Abstract KCNQ2 is a member of the voltage-gated potassium (Kv) channel family and regulates neuronal activity through potassium ion efflux. Pathogenic variants of KCNQ2 induce aberrant neuronal activity and cause two types of epilepsy: self-limited familial neonatal epilepsy (SLFNE) and developmental and epileptic encephalopathies (DEE). However, the molecular mechanism by which these pathogenic variants influence KCNQ2 expression remains unclear. Here, we show N-terminal and C-terminal fragments derived from mouse KCNQ2 (KCNQ2S−N and KCNQ2S−C, respectively), whose amounts differed significantly across variants compared with wild type, whereas those of full-length KCNQ2 (KCNQ2F) remained unchanged. Of particular interest, two variants at the same codon, Y284C and Y284D, which are associated with distinct clinical phenotypes—self-limited familial neonatal epilepsy (SLFNE) and developmental and epileptic encephalopathy (DEE), respectively—exerted opposite effects on the fragment: Y284C increased the amounts of both KCNQ2 fragments, whereas Y284D decreased it compared with the wild type. As both KCNQ2S−N and KCNQ2S−C were localized in the plasma membrane, both fragments were suggested to be post-translational products resulting from a cleavage of full-length KCNQ2. This novel post-translational cleavage was observed in neuronal cells and appears to be evolutionarily conserved. Although the role of this post-translational modification in epilepsy remains unknown, it may be elucidated through future studies.

ABSTRACT We described a 12‐year‐old boy with classic Fabry disease who was diagnosed through newborn screening. At age 6.2, he started agalsidase alfa based on evidence of subclinical organ involvement. At age 7.2, acroparesthesia subsequently developed. At age 10.9, after switching to agalsidase beta due to an insufficient clinical and biochemical response, his acroparesthesia resolved and plasma Lyso‐Gb3 decreased. This report broadens the clinical understanding of children with Fabry disease.

Newborn screening (NBS) for Fabry disease (FD) is highly effective at detecting FD prior to symptom onset. This initiative is currently being implemented worldwide. We previously reported results for 599,711 newborns from the first large-scale NBS program for FD in Japan, from August 2006 to December 2018. In this study, we provide additional data from January 2019 to September 2022. A total of 782,591 newborns were screened, and 29 variants, including 18 pathogenic variants and 11 variants of uncertain significance (VUS), were detected in 77 newborns (57 males and 20 females). Thirty-five male and 14 female newborns with pathogenic variants in GLA were identified. Twenty-two male and six female newborns with VUS in GLA were also identified. At the most recent follow-up, 5 of the 35 hemizygous patients manifested symptoms or signs and were receiving enzyme replacement therapy. The estimated frequency of patients with FD, including individuals with pathogenic variants or VUS identified in this study, was 1 in 7730, whereas that of patients with pathogenic variants was 1:12,436. FD-related cardiac and renal tissue damage are present before the onset of FD symptoms, such as limb pain. These findings highlight the importance of early diagnosis using NBS and regular monitoring.

