Long-term treatment with ganaxolone for seizures associated with cyclin-dependent kinase-like 5 deficiency disorder: Two-year open-label extension follow-up.

OBJECTIVE: In the placebo-controlled, double-blind phase of the Marigold study (NCT03572933), ganaxolone significantly reduced major motor seizure frequency (MMSF) in patients with cyclin-dependent kinase-like 5 deficiency disorder (CDD). We report 2-year safety and clinical outcomes data from the open-label extension (OLE) phase of Marigold. METHODS: Patients with CDD who completed the double-blind phase were eligible to continue in the OLE. Efficacy assessments included MMSF reduction from prerandomization baseline, responder rates, and Clinical Global Impression-Improvement scores, including assessment of seizure intensity and duration (CGI-CSID). Safety assessments included treatment-emergent adverse events (TEAEs) and TEAEs leading to discontinuation. RESULTS: Of 101 patients who enrolled in Marigold, 88 (87.1%) entered the OLE (median age = 5 years, 79.5% female). Median 28-day MMSF at baseline was 50.6. At 2 years in the OLE (months 22-24), MMSF was reduced by a median of 48.2% (n = 50); when missing data were imputed, median reduction in MMSF was 43.8% using a mixed effects model and 27.4% using a last observation carried forward model. During months 22-24, 23 of 50 (46.0%) patients experienced reductions in MMSF of ≥50%; 12 of 50 (24.0%) patients experienced MMSF reductions of ≥75%. During months 22-24, 40 of 49 (81.6%) patients were rated by caregivers as having improvement in seizure-related outcomes based on CGI-CSID scores. Thirty-seven patients discontinued ganaxolone due to lack of efficacy (n = 13), withdrawal by caregiver (n = 12), adverse event (n = 10), physician decision (n = 1), or death (n = 1; unrelated to study drug). The most common treatment-related TEAEs were somnolence (17.0%), seizure (11.4%), and decreased appetite (5.7%). Patients reported serious TEAEs (n = 28, 31.8%); those reported in ≥3% of patients were seizure (n = 6), pneumonia (n = 5), acute respiratory failure (n = 3), aspiration pneumonia (n = 3), and dehydration (n = 3). SIGNIFICANCE: Sustained reductions in MMSF at 2 years in the OLE support the efficacy of ganaxolone in seizures associated with CDD. Safety findings in the OLE were consistent with the double-blind phase.

Psychometric evaluation of clinician- and caregiver-reported clinical severity assessments for individuals with CDKL5 deficiency disorder.

OBJECTIVE: The CDKL5 Clinical Severity Assessment (CCSA) is a comprehensive, content-validated measurement tool capturing the diverse challenges of cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD), a genetically caused developmental epileptic encephalopathy (DEE). The CCSA is divided into clinician-reported (CCSA-Clinician) and caregiver-reported (CCSA-Caregiver) assessments. The aim of this study was to evaluate the factor structure of these measures through confirmatory factor analysis (CFA) and evaluate their validity and reliability. METHODS: Participants were recruited from the International CDKL5 Clinical Research Network to take part in an in-clinic CCSA-Clinician evaluation (n = 148) and/or complete the CCSA-Caregiver questionnaire (n = 198). CFA was used to determine domains, and factor loadings and validity were assessed. For the CCSA-Clinician, inter-rater reliability was assessed by nine CDD experienced clinicians via 14 pre-recorded evaluations. Eight clinicians re-viewed and re-scored the videos after 4 weeks to evaluate intra-rater reliability. The CCSA-Caregiver was completed on a second occasion by 34 caregivers after 2-4 weeks to assess test-retest reliability. RESULTS: CFA resulted in three domains for the CCSA-Clinician (motor and movement, communication, vision) and four domains for the CCSA-Caregiver (seizures, behavior, alertness, feeding), with good item loadings across both measures. Structural statistics, internal consistency, discriminant validity, and reliability were satisfactory for both measures, and scores were consistent between known groups. SIGNIFICANCE: This study provides strong evidence that the CCSA measures are suitable to assess the clinical severity of individuals with CDD, supporting their use in clinical trials. Further evaluation of responsiveness to change in a longitudinal assessment is planned. Use may also be appropriate in similar DEEs but would require validation in those populations.

Timing the clinical onset of epileptic spasms in infantile epileptic spasms syndrome: A tertiary health center's experience.

