Valerio Conti

Background and ObjectivesDisease-causing variants in the syntaxin-binding protein 1 (STXBP1) gene are among the most common genetic causes of developmental and epileptic encephalopathies and are as...

SLC35A2 encodes the Golgi uridine diphosphate galactose transporter, which is essential for glycosylation of glycoproteins and glycolipids. Variants in this gene, either germline or somatic, have emerged as causes of diverse neurological disorders ranging from congenital disorders of glycosylation (SLC35A2-CDG) to focal cortical malformations such as mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy (MOGHE). This review summarizes the molecular function of SLC35A2, clinical phenotypes of congenital and somatic variants, insights from functional assays and animal models, and therapeutic perspectives including galactose supplementation and precision medicine. We aim to provide an integrative synthesis of human genetics, neuropathology, glycomics, and translational approaches.

Brain somatic variants in the SLC35A2 gene, encoding for a Golgi galactose transporter, represent the major cause of mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy (MOGHE). Clinical features associated with MOGHE include early-onset epileptic encephalopathy, drug-resistant focal epilepsy with developmental delay, and intellectual disability. Half of somatic SLC35A2 variants identified in MOGHE patients are predicted to encode full-length SLC35A2 protein or stable protein products. We investigated the pathophysiological basis of MOGHE by analyzing the functional consequences of SLC35A2 pathogenetic variants in vitro and in vivo models. We assessed how different SLC35A2 variants impact protein stability and expression in transfected cellular models. We used in utero electroporation in the rat brain to model mosaic expression of SLC35A2 pathogenetic variants in the cerebral cortex and assessed their effect on neurons migration and morphology. We found that SLC35A2 variants identified in MOGHE patients variably impact on SLC35A2 protein expression. In utero expression of a SLC35A2 missense (p.G282A) or frameshift (p.F280Tfs*10) variants resulted in neuronal heterotopia in the white matter and impaired dendritogenesis at postnatal stages, suggesting a cell autonomous role for SLC35A2 in neuronal development. These phenotypes were recapitulated by in utero silencing of rat Slc35a2 gene. We successfully developed an in vivo mosaic model for the characterization of SLC35A2 variants identified in MOGHE patients and demonstrated that the expression of single SLC35A2 variants triggers the pathophysiological cascade associated with SLC35A2 dysfunction in neurons.

Vacuolar-type H⁺-ATPase (V-ATPase) is a multimeric complex present in a variety of cellular membranes that acts as an ATP-dependent proton pump and plays a key role in pH homeostasis and intracellular signalling pathways. In humans, 22 autosomal genes encode for a redundant set of subunits allowing the composition of diverse V-ATPase complexes with specific properties and expression. Sixteen subunits have been linked to human disease. Here we describe 26 patients harbouring 20 distinct pathogenic de novo missense ATP6V1A variants, mainly clustering within the ATP synthase α/β family-nucleotide-binding domain. At a mean age of 7 years (extremes: 6 weeks, youngest deceased patient to 22 years, oldest patient) clinical pictures included early lethal encephalopathies with rapidly progressive massive brain atrophy, severe developmental epileptic encephalopathies and static intellectual disability with epilepsy. The first clinical manifestation was early hypotonia, in 70%; 81% developed epilepsy, manifested as developmental epileptic encephalopathies in 58% of the cohort and with infantile spasms in 62%; 63% of developmental epileptic encephalopathies failed to achieve any developmental, communicative or motor skills. Less severe outcomes were observed in 23% of patients who, at a mean age of 10 years and 6 months, exhibited moderate intellectual disability, with independent walking and variable epilepsy. None of the patients developed communicative language. Microcephaly (38%) and amelogenesis imperfecta/enamel dysplasia (42%) were additional clinical features. Brain MRI demonstrated hypomyelination and generalized atrophy in 68%. Atrophy was progressive in all eight individuals undergoing repeated MRIs. Fibroblasts of two patients with developmental epileptic encephalopathies showed decreased LAMP1 expression, Lysotracker staining and increased organelle pH, consistent with lysosomal impairment and loss of V-ATPase function. Fibroblasts of two patients with milder disease, exhibited a different phenotype with increased Lysotracker staining, decreased organelle pH and no significant modification in LAMP1 expression. Quantification of substrates for lysosomal enzymes in cellular extracts from four patients revealed discrete accumulation. Transmission electron microscopy of fibroblasts of four patients with variable severity and of induced pluripotent stem cell-derived neurons from two patients with developmental epileptic encephalopathies showed electron-dense inclusions, lipid droplets, osmiophilic material and lamellated membrane structures resembling phospholipids. Quantitative assessment in induced pluripotent stem cell-derived neurons identified significantly smaller lysosomes. ATP6V1A-related encephalopathy represents a new paradigm among lysosomal disorders. It results from a dysfunctional endo-lysosomal membrane protein causing altered pH homeostasis. Its pathophysiology implies intracellular accumulation of substrates whose composition remains unclear, and a combination of developmental brain abnormalities and neurodegenerative changes established during prenatal and early postanal development, whose severity is variably determined by specific pathogenic variants.

