Genetically encoded impairment of neuronal KCC2 cotransporter function in human idiopathic generalized epilepsy.

The KCC2 cotransporter establishes the low neuronal Cl(-) levels required for GABAA and glycine (Gly) receptor-mediated inhibition, and KCC2 deficiency in model organisms results in network hyperexcitability. However, no mutations in KCC2 have been documented in human disease. Here, we report two non-synonymous functional variants in human KCC2, R952H and R1049C, exhibiting clear statistical association with idiopathic generalized epilepsy (IGE). These variants reside in conserved residues in the KCC2 cytoplasmic C-terminus, exhibit significantly impaired Cl(-)-extrusion capacities resulting in less hyperpolarized Gly equilibrium potentials (EG ly), and impair KCC2 stimulatory phosphorylation at serine 940, a key regulatory site. These data describe a novel KCC2 variant significantly associated with a human disease and suggest genetically encoded impairment of KCC2 functional regulation may be a risk factor for the development of human IGE.

Evaluation of a Cannabis Harm Reduction Intervention for People With First-Episode Psychosis: Protocol for a Pilot Multicentric Randomized Trial.

BACKGROUND: Cannabis use is highly prevalent in young people with first-episode psychosis (FEP). Most report cannabis use and are often diagnosed with a cannabis use disorder upon admission to specialized services for psychosis. Cannabis use in this population is associated with worse clinical and psychosocial outcomes, rendering it an important clinical target. Despite this, few cannabis-specific interventions have been developed for FEP and empirically evaluated through randomized controlled trials. Most evaluated interventions have targeted cannabis abstinence, with limited efficacy, but none have centered on harm reduction outcomes for people with FEP who use cannabis. Early intervention services (EIS), the standard of care for FEP, have not successfully addressed problematic cannabis use in people with FEP either. Clinical trials are needed to explore the potential of harm reduction strategies, although these should be preceded by robust pilot studies to establish optimal design and approaches. OBJECTIVE: Recognizing the need for harm reduction strategies for individuals with FEP who use cannabis and based on research on patients' preferences supporting harm reduction interventions, we developed a mobile app-based cannabis harm reduction intervention for this population. This intervention is called Cannabis Harm-reducing Application to Manage Practices Safely (CHAMPS). Here, we describe the protocol for a multicenter, 2-arm, parallel group, randomized pilot trial evaluating the acceptability of CHAMPS for people with FEP who use cannabis and the feasibility of conducting a full-scale trial in this population using CHAMPS. The impact on key clinical outcomes will also be explored. METHODS: This pilot trial aims to recruit 100 young people with FEP using cannabis from 6 Canadian EIS clinics. Participants will be randomized in a 1:1 ratio to CHAMPS+EIS or EIS-only. CHAMPS acceptability will be assessed using completion rates for the intervention arm. Trial feasibility will be assessed using a retention rate for randomized participants. Secondary outcomes will explore tendencies of change in the use of protective behavioral strategies and in motivation to change strategies. Exploratory outcomes include cannabis use-related problems, other substance use, the severity of dependence, psychotic symptoms, and health care service use. RESULTS: Recruitment began in December 2021. Data collection and analysis are expected to be completed in early 2024. Study results describing CHAMPS acceptability and trial feasibility will then be submitted for publication in a peer-reviewed journal. CONCLUSIONS: CHAMPS uniquely combines evidence-based approaches, patient perspectives, and mobile health technology to support harm reduction in people with FEP who use cannabis. Attaining adequate acceptability and feasibility through this trial may justify further exploration of harm reduction tools, particularly within the context of conducting a larger-scale randomized controlled trial. This pilot trial has the potential to advance knowledge for researchers and clinicians regarding a feasible and user-acceptable research design in the cannabis and early psychosis fields. TRIAL REGISTRATION: ClinicalTrials.gov NCT04968275, https://clinicaltrials.gov/ct2/show/NCT04968275. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/53094.

Reducing Cannabis Use in Young Adults With Psychosis Using iCanChange, a Mobile Health App: Protocol for a Pilot Randomized Controlled Trial (ReCAP-iCC).

