OBJECTIVE:Sudden unexpected death in epilepsy (SUDEP) is the leading cause of premature mortality in epilepsy. Genetic studies have identified that loss-of-function (LOF) KCNH2 variants are enriched in SUDEP patients, suggesting that they may act as a risk factor. KCNH2 encodes the KV11.1 channel, with LOF pathogenic variants a cause of long-QT syndrome (LQTS), increasing the risk of arrhythmia and sudden cardiac death. Here, we engineered preclinical rodent models that combine epilepsy-causing pathogenic variants with heterozygous Kcnh2 knockout mice to explore the impact of reduced KV11.1 channel function on mortality. METHODS:Both the Gabrg2R43Q/+ and Hcn1M294L/+ genetic mouse models of monogenic epilepsy were crossed with Kcnh2+/- mice. All genotypes were video-recorded post-weaning and time to death was measured. Additional mice underwent surgery to enable simultaneous electrocorticography and electrocardiography recordings. Atenolol was delivered in drinking water to a subset of mice. RESULTS:Both single mutant Gabrg2R43Q/+ and Hcn1M294L/+ mice displayed spontaneous seizures recapitulating the human phenotypes. Single mutant Kcnh2+/- mice exhibited an LQTS phenotype. Double mutant mice (Gabrg2R43Q/+/Kcnh2+/- and Hcn1M294L/+/Kcnh2+/-) had both seizure and prolonged QT interval phenotypes that were similar to their respective single mutant mice. Survival analysis revealed that Gabrg2R43Q/+/Kcnh2+/- and Hcn1M294L/+/Kcnh2+/- mice experienced a disproportionately higher rate of seizure-related death when compared to wild-type and their respective single mutant littermates. Oral administration of the cardiac-selective β-blocker atenolol significantly improved survival in Gabrg2R43Q/+/Kcnh2+/-, Hcn1M294L/+, and Hcn1M294L/+/Kcnh2+/- mice. Atenolol attenuated the sympathetic cardiac response to non-terminal seizures. SIGNIFICANCE:The data support the premise that LOF KCNH2 variants can contribute to SUDEP risk in a subset of epilepsy patients. Our findings also highlight the potential use of β-blockers as a prevention strategy in SUDEP.

OBJECTIVE:We analyzed the long-term safety and effectiveness of fenfluramine (FFA) in patients with Dravet syndrome (DS) in an open-label extension (OLE) study after participating in randomized controlled trials (RCTs) or commencing FFA de novo as adults. METHODS:Patients with DS who participated in one of three RCTs or were 19 to 35 years of age and started FFA de novo were included. Key endpoints were: incidence of treatment-emergent adverse events (TEAEs) in the safety population, and median percentage change in monthly convulsive seizure frequency (MCSF) from the RCT baseline to end of study (EOS) in the modified intent-to-treat (mITT) population. Post hoc analyses compared effectiveness in patients on concomitant stiripentol (STP) vs those not taking STP, and assessed safety (TEAEs) and effectiveness (Clinical Global Impression-Improvement [CGI-I] scale ratings) in patients enrolled as adults. RESULTS:A total of 374 patients, including 45 adults, received ≥1 FFA dose. Median FFA exposure was 824 days (range, 7-1280). TEAEs occurring in ≥10% of patients were pyrexia, nasopharyngitis, decreased appetite, seizure, decreased blood glucose, diarrhea, abnormal echocardiography (only physiologic regurgitation), upper respiratory tract infection, influenza, vomiting, and ear infection; no valvular heart disease or pulmonary arterial hypertension was observed over the OLE. In the mITT population (n = 324), median percentage change in MCSF from baseline to EOS was -66.8% (p < .001). The post hoc analyses of MCSF change from baseline to EOS in patients on concomitant STP (n = 75) was -36.2% vs -71.6% in those not on concomitant STP (n = 234) (p < .0001). In adult patients, 29 of 41 (70.7%) and 29 of 42 patients (69.1%) demonstrated clinically meaningful improvement on CGI-I at last visit as rated by caregivers and investigators, respectively. SIGNIFICANCE:Our OLE study of FFA in patients with DS confirmed previous positive findings and extended the exposure up to 3.5 years. No new or unexpected safety signals were observed and FFA demonstrated sustained and clinically meaningful reduction in MCSF.

Reanalysis of genomic data in rare disease is highly effective in increasing diagnostic yields but remains limited by manual approaches. Automation and optimization for high specificity will be necessary to ensure scalability, adoption and sustainability of iterative reanalysis. We developed a publicly available automated tool, Talos, and validated its performance using data from 1,089 individuals with rare genetic disease. Trio-based analysis identified 86% of known in-scope diagnoses, returning one variant per case on average. Variant burden reduced to one variant per 200 cases on iterative monthly reanalysis cycles. Application to an unselected cohort of 4,735 undiagnosed individuals identified 248 diagnoses (5.2% yield): 73 (29%) due to new gene-disease relationships, 56 (23%) due to new variant-level evidence, and 119 (48%) due to improved filtering and analysis strategies. Our automated, iterative reanalysis model, applied to thousands of rare disease patients, demonstrates the feasibility of delivering frequent, systematic reanalysis at scale.

