Pathogenic MTOR somatic variant causing focal cortical dysplasia drives hyperexcitability via overactivation of neuronal GluN2C N-methyl-D-aspartate receptors.

Post-zygotically acquired genetic variants, or somatic variants, that arise during cortical development have emerged as important causes of focal epilepsies, particularly those due to malformations of cortical development. Pathogenic somatic variants have been identified in many genes within the PI3K-AKT-mTOR-signalling pathway in individuals with hemimegalencephaly and focal cortical dysplasia (type II), and more recently in SLC35A2 in individuals with focal cortical dysplasia (type I) or non-dysplastic epileptic cortex. Given the expanding role of somatic variants across different brain malformations, we sought to delineate the landscape of somatic variants in a large cohort of patients who underwent epilepsy surgery with hemimegalencephaly or focal cortical dysplasia. We evaluated samples from 123 children with hemimegalencephaly (n = 16), focal cortical dysplasia type I and related phenotypes (n = 48), focal cortical dysplasia type II (n = 44), or focal cortical dysplasia type III (n = 15). We performed high-depth exome sequencing in brain tissue-derived DNA from each case and identified somatic single nucleotide, indel and large copy number variants. In 75% of individuals with hemimegalencephaly and 29% with focal cortical dysplasia type II, we identified pathogenic variants in PI3K-AKT-mTOR pathway genes. Four of 48 cases with focal cortical dysplasia type I (8%) had a likely pathogenic variant in SLC35A2. While no other gene had multiple disease-causing somatic variants across the focal cortical dysplasia type I cohort, four individuals in this group had a single pathogenic or likely pathogenic somatic variant in CASK, KRAS, NF1 and NIPBL, genes previously associated with neurodevelopmental disorders. No rare pathogenic or likely pathogenic somatic variants in any neurological disease genes like those identified in the focal cortical dysplasia type I cohort were found in 63 neurologically normal controls (P = 0.017), suggesting a role for these novel variants. We also identified a somatic loss-of-function variant in the known epilepsy gene, PCDH19, present in a small number of alleles in the dysplastic tissue from a female patient with focal cortical dysplasia IIIa with hippocampal sclerosis. In contrast to focal cortical dysplasia type II, neither focal cortical dysplasia type I nor III had somatic variants in genes that converge on a unifying biological pathway, suggesting greater genetic heterogeneity compared to type II. Importantly, we demonstrate that focal cortical dysplasia types I, II and III are associated with somatic gene variants across a broad range of genes, many associated with epilepsy in clinical syndromes caused by germline variants, as well as including some not previously associated with radiographically evident cortical brain malformations.

Background and ObjectivesSomatic and germline pathogenic variants in genes of the mammalian target of rapamycin (mTOR) signaling pathway are a common mechanism underlying a subset of focal malformations of cortical development (FMCDs) referred to as mTORopathies, which include focal cortical dysplasia (FCD) type II, subtypes of polymicrogyria, and hemimegalencephaly. Our objective is to screen resected FMCD specimens with mTORopathy features on histology for causal somatic variants in mTOR pathway genes, describe novel pathogenic variants, and examine the variant distribution in relation to neuroimaging, histopathologic classification, and clinical outcomes.MethodsWe performed ultra-deep sequencing using a custom HaloPlexHS Target Enrichment kit in DNA from 21 resected fresh-frozen histologically confirmed FCD type II, tuberous sclerosis complex, or hemimegalencephaly specimens. We mapped the variant alternative allele frequency (AAF) across the resected brain using targeted ultra-deep sequencing in multiple formalin-fixed paraffin-embedded tissue blocks. We also functionally validated 2 candidate somatic MTOR variants and performed targeted RNA sequencing to validate a splicing defect associated with a novel DEPDC5 variant.ResultsWe identified causal mTOR pathway gene variants in 66.7% (14/21) of patients, of which 13 were somatic with AAF ranging between 0.6% and 12.0%. Moreover, the AAF did not predict balloon cell presence. Favorable seizure outcomes were associated with genetically clear resection borders. Individuals in whom a causal somatic variant was undetected had excellent postsurgical outcomes. In addition, we demonstrate pathogenicity of the novel c.4373_4375dupATG and candidate c.7499T>A MTOR variants in vitro. We also identified a novel germline aberrant splice site variant in DEPDC5 (c.2802-1G>C).DiscussionThe AAF of somatic pathogenic variants correlated with the topographic distribution, histopathology, and postsurgical outcomes. Moreover, cortical regions with absent histologic FCD features had negligible or undetectable pathogenic variant loads. By contrast, specimens with frank histologic abnormalities had detectable pathogenic variant loads, which raises important questions as to whether there is a tolerable variant threshold and whether surgical margins should be clean, as performed in tumor resections. In addition, we describe 2 novel pathogenic variants, expanding the mTORopathy genetic spectrum. Although most pathogenic somatic variants are located at mutation hotspots, screening the full-coding gene sequence remains necessary in a subset of patients.

