Slc35a2 mosaic knockout impacts cortical development, dendritic arborisation, and neuronal firing.

Mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy (MOGHE) is an important cause of drug-resistant epilepsy. A significant subset of individuals diagnosed with MOGHE display somatic mosaicism for loss-of-function variants in SLC35A2, which encodes the UDP-galactose transporter. We developed a mouse model to investigate how disruption of this transporter leads to a malformation of cortical development. We used in utero electroporation and CRISPR/Cas9 to knockout Slc35a2 in a subset of layer 2/3 cortical neuronal progenitors in the developing brains of male and female fetal mice to model mosaic expression. Mosaic Slc35a2 knockout was verified through next-generation sequencing and immunohistochemistry of GFP-labelled transfected cells. Histology of brain tissue in mosaic Slc35a2 knockout mice revealed the presence of upper layer-derived cortical neurons in the white matter. Reconstruction of single filled neurons identified altered dendritic arborisation with Slc35a2 knockout neurons having increased complexity. Whole-cell electrophysiological recordings revealed that Slc35a2 knockout neurons display reduced action potential firing, increased afterhyperpolarisation duration and reduced burst-firing when compared with control neurons. Mosaic Slc35a2 knockout mice also exhibited significantly increased epileptiform spiking and increased locomotor activity. We successfully generated a mouse model of mosaic Slc35a2 deficiency, which recapitulates features of the human phenotype, including impaired neuronal migration. We show that knockout in layer 2/3 cortical neuron progenitors is sufficient to disrupt neuronal excitability, increase epileptiform activity and cause hyperactivity in mosaic mice. Our mouse model provides an opportunity to further investigate the disease mechanisms that contribute to MOGHE and facilitate the development of precision therapies.

Inherited PURA Pathogenic Variant Associated With a Mild Neurodevelopmental Disorder.

Objectives: Purine-rich element-binding protein alpha (PURA) regulates gene expression and is ubiquitously expressed with an enrichment in neural tissues. Pathogenic variants in PURA cause the neurodevelopmental disorder PURA syndrome that has a variable phenotype but typically comprises moderate-to-severe global developmental delay, intellectual disability, early-onset hypotonia and hypothermia, epilepsy, feeding difficulties, movement disorders, and subtle facial dysmorphism. Speech is reportedly absent in most, but the specific linguistic phenotype is not well described. Methods: We used genome sequencing to identify a pathogenic gene variant as part of a study of children ascertained for severe primary speech disorder in the absence of moderate or severe ID. Results: The novel PURA c.296G>T (p.Arg99Leu) pathogenic missense variant segregated in the female proband and her affected mother. The proband had dysarthria; phonological disorder; and severe receptive and expressive language impairment, borderline intellect, attention difficulties, oropharyngeal dysmotility, and dysmorphic facial features. Her mother had dysarthria, moderate receptive language impairment, and borderline intellect. Both the proband and her mother completed mainstream schooling with classroom support. Discussion: This is the first inherited PURA pathogenic germline variant in over 600 unrelated families documented on ClinVar or reported in the literature. PURA testing should be considered in families with primary speech disorder and borderline intellectual disability, given the specific genetic counseling implications.

Solving the Etiology of Developmental and Epileptic Encephalopathy with Spike-Wave Activation in Sleep (D/EE-SWAS).