Variants in spliceosomal small nuclear RNA (snRNA) genes RNU4-2 (ReNU syndrome), RNU5B-1, and RNU2-2 have recently been linked to dominant neurodevelopmental disorders (NDDs), revealing a major, previously overlooked role for noncoding snRNAs in human disease. Here, we systematically analysed 200 potentially functional snRNA genes in a French cohort comprising 26,911 individuals with rare disorders and through international collaborations. We identify de novo and biallelic variants in RNU2-2 associated with both dominant and recessive NDDs in 126 individuals from 108 unrelated families. Recessive RNU2-2 NDD is at least twice as frequent as the dominant NDD caused by n.4G>A and n.35A>G, and often arises from a de novo variant in trans with an inherited allele, reflecting the high mutability of snRNA genes. Dominant and recessive RNU2-2-NDDs share overlapping clinical features with frequent epilepsy. Blood transcriptomics and DNA methylation analyses revealed subtle, variant-specific effects on splicing and episignatures. Our findings support a gradient-of-impact model and a continuum between dominant and recessive inheritance, establishing RNU2-2 variants as a frequent cause of NDDs, nearly as prevalent as ReNU syndrome.

Valproate (VPA) use during pregnancy is associated with a wide range of birth defects and adverse neurodevelopmental outcomes, but not all exposed children are affected and there is evidence for a genetic predisposition. We hypothesised that genomic variants that impact on the binding affinity of transcription factors (TFs) are integral to VPA-associated teratogenicity and a plausible explanation for variance in interindividual risk. We interrogated maternal exomes from women recruited through international epilepsy genomics consortia. The variant burden within genes associated with 32 different birth defect types was higher for those exposed to VPA as compared to other antiseizure medications (OR 1·73 [95% CI 1·40 to 2·14], p = 2·25E-07). Variants in women exposed to VPA were predicted to impact the binding affinity of 359 TFs and network analysis of encoded proteins indicated that a master regulator, EP300, interacts with 42% (151/359) of all variant sensitive TFs. We then profiled coexpression between EP300 and other TFs in differentiating neurons derived from human embryonic stem cells (hESCs) exposed to VPA at 300µM and 700 µM, or unexposed, and a reference map generated using public data. We found strong overlap in EP300 -TF coexpressed pairs between the reference and all comparison groups (99%,900/911) but only 32% (134/422) of pairs observed in unexposed cells were evident following VPA exposure, and over half of all pairs (489/911) were observed in VPA-exposed cells only. Our findings suggest that VPA-induced disruption of EP300- related regulation is common across birth defect types and that genetic variation can modify subsequent transcriptional dysregulation, explaining why only some pregnancies are affected. The results have implications for the development of genetic risk biomarkers and safer drugs. ### Competing Interest Statement PP is supported by an Emerging Leadership Investigator Grant from the from the Australian National Health and Medical Research Council (APP2017651), The University of Melbourne, Monash University, the Austin Medical Research Foundation, and the Norman Beischer Medical Research Foundation. He has received speaker honoraria or consultancy fees to his institution from Chiesi, Eisai, LivaNova, Novartis, Sun Pharma, Supernus, and UCB Pharma, outside of the submitted work. He is on the board of International Registry of Antiepileptic Drugs and Pregnancy (EURAP), a non-profit organization that has received financial support from Accord, Angelini, Bial, EcuPharma, Eisai, Glenmark, GW Pharma, GlaxoSmithKline, Sanofi, SF Group, Teva, UCB, and Zentiva. He is Deputy Editor for Epilepsia Open. EP has received consultancy fees from Angelini, Arvelle, Sanofi group of companies, Shackelford Pharma, SKL Life Sciences and Takeda. He has participated on the board of Angelini, Arvelle, GW Phara, Janssen and Xenon Pharma. IES has served on scientific advisory boards for BioMarin, Chiesi, Eisai, Encoded Therapeutics, GlaxoSmithKline, Knopp Biosciences, Nutricia, Rogcon, Takeda Pharmaceuticals, UCB, Xenon Pharmaceuticals, Cerecin; has received speaker honoraria from GlaxoSmithKline, UCB, BioMarin, Biocodex, Chiesi, Liva Nova, Nutricia, Zuellig Pharma, Stoke Therapeutics and Eisai; has received funding for travel from UCB, Biocodex, GlaxoSmithKline, Biomarin, Encoded Therapeutics, Stoke Therapeutics and Eisai; has served as an investigator for Anavex Life Sciences, Cerevel Therapeutics, Eisai, Encoded Therapeutics, EpiMinder Inc, Epygenyx, ES-Therapeutics, GW Pharma, Marinus, Neurocrine BioSciences, Ovid Therapeutics, Takeda Pharmaceuticals, UCB, Ultragenyx, Xenon Pharmaceuticals, Zogenix and Zynerba; and has consulted for Care Beyond Diagnosis, Epilepsy Consortium, Atheneum Partners, Ovid Therapeutics, UCB, Zynerba Pharmaceuticals, BioMarin, Encoded Therapeutics and Biohaven Pharmaceuticals; and is a Non-Executive Director of Bellberry Ltd and a Director of the Australian Academy of Health and Medical Sciences and the Australian Council of Learned Academies Limited. She may accrue future revenue on pending patent WO61/010176 (filed: 2008): Therapeutic Compound; has a patent for SCN1A testing held by Bionomics Inc and licensed to various diagnostic companies; has a patent molecular diagnostic/theranostic target for benign familial infantile epilepsy (BFIE) [PRRT2] 2011904493 & 2012900190 and PCT/AU2012/001321 (TECH ID:2012-009). TOB has received consulting and/or research funding from UCB, Eisai, Supernus, Kinosis Pharmaceuticals, ES Therapeutics, and Government grant funding from the NHMRC, MRFF, DoD and NINDS. FJEV and the Raoul Wallenberg Australian Pregnancy Register of Antiepileptic Drugs have received funding from Epilepsy Action Australia, The Epilepsy Society of Australia, UCB, Eisai and Sanofi. ### Funding Statement The study was funded by NHMRC Project Grant (APP1059858) to TJOB, FJEV, PK, JC, SFB, and NHMRC Program Grant (APP1091593) to TJOB, SFB and IES, and EC grant 279062, EpiPGX (to EpiPGX consortium, BPCK, JC and SMS). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The study was approved by the Melbourne Health Human Research and Ethics Committee and written informed consent was obtained from all participants I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes RNA-seq data from our hESC assay are available from Gene Expression Omnibus (GEO) Accession Number GSE290300