OBJECTIVE: Lead time to treatment (clinical onset of epileptic spasms [ES] to initiation of appropriate treatment) is known to predict outcomes in infantile epileptic spasms syndrome (IESS). Timing the clinical onset of ES is crucial to establish lead time. We investigated how often ES onset could be established to the nearest week. We aimed to (1) ascertain the exact date or estimate the nearest week of ES onset and (2) compare clinical/demographic factors between patients where date of ES onset was determined or estimated to the nearest week and patients whose date of ES onset could not be estimated to the nearest week. Reasons for difficulties in estimating date of ES onset were explored. METHODS: Retrospective chart review of new onset IESS patients (January 2019-May 2022) extracted the date or week of the clinical onset of ES. Predictors of difficulty in date of ES onset estimation to the nearest week were examined by regression analysis. Sources contributing to difficulties determining date of ES onset were assessed after grouping into categories (provider-, caregiver-, disease-related). RESULTS: Among 100 patients, date of ES onset was estimated to the nearest week in 47%. On univariable analysis, age at diagnosis (p = .021), development delay (p = .007), developmental regression/stagnation (p = .021), ES intermixed with other seizures (p = .011), and nonclustered ES at onset (p = .005) were associated with difficulties estimating date of ES onset. On multivariable analysis, failure to establish date of ES onset was related to ES intermixed with other seizures (p = .004) and nonclustered ES at onset (p = .003). Sources contributing to difficulties determining date of ES onset included disease-related factors (ES characteristics, challenges interpreting electroencephalograms) and provider/caregiver-related factors (delayed diagnosis). SIGNIFICANCE: Difficulties with estimation of lead time (due to difficulties timing ES onset) can impact clinical care (prognostication), as even small increments in lead time duration can have adverse developmental consequences.

CDKL5 deficiency disorder and other infantile-onset genetic epilepsies.

AIM: To differentiate phenotypic features of individuals with CDKL5 deficiency disorder (CDD) from those of individuals with other infantile-onset epilepsies. METHOD: We performed a retrospective cohort study and ascertained individuals with CDD and comparison individuals with infantile-onset epilepsy who had epilepsy gene panel testing. We reviewed records, updated variant classifications, and compared phenotypic features. Wilcoxon rank-sum tests and χ2 or Fisher's exact tests were performed for between-cohort comparisons. RESULTS: We identified 137 individuals with CDD (110 females, 80.3%; median age at last follow-up 3 year 11 months) and 313 individuals with infantile-onset epilepsies (156 females, 49.8%; median age at last follow-up 5 years 2 months; 35% with genetic diagnosis). Features reported significantly more frequently in the CDD group than in the comparison cohort included developmental and epileptic encephalopathy (81% vs 66%), treatment-resistant epilepsy (95% vs 71%), sequential seizures (46% vs 6%), epileptic spasms (66% vs 42%, with hypsarrhythmia in 30% vs 48%), regression (52% vs 29%), evolution to Lennox-Gastaut syndrome (23% vs 5%), diffuse hypotonia (72% vs 36%), stereotypies (69% vs 11%), paroxysmal movement disorders (29% vs 17%), cerebral visual impairment (94% vs 28%), and failure to thrive (38% vs 22%). INTERPRETATION: CDD, compared with other suspected or confirmed genetic epilepsies presenting in the first year of life, is more often characterized by a combination of treatment-resistant epilepsy, developmental and epileptic encephalopathy, sequential seizures, spasms without hypsarrhythmia, diffuse hypotonia, paroxysmal movement disorders, cerebral visual impairment, and failure to thrive. Defining core phenotypic characteristics will improve precision diagnosis and treatment.

Anaerobic gut fungi are an untapped reservoir of natural products.