OBJECTIVE: To understand the etiological landscape and phenotypic differences between 2 developmental and epileptic encephalopathy (DEE) syndromes: DEE with spike-wave activation in sleep (DEE-SWAS) and epileptic encephalopathy with spike-wave activation in sleep (EE-SWAS). METHODS: All patients fulfilled International League Against Epilepsy (ILAE) DEE-SWAS or EE-SWAS criteria with a Core cohort (n = 91) drawn from our Epilepsy Genetics research program, together with 10 etiologically solved patients referred by collaborators in the Expanded cohort (n = 101). Detailed phenotyping and analysis of molecular genetic results were performed. We compared the phenotypic features of individuals with DEE-SWAS and EE-SWAS. Brain-specific gene co-expression analysis was performed for D/EE-SWAS genes. RESULTS: We identified the etiology in 42/91 (46%) patients in our Core cohort, including 29/44 (66%) with DEE-SWAS and 13/47 (28%) with EE-SWAS. A genetic etiology was identified in 31/91 (34%). D/EE-SWAS genes were highly co-expressed in brain, highlighting the importance of channelopathies and transcriptional regulators. Structural etiologies were found in 12/91 (13%) individuals. We identified 10 novel D/EE-SWAS genes with a range of functions: ATP1A2, CACNA1A, FOXP1, GRIN1, KCNMA1, KCNQ3, PPFIA3, PUF60, SETD1B, and ZBTB18, and 2 novel copy number variants, 17p11.2 duplication and 5q22 deletion. Although developmental regression patterns were similar in both syndromes, DEE-SWAS was associated with a longer duration of epilepsy and poorer intellectual outcome than EE-SWAS. INTERPRETATION: DEE-SWAS and EE-SWAS have highly heterogeneous genetic and structural etiologies. Phenotypic analysis highlights valuable clinical differences between DEE-SWAS and EE-SWAS which inform clinical care and prognostic counseling. Our etiological findings pave the way for the development of precision therapies. ANN NEUROL 2024.

Language and communication functioning in children and adolescents with agenesis of the corpus callosum.

The corpus callosum, the largest white matter inter-hemispheric pathway, is involved in language and communication. In a cohort of 15 children and adolescents (8-15 years) with developmental absence of the corpus callosum (AgCC), this study aimed to describe language and everyday communication functioning, and explored the role of anatomical factors, social risk, and non-verbal IQ in these outcomes. Standardised measures of language and everyday communication functioning, intellectual ability and social risk were used. AgCC classification and anterior commissure volume, a potential alternative pathway, were extracted from T1-weighted images. Participants with AgCC showed reduced receptive and expressive language compared with test norms, and high rates of language and communication impairments. Complete AgCC, higher social risk and lower non-verbal IQ were associated with communication difficulties. Anterior commissure volume was not associated with language and communication. Recognising heterogeneity in language and communication functioning enhances our understanding and suggests specific focuses for potential interventions.

De novo variants in the non-coding spliceosomal snRNA gene RNU4-2 are a frequent cause of syndromic neurodevelopmental disorders.

Around 60% of individuals with neurodevelopmental disorders (NDD) remain undiagnosed after comprehensive genetic testing, primarily of protein-coding genes1. Increasingly, large genome-sequenced cohorts are improving our ability to discover new diagnoses in the non-coding genome. Here, we identify the non-coding RNA RNU4-2 as a novel syndromic NDD gene. RNU4-2 encodes the U4 small nuclear RNA (snRNA), which is a critical component of the U4/U6.U5 tri-snRNP complex of the major spliceosome2. We identify an 18 bp region of RNU4-2 mapping to two structural elements in the U4/U6 snRNA duplex (the T-loop and Stem III) that is severely depleted of variation in the general population, but in which we identify heterozygous variants in 119 individuals with NDD. The vast majority of individuals (77.3%) have the same highly recurrent single base-pair insertion (n.64_65insT). We estimate that variants in this region explain 0.41% of individuals with NDD. We demonstrate that RNU4-2 is highly expressed in the developing human brain, in contrast to its contiguous counterpart RNU4-1 and other U4 homologs, supporting RNU4-2's role as the primary U4 transcript in the brain. Overall, this work underscores the importance of non-coding genes in rare disorders. It will provide a diagnosis to thousands of individuals with NDD worldwide and pave the way for the development of effective treatments for these individuals.

Why did my seizures start now? Influences of lesion connectivity and genetic etiology on age at seizure onset in focal epilepsy.