A large number of cases with Dravet syndrome (DS) has been attributed to SCN1A loss of function (LOF), whereas SCN1A gain-of-function (GOF) causes early infantile developmental and epileptic encephalopathy (EIDEE) and familial hemiplegic migraine 3. We retrospectively analyzed 37 individuals with SCN1A pathogenic variants at our institute between January 2012 and October 2024 to investigate phenotype-function correlations. Variant functions were classified as LOF, GOF, or mixed, based on existing patch-clamp data, paralog sodium channel experimental findings, and in silico prediction tools. Clinical characteristics, antiseizure medication (ASM) responses, and variant location were compared. Nine variants were novel. One variant with insufficient data for functional prediction was excluded. Of the 36 cases with predictable functions, five cases (14%) were classified as GOF/mixed (DS = 4, EIDEE = 1) and 31 (86%) as LOF (DS = 31). GOF/mixed-DS had earlier epilepsy onset but otherwise resembled LOF-DS. Sodium channel blocking ASMs (SCB-ASMs) did not exacerbate seizures in GOF/mixed DS cases, with carbamazepine reducing seizures in one case. GOF/mixed variants clustered in the intracellular S6 segment, whereas LOF variants clustered in the S5-S6 pore loop. These findings highlight a potential GOF effect for certain DS cases, suggesting that SCB-ASMs may be effective for GOF/mixed DS. This underscores the importance of functional characterization for tailored therapy, warranting further research to confirm and extend these results. PLAIN LANGUAGE SUMMARY: Dravet syndrome is a severe epilepsy that usually begins in infancy and is linked to changes in a gene called SCN1A. Most cases are caused by gene changes that reduce function, but in some cases, the gene may become overactive. In this study, we found that some patients with Dravet syndrome had these overactive changes and still showed typical symptoms. We found that people with overactive SCN1A function might respond differently to certain medications.

Toxins as channel probes, small guanidinium alkaloids, such as tetrodotoxin and saxitoxin, canonical pore occlusion in voltage-gated Na+ channels. Cystine-rich peptides from spiders, scorpions, cone snails, and sea anemones, which act as pore blockers or gating modifiers targeting voltage-sensing domains. Recent structural and electrophysiological studies have identified specific binding sites on ion channels, including the S5–S6 pore loops, outer vestibule and turret regions, and S3–S4 “paddle” motifs in NaV, Kv, and CaV channels. These discrete binding epitopes are recognized by different peptide toxins, enabling isoform- and state-specific modulation; for example, μ-conotoxins bind the NaV pore, whereas charybdotoxin and agitoxin target the Kv outer vestibule. Beyond mechanistic insights, peptide toxins inspire translational strategies, including emerging therapies for retinal degenerative diseases. Photopharmacology using chemical photoswitches allows reversible, light-controlled modulation of ion channels in retinal ganglion cells without genetic manipulation or cell transplantation. Although BENAQ was discovered by small-molecule screening rather than toxin-guided design, its ion channel control demonstrates the potential of toxin-based molecular determinants for engineering synthetic compounds. This review thus integrates structural, functional, and translational perspectives, emphasizing the versatility of animal-derived peptide toxins as molecular probes and as blueprints for precision ion channel modulation in health and disease.

BACKGROUND:Inherited glycosylphosphatidylinositol (GPI) deficiencies are a heterogeneous group of inherited disorders of glycosylation, caused by mutations in genes involved in GPI-anchored proteins (GPI-AP) biosynthesis. PIGW is a gene known to be involved in the early steps of the GPI-anchor biosynthesis, as well as functional studies for most patients. Biallelic mutations in PIGW have been previously linked to hyperphosphatasia with mental retardation syndrome 5, also known as glycosylphosphatidylinositol biosynthesis defect 11 (GPIBD11). METHODS:We report seven individuals, including two fetuses from six unrelated families. Whole exome sequencing and chromosome analysis were performed, with variant interpretation based on ACMG and AMP guidelines. Magnetic resonance imaging (MRI) was also conducted on some of the patients. Blood samples were collected from patients to analyze GPI-AP expression using flow cytometry on markers like CD16, CD24, and FLAER. Functional analyses were performed using PIGW KO HEK 293 cells generated with CRISPR/Cas9 technology. The cells were transfected with rat Pigw cDNA that contained patient variants. The restoration of GPI-AP expression was measured by flow cytometry. Western blotting was used to assess protein expression. RESULTS:Affected patients exhibited a wide range of clinical features. Some patients presented classic GPIBD11 symptoms like developmental delay, hyperphosphatasia, and intellectual disability. Other patients showed atypical or milder phenotypes. The magnetic resonance imaging scans revealed variable neurological abnormalities in the affected individuals. Whole exome sequencing results identified PIGW mutations in all patients, which confirms the genetic basis of the disorder. Flow cytometry analysis of blood samples from patients P1, P4, and P5 using various markers showed a significant reduction in GPI-AP expression. The CD16 marker decreased to 1.8% in P1 and 21% in P5 compared to controls. CD24 was reduced to 22% in P1 granulocytes. Also, a minor decrease in CD14 on monocytes was observed in P4, as well as a slight reduction in the expression of FLAER in lymphocytes. Functional studies on PIGW-deficient CHO cells and HEK293 cells, using flow cytometry, showed that GPI-AP expression is affected by the PIGW variants. Western blotting showed reduced PIGW protein expression, except for P153L and R36G, which were similar to wild-type levels. CONCLUSIONS:To date, six patients and two fetuses with biallelic variants in PIGW have been reported. Here, we describe five new patients and two fetuses harboring homozygous or compound heterozygous variants in the PIGW gene. Our results illustrate the clinical variability of GPIBD11, highlighting the importance of broad genomic sequencing assays for patients who do not show typical symptoms. Therefore, our study expands the clinical and molecular spectrum of PIGW-associated disorder.