Anaerobic fungi (class Neocallimastigomycetes) thrive as low-abundance members of the herbivore digestive tract. The genomes of anaerobic gut fungi are poorly characterized and have not been extensively mined for the biosynthetic enzymes of natural products such as antibiotics. Here, we investigate the potential of anaerobic gut fungi to synthesize natural products that could regulate membership within the gut microbiome. Complementary 'omics' approaches were combined to catalog the natural products of anaerobic gut fungi from four different representative species: Anaeromyces robustus (Arobustus), Caecomyces churrovis (Cchurrovis), Neocallimastix californiae (Ncaliforniae), and Piromyces finnis (Pfinnis). In total, 146 genes were identified that encode biosynthetic enzymes for diverse types of natural products, including nonribosomal peptide synthetases and polyketide synthases. In addition, N. californiae and C. churrovis genomes encoded seven putative bacteriocins, a class of antimicrobial peptides typically produced by bacteria. During standard laboratory growth on plant biomass or soluble substrates, 26% of total core biosynthetic genes in all four strains were transcribed. Across all four fungal strains, 30% of total biosynthetic gene products were detected via proteomics when grown on cellobiose. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) characterization of fungal supernatants detected 72 likely natural products from A. robustus alone. A compound produced by all four strains of anaerobic fungi was putatively identified as the polyketide-related styrylpyrone baumin. Molecular networking quantified similarities between tandem mass spectrometry (MS/MS) spectra among these fungi, enabling three groups of natural products to be identified that are unique to anaerobic fungi. Overall, these results support the finding that anaerobic gut fungi synthesize natural products, which could be harnessed as a source of antimicrobials, therapeutics, and other bioactive compounds.

Benchmark dose profiles for bivariate exposures.

While benchmark dose (BMD) methodology is well-established for settings with a single exposure, these methods cannot easily handle multidimensional exposures with nonlinear effects. We propose a framework for BMD analysis to characterize the joint effect of a two-dimensional exposure on a continuous outcome using a generalized additive model while adjusting for potential confounders via propensity scores. This leads to a dose-response surface which can be summarized in two dimensions by a contour plot in which combinations of exposures leading to the same expected effect are identified. In our motivating study of prenatal alcohol exposure, cognitive deficits in children are found to be associated with both the frequency of drinking as well as the amount of alcohol consumed on each drinking day during pregnancy. The general methodological framework is useful for a broad range of settings, including combinations of environmental stressors, such as chemical mixtures, and in explorations of the impact of dose rate rather than simply cumulative exposure on adverse outcomes.

Rat bronchoalveolar lavage proteome changes following e-cigarette aerosol exposures.

E-cigarette liquids are complex mixtures of chemicals consisting of humectants, such as propylene glycol (PG) and vegetable glycerin (VG), with nicotine or flavorings added. Published literature emphasizes the toxicity of e-cigarette aerosols with flavorings whereas much less attention has been given to the biologic effects of humectants. The purpose of the current study was to provide a comprehensive view of the acute biologic effects of e-cigarette aerosols on rat bronchoalveolar lavage (BAL) using mass spectrometry-based global proteomics. Sprague-Dawley rats were exposed to e-cigarette aerosol for 3 h/day for three consecutive days. Groups included: PG/VG alone, PG/VG + 2.5% nicotine (N), or PG/VG + N + 3.3% vanillin (V). Right lung lobes were lavaged for BAL and supernatants prepared for proteomics. Extracellular BAL S100A9 concentrations and BAL cell staining for citrullinated histone H3 (citH3) were also performed. From global proteomics, ∼2,100 proteins were identified from rat BAL. The greatest change in number of BAL proteins occurred with PG/VG exposures alone compared with controls with biological pathways enriched for acute phase responses, extracellular trap formation, and coagulation. Extracellular BAL S100A9 concentrations and the number of citH3 + BAL cells also increased significantly in PG/VG and PG/VG + 2.5% N. In contrast to PG/VG or PG/VG + N, the addition of vanillin to PG/VG + N increased BAL neutrophilia and downregulated lipid transport proteins. In summary, global proteomics support e-cigarette aerosol exposures to PG/VG alone as having a significant biologic effect on the lung independent of nicotine or flavoring with increased markers of extracellular trap formation.

New insights into the natural history of bronchopulmonary dysplasia from proteomics and multiplexed immunohistochemistry.