Focal cortical dysplasia (FCD) is a common cause of pharmacoresistant human epilepsy. FCD has frequently been discussed as a "forme fruste" of tuberous sclerosis complex (TSC) because of the radiologic and histologic resemblance of dysplastic areas to tubers in TSC. Mutations or a germ-line predisposition in terms of increased polymorphisms in the TSC genes have been presumed to influence the pathogenesis of FCD. A detailed genotype-phenotype analysis of these patients has not been performed so far. In this study, 33 patients with FCD (among them 23 with FCD type 2 and 4 patients with multifocal FCD) were investigated (1) clinically as to dermatologic manifestations, retinal hamartoma, cardial rhabdomyoma, and renal angiomyolipoma, and (2) genetically by considering lesional brain tissue and blood using single strand conformation polymorphism (SSCP) electrophoresis and sequencing of the TSC1 and TSC2 genes. In the clinical examinations, no subtle features of TSC could be detected in this large group of patients with FCD, pointing to the fact that this is a different patient group without clinical overlap. Several sequence alterations were found in the TSC1 and TSC2 genes in both lesional brain tissue and blood of FCD patients, however, in similar frequencies to that of the normal population. Moreover, most of these sequence alterations were silent. This study shows that FCD-even multifocal FCD-is not caused by mutations in the TSC genes and seems not to be promoted by polymorphisms in the TSC genes. Therefore, FCD cannot be regarded as a "forme fruste" of TSC.

To describe a child meeting diagnostic criteria for tuberous sclerosis complex (TSC) carrying a pathogenic somatic variant in RHEB, but no pathogenic variants in the 2 known TSC genes, TSC1 or TSC2. We present the clinical and imaging findings in a child presenting with drug-resistant focal seizures and multiple cortical tubers, a subependymal giant cell astrocytoma and multiple subependymal nodules in 1 cerebral hemisphere. Targeted panel sequencing and exome sequencing were performed on genomic DNA derived from blood and resected tuber tissue. The child satisfied clinical diagnostic criteria for TSC, having 3 major features, only 2 of which are required for diagnosis. Genetic testing did not identify pathogenic variants or copy number variations in TSC1 or TSC2 but identified a pathogenic somatic RHEB variant (NM_005614.4:c.104_105delACinsTA [p.Tyr35Leu]) in the cortical tuber. RHEB is a partner of the TSC1/2 complex in the mechanistic target of rapamycin pathway. Somatic variants in RHEB are associated with focal cortical dysplasia and hemimegalencephaly. We propose that variants in RHEB may explain some of the genetically undiagnosed TSC cases and may be the third gene for TSC, or TSC3.