OBJECTIVE: Patients with focal, lesional epilepsy present with seizures at variable ages. Larger lesion size and overlap with sensorimotor or default mode network (DMN) have been associated with younger age at seizure onset in cohorts with mixed types of focal cortical dysplasia (FCD). Here, we studied determinants of age at seizure onset in patients with bottom-of-sulcus dysplasia (BOSD), a discrete type of FCD with highly localized epileptogenicity. METHODS: Eighty-four patients (77% operated) with BOSD were studied. Demographic, histopathologic, and genetic findings were recorded. BOSD volume and anatomical, primary versus association, rostral versus caudal, and functional network locations were determined. Normative functional connectivity analyses were performed using each BOSD as a region of interest in resting-state functional magnetic resonance imaging data of healthy children. Variables were correlated with age at seizure onset. RESULTS: Median age at seizure onset was 5.4 (interquartile range = 2-7.9) years. Of 50 tested patients, 22 had somatic and nine had germline pathogenic mammalian target of rapamycin (mTOR) pathway variants. Younger age at seizure onset was associated with greater BOSD volume (p = .002), presence of a germline pathogenic variant (p = .04), DMN overlap (p = .04), and increased functional connectivity with the DMN (p < .05, false discovery rate corrected). Location within sensorimotor cortex and networks was not associated with younger age at seizure onset in our relatively small but homogenous cohort. SIGNIFICANCE: Greater lesion size, pathogenic mTOR pathway germline variants, and DMN connectivity are associated with younger age at seizure onset in small FCD. Our findings strengthen the suggested role of DMN connectivity in the onset of FCD-related focal epilepsy and reveal novel contributions of genetic etiology.

Diagnostic utility of exome sequencing followed by research reanalysis in human brain malformations.

This study aimed to determine the diagnostic yield of singleton exome sequencing and subsequent research-based trio exome analysis in children with a spectrum of brain malformations seen commonly in clinical practice. We recruited children ≤ 18 years old with a brain malformation diagnosed by magnetic resonance imaging and consistent with an established list of known genetic causes. Patients were ascertained nationally from eight tertiary paediatric centres as part of the Australian Genomics Brain Malformation Flagship. Chromosome microarray was required for all children, and those with pathogenic copy number changes were excluded. Cytomegalovirus polymerase chain reaction on neonatal blood spots was performed on all children with polymicrogyria with positive patients excluded. Singleton exome sequencing was performed through a diagnostic laboratory and analysed using a clinical exome sequencing pipeline. Undiagnosed patients were followed up in a research setting, including reanalysis of the singleton exome data and subsequent trio exome sequencing. A total of 102 children were recruited. Ten malformation subtypes were identified with the commonest being polymicrogyria (36%), pontocerebellar hypoplasia (14%), periventricular nodular heterotopia (11%), tubulinopathy (10%), lissencephaly (10%) and cortical dysplasia (9%). The overall diagnostic yield for the clinical singleton exome sequencing was 36%, which increased to 43% after research follow-up. The main source of increased diagnostic yield was the reanalysis of the singleton exome data to include newly discovered gene-disease associations. One additional diagnosis was made by trio exome sequencing. The highest phenotype-based diagnostic yields were for cobblestone malformation, tubulinopathy and lissencephaly and the lowest for cortical dysplasia and polymicrogyria. Pathogenic variants were identified in 32 genes, with variants in 6/32 genes occurring in more than one patient. The most frequent genetic diagnosis was pathogenic variants in TUBA1A. This study shows that over 40% of patients with common brain malformations have a genetic aetiology identified by exome sequencing. Periodic reanalysis of exome data to include newly identified genes was of greater value in increasing diagnostic yield than the expansion to trio exome. This study highlights the genetic and phenotypic heterogeneity of brain malformations, the importance of a multidisciplinary approach to diagnosis and the large number of patients that remain without a genetic diagnosis despite clinical exome sequencing and research reanalysis.

Ectopic HCN4 Provides a Target Biomarker for the Genetic Spectrum of mTORopathies.