Children with epilepsy and identified as responders to antiseizure medications (ASMs) were found to present markedly higher ghrelin plasma levels when compared to drug-resistant patients. However, it was undetermined if this phenotype could be influenced by the ASMs. Here, we prospectively investigated total ghrelin and des-acyl ghrelin (DAG) plasma levels by enzyme-linked immunosorbent assay before and after ASM administration. Inclusion criteria were: (i) subject with a suspicion of epilepsy; (ii) age ranging from 0 to 16 years; and (iii) informed consent signed by parents or caregivers. Exclusion criteria were acute or chronic metabolic disorders with occasional convulsions but without epilepsy. Fifty patients were followed over a period of one year in Italian neuropediatric centers. Apart from a few exceptions, the majority of children were responsive to ASMs. No differences were found in total ghrelin and DAG levels before and after the treatment, but total ghrelin levels were significantly lower in children with generalized epilepsy compared to those with combined focal and generalized epilepsy. Moreover, the ghrelin-to-DAG ratio was also markedly lower in generalized epilepsies compared to all the other types of epilepsy. Finally, ghrelin was unchanged by ASMs, including the first (e.g., carbamazepine), second (levetiracetam), and third (lacosamide) generation of anticonvulsants.

Focal malformations of cortical development (FMCD) including focal cortical dysplasia (FCD), hemimegalencephaly (HMEG) and megalencephaly (MEG), constitute a spectrum of neurodevelopmental disorders associated with brain overgrowth, cellular and architectural dysplasia, intractable epilepsy, autism, and intellectual disability. Importantly, FCD is the most common cause of intractable pediatric focal epilepsy. Gain and loss of function mutations in the PI3K-AKT-MTOR pathway have been identified in this spectrum, with variable levels of mosaicism and tissue distribution. In this study, we aimed to assess droplet digital Polymerase Chain Reaction (ddPCR) as a first-tier molecular diagnostic method, as well as define genotype-phenotype relationships among the most common PI3K-AKT-MTOR pathway mutations in FMCD. A total of 144 specimens, including 113 brain samples, were collected from 58 individuals with intractable focal epilepsy phenotypes including FCD, MEG, HMEG and other types of developmental cortical lesions. We designed an ultra-deep and highly sensitive molecular diagnostic panel using ddPCR for six of the most common mutations in three PI3K-AKT-MTOR pathway genes, namely PIK3CA (p.E542K, p.E545K, p.H1047R), AKT3 (p.E17K) and MTOR (p.S2215F, p.S2215Y). We quantified the level of mosaicism across all samples and correlated genotypes with key phenotype, neuroimaging and neuropathological data. Pathogenic variants were identified in 17 individuals, with an overall molecular solve rate of %. Variant allele fractions (VAF) ranged from 0.1% to 22.67% across all positive samples. Our data shows that MTOR mutations are mostly associated with FCD, whereas PIK3CA mutations are more frequent in the HMEG-DMEG spectrum. The presence of one of these common PI3K-AKT-MTOR-mutations correlated with earlier onset of seizures. However, levels of mosaicism did not correlate with the severity of the cortical malformation by neuroimaging or neuropathological examination. Interestingly, we could not identify the six most common pathogenic variants in other types of cortical lesions (e.g., polymicrogyria or mesial temporal sclerosis) suggesting that PI3K-AKT-MTOR mutations are specifically causal in the FCD-HMEG-MEG spectrum. Finally, our data suggest that ultra-deep targeted molecular analysis for the most common PI3K-AKT-MTOR mutations via ddPCR is an effective molecular diagnostic approach for FMCD phenotypes with a good diagnostic yield when paired with neuroimaging and neuropathology evaluations. The high sensitivity and low DNA input requirements suggests that ddPCR is an effective molecular diagnostic tool for disorders caused by somatic mutations with a narrow mutational spectrum, including specific subtypes of pediatric epilepsy surgical phenotypes such as FCD and HMEG.

