Chloride deregulation and GABA depolarization in MTOR related malformations of cortical development.

Focal Cortical Dysplasia, Hemimegalencephaly and Cortical Tuber are pediatric epileptogenic malformations of cortical development (MCDs) frequently pharmaco-resistant and mostly surgically treated by the resection of epileptic cortex. Availability of cortical resection samples allowed significant mechanistic discoveries directly from human material. Causal brain somatic or germline mutations in the AKT/PI3K/DEPDC5/MTOR genes were identified. GABAa mediated paradoxical depolarization, related to altered chloride (Cl-) homeostasis, was shown to participate to ictogenesis in human pediatric MCDs. However, the link between genomic alterations and neuronal hyperexcitability is still unclear. Here we studied the post translational interactions between the mTOR pathway and the regulation of cation-chloride cotransporters (CCC), KCC2 and NKCC1, that are largely responsible for controlling intracellular Cl- and ultimately GABAergic transmission. For this study, 35 children (25 MTORopathies and 10 pseudo controls, diagnosed by histology plus genetic profiling) were operated for drug resistant epilepsy. Postoperative cortical tissues were recorded on multielectrode array (MEA) to map epileptic activities. CCC expression level and phosphorylation status of the WNK1/SPAK-OSR1 pathway was measured during basal conditions and after pharmacological modulation. Direct interactions between mTOR and WNK1 pathway components were investigated by immunoprecipitation. Membranous incorporation of MCD samples in Xenopus laevis oocytes enabled Cl- conductance and equilibrium potential (EGABA) for GABA measurement. Of the 25 clinical cases, half harbored a somatic mutation in the mTOR pathway, while pS6 expression was increased in all MCD samples. Spontaneous interictal discharges were recorded in 65% of the slices. CCC expression was altered in MCDs, with a reduced KCC2/NKCC1 ratio and decreased KCC2 membranous expression. CCC expression was regulated by the WNK1/SPAK-OSR1 kinases through direct phosphorylation of Thr906 on KCC2, that was reversed by WNK1 and SPAK antagonists (NEM and Staurosporine). mSIN1 subunit of MTORC2 was found to interact with SPAK-OSR1 and WNK1. Interactions between these key epileptogenic pathways could be reversed by the mTOR specific antagonist Rapamycin, leading to a dephosphorylation of CCCs and recovery of the KCC2/NKCC1 ratio. The functional effect of such recovery was validated by the restoration of the depolarizing shift in EGABA by rapamycin, measured after incorporation of MCD membranes to X. laevis oocytes, in line with a reestablishment of normal ECl-. Our study deciphers a protein interaction network through a phosphorylation cascade between MTOR and WNK1/SPAK-OSR1 leading to chloride cotransporters deregulation, increased neuronal chloride levels and GABAa dysfunction in malformations of Cortical Development, linking genomic defects and functional effects and paving the way to target epilepsy therapy.

Distinct Clinical Courses and Shortened Lifespans in Childhood-Onset DNA Polymerase Gamma Deficiency.