Bronchopulmonary dysplasia (BPD) is a disease of prematurity related to the arrest of normal lung development. The objective of this study was to better understand how proteome modulation and cell-type shifts are noted in BPD pathology. Pediatric human donors aged 1-3 yr were classified based on history of prematurity and histopathology consistent with "healed" BPD (hBPD, n = 3) and "established" BPD (eBPD, n = 3) compared with respective full-term born (n = 6) age-matched term controls. Proteins were quantified by tandem mass spectroscopy with selected Western blot validations. Multiplexed immunofluorescence (MxIF) microscopy was performed on lung sections to enumerate cell types. Protein abundances and MxIF cell frequencies were compared among groups using ANOVA. Cell type and ontology enrichment were performed using an in-house tool and/or EnrichR. Proteomics detected 5,746 unique proteins, 186 upregulated and 534 downregulated, in eBPD versus control with fewer proteins differentially abundant in hBPD as compared with age-matched term controls. Cell-type enrichment suggested a loss of alveolar type I, alveolar type II, endothelial/capillary, and lymphatics, and an increase in smooth muscle and fibroblasts consistent with MxIF. Histochemistry and Western analysis also supported predictions of upregulated ferroptosis in eBPD versus control. Finally, several extracellular matrix components mapping to angiogenesis signaling pathways were altered in eBPD. Despite clear parsing by protein abundance, comparative MxIF analysis confirms phenotypic variability in BPD. This work provides the first demonstration of tandem mass spectrometry and multiplexed molecular analysis of human lung tissue for critical elucidation of BPD trajectory-defining factors into early childhood.NEW & NOTEWORTHY We provide new insights into the natural history of bronchopulmonary dysplasia in donor human lungs after the neonatal intensive care unit hospitalization. This study provides new insights into how the proteome and histopathology of BPD changes in early childhood, uncovering novel pathways for future study.

Ezogabine impacts seizures and development in patients with KCNQ2 developmental and epileptic encephalopathy.

Genetic variants in KCNQ2 are associated with a range of epilepsies, from self- limited (familial) neonatal-infantile epilepsy to developmental and epileptic encephalopathy (DEE). We retrospectively reviewed clinical data from eight patients with KCNQ2-related DEE who were treated with ezogabine. Treatment was initiated at a median age of 8 months (range, 7 weeks to 2.5 years) and continued for a median of 2.6 years (range, 7 months to 4.5 years). Five individuals had daily seizures at baseline and experienced at least 50% seizure reduction with treatment, sustained in four. One individual with two to four yearly seizures improved to rare events. Two individuals were seizure-free; treatment targeted cognition and development. Developmental improvements were reported in all eight patients. Weaning of ezogabine was associated with increased seizure frequency (N = 4), agitation and irritability (N = 2), poor sleep (N = 1), and developmental regression (N = 2). These data suggest that treatment with ezogabine is effective at reducing seizure burden and is associated with improved development. Minimal side effects were observed. Weaning was associated with increased seizures and behavioral disturbances in a subset. An approach targeting potassium channel dysfunction with ezogabine is warranted in patients with KCNQ2-related DEE.

Epileptic spasms in CDKL5 deficiency disorder: Delayed treatment and poor response to first-line therapies.

OBJECTIVE: We aimed to assess the treatment response of infantile-onset epileptic spasms (ES) in CDKL5 deficiency disorder (CDD) vs other etiologies. METHODS: We evaluated patients with ES from the CDKL5 Centers of Excellence and the National Infantile Spasms Consortium (NISC), with onset from 2 months to 2 years, treated with adrenocorticotropic hormone (ACTH), oral corticosteroids, vigabatrin, and/or the ketogenic diet. We excluded children with tuberous sclerosis complex, trisomy 21, or unknown etiology with normal development because of known differential treatment responses. We compared the two cohorts for time to treatment and ES remission at 14 days and 3 months. RESULTS: We evaluated 59 individuals with CDD (79% female, median ES onset 6 months) and 232 individuals from the NISC database (46% female, median onset 7 months). In the CDD cohort, seizures prior to ES were common (88%), and hypsarrhythmia and its variants were present at ES onset in 34%. Initial treatment with ACTH, oral corticosteroids, or vigabatrin started within 1 month of ES onset in 27 of 59 (46%) of the CDD cohort and 182 of 232 (78%) of the NISC cohort (p < .0001). Fourteen-day clinical remission of ES was lower for the CDD group (26%, 7/27) than for the NISC cohort (58%, 106/182, p = .0002). Sustained ES remission at 3 months occurred in 1 of 27 (4%) of CDD patients vs 96 of 182 (53%) of the NISC cohort (p < .0001). Comparable results were observed with longer lead time (≥1 month) or prior treatment. Ketogenic diet, used within 3 months of ES onset, resulted in ES remission at 1 month, sustained at 3 months, in at least 2 of 13 (15%) individuals with CDD. SIGNIFICANCE: Compared to the broad group of infants with ES, children with ES in the setting of CDD often experience longer lead time to treatment and respond poorly to standard treatments. Development of alternative treatments for ES in CDD is needed.

Assessing the landscape of STXBP1-related disorders in 534 individuals.

