Multiorgan manifestations of COL4A1 and COL4A2 variants and proposal for a clinical management protocol.

COL4A1/2 variants are associated with highly variable multiorgan manifestations. Depicting the whole clinical spectrum of COL4A1/2-related manifestations is challenging, and there is no consensus on management and preventative strategies. Based on a systematic review of current evidence on COL4A1/2-related disease, we developed a clinical questionnaire that we administered to 43 individuals from 23 distinct families carrying pathogenic variants. In this cohort, we extended ophthalmological and cardiological examinations to asymptomatic individuals and those with only limited or mild, often nonspecific, clinical signs commonly occurring in the general population (i.e., oligosymptomatic). The most frequent clinical findings emerging from both the literature review and the questionnaire included stroke (203/685, 29.6%), seizures or epilepsy (199/685, 29.0%), intellectual disability or developmental delay (168/685, 24.5%), porencephaly/schizencephaly (168/685, 24.5%), motor impairment (162/685, 23.6%), cataract (124/685, 18.1%), hematuria (63/685, 9.2%), and retinal arterial tortuosity (58/685, 8.5%). In oligosymptomatic and asymptomatic carriers, ophthalmological investigations detected retinal vascular tortuosity (5/13, 38.5%), dysgenesis of the anterior segment (4/13, 30.8%), and cataract (2/13, 15.4%), while cardiological investigations were unremarkable except for mild ascending aortic ectasia in 1/8 (12.5%). Our multimodal approach confirms highly variable penetrance and expressivity in COL4A1/2-related conditions, even at the intrafamilial level with neurological involvement being the most frequent and severe finding in both children and adults. We propose a protocol for prevention and management based on individualized risk estimation and periodic multiorgan evaluations.

Unilateral deep brain stimulation (DBS) of nucleus ventralis intermedius thalami (Vim) for the treatment of post-traumatic tremor in children: a multicentre experience.

PURPOSE: Deep brain stimulation (DBS) of nucleus ventralis intermedius thalami (Vim) is a validated technique for the treatment of essential tremor (ET) in adults. Conversely, its use for post traumatic tremor (PTT) and in paediatric patients is still debated. We evaluated the efficacy of Vim-DBS for lesional tremor in three paediatric patients with drug-resistant post-traumatic unilateral tremor. METHODS: We retrospectively collected data regarding three patients with unilateral tremor due to severe head injury, with no MRI evidence of basal ganglia lesions. The three patients underwent stereotactic frame-based robot-assisted DBS of Vim contralateral to the tremor side. RESULTS: Mean follow-up was 48 months (range: 36-60 months). Tremor was reduced in all patients with a better control of voluntary movements and improvement of functional status (mean FIM scale improvement + 7 points). No surgical complications occurred. CONCLUSION: Unilateral contralateral DBS of Vim could be efficacious in post-traumatic tremor, even in paediatric patients and should be offered in PTT drug-resistant patients.

Monoallelic variation in DHX9, the gene encoding the DExH-box helicase DHX9, underlies neurodevelopment disorders and Charcot-Marie-Tooth disease.

DExD/H-box RNA helicases (DDX/DHX) are encoded by a large paralogous gene family; in a subset of these human helicase genes, pathogenic variation causes neurodevelopmental disorder (NDD) traits and cancer. DHX9 encodes a BRCA1-interacting nuclear helicase regulating transcription, R-loops, and homologous recombination and exhibits the highest mutational constraint of all DDX/DHX paralogs but remains unassociated with disease traits in OMIM. Using exome sequencing and family-based rare-variant analyses, we identified 20 individuals with de novo, ultra-rare, heterozygous missense or loss-of-function (LoF) DHX9 variant alleles. Phenotypes ranged from NDDs to the distal symmetric polyneuropathy axonal Charcot-Marie-Tooth disease (CMT2). Quantitative Human Phenotype Ontology (HPO) analysis demonstrated genotype-phenotype correlations with LoF variants causing mild NDD phenotypes and nuclear localization signal (NLS) missense variants causing severe NDD. We investigated DHX9 variant-associated cellular phenotypes in human cell lines. Whereas wild-type DHX9 was restricted to the nucleus, NLS missense variants abnormally accumulated in the cytoplasm. Fibroblasts from an individual with an NLS variant also showed abnormal cytoplasmic DHX9 accumulation. CMT2-associated missense variants caused aberrant nucleolar DHX9 accumulation, a phenomenon previously associated with cellular stress. Two NDD-associated variants, p.Gly411Glu and p.Arg761Gln, altered DHX9 ATPase activity. The severe NDD-associated variant p.Arg141Gln did not affect DHX9 localization but instead increased R-loop levels and double-stranded DNA breaks. Dhx9-/- mice exhibited hypoactivity in novel environments, tremor, and sensorineural hearing loss. All together, these results establish DHX9 as a critical regulator of mammalian neurodevelopment and neuronal homeostasis.