要旨:Dravet症候群に対するfenfluramine(FFA)療法の有効性と安全性を評価した。対象は2022年11月から2023年8月の間に当センターでFFAを投与したDravet症候群5例とした。全例で焦点起始両側強直間代発作(focal to bilateral tonic clonic seizure:FBTCS)が主体の発作型であり、FFA療法前のFBTCS頻度は中央値4(2~12)回/月であった。FFA開始年齢の中央値は7.6(2.4~10.9)歳、FFA維持投与量は0.31(0.13~0.38)mg/kg/日であった。FFA投与後、6カ月時点のFBTCSの平均発作頻度は中央値0(0~3)回/月に減少し、FBTCSの最長発作抑制期間は中央値51(30~180)日に改善した。Dravet症候群のてんかん発作に対してFFAは有用であり、治療抵抗性を示す場合には治療選択順位を上げることが望ましい。

Identifying genetic risk factors for highly heterogeneous disorders such as epilepsy remains challenging. Here we present, to our knowledge, the largest whole-exome sequencing study of epilepsy to date, with more than 54,000 human exomes, comprising 20,979 deeply phenotyped patients from multiple genetic ancestry groups with diverse epilepsy subtypes and 33,444 controls, to investigate rare variants that confer disease risk. These analyses implicate seven individual genes, three gene sets and four copy number variants at exome-wide significance. Genes encoding ion channels show strong association with multiple epilepsy subtypes, including epileptic encephalopathies and generalized and focal epilepsies, whereas most other gene discoveries are subtype specific, highlighting distinct genetic contributions to different epilepsies. Combining results from rare single-nucleotide/short insertion and deletion variants, copy number variants and common variants, we offer an expanded view of the genetic architecture of epilepsy, with growing evidence of convergence among different genetic risk loci on the same genes. Top candidate genes are enriched for roles in synaptic transmission and neuronal excitability, particularly postnatally and in the neocortex. We also identify shared rare variant risk between epilepsy and other neurodevelopmental disorders. Our data can be accessed via an interactive browser, hopefully facilitating diagnostic efforts and accelerating the development of follow-up studies.

Mucopolysaccharidosis II (MPS II) is an X-linked, recessive, inborn metabolic disorder caused by defects in iduronate-2-sulfatase (IDS). The age at onset, disease severity, and rate of progression vary significantly among patients. This disease is classified into severe or mild forms depending on neurological symptom involvement. The severe form is associated with progressive cognitive decline while the mild form is predominantly associated with somatic features. Newborn screening (NBS) for MPS II has been performed since December 2016, mainly in Kyushu, Japan, where 197,700 newborns were screened using a fluorescence enzyme activity assay of dried blood spots. We diagnosed one newborn with MPS II with lower IDS activity, elevated urinary glycosaminoglycans, and a novel variant of the IDS gene. In the future, NBS for MPS II is expected to be performed in many regions of Japan and will contribute to the detection of more patients with MPS II, which is crucial to the early treatment of the disorder.

