We identified a de novo missense mutation, P302L, in the γ-aminobutyric acid type A (GABAA) receptor γ2 subunit gene GABRG2 in a patient with Dravet syndrome using targeted next-generation sequencing. The mutation was in the cytoplasmic portion of the transmembrane segment M2 of the γ2 subunit that faces the pore lumen. GABAA receptor α1 and β3 subunits were coexpressed with wild-type (wt) γ2L or mutant γ2L(P302L) subunits in HEK 293T cells and cultured mouse cortical neurons. We measured currents using whole-cell and single-channel patch clamp techniques, surface and total expression levels using surface biotinylation and Western blotting, and potential structural perturbations in mutant GABAA receptors using structural modeling. The γ2(P302L) subunit mutation produced an ∼90% reduction of whole-cell current by increasing macroscopic desensitization and reducing GABA potency, which resulted in a profound reduction of GABAA receptor-mediated miniature IPSCs (mIPSCs). The conductance of the receptor channel was reduced to 24% of control conductance by shifting the relative contribution of the conductance states from high- to low-conductance levels with only slight changes in receptor surface expression. Structural modeling of the GABAA receptor in the closed, open, and desensitized states showed that the mutation was positioned to slow activation, enhance desensitization, and shift channels to a low-conductance state by reshaping the hour-glass-like pore cavity during transitions between closed, open, and desensitized states. Our study revealed a novel γ2 subunit missense mutation (P302L) that has a novel pathogenic mechanism to cause defects in the conductance and gating of GABAA receptors, which results in hyperexcitability and contributes to the pathogenesis of the genetic epilepsy Dravet syndrome.

This study aimed to explore the efficacy and safety of pantethine in children with pantothenate kinase-associated neurodegeneration (PKAN).

GABAA receptors are brain inhibitory chloride ion channels. Here we show functional analyses and structural simulations for three de novo missense mutations in the GABAA receptor β3 subunit gene (GABRB3) identified in patients with early-onset epileptic encephalopathy (EOEE) and profound developmental delay. We sought to obtain insights into the molecular mechanisms that might link defects in GABAA receptor biophysics and biogenesis to patients with EOEE. The mutant residues are part of conserved structural domains such as the Cys-loop (L170R) and M2-M3 loop (A305V) that form the GABA binding/channel gating coupling junction and the channel pore (T288N), which are functionally coupled during receptor activation. The mutant coupling junction residues caused rearrangements and formation of new hydrogen bonds in the open state, while the mutant pore residue reshaped the pore cavity. Whereas mutant coupling junction residues uncoupled during activation and caused gain of function, the mutant pore residue favoured low conductance receptors and differential sensitivity to diazepam and loss of function. These data reveal novel molecular mechanisms by which EOEE-linked mutations affect GABAA receptor function.

To investigate the natural history and genotype-phenotype correlation of pantothenate kinase-associated neurodegeneration.

Neurodevelopment is a transcriptionally orchestrated process. Cyclin K, a regulator of transcription encoded by CCNK, is thought to play a critical role in the RNA polymerase II-mediated activities. However, dysfunction of CCNK has not been linked to genetic disorders. In this study, we identified three unrelated individuals harboring de novo heterozygous copy number loss of CCNK in an overlapping 14q32.3 region and one individual harboring a de novo nonsynonymous variant c.331A>G (p.Lys111Glu) in CCNK. These four individuals, though from different ethnic backgrounds, shared a common phenotype of developmental delay and intellectual disability (DD/ID), language defects, and distinctive facial dysmorphism including high hairline, hypertelorism, thin eyebrows, dysmorphic ears, broad nasal bridge and tip, and narrow jaw. Functional assay in zebrafish larvae showed that Ccnk knockdown resulted in defective brain development, small eyes, and curly spinal cord. These defects were partially rescued by wild-type mRNA coding CCNK but not the mRNA with the identified likely pathogenic variant c.331A>G, supporting a causal role of CCNK variants in neurodevelopmental disorders. Taken together, we reported a syndromic neurodevelopmental disorder with DD/ID and facial characteristics caused by CCNK variations, possibly through a mechanism of haploinsufficiency.

