Somatic variant analysis of resected brain tissue in epilepsy surgery patients.

We studied the distribution of germline and somatic variants in epilepsy surgery patients with (suspected) malformations of cortical development (MCD) who underwent surgery between 2015 and 2020 at University Medical Center Utrecht (the Netherlands) and pooled our data with four previously published cohort studies. Tissue analysis yielded a pathogenic variant in 203 of 663 (31%) combined cases. In 126 of 379 (33%) focal cortical dysplasia (FCD) type II cases and 23 of 37 (62%) hemimegalencephaly cases, a pathogenic variant was identified, mostly involving the mTOR signaling pathway. Pathogenic variants in 10 focal epilepsy genes were found in 48 of 178 (27%) FCDI/mild MCD/mMCD with oligodendroglial hyperplasia and epilepsy cases; 36 of these (75%) were SLC35A2 variants. Six of 69 (9%) patients without a histopathological lesion had a pathogenic variant in SLC35A2 (n = 5) or DEPDC5 (n = 1). A germline variant in blood DNA was confirmed in all cases with a pathogenic variant in tissue, with a variant allele frequency (VAF) of ~50%. In seven of 114 patients (6%) with a somatic variant in tissue, mosaicism in blood was detected. More than half of pathogenic somatic variants had a VAF < 5%. Further analysis of the correlation between genetic variants and surgical outcomes will improve patient counseling and may guide postoperative treatment decisions.

mTOR pathway: Insights into an established pathway for brain mosaicism in epilepsy.

The mechanistic target of rapamycin (mTOR) signaling pathway is an essential regulator of numerous cellular activities such as metabolism, growth, proliferation, and survival. The mTOR cascade recently emerged as a critical player in the pathogenesis of focal epilepsies and cortical malformations. The 'mTORopathies' comprise a spectrum of cortical malformations that range from whole brain (megalencephaly) and hemispheric (hemimegalencephaly) abnormalities to focal abnormalities, such as focal cortical dysplasia type II (FCDII), which manifest with drug-resistant epilepsies. The spectrum of cortical dysplasia results from somatic brain mutations in the mTOR pathway activators AKT3, MTOR, PIK3CA, and RHEB and from germline and somatic mutations in mTOR pathway repressors, DEPDC5, NPRL2, NPRL3, TSC1 and TSC2. The mTORopathies are characterized by excessive mTOR pathway activation, leading to a broad range of structural and functional impairments. Here, we provide a comprehensive literature review of somatic mTOR-activating mutations linked to epilepsy and cortical malformations in 292 patients and discuss the perspectives of targeted therapeutics for personalized medicine.

Defective lipid signalling caused by mutations in PIK3C2B underlies focal epilepsy.

Epilepsy is one of the most frequent neurological diseases, with focal epilepsy accounting for the largest number of cases. The genetic alterations involved in focal epilepsy are far from being fully elucidated. Here, we show that defective lipid signalling caused by heterozygous ultra-rare variants in PIK3C2B, encoding for the class II phosphatidylinositol 3-kinase PI3K-C2ß, underlie focal epilepsy in humans. We demonstrate that patients' variants act as loss-of-function alleles, leading to impaired synthesis of the rare signalling lipid phosphatidylinositol 3,4-bisphosphate, resulting in mTORC1 hyperactivation. In vivo, mutant Pik3c2b alleles caused dose-dependent neuronal hyperexcitability and increased seizure susceptibility, indicating haploinsufficiency as a key driver of disease. Moreover, acute mTORC1 inhibition in mutant mice prevented experimentally induced seizures, providing a potential therapeutic option for a selective group of patients with focal epilepsy. Our findings reveal an unexpected role for class II PI3K-mediated lipid signalling in regulating mTORC1-dependent neuronal excitability in mice and humans.

Detection of Brain Somatic Mutations in Cerebrospinal Fluid from Refractory Epilepsy Patients.

