mutations in genes crucial for brain function. Among these genes, GLS stands out due to its vital role in the central nervous system (CNS), with homozygous variants potentially causing DEE type 71. Using Whole Exome Sequencing (WES) on a patient exhibiting symptoms of epileptic encephalopathy, we identified a novel homozygous variant, NM_014905.5:c.1849G > T; p.(Asp617Tyr), in the GLS gene. The 5-year-old patient, born to consanguineous parents, presented with developmental delay, encephalopathy, frequent seizures, and hypotonia. Sanger sequencing further validated the GLS gene variant in both the patient and his family. Furthermore, our bioinformatics analysis indicated that this missense variant could lead to alteration of splicing, resulting in the activation of a cryptic donor site and potentially causing loss of protein function. Our finding highlights the pathogenic significance of the GLS gene, particularly in the context of brain disorders, specifically DEE71.

Early mortality in STXBP1-related disorders.

INTRODUCTION: Pathogenic variants in STXBP1 cause a spectrum of disorders mainly consisting of developmental and epileptic encephalopathy (DEE), often featuring drug-resistant epilepsy. An increased mortality risk occurs in individuals with drug-resistant epilepsy and DEE, with sudden unexpected death in epilepsy (SUDEP) often the major cause of death. This study aimed to identify the rate and causes of mortality in STXBP1-related disorders. METHODS: Through an international call, we analyzed data on individuals with STXBP1 pathogenic variants, who passed away from causes related to their disease. RESULTS: We estimated a mortality rate of 3.2% (31/966), based on the STXBP1 Foundation and the STXBP1 Global Connect registry. In total, we analyzed data on 40 individuals (23 males) harboring pathogenic STXBP1 variants, collected from different centers worldwide. They died at a median age of 13 years (range: 11 months-46 years). The most common cause of death was SUDEP (36%), followed by pulmonary infections and respiratory complications (33%). The incidence of SUDEP peaked in mid-childhood, while non-SUDEP causes were more frequent in early childhood or adulthood (p = 0.006). In the most severe phenotypes, death was related to non-SUDEP causes (p = 0.018). CONCLUSION: We found a mortality rate in STXBP1-related disorders similar to other DEEs, with an early age at death and SUDEP as well as pulmonary infections as the main cause of death. These findings assist in prognostic evaluation and genetic counseling for the families. They help to define the mortality risk of STXBP1-related disorders and implement preventative strategies.

De Novo Truncating Variants in the Last Exon of SEMA6B Cause Progressive Myoclonic Epilepsy.

De novo variants (DNVs) cause many genetic diseases. When DNVs are examined in the whole coding regions of genes in next-generation sequencing analyses, pathogenic DNVs often cluster in a specific region. One such region is the last exon and the last 50 bp of the penultimate exon, where truncating DNVs cause escape from nonsense-mediated mRNA decay [NMD(-) region]. Such variants can have dominant-negative or gain-of-function effects. Here, we first developed a resource of rates of truncating DNVs in NMD(-) regions under the null model of DNVs. Utilizing this resource, we performed enrichment analysis of truncating DNVs in NMD(-) regions in 346 developmental and epileptic encephalopathy (DEE) trios. We observed statistically significant enrichment of truncating DNVs in semaphorin 6B (SEMA6B) (p value: 2.8 × 10-8; exome-wide threshold: 2.5 × 10-6). The initial analysis of the 346 individuals and additional screening of 1,406 and 4,293 independent individuals affected by DEE and developmental disorders collectively identified four truncating DNVs in the SEMA6B NMD(-) region in five individuals who came from unrelated families (p value: 1.9 × 10-13) and consistently showed progressive myoclonic epilepsy. RNA analysis of lymphoblastoid cells established from an affected individual showed that the mutant allele escaped NMD, indicating stable production of the truncated protein. Importantly, heterozygous truncating variants in the NMD(+) region of SEMA6B are observed in general populations, and SEMA6B is most likely loss-of-function tolerant. Zebrafish expressing truncating variants in the NMD(-) region of SEMA6B orthologs displayed defective development of brain neurons and enhanced pentylenetetrazole-induced seizure behavior. In summary, we show that truncating DNVs in the final exon of SEMA6B cause progressive myoclonic epilepsy.

Zebrafish dnm1a gene plays a role in the formation of axons and synapses in the nervous tissue.