BACKGROUND: Cannabis use is the most prevalent among adolescents and young adults; frequent consumption is associated with cannabis use disorder (CUD) and psychosis, with a high prevalence (up to 50%) of CUD in individuals with first-episode psychosis (FEP). Early Intervention Services (EIS) for psychosis include face-to-face psychosocial interventions for CUD, because reducing or discontinuing cannabis use improves clinical and health care service use outcomes. However, multiple barriers (eg, staff availability and limited access to treatment) can hinder the implementation of these interventions. Mobile health (mHealth) interventions may help circumvent some of these barriers; however, to date, no study has evaluated the effects of mHealth psychological interventions for CUD in individuals with FEP. OBJECTIVE: This study describes the protocol for a pilot randomized controlled trial using a novel mHealth psychological intervention (iCanChange [iCC]) to address CUD in young adults with FEP. iCC was developed based on clinical evidence showing that in individuals without psychosis, integrating the principles of cognitive behavioral therapy, motivational interviewing, and behavioral self-management approaches are effective in improving cannabis use-related outcomes. METHODS: Consenting individuals (n=100) meeting the inclusion criteria (eg, aged 18-35 years with FEP and CUD) will be randomly allocated in a 1:1 ratio to the intervention (iCC+modified EIS) or control (EIS) group. The iCC is fully automatized and contains 21 modules that are completed over a 12-week period and 3 booster modules available during the 3-month follow-up period. Validated self-report measures will be taken via in-person assessments at baseline and at 6, 12 (end point), and 24 weeks (end of trial); iCC use data will be collected directly from the mobile app. Primary outcomes are intervention completion and trial retention rates, and secondary outcomes are cannabis use quantity, participant satisfaction, app use, and trial recruiting parameters. Exploratory outcomes include severity of psychotic symptoms and CUD severity. For primary outcomes, we will use the chi-square test using data collected at week 12. We will consider participation in iCC acceptable if ≥50% of the participants complete at least 11 out of 21 intervention modules and the trial feasible if attrition does not reach 50%. We will use analysis of covariance and mixed-effects models for secondary outcomes and generalized estimating equation multivariable analyses for exploratory outcomes. RESULTS: Recruitment began in July 2022, and data collection is anticipated to be completed in July 2024. The main results are expected to be submitted for publication in 2024. We will engage patient partners and other stakeholders in creating a multifaceted knowledge translation plan to reach a diverse audience. CONCLUSIONS: If feasible, this study will provide essential data for a larger-scale efficacy trial of iCC on cannabis use outcomes in individuals with FEP and CUD. TRIAL REGISTRATION: ClinicalTrials.gov NCT05310981; https://www.clinicaltrials.gov/ct2/show/NCT05310981. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/40817.

Novel α1 and γ2 GABAA receptor subunit mutations in families with idiopathic generalized epilepsy.

Epilepsy is a heterogeneous neurological disease affecting approximately 50 million people worldwide. Genetic factors play an important role in both the onset and severity of the condition, with mutations in several ion-channel genes being implicated, including those encoding the GABA(A) receptor. Here, we evaluated the frequency of additional mutations in the GABA(A) receptor by direct sequencing of the complete open reading frame of the GABRA1 and GABRG2 genes from a cohort of French Canadian families with idiopathic generalized epilepsy (IGE). Using this approach, we have identified three novel mutations that were absent in over 400 control chromosomes. In GABRA1, two mutations were found, with the first being a 25-bp insertion that was associated with intron retention (i.e. K353delins18X) and the second corresponding to a single point mutation that replaced the aspartate 219 residue with an asparagine (i.e. D219N). Electrophysiological analysis revealed that K353delins18X and D219N altered GABA(A) receptor function by reducing the total surface expression of mature protein and/or by curtailing neurotransmitter effectiveness. Both defects would be expected to have a detrimental effect on inhibitory control of neuronal circuits. In contrast, the single point mutation identified in the GABRG2 gene, namely P83S, was indistinguishable from the wildtype subunit in terms of surface expression and functionality. This finding was all the more intriguing as the mutation exhibited a high degree of penetrance in three generations of one French Canadian family. Further experimentation will be required to understand how this mutation contributes to the occurrence of IGE in these individuals.

Screening of GABRB3 in French-Canadian families with idiopathic generalized epilepsy.

Mutations in the GABRB3 have been recently associated with childhood absence epilepsy (CAE) in families from Honduras and Mexico. In this study, we aimed to determine the frequency of mutation in this gene in our cohort of families with CAE and other related idiopathic generalized epilepsy (IGE) syndromes. We screened the open reading frame of GABRB3 in 183 French-Canadian individuals with IGE, including 88 with CAE. A total of nine single nucleotide polymorphisms (SNPs) have been identified,five of which are novel. The previously described P11S missense mutation was found in three affected and one unaffected individuals from a French-Canadian family. However, the P11S variant was also found in one of our 190 control individuals of French-Canadian origin, suggesting that this variant is rather a rare polymorphism in this population. Further screening of other IGE cohorts from various ethnic origins would help to confirm the association between this rare functional variant and epilepsy.

Rare coding variants in genes encoding GABAA receptors in genetic generalised epilepsies: an exome-based case-control study.