Background and Objectives: Pathogenic variants in PI3K-AKT-mTOR pathway and GATOR1 complex genes resulting in hyperactivation of mechanistic target of rapamycin (mTOR) complex 1 are a major cause of drug-resistant epilepsy and focal cortical malformations (FCM). Resective neurosurgery is often required to achieve seizure control in patients with mTORopathies due to lack of effectiveness of nonsurgical therapies, including antiseizure medication and mTOR inhibitors. Elevated hyperpolarization-activated cyclic nucleotide-gated potassium channel isoform 4 (HCN4) has been proposed as a key marker in some mTOR-related brain malformations. This study aimed to investigate HCN4 as a biomarker in the brain across the genetic spectrum of mTORopathies in humans. Methods: Our study investigated the relative steady-state levels and cellular localization of HCN4 in resected human brain tissue from 18 individuals with mTORopathies (3 individuals with tuberous sclerosis complex (TSC) due to TSC2 variants, 5 individuals with focal cortical dysplasia type IIA (FCD IIA) due to genetic variants in MTOR, AKT3, and PIK3CA, and 10 individuals with FCD IIB due to variants in TSC1, MTOR, RHEB, DEPDC5, or NPRL3). Results: Elevated HCN4 was observed to be highly restricted to abnormal cell types (dysmorphic neurons and balloon cells) in brain tissue from all mTORopathy tissues (p < 0.0001) compared with those in controls, regardless of genetic cause or variant allele frequency. Elevated HCN4 was not observed in controls or individuals with non-mTOR-related focal epilepsy due to pathogenic variants in ATP1A3, SLC35A2, or FGFR1. Discussion: HCN4 provides a biomarker for the genetic spectrum of mTORopathies and may present a potential therapeutic target for seizure control in mTOR-related epilepsy.

The clinical, imaging, pathological and genetic landscape of bottom-of-sulcus dysplasia.

Bottom-of-sulcus dysplasia (BOSD) is increasingly recognized as a cause of drug-resistant, surgically-remediable, focal epilepsy, often in seemingly MRI-negative patients. We describe the clinical manifestations, morphological features, localization patterns and genetics of BOSD, with the aims of improving management and understanding pathogenesis. We studied 85 patients with BOSD diagnosed between 2005-2022. Presenting seizure and EEG characteristics, clinical course, genetic findings and treatment response were obtained from medical records. MRI (3 T) and 18F-FDG-PET scans were reviewed systematically for BOSD morphology and metabolism. Histopathological analysis and tissue genetic testing were performed in 64 operated patients. BOSD locations were transposed to common imaging space to study anatomical location, functional network localization and relationship to normal MTOR gene expression. All patients presented with stereotyped focal seizures with rapidly escalating frequency, prompting hospitalization in 48%. Despite 42% patients having seizure remissions, usually with sodium channel blocking medications, most eventually became drug-resistant and underwent surgery (86% seizure-free). Prior developmental delay was uncommon but intellectual, language and executive dysfunction were present in 24%, 48% and 29% when assessed preoperatively, low intellect being associated with greater epilepsy duration. BOSDs were missed on initial MRI in 68%, being ultimately recognized following repeat MRI, 18F-FDG-PET or image postprocessing. MRI features were grey-white junction blurring (100%), cortical thickening (91%), transmantle band (62%), increased cortical T1 signal (46%) and increased subcortical FLAIR signal (26%). BOSD hypometabolism was present on 18F-FDG-PET in 99%. Additional areas of cortical malformation or grey matter heterotopia were present in eight patients. BOSDs predominated in frontal and pericentral cortex and related functional networks, mostly sparing temporal and occipital cortex, and limbic and visual networks. Genetic testing yielded pathogenic mTOR pathway variants in 63% patients, including somatic MTOR variants in 47% operated patients and germline DEPDC5 or NPRL3 variants in 73% patients with familial focal epilepsy. BOSDs tended to occur in regions where the healthy brain normally shows lower MTOR expression, suggesting these regions may be more vulnerable to upregulation of MTOR activity. Consistent with the existing literature, these results highlight (i) clinical features raising suspicion of BOSD; (ii) the role of somatic and germline mTOR pathway variants in patients with sporadic and familial focal epilepsy associated with BOSD; and (iii) the role of 18F-FDG-PET alongside high-field MRI in detecting subtle BOSD. The anatomical and functional distribution of BOSDs likely explain their seizure, EEG and cognitive manifestations and may relate to relative MTOR expression.