Using Cluster Analysis to Overcome the Limits of Traditional Phenotype-Genotype Correlations: The Example of RYR1-Related Myopathies.

Thanks to advances in gene sequencing, RYR1-related myopathy (RYR1-RM) is now known to manifest itself in vastly heterogeneous forms, whose clinical interpretation is, therefore, highly challenging. We set out to develop a novel unsupervised cluster analysis method in a large patient population. The objective was to analyze the main RYR1-related characteristics to identify distinctive features of RYR1-RM and, thus, offer more precise genotype-phenotype correlations in a group of potentially life-threatening disorders. We studied 600 patients presenting with a suspicion of inherited myopathy, who were investigated using next-generation sequencing. Among them, 73 index cases harbored variants in RYR1. In an attempt to group genetic variants and fully exploit information derived from genetic, morphological, and clinical datasets, we performed unsupervised cluster analysis in 64 probands carrying monoallelic variants. Most of the 73 patients with positive molecular diagnoses were clinically asymptomatic or pauci-symptomatic. Multimodal integration of clinical and histological data, performed using a non-metric multi-dimensional scaling analysis with k-means clustering, grouped the 64 patients into 4 clusters with distinctive patterns of clinical and morphological findings. In addressing the need for more specific genotype-phenotype correlations, we found clustering to overcome the limits of the "single-dimension" paradigm traditionally used to describe genotype-phenotype relationships.

Early Immunotherapy and Longer Corticosteroid Treatment Are Associated With Lower Risk of Relapsing Disease Course in Pediatric MOGAD.

BACKGROUND AND OBJECTIVES: We sought to identify early factors associated with relapse and outcome in paediatric-onset myelin oligodendrocyte glycoprotein antibody-associated disorders (MOGAD). METHODS: In a multicenter retrospective cohort of pediatric MOGAD (≤18 years), onset features and treatment were compared in patients with monophasic vs relapsing disease (including cases with follow-up ≥12 months after onset or relapse at any time) and in patients with final Expanded Disability Status Scale (EDSS) 0 vs ≥1 at last follow-up (including cases with follow-up >3 months after last event or EDSS0 at any time). Multivariable logistic regression models were used to evaluate factors associated with relapsing disease course and EDSS ≥ 1 at final follow-up. RESULTS: Seventy-five children were included (median onset age 7 years; median 30 months of follow-up). Presentation with acute disseminated encephalomyelitis was more frequent in children aged 8 years or younger (66.7%, 28/42) than in older patients (30.3%, 10/33) (p = 0.002), whereas presentation with optic neuritis was more common in children older than 8 years (57.6%, 19/33) than in younger patients (21.4%, 9/42) (p = 0.001). 40.0% (26/65) of patients relapsed. Time to first relapse was longer in children aged 8 years or younger than in older patients (median 18 vs 4 months) (p = 0.013). Factors at first event independently associated with lower risk of relapsing disease course were immunotherapy <7 days from onset (6.7-fold reduced odds of relapsing course, OR 0.15, 95% CI 0.03-0.61, p = 0.009), corticosteroid treatment for ≥5 weeks (6.7-fold reduced odds of relapse, OR 0.15, 95% CI 0.03-0.80, p = 0.026), and abnormal optic nerves on onset MRI (12.5-fold reduced odds of relapse, OR 0.08, 95% CI 0.01-0.50, p = 0.007). 21.1% (15/71) had EDSS ≥ 1 at final follow-up. Patients with a relapsing course had a higher proportion of final EDSS ≥ 1 (37.5%, 9/24) than children with monophasic disease (12.8%, 5/39) (p = 0.022, univariate analysis). Each 1-point increment in worst EDSS at onset was independently associated with 6.7-fold increased odds of final EDSS ≥ 1 (OR 6.65, 95% CI 1.33-33.26, p = 0.021). DISCUSSION: At first attack of pediatric MOGAD, early immunotherapy, longer duration of corticosteroid treatment, and abnormal optic nerves on MRI seem associated with lower risk of relapse, whereas higher disease severity is associated with greater risk of final disability (EDSS ≥ 1).

