Carla Marini

Hyperpolarization-activated cyclic nucleotide-gated 1 (HCN1) channels mediate the cationic I _h current, a fundamental regulator of neuronal excitability and synaptic integration, and are predominantly expressed in the cortex and hippocampus. Although HCN1 variants have been increasingly identified in patients with epilepsy, from milder forms to developmental and epileptic encephalopathies (DEE), genotype–phenotype correlations remain poorly defined. Here, we used patch-clamp electrophysiology and confocal imaging in HEK293 cells to functionally characterize 43 HCN1 variants identified in patients with neurodevelopmental disorders, with or without epilepsy. Based on biophysical properties, we established a classification system comprising four distinct functional classes: I) low or no current; II) hyperpolarizing shift in voltage dependence; III) depolarizing shift in voltage dependence; and IV) generation of an instantaneous current. The molecular basis for this classification was further interpreted using available cryo-EM structures of HCN1. Integration with clinical data from 50 patients revealed a striking correlation between functional class and clinical presentation. Loss-of-function variants prevailed among individuals with milder phenotypes or without epilepsy, with the latter distinctively clustering within the channel selectivity filter. In contrast, gain-of-function and mixed variants were predominantly associated with DEE. Finally, we demonstrate that allosteric modulators, including peptides NB6 and TRIP8b _nano and the small molecule J&J12e, effectively normalize channel function across three of the four classes in vitro. These findings establish a biophysical framework for interpreting HCN1 variants, demonstrating that the direction of channel dysfunction is a major determinant of epilepsy risk and severity, and providing a conceptual foundation for future class-based precision medicine approaches.

Pathogenic variants in HCN1 are associated with a spectrum of epilepsies, including drug‐resistant developmental and epileptic encephalopathies. However, evidence to guide antiseizure medication (ASM) selection in HCN1 ‐related epilepsies remains limited. In this intentionally selected, retrospective case series, we identified seven patients with confirmed pathogenic or likely pathogenic HCN1 variants associated with gain‐of‐function effects. In all cases, treatment with sodium‐channel‐blocking ASMs including phenytoin, lamotrigine, oxcarbazepine, and lacosamide was associated with seizure worsening, with clinical improvement after drug discontinuation. These findings identify a clinically actionable gene–drug interaction in HCN1 ‐related epilepsies and support a mechanism‐based approach to ASM selection. Our data provide translational evidence to inform precision treatment strategies and help avoid potentially harmful therapies in affected children.

Genetic factors are major contributors to pediatric epilepsy, but their role in status epilepticus (SE) remains incompletely defined. We aimed to characterize the clinical and genetic landscape of pediatric epilepsy complicated by SE in a large cohort, and to identify clinical features associated with genetic etiologies. We conducted a retrospective, multicenter, exploratory cohort study by selecting patients aged 1 month-18 years who experienced SE from the Italian Pediatric Status Epilepticus (IPSE) group database (2010-2022). SE and epilepsy syndromes were defined according to International League Against Epilepsy criteria. From the IPSE dataset, we included only patients with epilepsy whose etiology was classified as genetic (confirmed or presumed) or nongenetic (lesional, autoimmune, metabolic-acquired, infectious, or unknown). Clinical data were collected using standardized electronic case report forms. Of 1,071 children in the IPSE cohort, 790 with SE and epilepsy were included (median age at SE onset 3.9 years, interquartile range 1.3-8.1; 44% female). A genetic etiology was confirmed or presumed in 519 (66%). Compared with those with nongenetic etiologies (n = 271), patients with genetic epilepsies presented with SE at a younger age (median 3.4 vs 4.8 years, q = 0.003) and more often had epilepsy with both focal and generalized seizures (31% vs 13%, q < 0.001). The underlying genetic abnormality was identified in 222 patients (42.7%) including 179 with confirmed single-gene pathogenic variants and 43 chromosomal alterations. Ion channel genes were most frequent (38%), with SCN1A variants accounting for 18% of confirmed single-gene cases. Neurodevelopmental and mTOR pathway genes were also frequent contributors. Logistic regression showed that younger age at SE (odds ratio [OR] 0.92, 95% CI 0.85-0.98, p = 0.020) and epilepsy with both focal and generalized seizures (OR 4.00, 95% CI 2.09-7.85, p < 0.001) were independently associated with channelopathies. In this large real-world cohort, two-thirds of children with epilepsy and SE had a genetic etiology, most commonly channelopathies. Younger age at SE and epilepsy with both focal and generalized seizure were linked to genetic causes. Despite limitations in testing strategies and retrospective design, these findings highlight the importance of systematic genetic investigation in pediatric epilepsies presenting SE.