Disease-causing variants in STXBP1 are among the most common genetic causes of neurodevelopmental disorders. However, the phenotypic spectrum in STXBP1-related disorders is wide and clear correlations between variant type and clinical features have not been observed so far. Here, we harmonized clinical data across 534 individuals with STXBP1-related disorders and analysed 19 973 derived phenotypic terms, including phenotypes of 253 individuals previously unreported in the scientific literature. The overall phenotypic landscape in STXBP1-related disorders is characterized by neurodevelopmental abnormalities in 95% and seizures in 89% of individuals, including focal-onset seizures as the most common seizure type (47%). More than 88% of individuals with STXBP1-related disorders have seizure onset in the first year of life, including neonatal seizure onset in 47%. Individuals with protein-truncating variants and deletions in STXBP1 (n = 261) were almost twice as likely to present with West syndrome and were more phenotypically similar than expected by chance. Five genetic hotspots with recurrent variants were identified in more than 10 individuals, including p.Arg406Cys/His (n = 40), p.Arg292Cys/His/Leu/Pro (n = 30), p.Arg551Cys/Gly/His/Leu (n = 24), p.Pro139Leu (n = 12), and p.Arg190Trp (n = 11). None of the recurrent variants were significantly associated with distinct electroclinical syndromes, single phenotypic features, or showed overall clinical similarity, indicating that the baseline variability in STXBP1-related disorders is too high for discrete phenotypic subgroups to emerge. We then reconstructed the seizure history in 62 individuals with STXBP1-related disorders in detail, retrospectively assigning seizure type and seizure frequency monthly across 4433 time intervals, and retrieved 251 anti-seizure medication prescriptions from the electronic medical records. We demonstrate a dynamic pattern of seizure control and complex interplay with response to specific medications particularly in the first year of life when seizures in STXBP1-related disorders are the most prominent. Adrenocorticotropic hormone and phenobarbital were more likely to initially reduce seizure frequency in infantile spasms and focal seizures compared to other treatment options, while the ketogenic diet was most effective in maintaining seizure freedom. In summary, we demonstrate how the multidimensional spectrum of phenotypic features in STXBP1-related disorders can be assessed using a computational phenotype framework to facilitate the development of future precision-medicine approaches.

Characterization of the GABRB2-Associated Neurodevelopmental Disorders.

OBJECTIVE: We aimed to characterize the phenotypic spectrum and functional consequences associated with variants in the gene GABRB2, coding for the γ-aminobutyric acid type A (GABAA ) receptor subunit ß2. METHODS: We recruited and systematically evaluated 25 individuals with variants in GABRB2, 17 of whom are newly described and 8 previously reported with additional clinical data. Functional analysis was performed using a Xenopus laevis oocyte model system. RESULTS: Our cohort of 25 individuals from 22 families with variants in GABRB2 demonstrated a range of epilepsy phenotypes from genetic generalized epilepsy to developmental and epileptic encephalopathy. Fifty-eight percent of individuals had pharmacoresistant epilepsy; response to medications targeting the GABAergic pathway was inconsistent. Developmental disability (present in 84%) ranged from mild intellectual disability to severe global disability; movement disorders (present in 44%) included choreoathetosis, dystonia, and ataxia. Disease-associated variants cluster in the extracellular N-terminus and transmembrane domains 1-3, with more severe phenotypes seen in association with variants in transmembrane domains 1 and 2 and the allosteric binding site between transmembrane domains 2 and 3. Functional analysis of 4 variants in transmembrane domains 1 or 2 (p.Ile246Thr, p.Pro252Leu, p.Ile288Ser, p.Val282Ala) revealed strongly reduced amplitudes of GABA-evoked anionic currents. INTERPRETATION: GABRB2-related epilepsy ranges broadly in severity from genetic generalized epilepsy to developmental and epileptic encephalopathies. Developmental disability and movement disorder are key features. The phenotypic spectrum is comparable to other GABAA receptor-encoding genes. Phenotypic severity varies by protein domain. Experimental evidence supports loss of GABAergic inhibition as the mechanism underlying GABRB2-associated neurodevelopmental disorders. ANN NEUROL 2021;89:573-586.

Wide range of phenotypic severity in individuals with late truncations unique to the predominant CDKL5 transcript in the brain.

Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is caused by heterozygous or hemizygous variants in CDKL5 and is characterized by refractory epilepsy, cognitive and motor impairments, and cerebral visual impairment. CDKL5 has multiple transcripts, of which the longest transcripts, NM_003159 and NM_001037343, have been used historically in clinical laboratory testing. However, the transcript NM_001323289 is the most highly expressed in brain and contains 170 nucleotides at the 3' end of its last exon that are noncoding in other transcripts. Two truncating variants in this region have been reported in association with a CDD phenotype. To clarify the significance and range of phenotypes associated with late truncating variants in this region of the predominant transcript in the brain, we report detailed information on two individuals, updated clinical information on a third individual, and a summary of published and unpublished individuals reported in ClinVar. The two new individuals (one male and one female) each had a relatively mild clinical presentation including periods of pharmaco-responsive epilepsy, independent walking and limited purposeful communication skills. A previously reported male continued to have a severe phenotype. Overall, variants in this region demonstrate a range of clinical severity consistent with reports in CDD but with the potential for milder presentation.

Proteomic Profiling of the Substantia Nigra to Identify Determinants of Lewy Body Pathology and Dopaminergic Neuronal Loss.

Proteinaceous aggregates containing α-synuclein protein called Lewy bodies in the substantia nigra is a hallmark of Parkinson's disease. The molecular mechanisms of Lewy body formation and associated neuronal loss remain largely unknown. To gain insights into proteins and pathways associated with Lewy body pathology, we performed quantitative profiling of the proteome. We analyzed substantia nigra tissue from 51 subjects arranged into three groups: cases with Lewy body pathology, Lewy body-negative controls with matching neuronal loss, and controls with no neuronal loss. Using a label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach, we characterized the proteome both in terms of protein abundances and peptide modifications. Statistical testing for differential abundance of the most abundant 2963 proteins, followed by pathway enrichment and Bayesian learning of the causal network structure, was performed to identify likely drivers of Lewy body formation and dopaminergic neuronal loss. The identified pathways include (1) Arp2/3 complex-mediated actin nucleation; (2) synaptic function; (3) poly(A) RNA binding; (4) basement membrane and endothelium; and (5) hydrogen peroxide metabolic process. According to the data, the endothelial/basement membrane pathway is tightly connected with both pathologies and likely to be one of the drivers of neuronal loss. The poly(A) RNA-binding proteins, including the ones relevant to other neurodegenerative disorders (e.g., TDP-43 and FUS), have a strong inverse correlation with Lewy bodies and may reflect an alternative mechanism of nigral neurodegeneration.

A Recurrent De Novo PACS2 Heterozygous Missense Variant Causes Neonatal-Onset Developmental Epileptic Encephalopathy, Facial Dysmorphism, and Cerebellar Dysgenesis.

Developmental and epileptic encephalopathies (DEEs) represent a large clinical and genetic heterogeneous group of neurodevelopmental diseases. The identification of pathogenic genetic variants in DEEs remains crucial for deciphering this complex group and for accurately caring for affected individuals (clinical diagnosis, genetic counseling, impacting medical, precision therapy, clinical trials, etc.). Whole-exome sequencing and intensive data sharing identified a recurrent de novo PACS2 heterozygous missense variant in 14 unrelated individuals. Their phenotype was characterized by epilepsy, global developmental delay with or without autism, common cerebellar dysgenesis, and facial dysmorphism. Mixed focal and generalized epilepsy occurred in the neonatal period, controlled with difficulty in the first year, but many improved in early childhood. PACS2 is an important PACS1 paralog and encodes a multifunctional sorting protein involved in nuclear gene expression and pathway traffic regulation. Both proteins harbor cargo(furin)-binding regions (FBRs) that bind cargo proteins, sorting adaptors, and cellular kinase. Compared to the defined PACS1 recurrent variant series, individuals with PACS2 variant have more consistently neonatal/early-infantile-onset epilepsy that can be challenging to control. Cerebellar abnormalities may be similar but PACS2 individuals exhibit a pattern of clear dysgenesis ranging from mild to severe. Functional studies demonstrated that the PACS2 recurrent variant reduces the ability of the predicted autoregulatory domain to modulate the interaction between the PACS2 FBR and client proteins, which may disturb cellular function. These findings support the causality of this recurrent de novo PACS2 heterozygous missense in DEEs with facial dysmorphim and cerebellar dysgenesis.

Effects of prenatal alcohol exposure on cognitive and behavioral development: Findings from a hierarchical meta-analysis of data from six prospective longitudinal U.S. cohorts.