Patients with epilepsy have not benefitted equally from the availability of next-generation sequencing. Although the diagnostic yield in epileptic encephalopathies is up to 50%, it is only approximately 12% in non-acquired focal epilepsies (NAFE).1 However, somatic mosaicism has emerged as an important cause of genetic causation in NAFE. This is hardly surprising, considering the scale of cellular divisions and differentiations during the human brain development that provides ample opportunities for the occurrence of brain-restricted somatic variation. For example, depending on the developmental timing and a progenitor lineage, pathogenic variants leading to mammalian target of rapamycin pathway activation result in a spectrum of brain malformations, such as focal cortical dysplasia (FCD) type 2 or hemimegalencephaly.2,3 Of interest, these developmental lesions seem to display a mutant allele gradient and a dose effect; approximately 13% of mutant alleles were observed in FCD type 2 compared with 40% in hemimegalencephaly, and the highest variant allele fraction seems to be in the epicenter of a lesion.3,4 Although research has elucidated mechanisms underlying some malformations of cortical development, molecular etiology of other FCD types has remained obscured. N-glycosylation defects linked to pathogenic variants in the Solute Carrier Family 35 Member A2 gene ( SLC35A2 ) have emerged as causal to an X-linked early onset epileptic encephalopathy typically manifested with infantile spasms or West syndrome in women or in mosaic men and most recently, they were also identified in approximately 30% of NAFE because of FCD type 1.5,6 SLC35A2 encodes a uridine diphosphate galactose transporter (UGT). A defect in the UGT results in a reduced galactosylation of N-glycosylated proteins, glycosphingolipids, and proteoglycans, which affects neuronal migration, axon guidance, and synaptic physiology.7 In this issue of Neurology ® Genetics , Miller et al.,8 attempted to explore the impact of SLC35A2 gene insufficiency on neuronal excitability. Evaluation of a 3-year-old boy with West syndrome and generalized spasms showed that the ictal electrographic correlates demonstrated a consistent emphasis over the left posterior head quadrant, a region further implicated by a subtle signal abnormality on brain MRI and a hypometabolism on a fluorodeoxyglucose positron emission tomography. Invasive monitoring with subdural grids and strips showed a varied density of interictal activity in the sampled areas and a broad ictal involvement of all subdural contacts. The patient underwent a multilobar resection, a neuropathologic examination confirmed FCD type 1c,2 and exome sequencing identified a somatic, brain tissue-restricted, previously reported pathogenic variant in the SLC35A2 gene (NM_005660.3:c.634-635delTC, p.Ser212LeufsTer9). The variant allele ranged from 0.6% to 27.7% in an exome and from 4.2% to 19.5% in a targeted variant validation across resected regions. The authors observed a correlation between interictal spike density and the pathogenic allele burden. The highest variant fraction (19.5%) and interictal spike density (32 spikes per minute) were in the hippocampal tissue, whereas the area of the inferior occipital gyrus showed the lowest spike frequency (5.7 spikes per minute) and a lesser variant allele proportion (4.2%–6.5%). This gene identification and validation study underscores the value for genetic diagnostic profiling of a resected brain tissue in patients with a nondiagnostic genetic testing of their peripheral samples. It adds to the growing number of cases where focal, genetically predisposed abnormalities manifest in generalized spasms. This lends some explanation for the surprisingly low (15%) yield of genetic testing of a peripheral DNA in this patient population.9 A report by Miller et al.8 further validates the SLC35A2 gene as causal in FCD type 1 and lends support to experimental data linking disordered N-glycosylation to FCD type 1 and neuronal hyperexcitability and seizures.10 It also attempts to link clinical markers of neuronal hyperexcitability to the burden of SLC35A2 variation. However, many questions remain; first, there is the currently unresolved relationship between a mutant variant load and the resulting phenotype. In a recent case series, Vals et al. failed to see a correlation between the phenotype severity and the estimated quantity of pathogenic SLC35A2 variants.5 Of interest in the current study, variant proportions did not correlate with the messenger RNA expression of the alternative allele, suggesting the possibility of a nonsense-mediated decay. Because the UGT protein was not assessed, relationship among the varied proportions of the alternative allele to UGT quantities or function remain unclear. In addition, the imaging suggested an extensive area of signal abnormality and one would assume a corresponding structural distortion of cortical architecture. Curiously, the interictal spiking was restricted to a few defined areas within much larger regions sampled in this study. Future experimental work will have to clarify the potential impact of different SLC35A2 pathogenic variants on neuronal structure and cellular and network function. The reported correlation among the spike frequency and the SLC35A2 pathogenic variant proportions is interesting but will need to be confirmed in a sufficiently large case series with well-defined ictal onset and extended sampling of ictal and interictal data. Future studies will also need to pay attention to the properties and densities of spikes and variants at the margins of the resected lesion and explain their relevance for the outcome. Although it is tempting to ponder the reported relationship among the pathogenic SLC35A2 variant proportions and the severity of imaging abnormalities, the absence of an unbiased quantification of the visualized lesion makes this correlation problematic to accept. Rather, it poses a research opportunity for the future. In summary, the case report by Miller et al.8 adds to the growing recognition of brain-restricted somatic mosaicism as causal to infantile spasms and NAFE. It provides additional evidence connecting the SLC35A2 gene to FCD type 1, thus opening opportunities to study glycosylation pathways in relationship to malformations of cortical development. It is also likely to stimulate future research linking SLC35A2 -related genetic defects with the development of structural lesions and their functional manifestations on cellular and network levels.