Classical dynamins (DNMs) are GTPase proteins engaged in endocytosis, a fundamental process for cargo internalization from the plasma membrane. In mammals, three DNM genes are present with different expression patterns. DNM1 is expressed at high levels in neurons, where it takes place in the recycling of synaptic vesicles; DNM2 is ubiquitously expressed, while DNM3 is found in the brain and in the testis. Due to the conservation of genes in comparison to mammals, we took advantage of a zebrafish model for functional characterization of dnm1a, ortholog of mammalian DNM1. Our data strongly demonstrated that dnm1a has a nervous tissue-specific expression pattern and plays a role in the formation of both axon and synapse. This is the first in vivo study that collects evidence about the effects of dnm1a loss of function in zebrafish, thus providing a new excellent model to be used in different scientific fields.

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

Missense variants of hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channels cause variable phenotypes, ranging from mild generalized epilepsy to developmental and epileptic encephalopathy (DEE). Although 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 a ketogenic diet, with a significant and long-lasting reduction of episodes. 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.

Functional characterization and in vitro pharmacological rescue of KCNQ2 pore mutations associated with epileptic encephalopathy.

Mutations in the KCNQ2 gene encoding KV7.2 subunit that mediates neuronal M-current cause a severe form of developmental and epileptic encephalopathy (DEE). Electrophysiological evaluation of KCNQ2 mutations has been proved clinically useful in improving outcome prediction and choosing rational anti-seizure medications (ASMs). In this study we described the clinical characteristics, electrophysiological phenotypes and the in vitro response to KCNQ openers of five KCNQ2 pore mutations (V250A, N258Y, H260P, A265T and G290S) from seven patients diagnosed with KCNQ2-DEE. The KCNQ2 variants were transfected into Chinese hamster ovary (CHO) cells alone, in combination with KCNQ3 (1:1) or with wild-type KCNQ2 (KCNQ2-WT) and KCNQ3 in a ratio of 1:1:2, respectively. Their expression and electrophysiological function were assessed. When transfected alone or in combination with KCNQ3, none of these mutations affected the membrane expression of KCNQ2, but most failed to induce a potassium current except A265T, in which trace currents were observed when co-transfected with KCNQ3. When co-expressed with KCNQ2-WT and KCNQ3 (1:1:2), the currents at 0 mV of these mutations were decreased by 30%-70% compared to the KCNQ2/3 channel, which could be significantly rescued by applying KCNQ openers including the approved antiepileptic drug retigabine (RTG, 10 µM), as well as two candidates subjected to clinical trials, pynegabine (HN37, 1 µM) and XEN1101 (1 µM). These newly identified pathologic variants enrich the KCNQ2-DEE mutation hotspots in the pore-forming domain. This electrophysiological study provides a rational basis for personalized therapy with KCNQ openers in DEE patients carrying loss-of-function (LOF) mutations in KCNQ2.

Bi-allelic Loss-of-Function CACNA1B Mutations in Progressive Epilepsy-Dyskinesia.

The occurrence of non-epileptic hyperkinetic movements in the context of developmental epileptic encephalopathies is an increasingly recognized phenomenon. Identification of causative mutations provides an important insight into common pathogenic mechanisms that cause both seizures and abnormal motor control. We report bi-allelic loss-of-function CACNA1B variants in six children from three unrelated families whose affected members present with a complex and progressive neurological syndrome. All affected individuals presented with epileptic encephalopathy, severe neurodevelopmental delay (often with regression), and a hyperkinetic movement disorder. Additional neurological features included postnatal microcephaly and hypotonia. Five children died in childhood or adolescence (mean age of death: 9 years), mainly as a result of secondary respiratory complications. CACNA1B encodes the pore-forming subunit of the pre-synaptic neuronal voltage-gated calcium channel Cav2.2/N-type, crucial for SNARE-mediated neurotransmission, particularly in the early postnatal period. Bi-allelic loss-of-function variants in CACNA1B are predicted to cause disruption of Ca2+ influx, leading to impaired synaptic neurotransmission. The resultant effect on neuronal function is likely to be important in the development of involuntary movements and epilepsy. Overall, our findings provide further evidence for the key role of Cav2.2 in normal human neurodevelopment.

Genetic Advancements in Infantile Epileptic Spasms Syndrome and Opportunities for Precision Medicine.