Objective To alert about the wide margin of unpredictability that distribution of somatic MTOR mosaicism may have in the brain and the risk for independent epileptogenesis arising from the seemingly healthy contralateral hemisphere after complete removal of epileptogenic focal cortical dysplasia (FCD). Methods Clinical, EEG, MRI, histopathology, and molecular genetics in 2 patients (1 and 2) treated with focal resections and subsequent complete hemispherectomy for epileptogenic FCD due to somatic MTOR mutations. Autoptic brain study of bilateral asymmetric hemispheric dysplasia and identification of alternative allele fraction (AAF) rates for AKT1 (patient 3). Results The strongly hyperactivating p.Ser2215Phe (patient 1) and p.Leu1460Pro (patient 2) MTOR mutations were at low-level AAF in the dysplastic tissue. After repeated resections and eventual complete hemispherectomy, both patients manifested intractable seizures arising from the contralateral, seemingly healthy hemisphere. In patient 3, the p.Glu17Lys AKT1 mutation exhibited random distribution and AAF rates in different tissues with double levels in the more severely dysplastic cerebral hemisphere. Conclusions Our understanding of the distribution of somatic mutations in the brain in relation to the type of malformation and its hypothesized time of origin may be faulty. Large studies may reveal that the risk of a first surgery being disappointing might be related more to the specific somatic mammalian target of rapamycin mutation identified than to completeness of resection and that the advantages of repeated resections after a first unsuccessful operation should be weighed against the risk of the contralateral hemisphere becoming in turn epileptogenic.

Malformations of cortical development (MCDs) are neurodevelopmental disorders that result from abnormal development of the cerebral cortex in utero. MCDs place a substantial burden on affected individuals, their families and societies worldwide, as these individuals can experience lifelong drug-resistant epilepsy, cerebral palsy, feeding difficulties, intellectual disability and other neurological and behavioural anomalies. The diagnostic pathway for MCDs is complex owing to wide variations in presentation and aetiology, thereby hampering timely and adequate management. In this article, the international MCD network Neuro-MIG provides consensus recommendations to aid both expert and non-expert clinicians in the diagnostic work-up of MCDs with the aim of improving patient management worldwide. We reviewed the literature on clinical presentation, aetiology and diagnostic approaches for the main MCD subtypes and collected data on current practices and recommendations from clinicians and diagnostic laboratories within Neuro-MIG. We reached consensus by 42 professionals from 20 countries, using expert discussions and a Delphi consensus process. We present a diagnostic workflow that can be applied to any individual with MCD and a comprehensive list of MCD-related genes with their associated phenotypes. The workflow is designed to maximize the diagnostic yield and increase the number of patients receiving personalized care and counselling on prognosis and recurrence risk.