A Schematic Approach to Defining the Prevalence of COL VI Variants in Five Years of Next-Generation Sequencing.

OBJECTIVE: To define the prevalence of variants in collagen VI genes through a next-generation sequencing (NGS) approach in undiagnosed patients with suspected neuromuscular disease and to propose a diagnostic flowchart to assess the real pathogenicity of those variants. METHODS: In the past five years, we have collected clinical and molecular information on 512 patients with neuromuscular symptoms referred to our center. To pinpoint variants in COLVI genes and corroborate their real pathogenicity, we sketched a multistep flowchart, taking into consideration the bioinformatic weight of the gene variants, their correlation with clinical manifestations and possible effects on protein stability and expression. RESULTS: In Step I, we identified variants in COLVI-related genes in 48 patients, of which three were homozygous variants (Group 1). Then, we sorted variants according to their CADD score, clinical data and complementary studies (such as muscle and skin biopsy, study of expression of COLVI on fibroblast or muscle and muscle magnetic resonance). We finally assessed how potentially pathogenic variants (two biallelic and 12 monoallelic) destabilize COL6A1-A2-A3 subunits. Overall, 15 out of 512 patients were prioritized according to this pipeline. In seven of them, we confirmed reduced or absent immunocytochemical expression of collagen VI in cultured skin fibroblasts or in muscle tissue. CONCLUSIONS: In a real-world diagnostic scenario applied to heterogeneous neuromuscular conditions, a multistep integration of clinical and molecular data allowed the identification of about 3% of those patients harboring pathogenetic collagen VI variants.

Seizure Outcome of Temporal Lobe Epilepsy Surgery in Adults and Children: A Systematic Review and Meta-Analysis.

BACKGROUND: Temporal lobe epilepsy (TLE) surgery is associated with the best seizure outcome in adults, although its long-term results remain suboptimal. Retrospective pediatric studies suggest better figures whose determinants are poorly understood. OBJECTIVE: To conduct a systematic review and meta-analysis of studies on the efficacy of TLE surgery in children (age younger than 18 years) and adults. METHODS: We searched MEDLINE, Embase, and Cochrane Library for TLE surgery original research from January 1, 1990, until May 12, 2020. The outcome measures were seizure freedom since surgery and seizure freedom either at last or longest follow-up. We meta-analyzed the proportion of children and adults achieving either Engel I/International League Against Epilepsy (ILAE) 1 or Engel IA/ILAE 1A outcome by follow-up duration, type of surgery, histopathology, neuroimaging, quality of the studies, and publication period. We used a random effects model with Freeman-Tukey double arcsine transformation of proportions. RESULTS: From 40 409 records identified, we included 277 studies (30 848 patients). The proportions of patients achieving Engel I/ILAE 1 and Engel IA/ILAE 1A outcomes were 0.74 (95% CI, 0.69-0.78) and 0.61 (0.48-0.74) for children and 0.69 (0.67-0.71) and 0.56 (0.52-0.60) for adults. Histopathology significantly influenced Engel I/ILAE 1 outcome in adults but not in children ( P < .0001), while the type of surgery significantly influenced Engel I/ILAE 1 outcome in children but not in adults. CONCLUSION: The proportion of seizure freedom after TLE surgery was higher in children, although not significantly. Histopathology and the surgical approach can influence seizure outcome, with age-related variability.

Prospective Evaluation of Ghrelin and Des-Acyl Ghrelin Plasma Levels in Children with Newly Diagnosed Epilepsy: Evidence for Reduced Ghrelin-to-Des-Acyl Ghrelin Ratio in Generalized Epilepsies.