Infantile epileptic spasms syndrome is a severe epilepsy of infancy that is often associated with focal malformations of cortical development. This study aimed to elucidate the genetic landscape and histopathologic aetiologies of infantile epileptic spasms syndrome due to focal malformations of cortical development requiring surgery. Fifty-nine children with a history of infantile epileptic spasms syndrome and focal malformations of cortical development on MRI were studied. Genetic testing of resected brain tissue was performed by high-coverage targeted panel sequencing or exome sequencing. Histopathology and MRI were reviewed, and integrated clinico-pathological diagnoses were established. A genetic diagnosis was achieved in 47 children (80% of cohort). Germline pathogenic variants were identified in 27/59 (46%) children, in TSC2 (x19), DEPDC5 (x2), CDKL5 (x2), NPRL3 (x1), FGFR1 (x1), TSC1 (x1), and one child with both a TUBB2A/TUBB2B deletion and a pathogenic variant in COL4A1 (x1). Pathogenic brain somatic variants were identified in 21/59 (36%) children, in SLC35A2 (x9), PIK3CA (x3), AKT3 (x2), TSC2 (x2), MTOR (x2), OFD1 (x1), TSC1 (x1) and DEPDC5 (x1). One child had ‘two-hit’ diagnosis, with both germline and somatic pathogenic DEPDC5 variants in trans. Multimodal data integration resulted in clinical diagnostic reclassifications in 24% of children, emphasizing the importance of combining genetic, histopathologic and imaging findings. Mammalian target of rapamycin pathway variants were identified in most children with tuberous sclerosis or focal cortical dysplasia type II. All nine children with somatic SLC35A2 variants in brain were reclassified to mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy. Somatic mosaicism was a major cause of focal cortical dysplasia type II/hemimegalencephaly (81%) and mild malformation of cortical development with oligodendroglial hyperplasia (100%). The genetic landscape of infantile epileptic spasms syndrome due to focal malformations comprises germline and somatic variants in a range of genes, with mTORopathies and SLC35A2-related mild malformation of cortical development with oligodendroglial hyperplasia being the major causes. Multimodal data integration incorporating genetic data aids in optimizing diagnostic pathways and can guide surgical decision-making and inform future research and therapeutic interventions.