Brain mosaic mutations are a major cause of refractory focal epilepsies with cortical malformations such as focal cortical dysplasia, hemimegalencephaly, malformation of cortical development with oligodendroglial hyperplasia in epilepsy, and ganglioglioma. Here, we collected cerebrospinal fluid (CSF) during epilepsy surgery to search for somatic variants in cell-free DNA (cfDNA) using targeted droplet digital polymerase chain reaction. In 3 of 12 epileptic patients with known somatic mutations previously identified in brain tissue, we here provide evidence that brain mosaicism can be detected in the CSF-derived cfDNA. These findings suggest future opportunities for detecting the mutant allele driving epilepsy in CSF. ANN NEUROL 2021;89:1248-1252.

Cortical Dysplasia and the mTOR Pathway: How the Study of Human Brain Tissue Has Led to Insights into Epileptogenesis.

Type II focal cortical dysplasia (FCD) is a neuropathological entity characterised by cortical dyslamination with the presence of dysmorphic neurons only (FCDIIA) or the presence of both dysmorphic neurons and balloon cells (FCDIIB). The year 2021 marks the 50th anniversary of the recognition of FCD as a cause of drug resistant epilepsy, and it is now the most common reason for epilepsy surgery. The causes of FCD remained unknown until relatively recently. The study of resected human FCD tissue using novel genomic technologies has led to remarkable advances in understanding the genetic basis of FCD. Mechanistic parallels have emerged between these non-neoplastic lesions and neoplastic disorders of cell growth and differentiation, especially through perturbations of the mammalian target of rapamycin (mTOR) signalling pathway. This narrative review presents the advances through which the aetiology of FCDII has been elucidated in chronological order, from recognition of an association between FCD and the mTOR pathway to the identification of somatic mosaicism within FCD tissue. We discuss the role of a two-hit mechanism, highlight current challenges and future directions in detecting somatic mosaicism in brain and discuss how knowledge of FCD may inform novel precision treatments of these focal epileptogenic malformations of human cortical development.

Toward a better definition of focal cortical dysplasia: An iterative histopathological and genetic agreement trial.

OBJECTIVE: Focal cortical dysplasia (FCD) is a major cause of difficult-to-treat epilepsy in children and young adults, and the diagnosis is currently based on microscopic review of surgical brain tissue using the International League Against Epilepsy classification scheme of 2011. We developed an iterative histopathological agreement trial with genetic testing to identify areas of diagnostic challenges in this widely used classification scheme. METHODS: Four web-based digital pathology trials were completed by 20 neuropathologists from 15 countries using a consecutive series of 196 surgical tissue blocks obtained from 22 epilepsy patients at a single center. Five independent genetic laboratories performed screening or validation sequencing of FCD-relevant genes in paired brain and blood samples from the same 22 epilepsy patients. RESULTS: Histopathology agreement based solely on hematoxylin and eosin stainings was low in Round 1, and gradually increased by adding a panel of immunostainings in Round 2 and the Delphi consensus method in Round 3. Interobserver agreement was good in Round 4 (kappa = .65), when the results of genetic tests were disclosed, namely, MTOR, AKT3, and SLC35A2 brain somatic mutations in five cases and germline mutations in DEPDC5 and NPRL3 in two cases. SIGNIFICANCE: The diagnoses of FCD 1 and 3 subtypes remained most challenging and were often difficult to differentiate from a normal homotypic or heterotypic cortical architecture. Immunohistochemistry was helpful, however, to confirm the diagnosis of FCD or no lesion. We observed a genotype-phenotype association for brain somatic mutations in SLC35A2 in two cases with mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy. Our results suggest that the current FCD classification should recognize a panel of immunohistochemical stainings for a better histopathological workup and definition of FCD subtypes. We also propose adding the level of genetic findings to obtain a comprehensive, reliable, and integrative genotype-phenotype diagnosis in the near future.

The landscape of epilepsy-related GATOR1 variants.