Children with epilepsy and identified as responders to antiseizure medications (ASMs) were found to present markedly higher ghrelin plasma levels when compared to drug-resistant patients. However, it was undetermined if this phenotype could be influenced by the ASMs. Here, we prospectively investigated total ghrelin and des-acyl ghrelin (DAG) plasma levels by enzyme-linked immunosorbent assay before and after ASM administration. Inclusion criteria were: (i) subject with a suspicion of epilepsy; (ii) age ranging from 0 to 16 years; and (iii) informed consent signed by parents or caregivers. Exclusion criteria were acute or chronic metabolic disorders with occasional convulsions but without epilepsy. Fifty patients were followed over a period of one year in Italian neuropediatric centers. Apart from a few exceptions, the majority of children were responsive to ASMs. No differences were found in total ghrelin and DAG levels before and after the treatment, but total ghrelin levels were significantly lower in children with generalized epilepsy compared to those with combined focal and generalized epilepsy. Moreover, the ghrelin-to-DAG ratio was also markedly lower in generalized epilepsies compared to all the other types of epilepsy. Finally, ghrelin was unchanged by ASMs, including the first (e.g., carbamazepine), second (levetiracetam), and third (lacosamide) generation of anticonvulsants.

Asymmetry in sleep spindles and motor outcome in infants with unilateral brain injury.

AIM: To determine whether interhemispheric difference in sleep spindles in infants with perinatal unilateral brain injury could link to a pathological network reorganization that underpins the development of unilateral cerebral palsy (CP). METHOD: This was a multicentre retrospective study of 40 infants (19 females, 21 males) with unilateral brain injury. Sleep spindles were detected and quantified with an automated algorithm from electroencephalograph records performed at 2 months to 5 months of age. The clinical outcomes after 18 months were compared to spindle power asymmetry (SPA) between hemispheres in different brain regions. RESULTS: We found a significantly increased SPA in infants who later developed unilateral CP (n=13, with the most robust interhemispheric difference seen in the central spindles. The best individual-level prediction of unilateral CP was seen in the centro-occipital spindles with an overall accuracy of 93%. An empiric cut-off level for SPA at 0.65 gave a positive predictive value of 100% and a negative predictive value of 93% for later development of unilateral CP. INTERPRETATION: Our data suggest that automated analysis of interhemispheric SPA provides a potential biomarker of unilateral CP at a very early age. This holds promise for guiding the early diagnostic process in infants with a perinatally identified brain injury. WHAT THIS PAPER ADDS: Unilateral perinatal brain injury may affect the development of electroencephalogram (EEG) sleep spindles. Interhemispheric asymmetry in sleep spindles can be quantified with automated EEG analysis. Spindle power asymmetry can be a potential biomarker of unilateral cerebral palsy.

Expanding the genetic and phenotypic spectrum of CHD2-related disease: From early neurodevelopmental disorders to adult-onset epilepsy.

CHD2 encodes the chromodomain helicase DNA-binding protein 2, an ATP-dependent enzyme that acts as a chromatin remodeler. CHD2 pathogenic variants have been associated with various early onset phenotypes including developmental and epileptic encephalopathy, self-limiting or pharmacoresponsive epilepsies and neurodevelopmental disorders without epilepsy. We reviewed 84 previously reported patients carrying 76 different CHD2 pathogenic or likely pathogenic variants and describe 18 unreported patients carrying 12 novel pathogenic or likely pathogenic variants, two recurrent likely pathogenic variants (in two patients each), three previously reported pathogenic variants, one gross deletion. We also describe a novel phenotype of adult-onset pharmacoresistant epilepsy, associated with a novel CHD2 missense likely pathogenic variant, located in an interdomain region. A combined review of previously published and our own observations indicates that although most patients (72.5%) carry truncating CHD2 pathogenic variants, CHD2-related phenotypes encompass a wide spectrum of conditions with developmental delay/intellectual disability (ID), including prominent language impairment, attention deficit hyperactivity disorder and autistic spectrum disorder. Epilepsy is present in 92% of patients with a median age at seizure onset of 2 years and 6 months. Generalized epilepsy types are prevalent and account for 75.5% of all epilepsies, with photosensitivity being a common feature and adult-onset nonsyndromic epilepsy a rare presentation. No clear genotype-phenotype correlation has emerged.