DNA methylation signatures ("episignatures") can be used as biomarkers of genetic aberrations, clinical phenotypes, and environmental exposures in rare diseases. Episignatures are utilized in molecular diagnostics and can clarify variants of uncertain significance. A growing number of disease genes, including epilepsy genes, exhibit robust and reproducible episignatures. However, whether SCN1A, the most prominent epilepsy gene, has one or more episignatures has not yet been determined. We generated genome-wide DNA methylation data and performed episignature analysis on 64 individuals with Dravet syndrome due to pathogenic loss-of-function (LOF) variants in SCN1A and seven individuals with early infantile SCN1A developmental and epileptic encephalopathy due to pathogenic gain-of-function (GOF) variants in SCN1A, relative to a large reference database of controls and rare disease episignature-positive cohorts. We analyzed all samples with LOF variants together and performed separate analyses for missense, nonsense, and GOF variant cohorts. A reproducible blood-derived episignature was not evident in any of the cohorts using current analytical approaches and reference data.

Cerebral cavernous malformations (CCMs) are intracranial vascular lesions associated with risk of haemorrhages and seizures. While the majority are sporadic and often associated with somatic variants in PIK3CA and MAP3K3, around 20% are familial with germline variants in one of three CCM genes-KRIT1/CCM1, CCM2 and PDCD10/CCM3. We performed comprehensive phenotyping and genetic analysis of nine multiplex families and ten sporadic individuals with CCM. In the familial cases, initial standard analyses had a low yield, we therefore searched for small copy number changes and deep intronic variants. Subsequently, pathogenic germline variants in KRIT1/CCM1 or CCM2 were identified in all 9 multiplex families. Single or multiple exon deletions or splice site variants in KRIT1/CCM1 were found in 3/9 families. Where cavernous malformation tissue was available, second hit somatic PIK3CA variants were identified in 4/7 individuals. These 4 individuals were from separate families with germline KRIT1/CCM1 variants. In 8/10 sporadic cases, we detected recurrent pathogenic somatic PIK3CA, MAP3K3 or CCM2 variants. All familial cases had multiple CCMs, whereas the sporadic cases had a single lesion only, which was in the temporal lobe in 9/10 individuals. Our comprehensive approach interrogating deep intronic variants combined with detection of small copy number variants warrants implementation in standard clinical genetic testing pipelines to increase diagnostic yield. We also build on the established second hit germline and somatic variant mechanism in some CCM lesions. Genetic diagnosis has clinical implications such as reproductive counselling and provides potential eligibility for precision medicine therapies to treat rapidly growing CCMs.

BACKGROUND:Sudden Unexpected Death in Epilepsy (SUDEP) is a rare and tragic outcome in epilepsy, identified by those with the condition as their most serious concern. Although several clinical factors are associated with elevated SUDEP risk, mechanisms underlying SUDEP are poorly understood, making individual risk prediction challenging, especially early in the disease course. We hypothesised that common genetic variation contributes to SUDEP risk. METHODS:Genetic data from people who had succumbed to SUDEP was compared to data from people with epilepsy who had not succumbed to SUDEP and from healthy controls. Polygenic risk scores (PRSs) for longevity, intelligence and epilepsy were compared across cohorts. Reactome pathways and gene ontology terms implicated by the contributing single nucleotide polymorphisms (SNPs) were explored. In the subset of SUDEP cases with the necessary data available, a risk score was calculated using an existing risk prediction tool (SUDEP-3); the added value to this prediction of SNP-based genomic information was evaluated. FINDINGS:Only European-ancestry participants were included. 161 SUDEP cases were compared to 768 cases with epilepsy and 1153 healthy controls. PRS for longevity was significantly reduced in SUDEP cases compared to disease (P = 0·0096) and healthy controls (P = 0·0016), as was PRS for intelligence (SUDEP cases compared to disease (P = 0·0073) and healthy controls (P = 0·00024)). The PRS for epilepsy did not differ between SUDEP cases and disease controls (P = 0·76). SNP-determined pathway and gene ontology analysis highlighted those related to inter-neuronal communication as amongst the most enriched in SUDEP. Addition of PRS for longevity and intelligence to SUDEP-3 scores improved risk prediction in a subset of cases (38) and controls (703), raising the area-under-the-curve in a receiver-operator characteristic from 0·699 using SUDEP-3 alone to 0·913 when PRSs were added. INTERPRETATION:Common genetic variation contributes to SUDEP risk, offering new approaches to improve risk prediction and to understand underlying mechanisms. FUNDING:The Amelia Roberts Fund; CURE Epilepsy; Epilepsy Society, UK; Finding A Cure for Epilepsy and Seizures (FACES).