Background and Objectives: DNA polymerase subunit gamma (POLG) deficiency is likely the most frequent cause of nuclear-encoded mitochondrial disorders. POLG-related disorders reportedly constitute a spectrum of overlapping phenotypes from infancy to late adulthood. We retrospectively reviewed natural histories for 40 children carrying biallelic pathogenic POLG variants. Methods: The patients were identified by the French coordinating center for mitochondrial disorders (CARAMMEL), making this a large monocentric series on childhood-onset POLG deficiency. Results: Three patterns of clinical course and survival were observed, distinguished by main category of symptoms: neurologic, hepatic, and gastrointestinal. A total of 24 patients needed urgent neurointensive care for tonic-clonic seizures, myoclonic epilepsy, and status epilepticus, occasionally precipitated by valproate administration. Other neurologic symptoms included dystonia, cerebellar ataxia, and peripheral neuropathy. We report 6 POLG-deficient patients with polyradiculoneuropathy mimicking subacute Guillain-Barré syndrome and provide postgadolinium MRI evidence of diffuse cranial nerve root and cauda equina enhancement, suggesting these disorders have an inflammatory component. Children presenting with enteral nervous system involvement had vomiting, gastroparesis, and chronic intestinal pseudo-obstruction. They had later ages of onset and lived much longer. Primarily, hepatic presentations had the earliest onset and shortest survivals. Secondary hepatic failure was frequently precipitated by valproate administration given before diagnosis to patients with focal impaired awareness seizures or absence of seizures. These POLG deficiencies were often fatal, with age at death ranging from 3 months to 10 years, with a significant difference in survival between the 3 clinical forms; 6 of the 40 children did survive. No genotype-phenotype correlations were found for the 3 clinical course types. Discussion: The study demonstrates the prevalence of neurologic presentation and the extent of central, peripheral, and autonomous nervous system involvement in 60% of patients. Most of the patients with early onset and rapidly fatal hepatic failure did not live long enough to develop neurologic symptoms. The study revealed a new clinical form of POLG deficiency presenting with neurodigestive symptoms with longer lifespan. We also propose that POLG deficiency should be considered in children presenting with unexplained polyradiculoneuropathy, demyelinating neuropathy, and elevated CSF protein. Finally, valproate administration remains a notable cause of avoidable death in POLG-deficient patients.

Genetic testing, another important tool in presurgical evaluation of focal epilepsies in childhood.

Epilepsy surgery may be a curative therapy for patients with drug-resistant epilepsies when focal lesions or foci are identified. Genetic testing is not yet routinely included in many presurgical evaluation programs although recent evidence support that finding a germline genetic mutation could help to better delineate the patient candidacy to surgery and provide valuable information on the expected surgery outcome. In this study, we report nine patients presenting drug-resistant focal epilepsy enrolled in presurgical evaluation. We show how the identification of genetic pathogenic variant in epilepsy known genes led to the interruption of the presurgical work-up and ruled out surgery in 7 of them. We observed that the co-existence of some recurrent clinical characteristics as early seizures' onset, frequent precipitating factors including fever, and developmental delay or intellectual disability may be useful markers for germline genetic pathogenic variants. In this group, genetic assessment should be mandatory during presurgical work up, mainly in patients with negative magnetic resonance imaging (MRI) or doubtful structural lesions. The integration of next generation targeted sequencing into the presurgical evaluation can improve the selection of candidates for resective surgery and fosters a personalized medicine approach with a better outcome. PLAINE LANGUAGE ABSTRACT: Genetic testing is not yet systematically included in the pre-surgical assessment of patients with drug-resistant focal epilepsies. In this study, through the description of nine patients, we underline how the integration of genomics into the presurgical work up can help in evaluating the patient candidacy to surgery and provide valuable information on expected surgery outcome.

Primary mitochondrial disorders and mimics: Insights from a large French cohort.

OBJECTIVE: The objective of this study was to evaluate the implementation of NGS within the French mitochondrial network, MitoDiag, from targeted gene panels to whole exome sequencing (WES) or whole genome sequencing (WGS) focusing on mitochondrial nuclear-encoded genes. METHODS: Over 2000 patients suspected of Primary Mitochondrial Diseases (PMD) were sequenced by either targeted gene panels, WES or WGS within MitoDiag. We described the clinical, biochemical, and molecular data of 397 genetically confirmed patients, comprising 294 children and 103 adults, carrying pathogenic or likely pathogenic variants in nuclear-encoded genes. RESULTS: The cohort exhibited a large genetic heterogeneity, with the identification of 172 distinct genes and 253 novel variants. Among children, a notable prevalence of pathogenic variants in genes associated with oxidative phosphorylation (OXPHOS) functions and mitochondrial translation was observed. In adults, pathogenic variants were primarily identified in genes linked to mtDNA maintenance. Additionally, a substantial proportion of patients (54% (42/78) and 48% (13/27) in children and adults, respectively), undergoing WES or WGS testing displayed PMD mimics, representing pathologies that clinically resemble mitochondrial diseases. INTERPRETATION: We reported the largest French cohort of patients suspected of PMD with pathogenic variants in nuclear genes. We have emphasized the clinical complexity of PMD and the challenges associated with recognizing and distinguishing them from other pathologies, particularly neuromuscular disorders. We confirmed that WES/WGS, instead of panel approach, was more valuable to identify the genetic basis in patients with "possible" PMD and we provided a genetic testing flowchart to guide physicians in their diagnostic strategy.