Inhibitory control in children with agenesis of the corpus callosum compared with typically developing children.

OBJECTIVES: The developmental absence (agenesis) of the corpus callosum (AgCC) is a congenital brain malformation associated with risk for a range of neuropsychological difficulties. Inhibitory control outcomes, including interference control and response inhibition, in children with AgCC are unclear. This study examined interference control and response inhibition: 1) in children with AgCC compared with typically developing (TD) children, 2) in children with different anatomical features of AgCC (complete vs. partial, isolated vs. complex), and 3) associations with white matter volume and microstructure of the anterior (AC) and posterior commissures (PC) and any remnant corpus callosum (CC). METHODS: Participants were 27 children with AgCC and 32 TD children 8-16 years who completed inhibitory control assessments and brain MRI to define AgCC anatomical features and measure white matter volume and microstructure. RESULTS: The AgCC cohort had poorer performance and higher rates of below average performance on inhibitory control measures than TD children. Children with complex AgCC had poorer response inhibition performance than children with isolated AgCC. While not statistically significant, there were select medium to large effect sizes for better inhibitory control associated with greater volume and microstructure of the AC and PC, and with reduced volume and microstructure of the remnant CC in partial AgCC. CONCLUSIONS: This study provides evidence of inhibitory control difficulties in children with AgCC. While the sample was small, the study found preliminary evidence that the AC (f2=.18) and PC (f2=.30) may play a compensatory role for inhibitory control outcomes in the absence of the CC.

Aicardi Syndrome Is a Genetically Heterogeneous Disorder.

Aicardi Syndrome (AIC) is a rare neurodevelopmental disorder recognized by the classical triad of agenesis of the corpus callosum, chorioretinal lacunae and infantile epileptic spasms syndrome. The diagnostic criteria of AIC were revised in 2005 to include additional phenotypes that are frequently observed in this patient group. AIC has been traditionally considered as X-linked and male lethal because it almost exclusively affects females. Despite numerous genetic and genomic investigations on AIC, a unifying X-linked cause has not been identified. Here, we performed exome and genome sequencing of 10 females with AIC or suspected AIC based on current criteria. We identified a unique de novo variant, each in different genes: KMT2B, SLF1, SMARCB1, SZT2 and WNT8B, in five of these females. Notably, genomic analyses of coding and non-coding single nucleotide variants, short tandem repeats and structural variation highlighted a distinct lack of X-linked candidate genes. We assessed the likely pathogenicity of our candidate autosomal variants using the TOPflash assay for WNT8B and morpholino knockdown in zebrafish (Danio rerio) embryos for other candidates. We show expression of Wnt8b and Slf1 are restricted to clinically relevant cortical tissues during mouse development. Our findings suggest that AIC is genetically heterogeneous with implicated genes converging on molecular pathways central to cortical development.

Pathogenic RHEB Somatic Variant in a Child With Tuberous Sclerosis Complex Without Pathogenic Variants in TSC1 or TSC2.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. DISCUSSION: 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.

Australian Genomics: Outcomes of a 5-year national program to accelerate the integration of genomics in healthcare.