PURPOSE: To define the phenotypic and mutational spectrum of epilepsies related to DEPDC5, NPRL2 and NPRL3 genes encoding the GATOR1 complex, a negative regulator of the mTORC1 pathway METHODS: We analyzed clinical and genetic data of 73 novel probands (familial and sporadic) with epilepsy-related variants in GATOR1-encoding genes and proposed new guidelines for clinical interpretation of GATOR1 variants. RESULTS: The GATOR1 seizure phenotype consisted mostly in focal seizures (e.g., hypermotor or frontal lobe seizures in 50%), with a mean age at onset of 4.4 years, often sleep-related and drug-resistant (54%), and associated with focal cortical dysplasia (20%). Infantile spasms were reported in 10% of the probands. Sudden unexpected death in epilepsy (SUDEP) occurred in 10% of the families. Novel classification framework of all 140 epilepsy-related GATOR1 variants (including the variants of this study) revealed that 68% are loss-of-function pathogenic, 14% are likely pathogenic, 15% are variants of uncertain significance and 3% are likely benign. CONCLUSION: Our data emphasize the increasingly important role of GATOR1 genes in the pathogenesis of focal epilepsies (>180 probands to date). The GATOR1 phenotypic spectrum ranges from sporadic early-onset epilepsies with cognitive impairment comorbidities to familial focal epilepsies, and SUDEP.

Correction to: The landscape of epilepsy-related GATOR1 variants.

The original version of this Article contained an error in the author list where the corresponding author Stéphanie Baulac was repeated twice. This has now been corrected in the HTML, the PDF was correct at the time of publication.

Correction: The landscape of epilepsy-related GATOR1 variants.

The original version of this article contained an error in the spelling of the author Erik H. Niks, which was incorrectly given as Erik Niks. This has now been corrected in both the PDF and HTML versions of the article.

Dissecting the genetic basis of focal cortical dysplasia: a large cohort study.

Genetic malformations of cortical development (MCDs), such as mild MCDs (mMCD), focal cortical dysplasia (FCD), and hemimegalencephaly (HME), are major causes of severe pediatric refractory epilepsies subjected to neurosurgery. FCD2 are characterized by neuropathological hallmarks that include enlarged dysmorphic neurons (DNs) and balloon cells (BCs). Here, we provide a comprehensive assessment of the contribution of germline and somatic variants in a large cohort of surgical MCD cases. We enrolled in a monocentric study 80 children with drug-resistant epilepsy and a postsurgical neuropathological diagnosis of mMCD, FCD1, FCD2, or HME. We performed targeted gene sequencing ( ≥ 2000X read depth) on matched blood-brain samples to search for low-allele frequency variants in mTOR pathway and FCD genes. We were able to elucidate 29% of mMCD/FCD1 patients and 63% of FCD2/HME patients. Somatic loss-of-function variants in the N-glycosylation pathway-associated SLC35A2 gene were found in mMCD/FCD1 cases. Somatic gain-of-function variants in MTOR and its activators (AKT3, PIK3CA, RHEB), as well as germline, somatic and two-hit loss-of-function variants in its repressors (DEPDC5, TSC1, TSC2) were found exclusively in FCD2/HME cases. We show that panel-negative FCD2 cases display strong pS6-immunostaining, stressing that all FCD2 are mTORopathies. Analysis of microdissected cells demonstrated that DNs and BCs carry the pathogenic variants. We further observed a correlation between the density of pathological cells and the variant-detection likelihood. Single-cell microdissection followed by sequencing of enriched pools of DNs unveiled a somatic second-hit loss-of-heterozygosity in a DEPDC5 germline case. In conclusion, this study indicates that mMCD/FCD1 and FCD2/HME are two distinct genetic entities: while all FCD2/HME are mosaic mTORopathies, mMCD/FCD1 are not caused by mTOR-pathway-hyperactivating variants, and ~ 30% of the cases are related to glycosylation defects. We provide a framework for efficient genetic testing in FCD/HME, linking neuropathology to genetic findings and emphasizing the usefulness of molecular evaluation in the pediatric epileptic neurosurgical population.

Mutations in the mammalian target of rapamycin pathway regulators NPRL2 and NPRL3 cause focal epilepsy.