Background: ATP1A1 encodes an α1 isoform of Na+/K+-ATPase, which is expressed abundantly in kidneys and central nervous system. ATP1A1 variants may cause Na+/K+-ATPase loss of function and lead to a wide spectrum of phenotypes. This study aims to summarize the clinical and genetic features of ATP1A1 de novo mutation-related disorders and explore the potential correlations between phenotypes and genotypes. Methods: We analyzed two new cases harboring novel de novo ATP1A1 variants and reviewed all reported cases. Results: Both our probands had developmental delay, patient 1 accompanied with sleep disorders, irritability, and patient 2 with refractory seizures. They each had a novel de novo heterozygous missense variant, c.2797G>A[p.Asp933Asn] (NM_000701) and c.2590G>A[p.Gly864Arg] (NM_000701) respectively. Four patients with de novo ATP1A1 variants have been reported in two previous papers. Among them, three patients had refractory seizures and one patient had complex hereditary spastic paraplegia (HSP). Therefore, all six patients had developmental delay, and four of them had epilepsy. All variants located in the transmembrane regions M3, M4, M7, and M8 of ATP1A1 protein. Four patients with mutations in M3 and M7 had more severe phenotypes, including developmental delay and epileptic encephalopathy, three of them with hypomagnesemia, whereas two patients with mutations in M4 and M8 had milder phenotypes, only with mild developmental delay, without seizures or hypomagnesemia. Correcting hypomagnesemia had not controlled those seizures. Conclusions: Two novel de novo ATP1A1 variants identified in two patients here enriched the genotypic and phenotypic spectrum of ATP1A1 mutation-related disorder. Our findings suggest that hypomagnesemia in this disorder might relate to more severe phenotype and indicate more severe Na+/K+-ATPase dysfunction. Variations in M3 and M7 transmembrane regions were related to more severe phenotype than those in M4 and M8, which suggested that variations in M3 and M7 might cause more severe ATP1A1 functional defect.

Leukoencephalopathy with vanishing white matter (VWM) is a rare autosomal recessive leukoencephalopathy resulting from mutations in EIF2B1-5, which encode subunits of eukaryotic translation initiation factor 2B (eIF2B). Studies have found that eIF2B mutation has a certain influence on embryonic brain development. So far, the effect of the eIF2B mutations on the dynamic process of brain development is not fully understood yet.

Hypomyelinating leukodystrophies (HLDs) are a rare group of heterogeneously genetic disorders characterized by persistent deficit of myelin observed on magnetic resonance imaging (MRI). To identify a new disease-associated gene of HLD, trio-based whole exome sequencing was performed for unexplained patients with HLD. Functional studies were performed to confirm the phenotypic effect of candidate protein variants. Two de novo heterozygous variants, c.227T>G p.(L76R) or c.227T>C p.(L76P) in TMEM163 were identified in two unrelated HLD patients. TMEM163 protein is a zinc efflux transporter localized within the plasma membrane, lysosomes, early endosomes, and other vesicular compartments. It has not been associated with hypomyelination. Functional zinc flux assays in HeLa cells stably-expressing TMEM163 protein variants, L76R and L76P, revealed distinct attenuation or enhancement of zinc efflux, respectively. Experiments using a zebrafish model with knockdown of tmem163a and tmem163b (morphants) showed that loss of tmem163 causes dysplasia of the larvae, locomotor disability and myelin deficit. Expression of human wild type TMEM163 mRNAs in morphants rescues the phenotype, while the TMEM163 L76P and L76R mutants aggravated the condition. Moreover, poor proliferation, elevated apoptosis of oligodendrocytes, and reduced oligodendrocytes and neurons were also observed in zebrafish morphants. Our findings suggest an unappreciated role for TMEM163 protein in myelin development and add TMEM163 to a growing list of genes associated with hypomyelination leukodystrophy.

Acitretin is an oral drug approved by the Food and Drug Administration that is commonly used to treat psoriasis. In recent years, acitretin has been identified as a candidate drug for the treatment of Alzheimer's disease, but its role in neuronal development is still unclear. In this study, the human neuroblastoma cell line SH-SY5Y was used as a model to study neuronal differentiation. We found that acitretin effectively promoted the differentiation of SH-SY5Y cells into neuronal cells and upregulated the expression of the neuronal marker β-III tubulin and the mature neuronal marker NFH. Differentially expressed genes were identified by RNA sequencing and analyzed by bioinformatics approaches. The results showed that genes associated with neuron development-related pathways, such as SSPO and KCNT1, had significant changes in expression. Analysis showed that PRKCA and CAMK2B may play important roles in the process by which acitretin promotes neurodevelopment. Through whole-cell patch clamping and a microelectrode array assay, we found that acitretin-treated neurons generated electrical spikes similar to those generated by mature neurons. This study provided evidence to support an accessible and safe model of neuron-like cells and verified that acitretin can promote the differentiation of neurons and has the potential to treat brain tumors and neurodevelopmental and neurodegenerative diseases.