Australian Genomics is a national collaborative partnership of more than 100 organizations piloting a whole-of-system approach to integrating genomics into healthcare, based on federation principles. In the first five years of operation, Australian Genomics has evaluated the outcomes of genomic testing in more than 5,200 individuals across 19 rare disease and cancer flagship studies. Comprehensive analyses of the health economic, policy, ethical, legal, implementation and workforce implications of incorporating genomics in the Australian context have informed evidence-based change in policy and practice, resulting in national government funding and equity of access for a range of genomic tests. Simultaneously, Australian Genomics has built national skills, infrastructure, policy, and data resources to enable effective data sharing to drive discovery research and support improvements in clinical genomic delivery.

WWOX developmental and epileptic encephalopathy: Understanding the epileptology and the mortality risk.

OBJECTIVE: WWOX is an autosomal recessive cause of early infantile developmental and epileptic encephalopathy (WWOX-DEE), also known as WOREE (WWOX-related epileptic encephalopathy). We analyzed the epileptology and imaging features of WWOX-DEE, and investigated genotype-phenotype correlations, particularly with regard to survival. METHODS: We studied 13 patients from 12 families with WWOX-DEE. Information regarding seizure semiology, comorbidities, facial dysmorphisms, and disease outcome were collected. Electroencephalographic (EEG) and brain magnetic resonance imaging (MRI) data were analyzed. Pathogenic WWOX variants from our cohort and the literature were coded as either null or missense, allowing individuals to be classified into one of three genotype classes: (1) null/null, (2) null/missense, (3) missense/missense. Differences in survival outcome were estimated using the Kaplan-Meier method. RESULTS: All patients experienced multiple seizure types (median onset = 5 weeks, range = 1 day-10 months), the most frequent being focal (85%), epileptic spasms (77%), and tonic seizures (69%). Ictal EEG recordings in six of 13 patients showed tonic (n = 5), myoclonic (n = 2), epileptic spasms (n = 2), focal (n = 1), and migrating focal (n = 1) seizures. Interictal EEGs demonstrated slow background activity with multifocal discharges, predominantly over frontal or temporo-occipital regions. Eleven of 13 patients had a movement disorder, most frequently dystonia. Brain MRIs revealed severe frontotemporal, hippocampal, and optic atrophy, thin corpus callosum, and white matter signal abnormalities. Pathogenic variants were located throughout WWOX and comprised both missense and null changes including five copy number variants (four deletions, one duplication). Survival analyses showed that patients with two null variants are at higher mortality risk (p-value = .0085, log-rank test). SIGNIFICANCE: Biallelic WWOX pathogenic variants cause an early infantile developmental and epileptic encephalopathy syndrome. The most common seizure types are focal seizures and epileptic spasms. Mortality risk is associated with mutation type; patients with biallelic null WWOX pathogenic variants have significantly lower survival probability compared to those carrying at least one presumed hypomorphic missense pathogenic variant.

Basal ganglia dysplasia and mTORopathy: A potential cause of postoperative seizures in focal cortical dysplasia.

Pathogenic somatic MTOR variants in the cerebral cortex are a frequent cause of focal cortical dysplasia (FCD). We describe a child with drug and surgery-resistant focal epilepsy due to FCD type II who developed progressive enlargement and T2 signal hyperintensity in the ipsilateral caudate and lentiform nuclei. Histopathology of caudate nucleus biopsies showed dysmorphic neurons, similar to those in resected cortex. Genetic analysis of frontal and temporal cortex and caudate nucleus identified a pathogenic somatic MTOR variant [NM_004958.4:c.4375G > C (p.Ala1459Pro)] that was not present in blood-derived gDNA. The mean variant allele frequency ranged from 0.4% to 3.2% in cerebral cortex and up to 5.4% in the caudate nucleus. The basal ganglia abnormalities suggest more widespread, potentially hemispheric dysplasia in this patient, consistent with the pathogenic variant occurring in early cerebral development. This finding provides a potential explanation for persistent seizures in some patients with seemingly complete resection of FCD or disconnection of a dysplastic hemisphere.

Intrinsic and secondary epileptogenicity in focal cortical dysplasia type II.