OBJECTIVE: Focal epilepsies are the most common form observed and have not generally been considered to be genetic in origin. Recently, we identified mutations in DEPDC5 as a cause of familial focal epilepsy. In this study, we investigated whether mutations in the mammalian target of rapamycin (mTOR) regulators, NPRL2 and NPRL3, also contribute to cases of focal epilepsy. METHODS: We used targeted capture and next-generation sequencing to analyze 404 unrelated probands with focal epilepsy. We performed exome sequencing on two families with multiple members affected with focal epilepsy and linkage analysis on one of these. RESULTS: In our cohort of 404 unrelated focal epilepsy patients, we identified five mutations in NPRL2 and five in NPRL3. Exome sequencing analysis of two families with focal epilepsy identified NPRL2 and NPRL3 as the top candidate-causative genes. Some patients had focal epilepsy associated with brain malformations. We also identified 18 new mutations in DEPDC5. INTERPRETATION: We have identified NPRL2 and NPRL3 as two new focal epilepsy genes that also play a role in the mTOR-signaling pathway. Our findings show that mutations in GATOR1 complex genes are the most significant cause of familial focal epilepsy identified to date, including cases with brain malformations. It is possible that deregulation of cellular growth control plays a more important role in epilepsy than is currently recognized.

GATOR1 complex: the common genetic actor in focal epilepsies.

The mammalian or mechanistic target of rapamycin (mTOR) signalling pathway has multiple roles in regulating physiology of the whole body and, particularly, the brain. Deregulation of mTOR signalling has been associated to various neurological conditions, including epilepsy. Mutations in genes encoding components of Gap Activity TOward Rags 1 (GATOR1) (DEPDC5, NPRL2 and NPRL3), a complex involved in the inhibition of the mTOR complex 1 (mTORC1), have been recently implicated in the pathogenesis of a wide spectrum of focal epilepsies (FEs), both lesional and non-lesional. The involvement of DEPDC5, NPRL2 and NRPL3 in about 10% of FEs is in contrast to the concept that specific seizure semiology points to the main involvement of a distinct brain area. The hypothesised pathogenic mechanism underlying epilepsy is the loss of the inhibitory function of GATOR1 towards mTORC1. The identification of the correct therapeutic strategy in patients with FE is challenging, especially in those with refractory epilepsy and/or malformations of cortical development (MCDs). In such cases, surgical excision of the epileptogenic zone is a curative option, although the long-term outcome is still undefined. The GATOR1/mTOR signalling represents a promising therapeutic target in FEs due to mutations in mTOR pathway genes, as in tuberous sclerosis complex, another MCD-associated epilepsy caused by mTOR signalling hyperactivation.

Development and characterization of a mucoadhesive sublingual formulation for pain control: extemporaneous oxycodone films in personalized therapy.

OBJECTIVE: The aim of this work was the development of mucoadhesive sublingual films, prepared using a casting method, for the administration of oxycodone. MATERIALS AND METHODS: A solvent casting method was employed to prepare the mucoadhesive films. A calibrated pipette was used to deposit single aliquots of different polymeric solutions on a polystyrene plate lid. Among the various tested polymers, hydroxypropylcellulose at low and medium molecular weight (HPC) and pectin at two different degrees of esterification (PC) were chosen for preparing solutions with good casting properties, capable of producing films suitable for mucosal application. RESULTS AND DISCUSSION: The obtained films showed excellent drug content uniformity and stability and rapid drug release, which, at 8 min, ranged from 60% to 80%. All films presented satisfactory mucoadhesive and mechanical properties, also confirmed by a test on healthy volunteers, who did not experience irritation or mucosa damages. Pectin films based on pectin at lower degrees of esterification have been further evaluated to study the influence of two different amounts of drug on the physicochemical properties of the formulation. A slight reduction in elasticity has been observed in films containing a higher drug dose; nevertheless, the formulation maintained satisfactory flexibility and resistance to elongation. CONCLUSIONS: HPC and PC sublingual films, obtained by a simple casting method, could be proposed to realize personalized hospital pharmacy preparations on a small scale.

Identity by descent fine mapping of familial adult myoclonus epilepsy (FAME) to 2p11.2-2q11.2.