Objective To summarize MRI findings of focal cortical dysplasia (FCD), analyze MRI characteristics of various pathological subtypes of focal cortical dysplasia. Methods Forty-four patients with FCD were collected. Their MRI findings were analyzed retrospectively. According to pathologic findings, these patients were divided into FCD type Ⅰ group and FCD type Ⅱ group. The following MR signs were observed in the two types of FCD: ( 1 ) Focal thickening of the cortex. ( 2 ) Blurring of the gray matter-white matter junction. ( 3 ) Tapering of white matter signal intensity alteration toward the ventricle on FLAIR and on T2WI. (4)Focal brain hypoplasia. (5)Increased signal intensity of gray matter on FLAIR. (6)Increased signal intensity of gray matter on T2 WI. ( 7 ) Increased signal intensity of subcortical white matter on FLAIR.(8) Increased signal intensity of subeortical white matter on T2WI. (9) Decreased signal intensity of subcortical white matter on T1 WI. The χ2 tests and corrected χ2 tests were used for comparison between the two groups. Results In the 44 cases, there were 30 cases with FCD type Ⅰ and 14 cases with FCD type Ⅱ. FCD was identified by MRI in 32 cases. Blurring of the gray-white matter junction is the most common sign of FCD (23 cases). There were 21 cases identified by MRI in FCD type Ⅰ group. Focal brain hypoplasia is a typical sign of FCD type Ⅰ , which was found in 11 cases in FCD type Ⅰ group but none in FCD type Ⅱ group. There was statistically significant difference between the two groups (continuity corrected χ2 =5. 0286,P =0. 0249) . In FCD type Ⅱ group, 11 cases were identified by MRI. Increased cortical thickness was found in 10 eases in FCD type Ⅱ group and 11 cases in FCD type Ⅰ group ( χ2 =4. 6234 ,P =0. 0315). Increased signal intensity of subcortical white matter on FLAIR was found in 9 cases in FCD type Ⅱ group and 7 cases in FCD type Ⅰ group (χ2 =6.9180,P =0.0085). Tapering of white matter signal intensity alteration toward the ventricle was found in 4 cases in FCD type Ⅱ group and none in FCD type Ⅰ group ( continuity corrected χ2 = 6. 2883, P = 0. 0122). The above-mentioned three MRI findings showed statistically significant difference between the two groups and were features of FCD type Ⅱ.All of the other MRI findings showed no statistically significant difference between the two groups. Conclusions Different pathological subtypes of FCD have different MRI characteristics. It is helpful to make preoperative diagnosis and planning. Key words: Cerebral cortex; Nervous system abnormalities; Magnetic resonance imaging

Background: The Notch signaling pathway is an important regulator of stem cell activity in various tissues, including the central nervous system. It has been implicated in neurodevelopmental processes, including neuronal differentiation and synaptic plasticity. Research suggests that its expression may be associated with certain epileptogenic lesions, particularly those with neurodevelopmental origin. The aim of this study was to investigate the expression of Notch-1 in brain biopsies from various cases of pharmacoresistant epilepsy. Methods: Here, we used immunohistochemistry staining to retrospectively analyze 128 developmental lesions associated with pharmacoresistant epilepsy, including 13 cases with focal cortical dysplasia (FCD) type I, 39 with FCD type II, 37 with hippocampal sclerosis (HS), 23 with FCD IIIc, 9 with mild malformations of cortical development (MCD), 4 cases with mild malformation of cortical development with oligodendroglial hyperplasia and epilepsy (MOGHE), and 3 with tuberous sclerosis (TS). The tissues were stained for Neurofilament protein, Vimentin, S-100 protein, NeuN, and GFAP, as well as the stem cell marker Notch-1. Tissue that stained positively for Notch-1 was further characterized. Results: A positive Notch-1 reaction was found in all cases of FCD type IIb and TS, where it appeared in balloon cells but not in dysmorphic neurons, and in a single case of meningioangiomatosis (FCD IIIc), where it stained spider-like cells. Notch-1-positive cells showed a stem-like, glio-neuronal precursor immunophenotype. No staining was observed in the remaining cases with FCD type I, type III, HS, mild MCD, and MOGHE. Conclusions: Notch-1 displays a distinct pattern of expression in some epileptogenic lesions, potentially highlighting a stem cell-like origin or neurodevelopmental abnormalities contributing to pharmacoresistant epilepsy; however, it is not a general marker of such lesions. Its differential expression may prove useful in distinguishing between different types of FCD or other cortical malformations, which could assist in both their diagnosis and potentially in the development of more targeted therapeutic approaches. Further studies with different stem cell markers are needed in this direction.