Infantile epileptic spasms syndrome (IESS) is a devastating developmental epileptic encephalopathy (DEE) consisting of epileptic spasms, as well as one or both of developmental regression or stagnation and hypsarrhythmia on EEG. A myriad of aetiologies are associated with the development of IESS; broadly, 60% of cases are thought to be structural, metabolic or infectious in nature, with the remainder genetic or of unknown cause. Epilepsy genetics is a growing field, and over 28 copy number variants and 70 single gene pathogenic variants related to IESS have been discovered to date. While not exhaustive, some of the most commonly reported genetic aetiologies include trisomy 21 and pathogenic variants in genes such as TSC1, TSC2, CDKL5, ARX, KCNQ2, STXBP1 and SCN2A. Understanding the genetic mechanisms of IESS may provide the opportunity to better discern IESS pathophysiology and improve treatments for this condition. This narrative review presents an overview of our current understanding of IESS genetics, with an emphasis on animal models of IESS pathogenesis, the spectrum of genetic aetiologies of IESS (i.e., chromosomal disorders, single-gene disorders, trinucleotide repeat disorders and mitochondrial disorders), as well as available genetic testing methods and their respective diagnostic yields. Future opportunities as they relate to precision medicine and epilepsy genetics in the treatment of IESS are also explored.

A roadmap to cure CHD2-related disorders.

Coalition to Cure CHD2 (CCC) is a patient advocacy group dedicated to improving the lives of those affected by CHD2-related disorders (CHD2-RD) by increasing education, building community, and accelerating research to uncover a cure. CHD2 is a chromatin remodeler that was identified in 2013 as being a genetic cause for developmental and epileptic encephalopathies. Pathogenic changes in CHD2 can cause treatment-resistant epilepsy, intellectual and developmental delays, and autism, and some individuals experience neurodevelopmental regression. There are currently no targeted therapies available for CHD2-related disorders. Haploinsufficiency of CHD2 is a causative mechanism of disease for individuals with pathogenic variants (primarily truncating) in CHD2. Recently, identification of individuals with deletion of nearby gene CHASERR, a regulator of CHD2 gene expression, has established dosage sensitivity in CHD2 and solidified the CHASERR gene as a potential therapeutic target for CHD2 levels. Through collaboration with our community and our scientific advisory board, CCC has created a Roadmap to Cure CHD2 as our guide toward a targeted cure that can benefit our community, with steps including (1) identifying and defining patients, (2) developing models of CHD2, (3) studying models of CHD2, (4) testing therapies, (5) involving patients, and (6) reaching a cure. Despite some of the challenges inherent in CHD2 research including establishing animal and cellular models that recapitulate the CHD2 clinical phenotype, identifying measurable outcomes and reliable biomarkers, or testing emerging therapeutic approaches, CCC continues to engage with our community to support ongoing research that aligns with our priorities. CCC sees new and exciting opportunities for additional research that can move our community toward our common goal of a cure that will improve the lives of individuals and their families now and in the future.

A roadmap to cure disorders caused by the CHD2 and CHASERR genes Coalition to Cure CHD2 (CCC) is a nonprofit founded in October 2020 to fund research towards a cure for individuals with CHD2-related disorders. The CHD2 gene was discovered as a genetic cause for epilepsy in 2013. Individuals with CHD2 typically experience seizures that can be resistant to treatment, intellectual disability, delayed development, autism, and other symptoms. The nearby CHASERR gene has been found to regulate CHD2 and is a possible therapeutic target. Individuals with a deletion of CHASERR have been identified - these individuals have too much CHD2 and more severe symptoms. CCC has created a Roadmap to Cure CHD2 as a guide for their journey towards a targeted cure for CHD2-related disorders. The steps in the roadmap include: (1) identify and define patients, (2) develop models of CHD2, (3) study models of CHD2, (4) test therapies, (5) involve patients, (6) reach a cure. CCC has worked with CHD2 families to identify family-level priorities for therapeutic development (e.g. seizures, behavior, etc), to capture the impact of disease through qualitative research, and to collect patient health data and tissue samples for scientific analysis. The development of CHD2 models, mouse models in particular, has been challenging as the mice do not develop seizures. Additional models are underway including frogs, zebrafish, and patient-derived cells. These models have provided crucial insight into the biology of CHD2 but scientific questions remain unanswered. A variety of therapeutic approaches have been proposed including novel treatments that directly target CHD2 biology as well as the repurposing of existing FDA-approved compounds. Establishing measurable outcomes, including biomarkers, and finding treatments that can reach the brain will be important. By continuing to follow this roadmap, the CCC believes that one day there will be a cure for CHD2-related disorders.

Biallelic pathogenic variants of PARS2 cause developmental and epileptic encephalopathy with spike-and-wave activation in sleep.