Objective: The transition from pediatric to adult healthcare services is a particularly sensitive phase for young adults with chronic conditions, including epilepsy. In European countries, there is a lack of standardized protocols for an appropriate and detailed transition path. This study aimed to investigate the experiences of both patients and clinicians during the transition process to adulthood, with the goal of contributing to a nationwide consensus document to standardize practices across Italy. Methods: This multicentric, observational, prospective, qualitative study included interviews with young people who turned 18 between 2018 and 2022 belonging to four regional epilepsy centers. In addition, pediatric and adult neurologists completed questionnaires addressing epilepsy type, comorbidities, daily life impact, and details of the transition process. Results: Thirty‐seven patients (92.5%) participated in the study, 34 of them began the transition process, and the remaining three included two patients who continued to receive care within pediatric services and one patient who was lost to follow up. At the time of the interview, 30 (88%) patients were still under the care of the public adult service, while one returned to the pediatric service, one was followed by a private epileptologist, and two were not under any specialist care. The study revealed significant variability in the duration of the preparatory phase, the time elapsed between referral and access to adult services, and the methods of communication/reporting. The caregivers of patients with epilepsy and associated disabilities reported greater difficulty accessing referrals for multidisciplinary and specialized care for rare or complex epilepsies in adulthood. Significance: While most patients successfully transitioned, the absence of uniform, structured protocols, in particular for patients with disabilities, created a significant difference in managing transition. The study strongly underscores the urgent need for standardized, shared national guidelines to ensure a consistent, equitable, and effective transition from pediatric to adult epilepsy care across Italy.

Objective: This study aimed to identify risk factors and develop a predictive scoring system for autoimmune‐associated epilepsy in subjects with autoimmune encephalitis presenting with new onset refractory status epilepticus (NORSE). Methods: This retrospective, multicenter, cohort study included subjects who presented with NORSE at the onset of autoimmune encephalitis and had at least 24 months of follow‐up after immunotherapy. The outcome was the development of autoimmune‐associated epilepsy, defined as persistent seizures despite adequate immunotherapy and absence of active inflammation. Factors independently associated with the outcome were identified through a backward stepwise selection. Adjusted regression coefficients of each independent predictor were transformed to produce a points‐based risk‐scoring system. Results: Seventy participants were included (median age = 24.2 years, 38.6% male). During a median follow‐up of 53 months, 54.3% of subjects developed autoimmune‐associated epilepsy. Status epilepticus duration ≥ 10 days (odds ratio [OR] = 31.14, 95% confidence interval [CI] = 2.12–456.87, p = .012), positivity for antibodies against surface antigens (OR = .12, 95% CI = .02–.85, p = .034), bitemporal magnetic resonance imaging (MRI) abnormalities suggestive of autoimmune encephalitis during acute stage (OR = 49.80, 95% CI = 2.95–841.77, p = .007), and interictal epileptiform discharges during electroencephalographic (EEG) follow‐up (OR = 71.32, 95% CI = 6.48–785.32, p < .001) were independently associated with the study outcome. The duration–antibodies–MRI–EEG (DAME) score was developed as an integer‐based scoring system predictive of autoimmune‐associated epilepsy. With an optimal cutoff of ≥3 points, it yielded a sensitivity of 86.8%, a specificity of 87.5%, and an overall accuracy of 87.1%. Significance: The DAME score could serve as a user‐friendly score to predict the risk of autoimmune‐associated epilepsy in patients with NORSE due to autoimmune encephalitis.

Objective: Variants in GABRG2 , encoding the γ2 subunit of the γ‐aminobutyric acid type A receptor, are linked to epilepsy phenotypes of varying severity, with gain‐of‐function (GoF) variants associated with the more severe phenotypes than loss‐of‐function variants. Here, we provide a comprehensive analysis of the early clinical features, motor, language, and eating abilities, and structural consequences of the recurrent GABRG2 p.(Ala106Thr) GoF variant, aiming to refine genotype–phenotype correlations and deepen the understanding of GoF‐associated GABRG2 disorders. Methods: Individuals were recruited through international collaborations, literature review, and patient advocacy groups. Clinical data were collected via standardized interviews and physician reports. Functional effects were assessed using electrophysiological recordings. Structural modeling was performed using homology‐based approaches to evaluate conformational changes. Results: We collected 23 unrelated individuals harboring the GABRG2 p.(Ala106Thr) variant. Symptoms began with early dysphagia (median onset age = 1 month), hypotonia, seizures (median onset age = 2 months), and hyperkinetic movement disorders (median onset age = 3 months). Epilepsy was present in 91% of the individuals, 71% featuring a developmental and epileptic encephalopathy; 33% of the individuals with epilepsy achieved seizure freedom during the follow‐up. All individuals exhibited moderate to severe cognitive/neurodevelopmental impairment, with profound deficits in motor, language, and other domains. Functional analysis confirmed a GoF effect of the p.(Ala106Thr) variant. By contrast, another variant affecting the same residue, p.(Ala106Pro), which is observed in population databases, was functionally characterized as neutral. Structural modeling indicated that the p.(Ala106Thr) variant may enhance receptor gating by facilitating a hydrogen bond between the extracellular and transmembrane domains, a mechanism not observed with the p.(Ala106Pro) variant. Significance: Although some individuals exhibited severe developmental impairment without epilepsy, our findings demonstrate that the recurrent GABRG2 p.(Ala106Thr) GoF variant is consistently associated with a severe neurodevelopmental phenotype. Despite its profound clinical impact, in silico tools consistently predict this variant as benign, highlighting a critical gap in current predictive models.