Objective To analyze the MR imaging features of epileptogenic focal cortical dysplasia (FCD)and to optimize the scanning protocols by correlating MRI appearance with pathological findings.Methods MRI findings and the relative scanning protocols in 36 patients with surgically and pathologically proved 40 lesions of FCD were retrospectively analyzed. According to Palmini classification system,all 40 lesions were pathologically categorized as FCD type Ⅰ (including FCD Ⅰ A and FCD Ⅰ B) and FCD type Ⅱ (including FCD Ⅱ A and FCD ⅡB ). The distribution of cerebral or dual lesions accompanied hippocampal sclerosis were observed.Differences of the distribution of cerebral in FCD type Ⅰ and FCD type Ⅱ were compared by using Fisher exact probabilities. MR scans in all patients consisted of routine and optimized protocols.Axial FSE T2WI,axial SET1WI and axial FLAIR were recognized as routine scanning protocols,while adding oblique coronal FSE T2WI and FLAIR were recognized as optimization scanning protocols.Both routine and optimization scanning protocols were performed in all patients.The conspicuity of main findings of FCD on different imaging plane and sequences of each protocol were assessed.The detection of cerebral or dual lesion and the accompanied hippocampal sclerosis were compared between the routine protocol and the optimized protocol by using McNemar test.Results Forty lesions were found in 36 cases with FCD,29 had temporal lobe lesion (72.5％ ),9 had frontal lobe lesion(22.5％ ) and 2 had parietal lobe lesion (5.0％).According to Palmini classification system,29 lesions in 27 patients (72.5％ ) were FCD type Ⅰ,11 lesions in 10 patients (27.5％) were FCD type Ⅱ lesions.There were 25 temporal lobe lesions in FCD type Ⅰ,while 4 in FCD type Ⅱ.There were statistically significant differences between FCD type Ⅰ group and FCD type Ⅱ group in the distribution of cerebral (P =0.002 ).Fourteen cases were found to have hippocampal sclerosis simultaneously,with 13 cases found in FCD type Ⅰ patients and 1 case in type Ⅱ patients.The detection rate of temporal lobe lesion was 65.5 ％ (19/29) and 44.8 ％ ( 13/29 ) respectively on optimized protocol and routine protocol.There was statistically significant difference ( x2 =4.167,P =0.031 ).The detection rate of hippocampal sclerosis was 85.7％ (12/14) and 42.9％ (6/14) respectively on optimized protocol and routine protocol respectively.There was statistically significant difference ( x2 =4.167,P =0.031 ).The detection rate of frontal lobe lesion showed no statistically significant difference between optimized protocols and routine protocols (x2 =0.304,P =1.000 ).Conclusions FCD was frequently involved the temporal lobe,followed by the frontal lobe.FCD type Ⅰ lesion was frequently found in the temporal lobe,with a higher incidence of concomitant hippocampal sclerosis. The optimized whole temporal lobe scanning with imaging plane perpendicular to the hippocampus long axis was a highly desired scanning protocol specifically for FCD,which is helpful for the detection of the FCD lesions. Key words: Cerebral cortex; Nervous system abnormalities; Magnetic resonance imaging

Focal cortical dysplasia (FCD) type 2 is the most common malformation of cortical development associated with pharmaco-resistant focal epilepsy and frequently located in the frontal cortex. Neuropathological hallmarks comprise abnormal cortical layering and enlarged, dysmorphic neuronal elements. Fundamentally altered local neuronal activity has been reported in human FCD type 2 epilepsy surgical biopsies. Of note, FCD type 2 emerges during brain development and forms complex connectivity architectures with surrounding neuronal networks. Local cortical microcircuits, particularly in frontal localization, are extensively modulated by monoaminergic axonal projections originating from the brainstem. Previous analysis of monoaminergic modulatory inputs in human FCD type 2 biopsies suggested altered density and distribution of these monoaminergic axons; however, a systematic investigation is still pending.Here, we perform a comprehensive analysis of dopaminergic (DA) innervation, in human FCD type 2 biopsies and in the medial prefrontal cortex (mPFC) of an FCD type 2 mouse model [mechanistic target of rapamyin (mTOR) hyperactivation model] during adolescent and adult stages. In addition, we analyse the expression of dopamine receptor transcripts via multiplex fluorescent RNA in situ hybridization in human specimens and the mPFC of this mouse model.In the mTOR hyperactivation mouse model, we observe a transient alteration of DA innervation density during adolescence and a trend towards decreased innervation in adulthood. In human FCD type 2 areas, the overall DA innervation density is decreased in adult patients compared with control areas from these patients. Moreover, the DA innervation shows an altered lamination pattern in the FCD type 2 area compared with the control area. Dopamine receptors 1 and 2 appear to be differentially expressed in the dysmorphic neurons in human samples and mTOR-mutant cells in mice compared with normally developed neurons.Intriguingly, our results suggest complex molecular and structural alterations putatively inducing impaired DA neurotransmission in FCD type 2. We hypothesize that this may have important implications for the development of these malformations and the manifestation of seizures.