Mechanically activated (MA) ion channels convert physical forces into electrical signals. Despite the importance of this function, the involvement of mechanosensitive ion channels in human disease is poorly understood. Here we report heterozygous missense mutations in the gene encoding the MA ion channel TMEM63A that result in an infantile disorder resembling a hypomyelinating leukodystrophy. Four unrelated individuals presented with congenital nystagmus, motor delay, and deficient myelination on serial scans in infancy, prompting the diagnosis of Pelizaeus-Merzbacher (like) disease. Genomic sequencing revealed that all four individuals carry heterozygous missense variants in the pore-forming domain of TMEM63A. These variants were confirmed to have arisen de novo in three of the four individuals. While the physiological role of TMEM63A is incompletely understood, it is highly expressed in oligodendrocytes and it has recently been shown to be a MA ion channel. Using patch clamp electrophysiology, we demonstrated that each of the modeled variants result in strongly attenuated stretch-activated currents when expressed in naive cells. Unexpectedly, the clinical evolution of all four individuals has been surprisingly favorable, with substantial improvements in neurological signs and developmental progression. In the three individuals with follow-up scans after 4 years of age, the myelin deficit had almost completely resolved. Our results suggest a previously unappreciated role for mechanosensitive ion channels in myelin development.

This study aimed to identify monogenic mutations from Chinese patients with childhood absence epilepsy (CAE) and summarize their characteristics. A total of 100 patients with CAE were recruited in Peking University First Hospital from 2005 to 2016 and underwent telephone and outpatient follow-up review. We used targeted disease-specific gene capture sequencing (involving 300 genes) to identify pathogenic variations for these patients. We identified three de novo epilepsy-related gene mutations, including missense mutations of SCN1A (c. 5399 T > A; p. Val1800Asp), SCN8A (c. 2371 G > T; p. Val791Phe), and CLCN2 (c. 481 G > A; p. Gly161Ser), from three patients, separately. All recruited patients presented typical CAE features and good prognosis. To date, CAE has been considered a complex disease caused by multiple susceptibility genes. In this study, we observed that 3% of typical CAE patients had a de novo mutation of a known monogenic epilepsy-related gene. Our study suggests that a significant proportion of typical CAE cases may be monogenic forms of epilepsy. For genetic generalized epilepsies, such as CAE, further studies are needed to clarify the contributions of de novo or inherited rare monogenic coding, noncoding and copy number variants.

To evaluate the effect of the COVID-19 pandemic on global access to care and practice patterns for children with epilepsy.

To summarize and extend the phenotypic characterization of Multiple Congenital Anomalies-Hypotonia-Seizures Syndrome, and to discuss genotype-phenotype correlations.

Dravet syndrome is a rare, catastrophic epileptic encephalopathy that begins in the first year of life, usually with febrile or afebrile hemiclonic or generalized tonic-clonic seizures followed by status epilepticus. De novo variants in genes that mediate synaptic transmission such as SCN1A and PCDH19 are often associated with Dravet syndrome. Recently, GABAA receptor subunit genes (GABRs) encoding α1 (GABRA1), β3 (GABRB3) and γ2 (GABRG2), but not β2 (GABRB2) or β1 (GABRB1), subunits are frequently associated with Dravet syndrome or Dravet syndrome-like phenotype. We performed next generation sequencing on 870 patients with Dravet syndrome and identified nine variants in three different GABRs. Interestingly, the variants were all in genes encoding the most common GABAA receptor, the α1β2γ2 receptor. Mutations in GABRA1 (c.644T>C, p. L215P; c.640C>T, p. R214C; c.859G>A; V287I; c.641G>A, p. R214H) and GABRG2 (c.269C>G, p. T90R; c.1025C>T, p. P342L) presented as de novo cases, while in GABRB2 two variants were de novo (c.992T>C, p. F331S; c.542A>T, p. Y181F) and one was autosomal dominant and inherited from the maternal side (c.990_992del, p.330_331del). We characterized the effects of these GABR variants on GABAA receptor biogenesis and channel function. We found that defects in receptor gating were the common deficiency of GABRA1 and GABRB2 Dravet syndrome variants, while mainly trafficking defects were found with the GABRG2 (c.269C>G, p. T90R) variant. It seems that variants in α1 and β2 subunits are less tolerated than in γ2 subunits, since variant α1 and β2 subunits express well but were functionally deficient. This suggests that all of these GABR variants are all targeting GABR genes that encode the assembled α1β2γ2 receptor, and regardless of which of the three subunits are mutated, variants in genes coding for α1, β2 and γ2 receptor subunits make them candidate causative genes in the pathogenesis of Dravet syndrome.