Objective: Highly purified cannabidiol (CBD) is approved as adjunctive therapy for seizures associated with Dravet syndrome (DS), Lennox–Gastaut syndrome (LGS), and tuberous sclerosis complex (TSC), although its role in other developmental and epileptic encephalopathies (DEEs) remains unexplored. The aim of this study was to assess the real‐world use, efficacy, and safety of CBD across a broad cohort of patients with DEEs, including off‐label indications. Methods: In this retrospective study, we evaluated 107 patients with DEEs treated with CBD for ≥3 months between 2020 and 2024. Data on seizure frequency, tolerability, retention, and non‐seizure outcomes were collected. Efficacy was defined as ≥50% or ≥75% seizure reduction. Statistical analyses explored predictors of response. Results: Patients had LGS (55.1%), DS (16.8%), TSC (8.4%), or other DEEs (19.6%), with a genetic etiology in 56.1%. At a median follow‐up of 20 months, 69% achieved ≥50% seizure reduction, and 21% achieved ≥75% reduction. Patients with LGS, TSC, and other DEEs showed higher efficacy and retention rates compared to DS. Genetic or unknown etiology was associated with better outcomes ( p = 0.011). Combination with valproate was associated with reduced efficacy ( p = 0.006), while combination with clobazam had no significant effect on efficacy nor safety. Non‐seizure improvements included increased alertness (56%), improved sleep quality (25%), and enhanced motor performance (14%). Adverse events occurred in 33.6%, mostly mild and transient; 9% discontinued due to side effects. Significances: This study confirms the effectiveness and good tolerability of CBD in a real‐world DEE population, including off‐label use. These findings support expanding CBD indications and underscore the need for prospective studies targeting both seizure and developmental outcomes. Plain language summary: This study looked at the use of cannabidiol (CBD), a cannabis‐based medicine, in 107 people with severe forms of epilepsy called developmental and epileptic encephalopathies (DEEs). With CBD add‐on, about two‐thirds of patients had fewer seizures, and some caregivers noticed improvements in alertness, sleep, or movement. Most patients tolerated the treatment well, but some experienced side effects, and a few had to stop taking it. While the results are promising, more research is needed to confirm how effective and safe CBD is for different types of DEEs, especially those not currently approved for treatment with CBD.

Objective: To characterize the clinical, electroencephalographic, and genetic features of epilepsies featuring absence seizures within monogenic etiology, highlighting the diagnostic, treatment and prognostic implications. Methods: We conducted a retrospective, multicenter study including patients with monogenic epilepsies and electroencephalography (EEG)–documented absence seizures. We analyzed clinical data, electroclinical findings, neurodevelopmental outcomes, and treatment responses through standardized questionnaires and medical records. We classified genetic variants according to American College of Medical Genetics and Genomics (ACMG) guidelines and performed univariate and multivariate analyses to identify predictors of developmental outcomes. Results: We included 160 patients (111 female; median age at last follow‐up: 13 years) with absence seizures and confirmed pathogenic or likely pathogenic monogenic variants. The most frequently implicated genes were SLC2A1, SLC6A1, SYNGAP1 , CHD2, and SCN1A . Four genes— HESX1, NCKAP1, SON, STARD9 —had not been previously associated with absence seizures. In 35% of patients, absence seizures were the only seizure type and in 67% were the initial manifestation. Atypical features included irregular EEG discharges (56%) eyelid myoclonia (42%), and automatisms (33%). Early‐onset (before age 3) seizures occurred in 58% and was significantly associated with atypical features ( p < .03). Using existing International League Against Epilepsy (ILAE) epilepsy syndrome classification, 60% of patients could not be classified. Developmental delay occurred in 54%, intellectual disability in 65%, and other neurodevelopmental comorbidities in 49%. Predictors of poor developmental outcomes included early developmental delay, drug‐resistant epilepsy, and early absence onset. We found no difference in the prevalence of drug resistance across the various genetic etiologies. The most effective medications for absence seizures included valproate, ethosuximide, benzodiazepines, and lamotrigine. Disease‐specific therapies (e.g., ketogenic diet in SLC2A1 , stiripentol/fenfluramine in SCN1A) were effective in select cases. Significance: Absence seizures are a common manifestation of different monogenic epilepsies, often associated with early onset, atypical clinical and/or EEG features, developmental delay or drug resistance. Classification models should incorporate genetic data alongside electroclinical features, especially as next‐generation sequencing is increasingly used.