Monoallelic KIF1A-related disorders: a multicenter cross sectional study and systematic literature review.

BACKGROUND: Monoallelic variants in the KIF1A gene are associated with a large set of clinical phenotypes including neurodevelopmental and neurodegenerative disorders, underpinned by a broad spectrum of central and peripheral nervous system involvement. METHODS: In a multicenter study conducted in patients presenting spastic gait or complex neurodevelopmental disorders, we analyzed the clinical, genetic and neuroradiological features of 28 index cases harboring heterozygous variants in KIF1A. We conducted a literature systematic review with the aim to comparing our findings with previously reported KIF1A-related phenotypes. RESULTS: Among 28 patients, we identified nine novel monoallelic variants, and one a copy number variation encompassing KIF1A. Mutations arose de novo in most patients and were prevalently located in the motor domain. Most patients presented features of a continuum ataxia-spasticity spectrum with only five cases showing a prevalently pure spastic phenotype and six presenting congenital ataxias. Seventeen mutations occurred in the motor domain of the Kinesin-1A protein, but location of mutation did not correlate with neurological and imaging presentations. When tested in 15 patients, muscle biopsy showed oxidative metabolism alterations (6 cases), impaired respiratory chain complexes II + III activity (3/6) and low CoQ10 levels (6/9). Ubiquinol supplementation (1gr/die) was used in 6 patients with subjective benefit. CONCLUSIONS: This study broadened our clinical, genetic, and neuroimaging knowledge of KIF1A-related disorders. Although highly heterogeneous, it seems that manifestations of ataxia-spasticity spectrum disorders seem to occur in most patients. Some patients also present secondary impairment of oxidative metabolism; in this subset, ubiquinol supplementation therapy might be appropriate.

Hypersensitivity to Rituximab in Children.

Biological agents have had an increased usage during the past years, also in pediatric population. Monoclonal antibodies can cause adverse drug reactions with different pathomechanisms, including type I IgE-mediated hypersensitivity reactions (HR). In this report, we describe 2 children who had a diagnosis of anaphylaxis to rituximab (RTX), confirmed by positive in vivo tests in both cases and elevated tryptase value in one case. We also made a review of the few cases of HR to RTX in pediatric population reported in literature and discuss differential diagnosis and utility of allergy investigations.

Patterns and predictors of language representation and the influence of epilepsy surgery on language reorganization in children and young adults with focal lesional epilepsy.

Mapping brain functions is crucial for neurosurgical planning in patients with drug-resistant seizures. However, presurgical language mapping using either functional or structural networks can be challenging, especially in children. In fact, most of the evidence on this topic derives from cross-sectional or retrospective studies in adults submitted to anterior temporal lobectomy. In this prospective study, we used fMRI and DTI to explore patterns of language representation, their predictors and impact on cognitive performances in 29 children and young adults (mean age at surgery: 14.6 ± 4.5 years) with focal lesional epilepsy. In 20 of them, we also assessed the influence of epilepsy surgery on language lateralization. All patients were consecutively enrolled at a single epilepsy surgery center between 2009 and 2015 and assessed with preoperative structural and functional 3T brain MRI during three language tasks: Word Generation (WG), Rhyme Generation (RG) and a comprehension task. We also acquired DTI data on arcuate fasciculus in 24 patients. We first assessed patterns of language representation (relationship of activations with the epileptogenic lesion and Laterality Index (LI)) and then hypothesized a causal model to test whether selected clinical variables would influence the patterns of language representation and the ensuing impact of the latter on cognitive performances. Twenty out of 29 patients also underwent postoperative language fMRI. We analyzed possible changes of fMRI and DTI LIs and their clinical predictors. Preoperatively, we found atypical language lateralization in four patients during WG task, in one patient during RG task and in seven patients during the comprehension task. Diffuse interictal EEG abnormalities predicted a more atypical language representation on fMRI (p = 0.012), which in turn correlated with lower attention (p = 0.036) and IQ/GDQ scores (p = 0.014). Postoperative language reorganization implied shifting towards atypical language representation. Abnormal postoperative EEG (p = 0.003) and surgical failures (p = 0.015) were associated with more atypical language lateralization, in turn correlating with worsened fluency. Neither preoperative asymmetry nor postoperative DTI LI changes in the arcuate fasciculus were observed. Focal lesional epilepsy associated with diffuse EEG abnormalities may favor atypical language lateralization and worse cognitive performances, which are potentially reversible after successful surgery.