BACKGROUND: Cognitive and behavioral sequelae of prenatal alcohol exposure (PAE) continue to be prevalent in the United States and worldwide. Because these sequelae are also common in other neurodevelopmental disorders, researchers have attempted to identify a distinct neurobehavioral profile to facilitate the differential diagnosis of fetal alcohol spectrum disorders (FASD). We used an innovative, individual participant meta-analytic technique to combine data from six large U.S. longitudinal cohorts to provide a more comprehensive and reliable characterization of the neurobehavioral deficits seen in FASD than can be obtained from smaller samples. METHODS: Meta-analyses were performed on data from 2236 participants to examine effects of PAE (measured as oz absolute alcohol/day (AA/day)) on IQ, four domains of cognition function (learning and memory, executive function, reading achievement, and math achievement), sustained attention, and behavior problems, after adjusting for potential confounders using propensity scores. RESULTS: The effect sizes for IQ and the four domains of cognitive function were strikingly similar to one another and did not differ at school age, adolescence, or young adulthood. Effect sizes were smaller in the more middle-class Seattle cohort and larger in the three cohorts that obtained more detailed and comprehensive assessments of AA/day. PAE effect sizes were somewhat weaker for parent- and teacher-reported behavior problems and not significant for sustained attention. In a meta-analysis of five aspects of executive function, the strongest effect was on set-shifting. CONCLUSIONS: The similarity in the effect sizes for the four domains of cognitive function suggests that PAE affects an underlying component or components of cognition involving learning and memory and executive function that are reflected in IQ and academic achievement scores. The weaker effects in the more middle-class cohort may reflect a more cognitively stimulating environment, a different maternal drinking pattern (lower alcohol dose/occasion), and/or better maternal prenatal nutrition. These findings identify two domains of cognition-learning/memory and set-shifting-that are particularly affected by PAE, and one, sustained attention, which is apparently spared.

Modelling and treating GRIN2A developmental and epileptic encephalopathy in mice.

NMDA receptors play crucial roles in excitatory synaptic transmission. Rare variants in GRIN2A encoding the GluN2A subunit are associated with a spectrum of disorders, ranging from mild speech and language delay to intractable neurodevelopmental disorders, including but not limited to developmental and epileptic encephalopathy. A de novo missense variant, p.Ser644Gly, was identified in a child with this disorder, and Grin2a knock-in mice were generated to model and extend understanding of this intractable childhood disease. Homozygous and heterozygous mutant mice exhibited altered hippocampal morphology at 2 weeks of age, and all homozygotes exhibited lethal tonic-clonic seizures by mid-third week. Heterozygous adults displayed susceptibility to induced generalized seizures, hyperactivity, repetitive and reduced anxiety behaviours, plus several unexpected features, including significant resistance to electrically-induced limbic seizures and to pentylenetetrazole induced tonic-clonic seizures. Multielectrode recordings of neuronal networks revealed hyperexcitability and altered bursting and synchronicity. In heterologous cells, mutant receptors had enhanced NMDA receptor agonist potency and slow deactivation following rapid removal of glutamate, as occurs at synapses. NMDA receptor-mediated synaptic currents in heterozygous hippocampal slices also showed a prolonged deactivation time course. Standard anti-epileptic drug monotherapy was ineffective in the patient. Introduction of NMDA receptor antagonists was correlated with a decrease in seizure burden. Chronic treatment of homozygous mouse pups with NMDA receptor antagonists significantly delayed the onset of lethal seizures but did not prevent them. These studies illustrate the power of using multiple experimental modalities to model and test therapies for severe neurodevelopmental disorders, while revealing significant biological complexities associated with GRIN2A developmental and epileptic encephalopathy.

Cerebral visual impairment in CDKL5 deficiency disorder: vision as an outcome measure.

AIM: To characterize the neuro-ophthalmological phenotype of cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) and assess visual acuity as a reproducible, quantitative outcome measure. METHOD: We retrospectively analyzed clinical data from patients with CDD. Complete neuro-ophthalmological assessments, including visual acuity, were evaluated. RESULTS: Of 26 patients (22 females, four males; median age 4y, interquartile range 2y 1mo-7y 10mo), cerebral visual impairment (CVI), defined as visual dysfunction in the absence of ocular or anterior visual pathway abnormalities, was diagnosed in all those over 2 years of age. Ophthalmological examinations revealed nystagmus in 10 patients and strabismus in 24 patients. Visual acuity was measured in 24 patients, by preferential looking in all and by sweep visual evoked potential in 13. Visual acuities were lower than age expectations and demonstrated improvement in the first 3 years. Adjusting for age and sex, average preferential looking visual acuity after 2 years of age was higher in patients with intact mobility than in those who were non-mobile. INTERPRETATION: CVI was observed in patients with CDD. Visual acuity improved over time and correlated with mobility. Visual acuity, as a quantifiable measure of visual function, should be considered as an outcome measure in pre-clinical and clinical studies for CDD. What this paper adds Cerebral visual impairment is highly prevalent in cyclin-dependent kinase-like 5 deficiency disorder (CDD). Visual acuity is a measurable quantitative outcome measure in CDD. Visual acuity in CDD correlates with gross motor ability.