Lessons from two series by physicians and caregivers' self-reported data in DDX3X-related disorders.

INTRODUCTION AND METHODS: We report two series of individuals with DDX3X variations, one (48 individuals) from physicians and one (44 individuals) from caregivers. RESULTS: These two series include several symptoms in common, with fairly similar distribution, which suggests that caregivers' data are close to physicians' data. For example, both series identified early childhood symptoms that were not previously described: feeding difficulties, mean walking age, and age at first words. DISCUSSION: Each of the two datasets provides complementary knowledge. We confirmed that symptoms are similar to those in the literature and provides more details on feeding difficulties. Caregivers considered that the symptom attention-deficit/hyperactivity disorder were most worrisome. Both series also reported sleep disturbance. Recently, anxiety has been reported in individuals with DDX3X variants. We strongly suggest that attention-deficit/hyperactivity disorder, anxiety, and sleep disorders need to be treated.

Monoallelic variation in DHX9, the gene encoding the DExH-box helicase DHX9, underlies neurodevelopment disorders and Charcot-Marie-Tooth disease.

DExD/H-box RNA helicases (DDX/DHX) are encoded by a large paralogous gene family; in a subset of these human helicase genes, pathogenic variation causes neurodevelopmental disorder (NDD) traits and cancer. DHX9 encodes a BRCA1-interacting nuclear helicase regulating transcription, R-loops, and homologous recombination and exhibits the highest mutational constraint of all DDX/DHX paralogs but remains unassociated with disease traits in OMIM. Using exome sequencing and family-based rare-variant analyses, we identified 20 individuals with de novo, ultra-rare, heterozygous missense or loss-of-function (LoF) DHX9 variant alleles. Phenotypes ranged from NDDs to the distal symmetric polyneuropathy axonal Charcot-Marie-Tooth disease (CMT2). Quantitative Human Phenotype Ontology (HPO) analysis demonstrated genotype-phenotype correlations with LoF variants causing mild NDD phenotypes and nuclear localization signal (NLS) missense variants causing severe NDD. We investigated DHX9 variant-associated cellular phenotypes in human cell lines. Whereas wild-type DHX9 was restricted to the nucleus, NLS missense variants abnormally accumulated in the cytoplasm. Fibroblasts from an individual with an NLS variant also showed abnormal cytoplasmic DHX9 accumulation. CMT2-associated missense variants caused aberrant nucleolar DHX9 accumulation, a phenomenon previously associated with cellular stress. Two NDD-associated variants, p.Gly411Glu and p.Arg761Gln, altered DHX9 ATPase activity. The severe NDD-associated variant p.Arg141Gln did not affect DHX9 localization but instead increased R-loop levels and double-stranded DNA breaks. Dhx9-/- mice exhibited hypoactivity in novel environments, tremor, and sensorineural hearing loss. All together, these results establish DHX9 as a critical regulator of mammalian neurodevelopment and neuronal homeostasis.

Clinical and radiological description of 120 pediatric stroke-like episodes.