BACKGROUND: Biallelic pathogenic variants in the mitochondrial prolyl-tRNA synthetase 2 gene (PARS2, OMIM * 612036) have been associated with Developmental and Epileptic Encephalopathy-75 (DEE-75, MIM #618437). This condition is typically characterized by early-onset refractory infantile spasms with hypsarrhythmia, intellectual disability, microcephaly, cerebral atrophy with hypomyelination, lactic acidemia, and cardiomyopathy. Most affected individuals do not survive beyond the age of 10 years. METHODS: We describe a patient with early-onset DEE, consistently showing an EEG pattern of Spike-and-Wave Activation in Sleep (SWAS) since childhood. The patient underwent extensive clinical, metabolic and genetic investigations, including whole exome sequencing (WES). RESULTS: WES analysis identified compound heterozygous variants in PARS2 that have been already reported as pathogenic. A literature review of PARS2-associated DEE, focusing mainly on the electroclinical phenotype, did not reveal the association of SWAS with pathogenic variants in PARS2. Notably, unlike previously reported cases with the same genotype, this patient had longer survival without cardiac involvement or lactic acidosis, suggesting potential genetic modifiers contributing to disease variability. CONCLUSION: These findings widen the genetic heterogeneity of DEE-SWAS, including PARS2 as a causative gene in this syndromic entity, and highlight the importance of prolonged sleep EEG recording for the recognition of SWAS as a possible electroclinical evolution of PARS2-related DEE.

Developmental changes in brain activity of heterozygous Scn1a knockout rats.

Introduction: Dravet syndrome (DS) is an infantile-onset developmental and epileptic encephalopathy characterized by an age-dependent evolution of drug-resistant seizures and poor developmental outcomes. Functional impairment of gamma-aminobutyric acid (GABA)ergic interneurons due to loss-of-function mutation of SCN1A is currently considered the main pathogenesis. In this study, to better understand the age-dependent changes in the pathogenesis of DS, we characterized the activity of different brain regions in Scn1a knockout rats at each developmental stage. Methods: We established an Scn1a knockout rat model and examined brain activity from postnatal day (P) 15 to 38 using a manganese-enhanced magnetic resonance imaging technique (MEMRI). Results: Scn1a heterozygous knockout (Scn1a +/-) rats showed a reduced expression of voltage-gated sodium channel alpha subunit 1 protein in the brain and heat-induced seizures. Neural activity was significantly higher in widespread brain regions of Scn1a +/- rats than in wild-type rats from P19 to P22, but this difference did not persist thereafter. Bumetanide, a Na+-K+-2Cl- cotransporter 1 inhibitor, mitigated hyperactivity to the wild-type level, although no change was observed in the fourth postnatal week. Bumetanide also increased heat-induced seizure thresholds of Scn1a +/- rats at P21. Conclusions: In Scn1a +/- rats, neural activity in widespread brain regions increased during the third postnatal week, corresponding to approximately 6 months of age in humans, when seizures most commonly develop in DS. In addition to impairment of GABAergic interneurons, the effects of bumetanide suggest a possible contribution of immature type A gamma-aminobutyric acid receptor signaling to transient hyperactivity and seizure susceptibility during the early stage of DS. This hypothesis should be addressed in the future. MEMRI is a potential technique for visualizing changes in basal brain activity in developmental and epileptic encephalopathies.

Synaptopathies in Developmental and Epileptic Encephalopathies: A Focus on Pre-synaptic Dysfunction.

The proper connection between the pre- and post-synaptic nervous cells depends on any element constituting the synapse: the pre- and post-synaptic membranes, the synaptic cleft, and the surrounding glial cells and extracellular matrix. An alteration of the mechanisms regulating the physiological synergy among these synaptic components is defined as "synaptopathy." Mutations in the genes encoding for proteins involved in neuronal transmission are associated with several neuropsychiatric disorders, but only some of them are associated with Developmental and Epileptic Encephalopathies (DEEs). These conditions include a heterogeneous group of epilepsy syndromes associated with cognitive disturbances/intellectual disability, autistic features, and movement disorders. This review aims to elucidate the pathogenesis of these conditions, focusing on mechanisms affecting the neuronal pre-synaptic terminal and its role in the onset of DEEs, including potential therapeutic approaches.

Monogenic developmental and epileptic encephalopathies of infancy and childhood, a population cohort from Norway.