OBJECTIVE:A growing body of evidence indicates a strong genetic overlap between developmental and epileptic encephalopathies (DEEs) and movement disorders. De novo loss-of-function variants in NUS1 have been recently identified in DEE cases. Herein, we report a large cohort of cases with pathogenic NUS1 variants and describe their clinical presentation and the details of the associated epilepsy and movement disorders. METHODS:Cases with NUS1-related disorders were identified through a multicentric international collaboration made possible by the GeneMatcher platform. Clinical data were acquired through retrospective case-note review. RESULTS:We identified 41 subjects carrying 38 different pathogenic or likely pathogenic heterozygous NUS1 variants. The majority of cases displayed developmental delays and intellectual disability of variable severity. Epilepsy was present in 68.3% of cases (28/41) with onset typically in early childhood. Strikingly, 87.8% of cases (36/41) presented with movement disorders and for 13 of these cases the movement disorder was not accompanied by epilepsy. The phenomenology of the movement disorders was complex with myoclonus observed in 68.3% of cases (28/41), either in isolation or in combination with dystonia, ataxia, and/or parkinsonism. Seven cases that otherwise did not have prominent movement disorders had mild incoordination and intention tremor, suggestive of cerebellar dysfunction. There was no observed genotype-phenotype correlation, suggesting that other genetic or acquired factors impact the clinical presentation. INTERPRETATION:Heterozygous NUS1 pathogenic variants cause a complex neurological disorder, variably featuring developmental and epileptic encephalopathies and a broad spectrum of movement disorders, which represent the major source of neurological disability for most cases. ANN NEUROL 2025;98:561-572.

Background Developmental and epileptic encephalopathies (DEE) are rare but severe neurodevelopmental disorders characterised by early-onset seizures often combined with developmental delay, behavioural and cognitive deficits. Treatment for DEEs is currently limited to seizure control and provides no benefits to the patients' developmental and cognitive outcomes. Genetic variants are the most common cause of DEE with KCNQ2 being one of the most frequently identified disease-causing genes. KCNQ2 encodes a voltage-gated potassium channel KV7.2 widely expressed in the central nervous system and critically involved in the regulation of neuronal excitability. In this study, we aimed to characterise a KCNQ2 variant (K556E) found in a female patient with DEE using a heterologous expression system and a knock-in mouse model. Methods Wild-type KCNQ2 or K556E variant were expressed in Chinese Hamster Ovary (CHO) cells (with or without KCNQ3) and their biophysical properties assessed using patch clamp recordings. We further engineered a new Kcnq2 DEE mouse model (K557E) based on the K556E variant and characterised it using behavioural, electrophysiological, and transcriptome analysis. Results A mild loss of function was observed only when the mutant channel was co-expressed with KCNQ3 in the heterologous system. The heterozygous knock-in mice showed a reduced survival rate and increased susceptibility to induced seizures. Electrophysiology recordings in brain slices revealed a hyperexcitable phenotype for cortical layer 2/3 pyramidal neurons with retigabine (KV7 channel opener) able to rescue both the increased sensitivity to chemically-induced seizures in vivo and neuronal excitability ex vivo. Whole-brain RNA sequencing revealed numerous differentially expressed genes and biological pathways pointing at dysregulation of early developmental processes. Conclusions Our study reports on a novel Kcnq2 DEE mouse model recapitulating aspects of the disease phenotype with the electrophysiological and transcriptome analysis providing insights into KCNQ2 DEE mechanisms that can be leveraged for future therapy development.