BACKGROUND AND PURPOSE: Stroke-like episodes (SLEs) are defined as acute onset of neurological symptoms mimicking a stroke and radiological lesions non-congruent to vascular territory. We aimed to analyze the acute clinical and radiological features of SLEs to determine their pathophysiology. METHODS: We performed a monocenter retrospective analysis of 120 SLEs in 60 children over a 20-year period. Inclusion criteria were compatible clinical symptoms and stroke-like lesions on brain magnetic resonance imaging (MRI; performed for all 120 events) with focal hyperintensity on diffusion-weighted imaging in a non-vascular territory. RESULTS: Three groups were identified: children with mitochondrial diseases (n = 22) involving mitochondrial DNA mutations (55%) or nuclear DNA mutations (45%); those with other metabolic diseases or epilepsy disorders (n = 22); and those in whom no etiology was found despite extensive investigations (n = 16). Age at first SLE was younger in the group with metabolic or epilepsy disorders (18 months vs. 128 months; p < 0.0001) and an infectious trigger was more frequent (69% vs. 20%; p = 0.0001). Seizures occurred in 75% of episodes, revealing 50% episodes of SLEs and mainly leading to status epilepticus (90%). Of the 120 MRI scans confirming the diagnosis, 28 were performed within a short and strict 48-h period and were further analyzed to better understand the underlying mechanisms. The scans showed primary cortical hyperintensity (n = 28/28) with decreased apparent diffusion coefficient in 52% of cases. Systematic hyperperfusion was found on spin labeling sequences when available (n = 18/18). CONCLUSION: Clinical and radiological results support the existence of a vicious circle based on two main mechanisms: energy deficit and neuronal hyperexcitability at the origin of SLE.

Novel ELAC2 Mutations in Individuals Presenting with Variably Severe Neurological Disease in the Presence or Absence of Cardiomyopathy.

Transcription of mitochondrial DNA generates long polycistronic precursors whose nucleolytic cleavage yields the individual mtDNA-encoded transcripts. In most cases, this cleavage occurs at the 5'- and 3'-ends of tRNA sequences by the concerted action of RNAseP and RNaseZ/ELAC2 endonucleases, respectively. Variants in the ELAC2 gene have been predominantly linked to severe to mild cardiomyopathy that, in its milder forms, is accompanied by variably severe neurological presentations. Here, we report five patients from three unrelated families. Four of the patients presented mild to moderate cardiomyopathy and one died at 1 year of age, one patient had no evidence of cardiomyopathy. The patients had variable neurological presentations that included intellectual disability, ataxia, refractory epilepsy, neuropathy and deafness. All patients carried previously unreported missense and nonsense variants. Enzymatic analyses showed multiple OXPHOS deficiencies in biopsies from two patients, whereas immunoblot analyses revealed a decreased abundance of ELAC2 in fibroblasts from three patients. Northern blot analysis revealed an accumulation of unprocessed mt-tRNAVal-precursor consistent with the role of ELAC2 in transcript processing. Our study expands the genetic spectrum of ELAC2-linked disease and suggests that cardiomyopathy is not an invariably present clinical hallmark of this pathology.

Genotypic and phenotypic spectrum of infantile liver failure due to pathogenic TRMU variants.

PURPOSE: This study aimed to define the genotypic and phenotypic spectrum of reversible acute liver failure (ALF) of infancy resulting from biallelic pathogenic TRMU variants and determine the role of cysteine supplementation in its treatment. METHODS: Individuals with biallelic (likely) pathogenic variants in TRMU were studied within an international retrospective collection of de-identified patient data. RESULTS: In 62 individuals, including 30 previously unreported cases, we described 47 (likely) pathogenic TRMU variants, of which 17 were novel, and 1 intragenic deletion. Of these 62 individuals, 42 were alive at a median age of 6.8 (0.6-22) years after a median follow-up of 3.6 (0.1-22) years. The most frequent finding, occurring in all but 2 individuals, was liver involvement. ALF occurred only in the first year of life and was reported in 43 of 62 individuals; 11 of whom received liver transplantation. Loss-of-function TRMU variants were associated with poor survival. Supplementation with at least 1 cysteine source, typically N-acetylcysteine, improved survival significantly. Neurodevelopmental delay was observed in 11 individuals and persisted in 4 of the survivors, but we were unable to determine whether this was a primary or a secondary consequence of TRMU deficiency. CONCLUSION: In most patients, TRMU-associated ALF was a transient, reversible disease and cysteine supplementation improved survival.

Bi-allelic loss-of-function variants in TMEM147 cause moderate to profound intellectual disability with facial dysmorphism and pseudo-Pelger-Huët anomaly.

