USP25 encodes ubiquitin-specific proteases 25, a key member of deubiquitinating enzyme family and is involved in neural fate determination. Although abnormal expression in Down\'s syndrome was reported previously, the specific role of USP25 in human diseases has not been defined. In this study, we performed trio-based whole exome sequencing in a cohort of 319 cases (families) with generalized epilepsy of unknown etiology. Five heterozygous USP25 variants including two de novo and three co-segregated variants were determined in eight individuals affected by generalized seizures and/or febrile seizures from five unrelated families. The frequency of USP25 variants showed a significantly high aggregation in this cohort compared to the East Asian population and all populations in the gnomAD database. The mean onset ages of febrile and afebrile seizures were 10 months (infancy) and 11.8 years (juvenile), respectively. The patients achieved seizure freedom except one had occasional nocturnal seizures at the last follow-up. Two patients exhibited intellectual disability. Usp25 was ubiquitously expressed in mouse brain with two peaks on embryonic days (E14‒E16) and postnatal day 21, respectively. Similarly, USP25 expressed in fetus/early childhood stage with a second peak at approximately 12‒20 years old in human brain, consistent with the seizure onset age at infancy and juvenile in the patients. To investigate the functional impact of USP25 deficiency in vivo, we established Usp25 knock-out mice, which showed increased seizure susceptibility compared to wild-type mice in pentylenetetrazol-induced seizure test. To explore the impact of USP25 variants, we employed multiple functional detections. In HEK293T cells, the severe phenotype associated variant (p.Gln889Ter) led to a significant reduction of mRNA and protein expressions but formed a stable truncated dimers with increment of deubiquitinating enzyme activities and abnormal cellular aggregations, indicating a gain-of-function effect. The p.Gln889Ter and p.Leu1045del increased neuronal excitability in mice brain, with a higher firing ability in p.Gln889Ter. These functional impairments align with the severity of the observed phenotypes, suggesting a genotype-phenotype correlation. Hence, a moderate association between USP25 and epilepsy was noted, indicating USP25 is potentially a predisposing gene for epilepsy. Our results from Usp25 null mice and the patient-derived variants indicated that USP25 would play epileptogenic role via loss-of-function or gain-of-function effects. The truncated variant p.Gln889Ter would have profoundly different effect on epilepsy. Together, our results underscore the significance of USP25 heterozygous variants in epilepsy, thereby highlighting the critical role of USP25 in the brain.

OBJECTIVE: The RELN gene encodes the secreted glycoprotein Reelin and has important functions in both developing and adult brains. In this study, we aimed to explore the association between the RELN and genetic generalized epilepsy (GGE).
METHODS: We performed whole-exome sequencing on a cohort of 92 patients with GGE. Based on amino acid sequence alignments, allele frequency, pedigree validation and computational modeling, the RELN variants were identified and clinical features of cases were summarized. Cell-based Reelin secretion assays were examined by Western blotting. Alterations of mutant Reelin transport through the secretion pathway were detected by immunofluorescence staining.
RESULTS: Three novel pathogenic RELN variants (3.26%; c.2260C>T/p.R754W, c.2914C>G/p.P972A and c.3029G>A/p.R1010H) were identified. All probands showed adolescence-onset generalized seizures characterized by generalized epileptiform discharges with normal EEG backgrounds, no or mild cognitive impairment, and responded well to anti-seizure medications. All these variants were located in the central regions from 1B to 2A consecutive repeats, and protein modeling demonstrated structural alterations in Reelin. Moreover, we found that these heterozygous missense variants significantly decreased the secretion of mutant proteins in HEK-293T cells, and this impairment was due to the altered transport of mutant Reelin in the secretion pathway.
CONCLUSIONS: These results suggest that RELN is potentially associated with GGE. The phenotype of GGE caused by RELN variants is relatively mild, and the pathogenic mechanism may involve a loss-of-function.

OBJECTIVE: Epilepsy with eyelid myoclonias(EMA) is a genetic generalized epilepsy (GGE) characterized by eyelid myoclonia, eye-closure sensitivity and photosensitivity. Data on EMA patients who specifically present with photoparoxysmal response on EEG is lacking. EMA is an under-recognized syndrome which is frequently misclassified as another GGE. The main objective of our research is to describe the occurrence of EMA versus other GGEs among patients with photoparoxysmal response and evaluate their distinguishing features.
METHODS: We retrospectively identified all patients who had photoparoxysmal response on EEGs performed at Cleveland clinic between 01/01/2012 and 12/31/2019. Initial epilepsy diagnosis and clinical data were collected. EEGs were reviewed for eyelid myoclonia and eye-closure-sensitivity which were used as main diagnostic clues for EMA. If clinical criteria was met, diagnosis was revised as EMA.
RESULTS: Of 249 patients with photoparoxysmal response, 70(28.1%) met EMA criteria. Sixty-two (88.6%) were females. Mean age of onset of epilepsy was 7 years (+7.9) and 120(48.2%) had other GGEs. Fifty-four (77.1%) patients with EMA were initially classified as another epilepsy. Initial diagnosis included CAE or JME in 40(57.1%) patients with EMA so we compared EMA with these syndromes. Female preponderance, drug refractoriness, older age of onset and generalized myoclonia were more common in EMA than CAE. Earlier age of onset, absence seizures, and lack of generalized myoclonic jerks were more common EMA than JME.
CONCLUSIONS: Our study demonstrates that EMA is under-recognized among GGE patients with photoparoxysmal response. It highlights distinguishing clinical and electrographic features which separate EMA from other GGEs. It emphasizes the diverse treatments utilized and the need for therapeutic options for patients with refractory EMA.

OBJECTIVE: The underlying pathophysiology of juvenile absence epilepsy (JAE) is unclear. Since cortical and subcortical brain regions are thought to be altered in genetic generalized epilepsy, the present study examined the resting-state functional network topology of the same regions in JAE.
METHODS: Electroencephalography and functional magnetic resonance imaging (EEG-fMRI) were performed on 18 JAE patients and 28 healthy controls (HCs). The topology of functional networks was analyzed using the graph-theoretic method. Both global and nodal network parameters were calculated, and parameters differing significantly between the two groups were correlated with clinical variables.
RESULTS: Both JAE patients and HCs had small-world functional network topological architectures. However, JAE patients showed higher values for the global parameters of clustering coefficient (Cp) and normalized characteristic path length (Lambda). At the nodal level, patients exhibited greater centrality at widespread cortices, including the left superior parietal gyrus, right superior temporal gyrus, right orbital part of middle frontal gyrus and bilateral supplementary motor area. Conversely, patients showed decreased nodal centrality predominantly in the limbic network, left thalamus and right caudate nucleus. Degree centrality in the right hippocampus and betweenness centrality in the right caudate nucleus positively correlated with epilepsy duration.
CONCLUSIONS: The global functional network of JAE shows small-world properties, but tends to be regular with higher segregation and lower integration. Regions in the basal ganglia-thalamo-cortical network have aberrant nodal centrality. The hippocampus and caudate nucleus may reorganize as epilepsy progresses. Our findings indicate the pathogenesis and compensatory mechanisms to seizure attacks and cognitive deficits of JAE.