BACKGROUND: Genetic generalised epilepsy is the most common type of inherited epilepsy. Despite a high concordance rate of 80% in monozygotic twins, the genetic background is still poorly understood. We aimed to investigate the burden of rare genetic variants in genetic generalised epilepsy. METHODS: For this exome-based case-control study, we used three different genetic generalised epilepsy case cohorts and three independent control cohorts, all of European descent. Cases included in the study were clinically evaluated for genetic generalised epilepsy. Whole-exome sequencing was done for the discovery case cohort, a validation case cohort, and two independent control cohorts. The replication case cohort underwent targeted next-generation sequencing of the 19 known genes encoding subunits of GABAA receptors and was compared to the respective GABAA receptor variants of a third independent control cohort. Functional investigations were done with automated two-microelectrode voltage clamping in Xenopus laevis oocytes. FINDINGS: Statistical comparison of 152 familial index cases with genetic generalised epilepsy in the discovery cohort to 549 ethnically matched controls suggested an enrichment of rare missense (Nonsyn) variants in the ensemble of 19 genes encoding GABAA receptors in cases (odds ratio [OR] 2·40 [95% CI 1·41-4·10]; pNonsyn=0·0014, adjusted pNonsyn=0·019). Enrichment for these genes was validated in a whole-exome sequencing cohort of 357 sporadic and familial genetic generalised epilepsy cases and 1485 independent controls (OR 1·46 [95% CI 1·05-2·03]; pNonsyn=0·0081, adjusted pNonsyn=0·016). Comparison of genes encoding GABAA receptors in the independent replication cohort of 583 familial and sporadic genetic generalised epilepsy index cases, based on candidate-gene panel sequencing, with a third independent control cohort of 635 controls confirmed the overall enrichment of rare missense variants for 15 GABAA receptor genes in cases compared with controls (OR 1·46 [95% CI 1·02-2·08]; pNonsyn=0·013, adjusted pNonsyn=0·027). Functional studies for two selected genes (GABRB2 and GABRA5) showed significant loss-of-function effects with reduced current amplitudes in four of seven tested variants compared with wild-type receptors. INTERPRETATION: Functionally relevant variants in genes encoding GABAA receptor subunits constitute a significant risk factor for genetic generalised epilepsy. Examination of the role of specific gene groups and pathways can disentangle the complex genetic architecture of genetic generalised epilepsy. FUNDING: EuroEPINOMICS (European Science Foundation through national funding organisations), Epicure and EpiPGX (Sixth Framework Programme and Seventh Framework Programme of the European Commission), Research Unit FOR2715 (German Research Foundation and Luxembourg National Research Fund).

Mutation burden of rare variants in schizophrenia candidate genes.

BACKGROUND: Schizophrenia (SCZ) is a very heterogeneous disease that affects approximately 1% of the general population. Recently, the genetic complexity thought to underlie this condition was further supported by three independent studies that identified an increased number of damaging de novo mutations DNM in different SCZ probands. While these three reports support the implication of DNM in the pathogenesis of SCZ, the absence of overlap in the genes identified suggests that the number of genes involved in SCZ is likely to be very large; a notion that has been supported by the moderate success of Genome-Wide Association Studies (GWAS). METHODS: To further examine the genetic heterogeneity of this disease, we resequenced 62 genes that were found to have a DNM in SCZ patients, and 40 genes that encode for proteins known to interact with the products of the genes with DNM, in a cohort of 235 SCZ cases and 233 controls. RESULTS: We found an enrichment of private nonsense mutations amongst schizophrenia patients. Using a kernel association method, we were able to assess for association for different sets. Although our power of detection was limited, we observed an increased mutation burden in the genes that have DNM.

Single-cell genetic expression of mutant GABAA receptors causing Human genetic epilepsy alters dendritic spine and GABAergic bouton formation in a mutation-specific manner.

Mutations in genes encoding for GABAA receptor subunits is a well-established cause of genetic generalized epilepsy. GABA neurotransmission is implicated in several developmental processes including neurite outgrowth and synapse formation. Alteration in excitatory/inhibitory synaptic activities plays a critical role in epilepsy, thus here we investigated whether mutations in α1 subunit of GABAA receptor may affect dendritic spine and GABAergic bouton formation. In particular, we examined the effects of three mutations of the GABRA1 gene (D219N, A322D and K353delins18X) that were found in a cohort of French Canadian families with genetic generalized epilepsy. We used a novel single-cell genetic approach, by preparing cortical organotypic cultures from GABRA1 (flox/flox) mice and simultaneously inactivating endogenous GABRA1 and transfecting mutant α1 subunits in single glutamatergic pyramidal cells and basket GABAergic interneurons by biolistic transfection. We found that GABRA1 (-/-) GABAergic cells showed reduced innervation field, which was rescued by co-expressing α1-A322D and α1-WT but not α1-D219N. We further found that the expression of the most severe GABRA1 missense mutation (α1-A322D) induced a striking increase of spine density in pyramidal cells along with an increase in the number of mushroom-like spines. In addition, α1-A322D expression in GABAergic cells slightly increased perisomatic bouton density, whereas other mutations did not alter bouton formation. All together, these results suggest that the effects of different GABAAR mutations on GABAergic bouton and dendritic spine formation are specific to the mutation and cannot be always explained by a simple loss-of-function gene model. The use of single cell genetic manipulation in organotypic cultures may provide a better understanding of the specific and distinct neural circuit alterations caused by different GABAA receptor subunit mutations and will help define the pathophysiology of genetic generalized epilepsy syndromes.