The ketogenic diet (KD) therapy is a primary treatment for drug-resistant epilepsy, and beta-hydroxybutyrate (BHB) is the main ketone produced during KD. However, the pattern of increase in BHB levels is not well understood, and the reference range for BHB need to be defined. The aim of this study was to evaluate the BHB levels in the first three months, especially one week, after KD initiation, and to explore the physiological reference range for BHB. In our study, a fasting initiation strategy was used for the majority of patients (252/300, 84

KIF1A-associated neurological disorder (KAND) is a genetic condition characterised by motor, cognitive and ophthalmologic features. The speech and language phenotype have not been systematically analysed. Here, we assess speech and language using observer- and clinician-reported outcomes, and performance outcome measures. 44 individuals (25 female) with KAND (median age 7 years, range 1-60 years) participated. Median age at diagnosis was 4 years (range 0.5-58 years). KIF1A variants were missense (41/44 individuals, 93%), intragenic deletion (2/44, 5%) and splice site (1/44, 2%). Age at first words was delayed (>12 months) in 38/44 (86%) individuals. At assessment, 28/44 (64%) combined words into sentences and all of the 20 individuals assessed had dysarthria. Apraxic speech features and phonological impairments occurred in children aged under 8 years. 36/37 (97%) participants had language impairment, with expressive language skills stronger than receptive (p = 0.02) and written (p = 0.03) language on the Vineland Adaptive Behaviour Scales. 7/32 (22%) caregivers reported speech and language regression. Mild to severe intellectual disability occurred in 31/33 (94%) individuals. 22/44 (50%) participants had used augmentative and alternative communication, such as key word sign or speech generating devices. Individuals had average social motivation skills in contrast to moderately impaired social cognition, communication and awareness on the Social Responsiveness Scale (p < 0.05). 16/44 (36%) had epilepsy and 40/44 (91%) had visual impairment, namely nystagmus (16/44, 36%), optic nerve atrophy and strabismus (both 12/44, 27%). Individuals with KAND frequently have speech and language disorders necessitating early and targeted speech and language interventions.