1-P-F-7. Analysis of ictal high-frequency oscillation patterns of intracranial EEGs in surgical epilepsy cases due to various etiologies

Tuberous sclerosis complex (TSC) and focal cortical dysplasia (FCD) are focal malformations of cortical development (FMCDs) that are highly associated with intractable epilepsy. TSC and FCD are mTORopathies caused by a spectrum of pathogenic variants in the mechanistic target of rapamycin (mTOR) pathway genes leading to differential activation of mTOR signaling. However, whether the degree of mTOR hyperactivity influences disease severity remains unclear. Here, we examined the effects of differential mTOR hyperactivity levels on epilepsy and associated neuropathology in a mouse model of TSC and FCD. Constitutively active Rheb (RhebCA), the canonical activator of mTOR complex 1 (mTORC1), was expressed in mouse embryos of either sex via in utero electroporation at low, intermediate, and high concentrations to induce different mTORC1 activity levels in developing cortical neurons. We found that RhebCA expression induced mTORC1 hyperactivation and increased neuronal soma size and misplacement in a dose-dependent manner. No seizures were detected in the low RhebCA mice, whereas the intermediate and high RhebCA mice displayed spontaneous, recurrent seizures that significantly increased with higher RhebCA concentrations. Seizures were associated with a global increase in microglial activation that was notably higher in the regions containing RhebCA-expressing neurons. These data demonstrate that neuronal mTOR hyperactivity levels influence the severity of epilepsy and associated neuropathology in experimental TSC and FCD. Overall, these findings highlight the importance of evaluating the outcome of individual variants on mTOR activity levels and support personalized medicine strategies based on patient variants and mTOR activity level for TSC, FCD, and potentially other mTORopathies.SIGNIFICANCE STATEMENT Tuberous sclerosis complex (TSC) and focal cortical dysplasia (FCD) are epileptogenic cortical malformations caused by pathogenic variants in mechanistic target of rapamycin (mTOR) pathway genes leading to differential mTOR hyperactivation. Here, we present novel findings that neuronal mTOR hyperactivity levels correlate with the severity of epilepsy and associated neuropathology in a mouse model of TSC and FCD. Our findings suggest the need to evaluate the outcome of individual variants on mTOR activity levels in clinical assessments and support personalized medicine strategies based on patient variants and mTOR activity level. Additionally, we present useful modifications to a previously described mouse model of TSC and FCD that allows for titration of seizure frequency and generation of a mild to severe epilepsy phenotype as applicable for preclinical drug testing and mechanistic studies.