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

Dravet syndrome (DS) is an infantile-onset epileptic encephalopathy. More than 80% of DS patients have a heterozygous mutation in SCN1A , which encodes a subunit of the voltage-gated sodium channel, Nav 1.1 , in neurons. The roles played by astrocytes, the most abundant glial cell type in the brain, have been investigated in the pathogenesis of epilepsy; however, the specific involvement of astrocytes in DS has not been clarified. In this study, we evaluated Ca 2+ signaling in astrocytes using genetically modified mice that have a loss-of-function mutation in Scn1a . We found that the slope of spontaneous Ca 2+ spiking was increased without a change in amplitude in Scn1a +/− astrocytes. In addition, ATP-induced transient Ca 2+ influx and the slope of Ca 2+ spiking were also increased in Scn1a +/− astrocytes. These data indicate that perturbed Ca 2+ dynamics in astrocytes may be involved in the pathogenesis of DS.

Molecular biological studies and electrophysiological data have demonstrated that acetylcholine (ACh) is the principal cochlear and vestibular efferent neurotransmitter among mammalians. However, the functional roles of ACh in type II vestibular hair cells (VHCs II) among mammalians are still unclear, with the exception of the well-known α9-containing nicotinic ACh receptor (α9-containing nAChR)-activated small conductance, calcium-dependent potassium current (SK) in cochlear hair cells and frog saccular hair cells. The activation of SK current was necessary for the calcium influx through the α9-containing nAChR. Recently, we have demonstrated that ACh-induced big conductance, calcium-dependent potassium current (BK) was present in VHCs II of the vestibular end-organ of guinea pig. In this study, the nature of calcium influx for the activation of ACh-induced BK current in saccular VHCs II of guinea pig was investigated. Following extracellular perfusion of ACh, saccular VHCs II displayed a sustained outward current, which was sensitive to iberiotoxin (IBTX). High concentration of apamin failed to inhibit the current amplitude of ACh-induced outward current. Intracellular application of Cs+ completely abolished the current evoked by ACh. ACh-induced current was potently inhibited by nifedipine, nimodipine, Cd2+ and Ni2+, respectively. The inhibition potency of these four calcium channel antagonists was nimodipine > nifedipine > cadmium > nickel. The L-type Ca2+ channels agonist, (−)-Bay-K 8644 mimicked the effect of ACh and activated an IBTX-sensitive current. In addition, partial VHCs II displayed a biphasic waveform. In conclusion, the present data showed that in the guinea pig saccular VHCs II, ACh-induced BK channel was coupled with the calcium channel, but not the receptor. The perfusion of ACh will drive the opening of calcium channels; the influx of calcium ions will then activate the BK current.

Syntaxin-binding protein1 (STXBP1) is a member of the Sec1/Munc18-1 protein family, which comprises important regulators of the secretory and synaptic vesicle fusion machinery underlying hormonal and neuronal transmission, respectively. STXBP1 pathogenic variants are associated with multiple neurological disorders. Herein, we present the case of a Japanese girl with a mutation in the STXBP1 gene, who was born at 40 weeks without neonatal asphyxia. At 15 days old, she developed epilepsy and generalized seizures. Around 88 days old, she presented with a series of nodding spasms, with the seizure frequency gradually increasing. Interictal EEG indicated hypsarrhythmia and she presented with developmental regression. At 1.5 years old, genetic testing was performed and mutational analysis revealed an STXBP1 gene mutation (c.875G > A: p.Arg292His). Accordingly, she was diagnosed with developmental and epileptic encephalopathy, presenting West syndrome’s clinical characteristics caused by the STXBP1 gene mutation. Although drug treatment has reduced the frequency of epileptic seizures, her development has remained regressive. The relationship between the location and type of genetic abnormality and the phenotype remains unclear. Future studies should investigate the genotype–phenotype correlation and the underlying pathophysiology to elucidate the causal relationships among the multiple phenotype-determining factors.