Post-mortem histopathology of a pediatric brain after bilateral DBS of GPI for status dystonicus: case report and review of the literature.

PURPOSE: To investigate the effects of deep brain stimulation (DBS) electrodes on the brain of a dystonic pediatric patient submitted to bilateral DBS of the globus pallidus internus (GPI). METHODS: An 8-year-old male patient underwent bilateral DBS of GPI for status dystonicus. He died 2 months later due to multiorgan failure triggered by bacterial pneumonia. A post-mortem pathological study of the brain was done. RESULTS: At visual inspection, no grossly apparent softening, hemorrhage, or necrosis of the brain adjacent to the DBS lead tracts was detected. High-power microscopic examination of the tissue surrounding the electrode trajectories showed lymphocyte infiltration, astrocytic gliosis, microglia, macrophages, and clusters of multinucleate giant cells. Significant astrocytosis was confirmed by GFAP staining in the electrode site. The T cell lymphocyte activity was overexpressed with activated macrophages detected with CD3, CD20, CD45, and CD68 stains respectively. There was no gliosis or leukocyte infiltration away from the surgical tracks of the electrodes. CONCLUSION: This is the first post-mortem examination of a child's brain after bilateral DBS of GPI. The comparison with adult post-mortem reports showed no significant differences and confirms the safety of DBS implantation in the pediatric population too.

Type I sialidosis, a normosomatic lysosomal disease, in the differential diagnosis of late-onset ataxia and myoclonus: An overview.

Lysosomal storage diseases (LSDs) are rare to extremely rare monogenic disorders. Their incidence, however, has probably been underestimated owing to their complex clinical manifestations. Sialidosis is a prototypical LSD inherited as an autosomal recessive trait and caused by mutations in the NEU1 gene that result in a deficiency of alpha-N-acetyl neuraminidase 1 (NEU1). Two basic forms of this disease, type I and type II, are known. The dysmorphic type II form features LSD symptoms including congenital hydrops, dysmorphogenetic traits, hepato-splenomegaly and severe intellectual disability. The diagnosis is more challenging in the normosomatic type I forms, whose clinical findings at onset include ocular defects, ataxia and generalized myoclonus. Here we report the clinical, biochemical and molecular analysis of five patients with sialidosis type I. Two patients presented novel NEU1 mutations. One of these patients was compound heterozygous for two novel NEU1 missense mutations: c.530A>T (p.Asp177Val) and c.1010A>G (p.His337Arg), whereas a second patient was compound heterozygous for a known mutation and a novel c.839G>A (p.Arg280Gln) mutation. We discuss the impact of these new mutations on the structural properties of NEU1. We also review available clinical reports of patients with sialidosis type I, with the aim of identifying the most frequent initial clinical manifestations and achieving more focused diagnoses.

Next-generation sequencing approach to hyperCKemia: A 2-year cohort study.

OBJECTIVE: Next-generation sequencing (NGS) was applied in molecularly undiagnosed asymptomatic or paucisymptomatic hyperCKemia to investigate whether this technique might allow detection of the genetic basis of the condition. METHODS: Sixty-six patients with undiagnosed asymptomatic or paucisymptomatic hyperCKemia, referred to tertiary neuromuscular centers over an approximately 2-year period, were analyzed using a customized, targeted sequencing panel able to investigate the coding exons and flanking intronic regions of 78 genes associated with limb-girdle muscular dystrophies, rhabdomyolysis, and metabolic and distal myopathies. RESULTS: A molecular diagnosis was reached in 33 cases, corresponding to a positive diagnostic yield of 50%. Variants of unknown significance were found in 17 patients (26%), whereas 16 cases (24%) remained molecularly undefined. The major features of the diagnosed cases were mild proximal muscle weakness (found in 27%) and myalgia (in 24%). Fourteen patients with a molecular diagnosis and mild myopathic features on muscle biopsy remained asymptomatic at a 24-month follow-up. CONCLUSIONS: This study of patients with undiagnosed hyperCKemia, highlighting the advantages of NGS used as a first-tier diagnostic approach in genetically heterogeneous conditions, illustrates the ongoing evolution of molecular diagnosis in the field of clinical neurology. Isolated hyperCKemia can be the sole feature alerting to a progressive muscular disorder requiring careful surveillance.