OBJECTIVE: Favorable seizure outcome is reported following resection of bottom-of-sulcus dysplasia (BOSD). We assessed the distribution of epileptogenicity and dysplasia in and around BOSD to better understand this clinical outcome and the optimal surgical approach. METHODS: We studied 27 children and adolescents with magnetic resonance imaging (MRI)-positive BOSD who underwent epilepsy surgery; 85% became seizure-free postresection (median = 5.0 years follow-up). All patients had resection of the dysplastic sulcus, and 11 had additional resection of the gyral crown (GC) or adjacent gyri (AG). Markers of epileptogenicity were relative cortical hypometabolism on preoperative 18 F-fluorodeoxyglucose (FDG) positron emission tomography (PET), and spiking, ripples, fast ripples, spike-high-frequency oscillation cross-rate, and phase amplitude coupling (PAC) on preresection and postresection electrocorticography (ECoG), all analyzed at the bottom-of-sulcus (BOS), top-of-sulcus (TOS), GC, and AG. Markers of dysplasia were increased cortical thickness on preoperative MRI, and dysmorphic neuron density and variant allele frequency of somatic MTOR mutations in resected tissue, analyzed at similar locations. RESULTS: Relative cortical metabolism was significantly reduced and ECoG markers were significantly increased at the BOS compared to other regions. Apart from spiking and PAC, which were greater at the TOS compared to the GC, there were no significant differences in PET and other ECoG markers between the TOS, GC, and AG, suggesting a cutoff of epileptogenicity at the TOS rather than a tapering gradient on the cortical surface. MRI and tissue markers of dysplasia were all maximal in the BOS, reduced in the TOS, and mostly absent in the GC. Spiking and PAC reduced significantly over the GC after resection of the dysplastic sulcus. SIGNIFICANCE: These findings support the concept that dysplasia and intrinsic epileptogenicity are mostly limited to the dysplastic sulcus in BOSD and support resection or ablation confined to the MRI-visible lesion as a first-line surgical approach. 18 F-FDG PET and ECoG abnormalities in surrounding cortex seem to be secondary phenomena.

Monoallelic and biallelic mutations in RELN underlie a graded series of neurodevelopmental disorders.

Reelin, a large extracellular protein, plays several critical roles in brain development and function. It is encoded by RELN, first identified as the gene disrupted in the reeler mouse, a classic neurological mutant exhibiting ataxia, tremors and a 'reeling' gait. In humans, biallelic variants in RELN have been associated with a recessive lissencephaly variant with cerebellar hypoplasia, which matches well with the homozygous mouse mutant that has abnormal cortical structure, small hippocampi and severe cerebellar hypoplasia. Despite the large size of the gene, only 11 individuals with RELN-related lissencephaly with cerebellar hypoplasia from six families have previously been reported. Heterozygous carriers in these families were briefly reported as unaffected, although putative loss-of-function variants are practically absent in the population (probability of loss of function intolerance = 1). Here we present data on seven individuals from four families with biallelic and 13 individuals from seven families with monoallelic (heterozygous) variants of RELN and frontotemporal or temporal-predominant lissencephaly variant. Some individuals with monoallelic variants have moderate frontotemporal lissencephaly, but with normal cerebellar structure and intellectual disability with severe behavioural dysfunction. However, one adult had abnormal MRI with normal intelligence and neurological profile. Thorough literature analysis supports a causal role for monoallelic RELN variants in four seemingly distinct phenotypes including frontotemporal lissencephaly, epilepsy, autism and probably schizophrenia. Notably, we observed a significantly higher proportion of loss-of-function variants in the biallelic compared to the monoallelic cohort, where the variant spectrum included missense and splice-site variants. We assessed the impact of two canonical splice-site variants observed as biallelic or monoallelic variants in individuals with moderately affected or normal cerebellum and demonstrated exon skipping causing in-frame loss of 46 or 52 amino acids in the central RELN domain. Previously reported functional studies demonstrated severe reduction in overall RELN secretion caused by heterozygous missense variants p.Cys539Arg and p.Arg3207Cys associated with lissencephaly suggesting a dominant-negative effect. We conclude that biallelic variants resulting in complete absence of RELN expression are associated with a consistent and severe phenotype that includes cerebellar hypoplasia. However, reduced expression of RELN remains sufficient to maintain nearly normal cerebellar structure. Monoallelic variants are associated with incomplete penetrance and variable expressivity even within the same family and may have dominant-negative effects. Reduced RELN secretion in heterozygous individuals affects only cortical structure whereas the cerebellum remains intact. Our data expand the spectrum of RELN-related neurodevelopmental disorders ranging from lethal brain malformations to adult phenotypes with normal brain imaging.