Neuropathology of resected brain tissue has revealed an association of focal cortical dysplasia (FCD) with drug-resistant epilepsy (DRE). Recent studies have shown that the mechanistic target of rapamycin (mTOR) pathway is hyperactivated in FCD as evidenced by increased phosphorylation of the ribosomal protein S6 (S6) at serine 240/244 (S(240/244) ), a downstream target of mTOR. Moreover, extracellular regulated kinase (ERK) has been shown to phosphorylate S6 at serine 235/236 (S(235/236) ) and tuberous sclerosis complex 2 (TSC2) at serine 664 (S(664) ) leading to hyperactive mTOR signaling. We evaluated ERK phosphorylation of S6 and TSC2 in two types of FCD (FCD I and FCD II) as a candidate mechanism contributing to mTOR pathway dysregulation. Tissue samples from patients with tuberous sclerosis (TS) served as a positive control. Immunostaining for phospho-S6 (pS6(240/244) and pS6(235/236) ), phospho-ERK (pERK), and phospho-TSC2 (pTSC2) was performed on resected brain tissue with FCD and TS. We found increased pS6(240/244) and pS6(235/236) staining in FCD I, FCD II and TS compared to normal-appearing tissue, while pERK and pTSC2 staining was increased only in FCD IIb and TS tissue. Our results suggest that both the ERK and mTOR pathways are dysregulated in FCD and TS; however, the signaling alterations are different for FCD I as compared to FCD II and TS.

Background and ObjectivesThe 2-hit model of genetic disease is well established in cancer, yet hasonly recently been reported to cause brain malformations associated withepilepsy. Pathogenic germline and somatic variants in genes in themechanistic target of rapamycin (mTOR) pathway have been implicated inseveral malformations of cortical development. We investigated the 2-hitmodel by performing genetic analysis and searching for germline and somaticvariants in genes in the mTOR and related pathways.MethodsWe searched for germline and somatic pathogenic variants in 2 brothers withdrug-resistant focal epilepsy and surgically resected focal corticaldysplasia (FCD) type IIA. Exome sequencing was performed on blood- andbrain-derived DNA to identify pathogenic variants, which were validated bydroplet digital PCR. In vitro functional assays of a somatic variant wereperformed.ResultsExome analysis revealed a novel, maternally inherited, germline pathogenictruncation variant (c.48delG; p.Ser17Alafs*70) inNPRL3 in both brothers. NPRL3 is aknown FCD gene that encodes a negative regulator of the mTOR pathway.Somatic variant calling in brain-derived DNA from both brothers revealed alow allele fraction somatic variant (c.338C>T; p.Ala113Val) in theWNT2 gene in 1 brother, confirmed by droplet digitalPCR. In vitro functional studies suggested a loss of WNT2 function as aconsequence of this variant. A second somatic variant has not yet been foundin the other brother.DiscussionWe identify a pathogenic germline mTOR pathway variant(NPRL3) and a somatic variant (WNT2)in the intersecting WNT signaling pathway, potentially implicating theWNT2 gene in FCD and supporting a dual-pathway 2-hitmodel. If confirmed in other cases, this would extend the 2-hit model topathogenic variants in different genes in critical, intersecting pathways ina malformation of cortical development. Detection of low allele fractionsomatic second hits is challenging but promises to unravel the moleculararchitecture of FCDs.

This study describes the existence and the clinical and electrophysiological features of multi-focal cortical dysplasia in epilepsy patients. Five patients with intractable focal epilepsy are reported. All patients underwent invasive presurgical video-electroencephalography monitoring. Localization of dysplastic areas was based on high-resolution magnetic resonance scanning, surface and intracranial electroencephalography. Four patients underwent epilepsy surgery. Histological findings in focal cortical dysplasia (FCD) were classified according to Palmini. In addition, genetic examinations were performed in order to assess possible mutations in the genes for tuberous sclerosis complex. In four patients, FCDs were located in the same hemisphere. One case presented with bilateral FCDs. In three patients seizures arose from two separate dysplastic areas and in one patient, one lesion showed only interictal activity. In one further patient, seizures started exclusively from the hippocampus. In two of three patients with removal of the FCDs, the histological subtype was identical (Palmini type 2) and in one patient, histology differed between the lesions. All operated patients became seizure-free. In patients with FCD type 2, germ-line mutations in the tuberous sclerosis complex genes were not detectable. Dysplastic brain regions may not be restricted to a single brain region. Areas of FCD can have different degrees of epileptogenicity, ranging from electrographic silence to interictal epileptic discharges and initial involvement in seizure generation. Based on genetic analysis and clinical features, multi-FCD in this patient series was not likely to be related to tuberous sclerosis.