CUX2 gene encodes a transcription factor that controls neuronal proliferation, dendrite branching and synapse formation, locating at the epilepsy-associated chromosomal region 12q24 that we previously identified by a genome-wide association study (GWAS) in Japanese population. A CUX2 recurrent de novo variant p.E590K has been described in patients with rare epileptic encephalopathies and the gene is a candidate for the locus, however the mutation may not be enough to generate the genome-wide significance in the GWAS and whether CUX2 variants appear in other types of epilepsies and physiopathological mechanisms are remained to be investigated. Here in this study, we conducted targeted sequencings of CUX2, a paralog CUX1 and its short isoform CASP harboring a unique C-terminus on 271 Japanese patients with a variety of epilepsies, and found that multiple CUX2 missense variants, other than the p.E590K, and some CASP variants including a deletion, predominantly appeared in patients with temporal lobe epilepsy (TLE). The CUX2 variants showed abnormal localization in human cell culture analysis. While wild-type CUX2 enhances dendritic arborization in fly neurons, the effect was compromised by some of the variants. Cux2- and Casp-specific knockout mice both showed high susceptibility to kainate, increased excitatory cell number in the entorhinal cortex, and significant enhancement in glutamatergic synaptic transmission to the hippocampus. CASP and CUX2 proteins physiologically bound to each other and co-expressed in excitatory neurons in brain regions including the entorhinal cortex. These results suggest that CUX2 and CASP variants contribute to the TLE pathology through a facilitation of excitatory synaptic transmission from entorhinal cortex to hippocampus.

Although astrocytes are involved in the pathogenesis of CNS diseases, how they induce synaptic abnormalities is unclear. Currently, in vitro pathological astrocyte cultures or animal models do not reproduce human disease phenotypes accurately. Induced pluripotent stemcells (iPSCs) are replacing animalmodels in pathological studies. We developed an autaptic culture (AC) system containing single neuron cultures grown on microislands of astrocytes. AC with human iPSCderived astrocytes (HiA) was established. We evaluated the effect of astrocytes on the synaptic functions of human-derived neurons. We found a significantly higher Na+ current amplitude, membrane capacitance, and number of synapses, as well as longer dendrites, in HiAACs compared with neuron monocultures. Furthermore, HiAs were involved in the formation and maturation of functional synapses that exhibited excitatory postsynaptic currents. This system can facilitate the study of CNS diseases and advance the development of drugs targeting glial cells.

Dravet syndrome is a well-established electro-clinical condition first described in 1978. A main genetic cause was identified with the discovery of a loss-of-function SCN1A variant in 2001. Mechanisms underlying the phenotypic variations have subsequently been a main topic of research. Various genetic modifiers of clinical severities have been elucidated through many rigorous studies on genotype-phenotype correlations and the recent advances in next generation sequencing technology. Furthermore, a deeper understanding of the regulation of gene expression and remarkable progress on genome-editing technology using the CRISPR-Cas9 system provide significant opportunities to overcome hurdles of gene therapy, such as enhancing NaV1.1 expression. This article reviews the current understanding of genetic pathology and the status of research toward the development of gene therapy for Dravet syndrome. This article is part of the Special Issue "Severe Infantile Epilepsies".