Familial adult myoclonus epilepsy (FAME) is a rare autosomal dominant disorder characterized by adult onset, involuntary muscle jerks, cortical myoclonus and occasional seizures. FAME is genetically heterogeneous with more than 70 families reported worldwide and five potential disease loci. The efforts to identify potential causal variants have been unsuccessful in all but three families. To date, linkage analysis has been the main approach to find and narrow FAME critical regions. We propose an alternative method, pedigree free identity-by-descent (IBD) mapping, that infers regions of the genome between individuals that have been inherited from a common ancestor. IBD mapping provides an alternative to linkage analysis in the presence of allelic and locus heterogeneity by detecting clusters of individuals who share a common allele. Succeeding IBD mapping, gene prioritization based on gene co-expression analysis can be used to identify the most promising candidate genes. We performed an IBD analysis using high-density single nucleotide polymorphism (SNP) array data followed by gene prioritization on a FAME cohort of ten European families and one Australian/New Zealander family; eight of which had known disease loci. By identifying IBD regions common to multiple families, we were able to narrow the FAME2 locus to a 9.78 megabase interval within 2p11.2-q11.2. We provide additional evidence of a founder effect in four Italian families and allelic heterogeneity with at least four distinct founders responsible for FAME at the FAME2 locus. In addition, we suggest candidate disease genes using gene prioritization based on gene co-expression analysis.

Autosomal dominant partial epilepsy with auditory features: a new locus on chromosome 19q13.11-q13.31.

OBJECTIVE: To clinically and genetically characterize a large Brazilian family with autosomal dominant partial epilepsy with auditory features (ADPEAF) not related to leucine-rich, glioma-inactivated 1 (LGI1) gene. METHODS: Seventy family members (four married-ins) participating in the study were assessed by a detailed clinical interview and a complete neurologic examination. Genetic mapping was conducted through autosome-wide single nucleotide polymorphism (SNP) genotyping and subsequent linkage analysis on 16 and haplotype analysis on 25 subjects, respectively. RESULTS: The pedigree comprised 15 affected members, of whom 11 were included in the study (male/female: 6/5; mean age 39.5 years). All but two (III:22 and IV:92) had focal seizures with auditory aura followed by secondary generalization in 44.4%. The mean age at onset of epilepsy seizures was 13.7 years. Initial autosome-wide SNP linkage analysis conducted on 12 subjects (8 affected) pointed to a single genomic region on chromosome 19 with a maximum multipoint logarithm of the odds (LOD) score of 2.60. Further refinement of this region through SNP and microsatellite genotyping on 16 subjects (11 affected) increased the LOD score to 3.41, thereby establishing 19q13.11-q13.31 as a novel ADPEAF locus. Haplotype analysis indicated that the underlying mutation is most likely located in a 9.74 Mb interval between markers D19S416 and D19S420. Sequence analysis of the most prominent candidate genes within this critical interval (SCN1B, LGI4, KCNK6, and LRFN1) did not reveal any mutation. SIGNIFICANCE: This study disclosed a novel ADPEAF locus on chromosome 19q13.11-q13.31, contributing to future identification of a second dominant gene for this epileptic syndrome. A PowerPoint slide summarizing this article is available for download in the Supporting Information section here.

Unveiling Niaprazine's Potential: Behavioral Insights into a Re-Emerging Anxiolytic Agent.

Ongoing global research actions seek to comprehensively understand the adverse impact of stress and anxiety on the physical and mental health of both human beings and animals. Niaprazine (NIA) is a chemical compound that belongs to the class of piperazine derivatives. This compound has recently gained renewed attention due to its potential therapeutic properties for treating certain conditions such as anxiety. Despite its potential benefits, the behavioral effects of NIA have not been thoroughly investigated. This study aimed to examine NIA's potential as an anti-anxiety and anti-stress agent. After administering either vehicle or NIA in their drinking water to mice for 14 days, we conducted behavioral analyses using the Marble Burying Test and the Elevated Plus Maze test. NIA-treated mice spend more time in the open arms and bury fewer marbles. Moreover, a stability study confirmed the linear relationship between NIA concentration and its response across concentrations encompassing the NIA mother solution and the NIA solutions administered to mice. Also, a preliminary synaptic toxicity analysis showed no direct damage to cortical nerve endings. Here, we show that NIA can modulate anxiety-related behaviors without significantly impacting exploratory activity or adverse effects. Our work describes new findings that contribute to the research on safer and more tolerable anxiety management options.

Development of Biotinylated Liposomes Encapsulating Metformin for Therapeutic Targeting of Inflammation-Based Diseases.