The evolution from nocturnal paroxysmal dystonia (NPD) to sleep-related hypermotor epilepsy (SHE) is a complex and fascinating journey, marked by numerous twists and discoveries.1 This topic was recently reviewed by Fotedar and Luders,2 who erroneously concluded that SHE is not an identifiable focal epilepsy syndrome as they believed that it is based on weak evidence. We wish to address errors in their analysis and offer a more balanced understanding of this important form of epilepsy. The authors2review more than 40 years of history largely through a lens based on electrophysiology and pre-surgical evaluation. They challenge the epileptic origin of SHE, previously termed nocturnal frontal lobe epilepsy (NFLE), now recognized as a well-characterized entity 3. Their chronological reconstruction, more comprehensively addressed in previous works,1, 4 seems arbitrary and incomplete, omitting key studies that have contributed significantly to the understanding of the epileptic origin of the syndrome. In particular, even before the debates on the true nature of NPD began, others had observed episodes similar to NPD in patients with confirmed epilepsy. From the 1970s, authorities in North America began to define frontal lobe epilepsy (nocturnal and diurnal), often misdiagnosed as psychiatric in origin (Figure 1).3, 5-18 The historical reconstruction presented in the review2 is also incomplete in its identification of three eras marked by landmark studies (1972–1993, 1994–1998, 1999 to present), paying cursory attention to a crucial event: the Consensus Conference in Bologna,17 which established diagnostic criteria for the syndrome (Table 1). The syndrome was subsequently accepted by the International League Against Epilepsy (ILAE) Commission on Terminology.3 The consensus conference method is recommended for addressing important clinical questions in the face of limited high-quality evidence. The main outcome, a consensus statement, represents the collective opinions of an expert panel, derived from systematic review and discussion of available evidence.19 The Bologna Consensus Conference was planned and completed between November 2013 and September 2014, using rigorous methods addressing conditions with limited evidence, such as rare diseases (see online appendix in Tinuper et al.17 for details). The final definition of the condition was reached through a transparent process that included predefined research questions, a systematic review for each question, an independent systematic mapping of the evidence,20 an assessment of the literature's quality with reliable tools,21, 22 and an open, structured debate of 2 days involving a workgroup of experts for each the three main topics (clinical history; electro-clinical features; etiologic and pathogenic background) and a multidisciplinary international panel jury including specialists in child and adult epilepsy, sleep medicine, neurosurgery, genetics, epidemiology, and research methodology. The analysis explicitly covered all controversies and gray areas highlighted by Fotedar and Luders2 (e.g., absence of a clear ictal rhythm does not exclude an epileptic origin, not all seizures in SHE are frontal in origin, and so on). Fotedar and Luders correctly delineate the change in terminology over decades but failed to note that terms such as paroxysmal arousal (PA),23 epileptic nocturnal wandering (ENW),24 and minor motor episodes or events (MMEs)25, 26 – have been long since abandoned.3, 17 We agree that not all episodes previously reported under the term "NPD" are unequivocally epileptic. However, there is robust evidence for an epileptic basis in many cases, based on consistent hypermotor seizure semiology observed in the same patient, both within the same night and over the years, supported by anatomo-electro-clinical data in some. The evolution of seizures with the same semiological onset but varying duration has led to seemingly distinct descriptors, ranging from very brief motor attacks (brief) to hypermotor seizures sometimes followed by prolonged complex ambulatory behavior (long). These have been subsequently recognized as part of the clinical spectrum of seizures in SHE, both within and between patients.1 Fotedar and Luders express frustration that influential neurologists in the mid-1990s led the community to believe that epilepsy was the basis for most sleep-related paroxysmal motor episodes.14-16 They argued that all of these episodes were automatically assumed to be seizures and alternative diagnoses were often dismissed. This was never the case. Indeed, it is essential to recognize the extensive work that was done to clarify the differential diagnosis between sleep-related seizures and other sleep disorders (e.g., parasomnias), which remains a challenging but critical distinction. In that period, in addition to further electroclinical studies, diagnostic tools such as questionnaires and algorithms were developed, to aid clinicians in minimizing diagnostic errors in either direction, assessing the diagnostic accuracy of semiological patterns observed on video27 or reported on clinical history.28-33 Fotedar and Luders emphasize that the frequency of definitive interictal epileptiform changes on the electroencephalography (EEG) recordings of pre-surgical SHE cases is considerably higher than that seen in familial cases of SHE and even in a series of sporadic cases. We agree with this observation, which is likely an artifact of ascertainment bias, EEG recording time, and etiology. Pre-surgical cases are studied because they are drug resistant and typically have days or weeks of day and night video-EEG monitoring, and may have lesions. Milder cases, especially those in families, may have a single routine EEG and may be in remission at the time they are evaluated and are typically non-lesional.15 The absence of ictal and interictal epileptiform abnormalities does not exclude a diagnosis of SHE, in severe cases (concentrated in surgical series) or milder ones. In surgical series, it is widely recognized that co-registration of the scalp and stereo EEG (SEEG) can show surprisingly little abnormality on scalp EEG even when SEEG is very active (Figure 2). The authors cite (Figure 2 in ref.2) a case with PAs16 to support their criticism that many of the published NPD cases lacked definitive ictal/interictal epileptiform changes. PAs, frequently occurring in patients with SHE, are characterized by abrupt trunk and limb movements that can resemble simple motor sleep phenomena and exhibit a pseudoperiodic pattern linked to K-complex bursts or Cyclic Alternating Pattern (CAP) recurrence.1 However, the Consensus Conference deemed PAs insufficient for diagnosing SHE due to their controversial nature, inconsistent nomenclature across SHE study groups, and the risk of unreliable clinical diagnosis when only minor motor events or few episodes are captured.1, 17 We also note the attempts by Fotedar and Luders to reinterpret EEG tracings from older publications, especially given the challenges of analyzing published figures, rather than the whole recording. We do not wish to address every critique related to interpretations of EEG records from the 1990s, but we want to highlight one specific case—figure 42 – which the authors cited as a paradigmatic example of "overreading." Although we will not delve into the objections about this EEG tracing (whose quality understandably falls short of 2024 standards), we would like to point out that the patient in question carried a pathogenic KCNT1 variant (Figure 2, Family B, subject III.2 in Heron et al.34). This patient subsequently underwent epilepsy surgery involving resection of the right mesiolateral frontal region, with histopathology confirming the presence of focal cortical dysplasia (Figure 1, Family B, subject III.2).35 Furthermore, the authors propose that epileptic sleep-related paroxysmal motor events require the presence of a magnetic resonance imaging (MRI) lesion (table 1 in ref.2). However, even with advances in MRI techniques, 40%–50% of patients with SHE have negative MRIs in surgical series of drug-resistant patients.18, 36 Fotedar and Luders point out that many of the cases of SHE may have originated outside the frontal lobe. This has been well recognized by many groups and emphasized at the Consensus Conference and, precisely for this reason, led to the change of name from NFLE to SHE.17 It is well established that up to 30% of patients with hypermotor seizures, once categorized as the hallmark of NFLE, actually have seizures originating from extrafrontal regions.17 Seizures may arise in areas including the insula,37 midline parietal cortex,38 and other regions.18, 39 Therefore, revisiting this well-established point adds little value to their article. In the initial part of their "critical review," Fotedar and Luders disingenuously imply that the finding of CHRNA4 pathogenic variants was not replicated. Their review of molecular genetics is outdated, incomplete, and inaccurate. For CHRNA4, its pathogenic role in SHE is cemented by identification in both families and de novo cases, in individuals of European, Lebanese, and Japanese ethnicities.40-42 Multiple groups over the last three decades have confirmed the presence of autosomal dominant pathogenic variants in a range of genes including those encoding nicotinic subunits (CHRNA4, CHRNB2, and CHRNA2), mechanistic target of rapamycin (mTOR) pathway proteins (DEPDC5, NPRL2, and NPRL3) and potassium sodium-activated channel subunit (KCNT1) (Table 2).34, 43-47 In a series of 103 SHE cases, 19% of familial and 7% of sporadic cases had a pathogenic variant in an established SHE gene.48 The mTOR pathway genes are sometimes associated with structural malformations visible on MRI and may be included in surgical series.49, 50 nAChRs genes43 CHRNA4 CHRNB2 CHRNA2 Cholinergic Receptor Nicotinic Alpha 4 Subunit Cholinergic Receptor Nicotinic Beta 2 Subunit Cholinergic Receptor Nicotinic Alpha 2 Subunit 20q13.33 1q21.3 8p21.2 AD AD AD MIM*118504 MIM*118507 MIM*118502 GATOR-1 genes DEPDC5 44, 45 NPRL2 46 NPRL3 47 DEP Domain Containing 5 NPR2- like Protein Nitrogen Permease Regulator-like 3 22q12.2-q12.3 3p21.31 16p13.3 AD AD AD MIM*614191 MIM*607072 MIM*600928 The authors later try to disconnect the molecular findings from the epileptology, implying that these validated genetic variants are associated with non-specific sleep-related paroxysmal motor episodes, rather than with SHE. The clinical relationship of SHE to parasomnias remains poorly understood but, at this time, there is no evidence associating these genes with other familial sleep disorders.51 There are several reports of patients with pathogenic variants in CHRNA4, clinical features of SHE, and ictal epileptiform changes48 including one case with SEEG documenting a widespread epileptogenic network (case 352). The same findings have been reported for a few patients with germ-line pathogenic variants in the mTOR pathway genes.53 Overlooking this well-supported evidence disregards significant advancements in understanding the genetic underpinnings of epilepsy. The authors proposed a four-dimensional classification system for paroxysmal motor sleep episodes based on semiology, naming the new entity "sleep-related paroxysmal motor episodes" (SPME). We believe that this definition lacks both clarity and utility in the terms required by current scientific standards for defining new diagnostic criteria (e.g., prognostic ability, reproducibility, accuracy, and favorable balance between benefits and harms in applying the new definition).54 The implications of not differentiating epileptic seizures from other sleep-related motor phenomena, resulting in incorrect diagnosis and management, are potentially dangerous in terms of morbidity, mortality risk, and impact on quality of life. In conclusion, although we certainly welcome continued discourse on SHE, it is important that these discussions be grounded in comprehensive, up-to-date evidence and not a rehash of debates that have long been resolved. We thank Dr. Lorenzo Ferri and Giulia Bruschi for their assistance in preparing the figures and Dr. Anna Scarabello and Dr. Lorenzo Muccioli for their contributions to the editing of the manuscript and the bibliographic review. Open access publishing facilitated by The University of Melbourne, as part of the Wiley - The University of Melbourne agreement via the Council of Australian University Librarians. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. Francesca Bisulli has served on scientific advisory boards for Jazz, Takeda Pharmaceuticals, Ethypharm, and UCB; has received speaker honoraria from Angelini, UCB, Jazz, and Eisai; has received funding for travel from Jazz, Eisai, Angelini, and UCB; has served as an investigator for UCB, Ultragenyx, Xenon Pharmaceuticals, Zogenix, and Zynerba; and has consulted for Xenon Pharmaceuticals and Takeda Pharmaceuticals. Samuel F. Berkovic has received unrestricted educational grants to his institution from UCB Pharma, Eisai, SEER, Chiesi, and LivaNova. He has served as a consultant for Praxis Precision Medicines and has received personal honoraria for lectures and presentations from Eisai and DeltaMed. He holds a patent on methods of treatment and diagnosis of epilepsy by detecting mutations in the SCN1A gene, which is held by Bionomics Inc. and licensed to Athena Diagnostics and Genetics Technologies Ltd., with institutional royalties. He serves as Chief Medical Officer for the Epilepsy Foundation (Victoria). Ingrid Scheffer has served on scientific advisory boards for BioMarin, Chiesi, Eisai, Encoded Therapeutics, GlaxoSmithKline, Knopp Biosciences, Nutricia, Takeda Pharmaceuticals, UCB, Xenon Pharmaceuticals, and Longboard Pharmaceuticals; has received speaker honoraria from GlaxoSmithKline, UCB, BioMarin, Biocodex, Chiesi, LivaNova, Nutricia, Zuellig Pharma, Stoke Therapeutics, Eisai, Akumentis, and Praxis; has received funding for travel from UCB, Biocodex, GlaxoSmithKline, Biomarin, Encoded Therapeutics, Stoke Therapeutics, Eisai, and Longboard Pharmaceuticals; has served as an investigator for Anavex Life Sciences, Cerevel Therapeutics, Eisai, Encoded Therapeutics, EpiMinder Inc., Epygenyx, ES-Therapeutics, GW Pharma, Longboard Pharmaceuticals, Marinus, Neurocrine BioSciences, Ovid Therapeutics, SK Life Science, Takeda Pharmaceuticals, UCB, Ultragenyx, Xenon Pharmaceuticals, Zogenix, and Zynerba; has consulted for Care Beyond Diagnosis, Epilepsy Consortium, Atheneum Partners, Ovid Therapeutics, UCB, Zynerba Pharmaceuticals, BioMarin, Encoded Therapeutics, Biohaven Pharmaceuticals, Stoke Therapeutics, Praxis; and is a Non-Executive Director of Bellberry Ltd. and a Director of the Australian Academy of Health and Medical Sciences. She may accrue future revenue on a pending patent WO61/010176 (filed: 2008): Therapeutic Compound; has a patent for SCN1A testing held by Bionomics Inc. and licensed to various diagnostic companies; and has a patent molecular diagnostic/theranostic target for benign familial infantile epilepsy (BFIE) [PRRT2] 2011904493 & 2 012 900 190 and PCT/AU2012/001321 (TECH ID:2012–009). Eduard Hirsch, Lino Nobili, Federica Provini, Paolo Tinuper, and Luca Vignatelli declare no disclosures related to this paper. Data sharing is not applicable to this article as no datasets were generated or analyzed.