Repetitive diacetyl vapor exposure promotes ubiquitin proteasome stress and precedes bronchiolitis obliterans pathology.

Bronchiolitis obliterans (BO) is a devastating lung disease seen commonly after lung transplant, following severe respiratory tract infection or chemical inhalation exposure. Diacetyl (DA; 2,3-butanedione) is a highly reactive alpha-diketone known to cause BO when inhaled, however, the mechanisms of how inhalation exposure leads to BO development remains poorly understood. In the current work, we combined two clinically relevant models for studying the pathogenesis of DA-induced BO: (1) an in vivo rat model of repetitive DA vapor exposures with recovery and (2) an in vitro model of primary human airway epithelial cells exposed to pure DA vapors. Rats exposed to 5 consecutive days 200 parts-per-million DA 6 h per day had worsening survival, persistent hypoxemia, poor weight gain, and histologic evidence of BO 14 days after DA exposure cessation. At the end of exposure, increased expression of the ubiquitin stress protein ubiquitin-C accumulated within DA-exposed rat lung homogenates and localized primarily to the airway epithelium, the primary site of BO development. Lung proteasome activity increased concurrently with ubiquitin-C expression after DA exposure, supportive of significant proteasome stress. In primary human airway cultures, global proteomics identified 519 significantly modified proteins in DA-exposed samples relative to controls with common pathways of the ubiquitin proteasome system, endosomal reticulum transport, and response to unfolded protein pathways being upregulated and cell-cell adhesion and oxidation-reduction pathways being downregulated. Collectively, these two models suggest that diacetyl inhalation exposure causes abundant protein damage and subsequent ubiquitin proteasome stress prior to the development of chemical-induced BO pathology.

Genetic diagnoses in epilepsy: The impact of dynamic exome analysis in a pediatric cohort.

OBJECTIVE: We evaluated the yield of systematic analysis and/or reanalysis of whole exome sequencing (WES) data from a cohort of well-phenotyped pediatric patients with epilepsy and suspected but previously undetermined genetic etiology. METHODS: We identified and phenotyped 125 participants with pediatric epilepsy. Etiology was unexplained at the time of enrollment despite clinical testing, which included chromosomal microarray (57 patients), epilepsy gene panel (n = 48), both (n = 28), or WES (n = 8). Clinical epilepsy diagnoses included developmental and epileptic encephalopathy (DEE), febrile infection-related epilepsy syndrome, Rasmussen encephalitis, and other focal and generalized epilepsies. We analyzed WES data and compared the yield in participants with and without prior clinical genetic testing. RESULTS: Overall, we identified pathogenic or likely pathogenic variants in 40% (50/125) of our study participants. Nine patients with DEE had genetic variants in recently published genes that had not been recognized as epilepsy-related at the time of clinical testing (FGF12, GABBR1, GABBR2, ITPA, KAT6A, PTPN23, RHOBTB2, SATB2), and eight patients had genetic variants in candidate epilepsy genes (CAMTA1, FAT3, GABRA6, HUWE1, PTCHD1). Ninety participants had concomitant or subsequent clinical genetic testing, which was ultimately explanatory for 26% (23/90). Of the 67 participants whose molecular diagnoses were "unsolved" through clinical genetic testing, we identified pathogenic or likely pathogenic variants in 17 (25%). SIGNIFICANCE: Our data argue for early consideration of WES with iterative reanalysis for patients with epilepsy, particularly those with DEE or epilepsy with intellectual disability. Rigorous analysis of WES data of well-phenotyped patients with epilepsy leads to a broader understanding of gene-specific phenotypic spectra as well as candidate disease gene identification. We illustrate the dynamic nature of genetic diagnosis over time, with analysis and in some cases reanalysis of exome data leading to the identification of disease-associated variants among participants with previously nondiagnostic results from a variety of clinical testing strategies.