Heterozygous PNPT1 Variants Cause Spinocerebellar Ataxia Type 25.

OBJECTIVE: Dominant spinocerebellar ataxias (SCA) are characterized by genetic heterogeneity. Some mapped and named loci remain without a causal gene identified. Here we applied next generation sequencing (NGS) to uncover the genetic etiology of the SCA25 locus. METHODS: Whole-exome and whole-genome sequencing were performed in families linked to SCA25, including the French family in which the SCA25 locus was originally mapped. Whole exome sequence data were interrogated in a cohort of 796 ataxia patients of unknown etiology. RESULTS: The SCA25 phenotype spans a slowly evolving sensory and cerebellar ataxia, in most cases attributed to ganglionopathy. A pathogenic variant causing exon skipping was identified in the gene encoding Polyribonucleotide Nucleotidyltransferase PNPase 1 (PNPT1) located in the SCA25 linkage interval. A second splice variant in PNPT1 was detected in a large Australian family with a dominant ataxia also mapping to SCA25. An additional nonsense variant was detected in an unrelated individual with ataxia. Both nonsense and splice heterozygous variants result in premature stop codons, all located in the S1-domain of PNPase. In addition, an elevated type I interferon response was observed in blood from all affected heterozygous carriers tested. PNPase notably prevents the abnormal accumulation of double-stranded mtRNAs in the mitochondria and leakage into the cytoplasm, associated with triggering a type I interferon response. INTERPRETATION: This study identifies PNPT1 as a new SCA gene, responsible for SCA25, and highlights biological links between alterations of mtRNA trafficking, interferonopathies and ataxia. ANN NEUROL 2022;92:122-137.

Sporadic hypothalamic hamartoma is a ciliopathy with somatic and bi-allelic contributions.

Hypothalamic hamartoma with gelastic seizures is a well-established cause of drug-resistant epilepsy in early life. The development of novel surgical techniques has permitted the genomic interrogation of hypothalamic hamartoma tissue. This has revealed causative mosaic variants within GLI3, OFD1 and other key regulators of the sonic-hedgehog pathway in a minority of cases. Sonic-hedgehog signalling proteins localize to the cellular organelle primary cilia. We therefore explored the hypothesis that cilia gene variants may underlie hitherto unsolved cases of sporadic hypothalamic hamartoma. We performed high-depth exome sequencing and chromosomal microarray on surgically resected hypothalamic hamartoma tissue and paired leukocyte-derived DNA from 27 patients. We searched for both germline and somatic variants under both dominant and bi-allelic genetic models. In hamartoma-derived DNA of seven patients we identified bi-allelic (one germline, one somatic) variants within one of four cilia genes-DYNC2I1, DYNC2H1, IFT140 or SMO. In eight patients, we identified single somatic variants in the previously established hypothalamic hamartoma disease genes GLI3 or OFD1. Overall, we established a plausible molecular cause for 15/27 (56%) patients. Here, we expand the genetic architecture beyond single variants within dominant disease genes that cause sporadic hypothalamic hamartoma to bi-allelic (one germline/one somatic) variants, implicate three novel cilia genes and reconceptualize the disorder as a ciliopathy.