Purpose: Leukoencephalopathy with vanishing white matter (VWM) is an autosomal recessive leukoencephalopathy caused by mutations in any of the five genes encoding the subunits of eukaryotic translation initiation factor 2B (eIF2B). The severity of the disease varies considerably, and its genotypic-phenotypic correlation is still unclear. Age of onset is the only independent clinical predictor for VWM severity. In this study, the correlation between genotype and age at onset of patients was investigated. Methods: Data were collected from patients with VWM in the available literature reports and from those diagnosed in Peking University First Hospital. The age of onset was divided into early-onset (≤4 years) and late-onset type (>4 years) for the analysis of the correlation between genotype and age of onset in patients with VWM. Results: A total of 341 patients were included, 281 were reported in 87 available articles and 60 were diagnosed in our center. A total of 180 different mutations were found, among which 86.1% were missense. The gene (EIF2B1-5) in which the mutation located, and the number of null alleles were not associated with age of onset in these patients. Certain mutations such as eIF2Bε[Arg195His] and eIF2Bε[Arg269Gln] that were predicted to have a serious influence on eIF2B structure were related to earlier age of onset. EIF2Bγ[Ala87Val] which was predicted to have a minimal influence on eIF2B structure, was related to later age of onset. Whereas eIF2Bβ[Glu213Gly], eIF2Bβ[Gly200Val] and eIF2Bε[Thr91Ala], also predicted having a small effect on the structure of eIF2B, did not show correlation with the age of onset. The onset age of patients with one or biallelic missense mutations located in the catalytic domain or other homologous domains in catalytic subunits (eIF2Bγ, ε) was earlier than that of patients with biallelic mutations located in the NT domain. Conclusion: The onset age of patients with different genotypes varied greatly. The degree of influence in protein structure of some missense mutations was correlated with phenotypic severity, but the results were not completely consistent. The combined effect of biallelic mutations, the role of regulatory genes, environmental stress and other potential factors on phenotypes need to be further explored.

Mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy (MOGHE) is a new and rare histopathological entity of cortical developmental malformations. The clinical characteristics of MOGHE remain challenging.

Neuroblastoma is a type of developmental childhood cancer that arises from the neural crest. It is the most common pediatric solid tumor in the world. AM580 is a powerful cyto-differentiating molecule on acute promyelocytic leukemia cells and induced pluripotent stem cells, but its effect on neuroblastoma is still unknown. In this study, the neuronal differentiation impact of AM580 was investigated using the human neuroblastoma cell line SH-SY5Y as a model. AM580 successfully stimulated the SH-SY5Y cells to develop into neuron-like cells. Functional enrichment analysis of RNAseq data revealed that differentially expressed genes (DEGs) were substantially enriched for GO keywords and KEGG pathways linked to neuron development. Some potassium ion channel genes associated with neuronal excitation, such as KCNT1, were shown to be upregulated. Through the MEA tests, we found the AM580-induced neurons possessed electrical spikes as mature neurons. AM580 also induced the neuronal marker β-tubulin III and mature neurons marker Neurofilament H. Our study proved that AM580 can promote the differentiation of neurons and has the potential to treat neuroblastoma, neurodevelopmental and neurodegenerative diseases.

LAMA2-related muscular dystrophy including LAMA2-related congenital muscular dystrophy (LAMA2-CMD) and autosomal recessive limb-girdle muscular dystrophy-23 (LGMDR23) is caused by LAMA2 pathogenic variants. We aimed to describe the natural history and establish genotype-phenotype correlations in a large cohort of Chinese patients with LAMA2-related muscular dystrophy.

Hypomyelinating disorders are a group of clinically and genetically heterogeneous diseases characterized by neurological deterioration with hypomyelination visible on brain MRI scans. This study was aimed to clarify the clinical and genetic features of HMDs in Chinese population.

Dravet syndrome (DS) and Lennox-Gastaut syndrome (LGS) are rare treatment-resistant childhood epilepsies classed as developmental and epileptic encephalopathies. ELEKTRA investigated the efficacy and safety of soticlestat (TAK-935) as adjunctive therapy in children with DS or LGS (NCT03650452).