Short tandem repeats (STRs) are common variations in human genomes that frequently expand or contract, causing genetic disorders, mainly when expanded. Traditional diagnostic methods for identifying these expansions, such as repeat-primed PCR and Southern blotting, are often labor-intensive, locus-specific, and are unable to precisely determine long repeat expansions. Sequencing-based methods, although capable of genome-wide detection, are limited by inaccuracy (short-read technologies) and high associated costs (long-read technologies). This study evaluated optical genome mapping (OGM) as an efficient, accurate approach for measuring STR lengths and assessing somatic stability in 85 samples with known pathogenic repeat expansions in DMPK, CNBP, and RFC1, causing myotonic dystrophy types 1 and 2 and cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS), respectively. Three workflows-manual de novo assembly, local guided assembly (local-GA), and a molecule distance script-were applied, of which the latter two were developed as part of this study to assess the repeat sizes and somatic repeat stability. OGM successfully identified 84/85 (98.8%) of the pathogenic expansions, distinguishing between wild-type and expanded alleles or between two expanded alleles in recessive cases, with greater accuracy than standard of care (SOC) for long repeats and no apparent upper size limit. Notably, OGM detected somatic instability in a subset of DMPK, CNBP, and RFC1 samples. These findings suggest OGM could advance diagnostic accuracy for large repeat expansions, providing a more comprehensive genome-wide assay for repeat expansion disorders by measuring exact repeat lengths and somatic instability across multiple loci simultaneously.