BACKGROUND Invasive candidiasis (IC) is a leading infectious cause of morbidity and mortality in premature infants. The objective of this study was to determine the prevalence of IC in newborns in the neonatal intensive care unit (NICU) of a tertiary hospital in Japan, and to identify specific predisposing factors for IC. METHODS We retrospectively collected data on demographics, clinical characteristics, and outcomes of infants with IC, who were discharged from a tertiary NICU in Japan between January 2009 and December 2020. We compared predisposing factors associated with the occurrence of early-onset IC (EOIC, < 72 hours) and late-onset IC (LOIC, ≥ 72 hours) with those of early-onset and late-onset bacterial sepsis, respectively. RESULTS Over 12 years, 3,549 infants were admitted to the NICU, including 344 extremely low birth weight (ELBW) infants. Eleven infants (including 9 ELBW infants) had IC (incidence 0.31%), and the mortality rate of IC was 0%. Four (36%) infants had EOIC and 7 (64%) had LOIC. All those with EOIC presented with skin lesions, and 86% with LOIC had thrombocytopenia. Maternal vaginal Candida colonization was a more specific predisposing factor for EOIC, while gestational age < 26 weeks, broad-spectrum antibiotic use, prior bacterial infection, prior gastrointestinal (GI) surgery, and GI diseases were more specific predisposing factors for LOIC. CONCLUSIONS The findings suggest that maternal vaginal Candida colonization and skin lesions in ELBW infants may contribute to early recognition of EOIC, and LOIC should be suspected if ELBW infants with several predisposing factors of LOIC have thrombocytopenia.

Astrocyte is a type of glial cell involved in synaptic transmission and the formation and maturation of synapses. Currently, the establishment of induced pluripotent stem cells (iPSCs) allows the differentiation of stem cells into various types of cells while preserving the patient phenotype. Therefore, patient iPSCs replace animal models in pathological analysis and drug discovery. Technological advances have provided access to human iPSC-derived astrocytes (HiAs). Furthermore, neurons co-cultured with pathological astrocytes have been used to study their morphology, protein levels, and spontaneous synaptic responses. However, these studies did not investigate detailed synaptic functions such as synaptic transmission evoked by electrical stimulation and morphological analysis at the single neuron level. In this study, we established autaptic cultures with HiAs (HiAs Autaptic Cultures, HiAACs), single neuron cultures grown in isolation on microislands of HiAs that form synapses exclusively with themselves. We found that neurons in HiAACs develop morphologically by co-culture with HiAs and form functional synapses that exhibit excitatory postsynaptic currents. Although we used healthy astrocytes in this study, HiAACs can be used to study various diseases by using patient-derived astrocytes in the future.This work was supported by funding from JSPS, AMED, MEXT, the Science Research Promotion Fund and The Fukuoka University Fund, and Kyushu University Hospital.

Objective. To examine the current medical and psychosocial status of patients with epilepsy, aiming to facilitate appropriate application of the Intractable/Rare Diseases Act of Japan. Methods. By analysing the cross-sectional data of patients registered in the tertiary hospital-based Epilepsy Syndrome Registry of Japan, we investigated the proportion of patients who met the severity criteria as defined by the Act (seizure frequency of at least once a month, or presence of intellectual/neurological/psychiatric symptoms, or both) and whether there are candidate syndrome/diseases to be added to the existing list in the Act. Results. In total, 2,209 patients were registered. After excluding self-limited/idiopathic epilepsies, 1,851 of 2,110 patients (87.7%) met the severity criteria. The patients were classified into eight main epilepsy syndromes (594 patients), 20 groups based on aetiology (1,078 patients), and three groups without known aetiology (427 patients). Most of the groups classified by syndrome or aetiology had high proportions of patients satisfying the severity criteria (>90%), but some groups had relatively low proportions (<80%) resulting from favourable outcome of surgical therapy. Several small groups with known syndrome/aetiology await detailed analysis based on a sufficiently large enough number of patients registered, some of whom may potentially be added to the list of the Act. Significance. The registry provides data to examine the usefulness of the severity criteria and list of diseases that are operationally defined by the Act. Most epilepsy patients with various syndromes/diseases and aetiology groups are covered by the Act but some are not, and the list of designated syndromes/diseases should be complemented by further amendments, as suggested by future research.