Automatic detection and sonification of nonmotor generalized onset epileptic seizures: Preliminary results.

Long-term video-EEG monitoring has improved diagnosis and treatment of epilepsy, especially in children. However, the amount of data neurophysiologists must analyze has grown remarkably. The main purpose of this paper is to provide a diagnostic support to speed up and ease EEG interpretation for a specific application concerning absence seizures, a type of non-motor generalized epileptic seizures. The proposed method consists of a pre-processing step where signals are filtered through the Stationary Wavelet Transform for the reduction of possible artefacts. Subsequently, a supervised automatic classification method is implemented for seizure detection, based on the Support Vector Machine Fine Gaussian method. Finally, a post-processing step is implemented in which spatial and temporal thresholds are defined for both online and offline application. In addition, a method that applies sonification techniques is developed. Sonification techniques could speed up the process of interpreting information, allowing rapid clinical intervention and a continuous monitoring of the event. The dataset consists of 30 EEG recordings performed in 24 children with absence seizures, clinically evaluated at the Meyer Children's Hospital in Firenze, Italy. The method shows encouraging results both in terms of balanced accuracy (about 96%) and latency times (1.25 s on average), which might make it suitable for online clinical trials. In fact, it was implemented in the perspective of a possible real-time application in clinical practice.

HCN1 mutation spectrum: from neonatal epileptic encephalopathy to benign generalized epilepsy and beyond.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels control neuronal excitability and their dysfunction has been linked to epileptogenesis but few individuals with neurological disorders related to variants altering HCN channels have been reported so far. In 2014, we described five individuals with epileptic encephalopathy due to de novo HCN1 variants. To delineate HCN1-related disorders and investigate genotype-phenotype correlations further, we assembled a cohort of 33 unpublished patients with novel pathogenic or likely pathogenic variants: 19 probands carrying 14 different de novo mutations and four families with dominantly inherited variants segregating with epilepsy in 14 individuals, but not penetrant in six additional individuals. Sporadic patients had epilepsy with median onset at age 7 months and in 36% the first seizure occurred during a febrile illness. Overall, considering familial and sporadic patients, the predominant phenotypes were mild, including genetic generalized epilepsies and genetic epilepsy with febrile seizures plus (GEFS+) spectrum. About 20% manifested neonatal/infantile onset otherwise unclassified epileptic encephalopathy. The study also included eight patients with variants of unknown significance: one adopted patient had two HCN1 variants, four probands had intellectual disability without seizures, and three individuals had missense variants inherited from an asymptomatic parent. Of the 18 novel pathogenic missense variants identified, 12 were associated with severe phenotypes and clustered within or close to transmembrane domains, while variants segregating with milder phenotypes were located outside transmembrane domains, in the intracellular N- and C-terminal parts of the channel. Five recurrent variants were associated with similar phenotypes. Using whole-cell patch-clamp, we showed that the impact of 12 selected variants ranged from complete loss-of-function to significant shifts in activation kinetics and/or voltage dependence. Functional analysis of three different substitutions altering Gly391 revealed that these variants had different consequences on channel biophysical properties. The Gly391Asp variant, associated with the most severe, neonatal phenotype, also had the most severe impact on channel function. Molecular dynamics simulation on channel structure showed that homotetramers were not conducting ions because the permeation path was blocked by cation(s) strongly complexed to the Asp residue, whereas heterotetramers showed an instantaneous current component possibly linked to deformation of the channel pore. In conclusion, our results considerably expand the clinical spectrum related to HCN1 variants to include common generalized epilepsy phenotypes and further illustrate how HCN1 has a pivotal function in brain development and control of neuronal excitability.