OBJECTIVE:To describe long-term safety and effectiveness of fenfluramine in pediatric and adult patients with Lennox-Gastaut syndrome (LGS) from the final analysis of an open-label extension (OLE) study. METHODS:Patients (aged 2-35y) who participated in the randomized controlled trial (RCT) were eligible to continue in this OLE (NCT03355209). Fenfluramine 0.2 mg/kg/day was initiated; after one month, titration up to 0.7 mg/kg/day (26 mg/day maximum) was allowed. Key endpoints: incidence of treatment-emergent adverse events (TEAEs), median percentage change from RCT baseline in frequency of seizures associated with a fall, improvement by caregivers and investigators on Clinical Global Impression-Improvement (CGI-I), change from baseline in Quality of Life in Childhood Epilepsy Questionnaire scores, and Hospital Anxiety and Depression Scale (HADS) in parents/caregivers. RESULTS:247 patients enrolled: 158 (64.0 %) patients completed this OLE. Mean ± SD age, 14.3 ± 7.6y; median fenfluramine exposure, 364d (range, 19-537); mean ± SD fenfluramine daily dose, 0.4 ± 0.1 mg/kg/day. TEAEs in ≥10 % of patients: decreased appetite, fatigue, nasopharyngitis, seizure, pyrexia; no valvular heart disease or pulmonary arterial hypertension cases. Median change in frequency of seizures associated with a fall from Month 2 to end of study: -31.1 % (n = 240; P < 0.0001); pediatric: -27.6 % (n = 170; P = 0.0005), adult: -40.0 % (n = 70; P < 0.0001). On last-visit CGI-I, caregivers and investigators rated 59.9 % and 57.0 % of patients as improved, respectively. At Month 12, mean overall patient quality of life and caregiver anxiety on HADS significantly improved from baseline. SIGNIFICANCE:These results support the long-term safety and effectiveness of fenfluramine in patients with LGS, with no new safety signals identified, and sustained reductions in seizures and improvement in global functioning observed.

CLN2 and CLN3 diseases, the most common types of Batten disease (also known as neuronal ceroid lipofuscinosis), are childhood dementias associated with progressive loss of speech, language and feeding skills. Here we delineate speech, language, non-verbal communication and feeding phenotypes in 33 individuals (19 females) with a median age of 9.5 years (range 3-28 years); 16 had CLN2 and 17 CLN3 disease; 8/15 (53%) participants with CLN2 and 8/17 (47%) participants with CLN3 disease had speech and language impairments prior to genetic diagnosis. At the time of study all participants, bar one, had language impairments. The remaining participant with typical language was tested at age 3 years, following pre-symptomatic enzyme replacement therapy (ERT) from age 9 months. CLN2 and CLN3 disease had different profiles. For CLN2 disease, all affected individuals showed language impairment with dysarthria; older individuals with classical disease progressively became non-verbal. For CLN3 disease, the presentation was more heterogeneous. Speech impairment was evident early in the disease course, with dysarthria (13/15, 87%), often manifesting as neurogenic stuttering (5/15, 33%). Participants with CLN2 disease had comparable expressive and receptive language skills (p > 0.99), yet participants with CLN3 disease had stronger expressive language than receptive language skills (p = 0.004). Speech, cognitive and language impairment and adaptive behaviour showed progressive decline in both diseases. Individuals with pre-symptomatic ERT or atypical CLN2 disease were less impaired. Challenging behaviours were common in CLN3 (11/17, 65%), but less frequent in CLN2 (4/16, 25%) disease. Individuals with Batten disease require tailored speech therapy incorporating communication partner training utilising environment adaptations and informal communication behaviours.