Introduction: Developmental and epileptic encephalopathies (DEE) is a group of epilepsies where the epileptic activity, seizures and the underlying neurobiology contributes to cognitive and behavioral impairments. Uncovering the causes of DEE is important in order to develop guidelines for treatment and follow-up. The aim of the present study was to describe the clinical picture and to identify genetic causes in a patient cohort with DEE without known etiology, from a Norwegian regional hospital. Methods: Systematic searches of medical records were performed at Drammen Hospital, Vestre Viken Health Trust, to identify patients with epilepsy in the period 1999-2018. Medical records were reviewed to identify patients with DEE of unknown cause. In 2018, patients were also recruited consecutively from treating physicians. All patients underwent thorough clinical evaluation and updated genetic diagnostic analyses. Results: Fifty-five of 2,225 patients with epilepsy had DEE of unknown etiology. Disease-causing genetic variants were found in 15/33 (45%) included patients. Three had potentially treatable metabolic disorders (SLC2A1, COQ4 and SLC6A8). Developmental comorbidity was higher in the group with a genetic diagnosis, compared to those who remained undiagnosed. Five novel variants in known genes were found, and the patient phenotypes are described. Conclusion: The results from this study illustrate the importance of performing updated genetic investigations and/or analyses in patients with DEE of unknown etiology. A genetic cause was identified in 45% of the patients, and three of these patients had potentially treatable conditions where available targeted therapy may improve patient outcome.

Case Report: Effect of Targeted Therapy With Carbamazepine in KCNQ2 Neonatal Epilepsy.

We present a family case of neonatal-onset KCNQ2-related epilepsy due to a novel intronic mutation. Three members of an Italian family (father and offspring) presented with neonatal-onset asymmetric tonic and clonic seizures with peculiar video-electroencephalography and aEEG features referring to sequential seizures. The father and the first son underwent standard of care treatments in line with current neonatal intensive care unit protocols, with a prolonged hospitalization before reaching full seizure control with carbamazepine. After the experience acquired with her family and the latest advances in the literature, the younger daughter was directly treated with carbamazepine, obtaining rapid seizure control and short hospitalization. They all had normal development. Carbamazepine is rarely administered as a first-line option in neonatal seizures. Recent evidence suggests that neonatal intensive care unit protocols should implement a trial with sodium channel blockers such as carbamazepine as first-option anti-seizure medication and a fast access to genetic testing in neonates with sequential seizures without structural brain injury or acute causes. Moreover, we report and discuss the laboratory studies performed on a novel causative intronic mutation in KCNQ2 (c.1525+5 G>A in IVS13), since pathogenicity may be difficult to prove for intronic variants.

Developmental and epileptic encephalopathies: recognition and approaches to care.

The term "developmental and epileptic encephalopathy" (DEE) refers to when cognitive functions are influenced by both seizure and interictal epileptiform activity and the neurobiological process behind the epilepsy. Many DEEs are related to gene variants and the onset is typically during early childhood. In this setting, neurocognition, whilst not improved by seizure control, may benefit from some precision therapies. In patients with non-progressive diseases with cognitive impairment and co-existing epilepsy, in whom the epileptiform activity does not affect or has minimal effect on function, the term "developmental encephalopathy" (DE) can be used. In contrast, for those patients with direct impact on cognition due to epileptic or epileptiform activity, the term "epileptic encephalopathy" (EE) is preferred, as most can revert to their normal or near normal baseline cognitive state with appropriate intervention. These children need aggressive treatment. Clinicians must tailor care towards individual needs and realistic expectations for each affected person; those with DE are unlikely to gain from aggressive antiseizure medication whilst those with EE will gain. Patients with DEE might benefit from a precision medicine approach in order to reduce the overall burden of epilepsy.

Screening Platforms for Genetic Epilepsies-Zebrafish, iPSC-Derived Neurons, and Organoids.

Recent advances in molecular and cellular engineering, such as human cell reprogramming, genome editing, and patient-specific organoids, have provided unprecedented opportunities for investigating human disorders in both animals and human-based models at an improved pace and precision. This progress will inevitably lead to the development of innovative drug-screening platforms and new patient-specific therapeutics. In this review, we discuss recent advances that have been made using zebrafish and human-induced pluripotent stem cell (iPSC)-derived neurons and organoids for modeling genetic epilepsies. We also provide our prospective on how these models can potentially be combined to build new screening platforms for antiseizure and antiepileptogenic drug discovery that harness the robustness and tractability of zebrafish models as well as the patient-specific genetics and biology of iPSC-derived neurons and organoids.