Inflammation is a physiological response to a damaging stimulus but sometimes can be the cause of the onset of neurodegenerative diseases, atherosclerosis, and cancer. These pathologies are characterized by the overexpression of inflammatory markers like endothelial adhesion molecules, such as Vascular Cell Adhesion Molecule-1 (VCAM-1). In the present work, the development of liposomes for therapeutic targeted delivery to inflamed endothelia is described. The idea is to exploit a three-step pretargeting system based on the biotin-avidin high-affinity interaction: the first step involves a previously described biotin derivative bearing a VCAM-1 binding peptide; in the second step, the avidin derivative NeutrAvidinTM, which strongly binds to the biotin moiety, is injected; the final step is the administration of biotinylated liposomes that would bind to NeutravidinTM immobilized onto VCAM-1 overexpressing endothelium. Stealth biotinylated liposomes, prepared via the thin film hydration method followed by extrusion and purification via size exclusion chromatography, have been thoroughly characterized for their chemico-physical and morphological features and loaded with metformin hydrochloride, a potential anti-inflammatory agent. The three-step system, tested in vitro on different cell lines via confocal microscopy, FACS analysis and metformin uptake, has proved its suitability for therapeutic applications.

Development of a Multilayer Film Including the Soluble Eggshell Membrane Fraction for the Treatment of Oral Mucosa Lesions.

BACKGROUND/OBJECTIVES: Oral diseases causing mucosal lesions are normally treated with local or systemic anti-inflammatory, analgesic and antimicrobial agents. The development of topical formulations, including wound-healing promoters, might speed up the recovery process, improving patients' quality of life, and reduce the risk of deterioration in health conditions. In this study, a mucoadhesive multilayer film, including a novel biocompatible substance (solubilized eggshell membrane, SESM), was rationally designed. METHODS: The SESM preparation procedure was optimized and its biological effects on cell proliferation and inflammation marker gene expression were evaluated in vitro; preformulation studies were conducted to identify the most promising polymers with film-forming properties; then, trilayer films, consisting of an outer layer including chlorhexidine digluconate as a model drug, a supporting layer and a mucoadhesive layer, incorporating SESM, were prepared using the casting method and their mechanical, adhesion and drug release control properties were evaluated. RESULTS: SESM proved to possess a notable wound-healing capacity, inducing a wound closure of 84% in 24 h without inhibiting blood clotting. The films revealed a maximum detachment force from porcine mucosa of approx. 1.7 kPa and maximum in vivo residence time of approx. 200-240 min; finally, they released up to 98% of the loaded drug within 4 h. CONCLUSIONS: The formulated trilayer films were found to possess adequate properties, making them potentially suitable for protecting oral lesions and favoring their rapid healing, while releasing antimicrobial substances that might be beneficial in reducing the risk of bacterial infections.

A Novel Hydrogel Sponge for Three-Dimensional Cell Culture.

BACKGROUND/OBJECTIVES: Three-dimensional (3D) cell culture technologies allow us to overcome the constraints of two-dimensional methods in different fields like biochemistry and cell biology and in pharmaceutical in vitro tests. In this study, a novel 3D hydrogel sponge scaffold, composed of a crosslinked polyacrylic acid forming a porous matrix, has been developed and characterized. METHODS: The scaffold was obtained via an innovative procedure involving thermal treatment followed by a salt-leaching step on a matrix-containing polymer along with a gas-forming agent. Based on experimental design for mixtures, a series of formulations were prepared to study the effect of the three components (polyacrylic acid, NaHCO3 and NaCl) on the scaffold mechanical properties, density, swelling behavior and morphological changes. Physical appearance, surface morphology, porosity, molecular diffusion, transparency, biocompatibility and cytocompatibility were also evaluated. RESULTS: The hydrogel scaffolds obtained show high porosity and good optical transparency and mechanical resistance. The scaffolds were successfully employed to culture several cell lines for more than 20 days. CONCLUSIONS: The developed scaffolds could be an important tool, as such or with a specific coating, to obtain a more predictive cellular response to evaluate drugs in preclinical studies or for testing chemical compounds, biocides and cosmetics, thus reducing animal testing.