The transmembrane protein TMEM147 has a dual function: first at the nuclear envelope, where it anchors lamin B receptor (LBR) to the inner membrane, and second at the endoplasmic reticulum (ER), where it facilitates the translation of nascent polypeptides within the ribosome-bound TMCO1 translocon complex. Through international data sharing, we identified 23 individuals from 15 unrelated families with bi-allelic TMEM147 loss-of-function variants, including splice-site, nonsense, frameshift, and missense variants. These affected children displayed congruent clinical features including coarse facies, developmental delay, intellectual disability, and behavioral problems. In silico structural analyses predicted disruptive consequences of the identified amino acid substitutions on translocon complex assembly and/or function, and in vitro analyses documented accelerated protein degradation via the autophagy-lysosomal-mediated pathway. Furthermore, TMEM147-deficient cells showed CKAP4 (CLIMP-63) and RTN4 (NOGO) upregulation with a concomitant reorientation of the ER, which was also witnessed in primary fibroblast cell culture. LBR mislocalization and nuclear segmentation was observed in primary fibroblast cells. Abnormal nuclear segmentation and chromatin compaction were also observed in approximately 20% of neutrophils, indicating the presence of a pseudo-Pelger-Huët anomaly. Finally, co-expression analysis revealed significant correlation with neurodevelopmental genes in the brain, further supporting a role of TMEM147 in neurodevelopment. Our findings provide clinical, genetic, and functional evidence that bi-allelic loss-of-function variants in TMEM147 cause syndromic intellectual disability due to ER-translocon and nuclear organization dysfunction.

Molecular and clinical descriptions of patients with GABAA receptor gene variants (GABRA1, GABRB2, GABRB3, GABRG2): A cohort study, review of literature, and genotype-phenotype correlation.

OBJECTIVE: γ-Aminobutyric acid (GABA)A -receptor subunit variants have recently been associated with neurodevelopmental disorders and/or epilepsy. The phenotype linked with each gene is becoming better known. Because of the common molecular structure and physiological role of these phenotypes, it seemed interesting to describe a putative phenotype associated with GABAA -receptor-related disorders as a whole and seek possible genotype-phenotype correlations. METHODS: We collected clinical, electrophysiological, therapeutic, and molecular data from patients with GABAA -receptor subunit variants (GABRA1, GABRB2, GABRB3, and GABRG2) through a national French collaboration using the EPIGENE network and compared these data to the one already described in the literature. RESULTS: We gathered the reported patients in three epileptic phenotypes: 15 patients with fever-related epilepsy (40%), 11 with early developmental epileptic encephalopathy (30%), 10 with generalized epilepsy spectrum (27%), and 1 patient without seizures (3%). We did not find a specific phenotype for any gene, but we showed that the location of variants on the transmembrane (TM) segment was associated with a more severe phenotype, irrespective of the GABAA -receptor subunit gene, whereas N-terminal variants seemed to be related to milder phenotypes. SIGNIFICANCE: GABAA -receptor subunit variants are associated with highly variable phenotypes despite their molecular and physiological proximity. None of the genes described here was associated with a specific phenotype. On the other hand, it appears that the location of the variant on the protein may be a marker of severity. Variant location may have important weight in the development of targeted therapeutics.

Phenotypic and genetic spectrum of ATP6V1A encephalopathy: a disorder of lysosomal homeostasis.

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/ß 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.

Mild MDPL in a patient with a novel de novo missense variant in the Cys-B region of POLD1.

DNA polymerase δ is one of the three main enzymes responsible for DNA replication. POLD1 heterozygous missense variants in the exonuclease domain result in a cancer predisposition phenotype. In contrast, heterozygous variants in POLD1 polymerase domain have more recently been shown to be the underlying basis of the distinct autosomal dominant multisystem lipodystrophy disorder, MDPL (mandibular hypoplasia, deafness, progeroid features, and lipodystrophy syndrome OMIM # 615381), most commonly a recurrent in-frame deletion of serine at position 604, accounting for 18 of the 21 reported cases of this condition. One patient with an unusually severe phenotype has been reported, caused by a de novo c. 3209 T > A, (p.(Ile1070Asn)) variant in the highly conserved CysB motif in the C-terminal of the POLD1 protein. This region has recently been shown to bind an iron-sulphur cluster of the 4Fe-4S type. This report concerns a novel de novo missense variant in the CysB region, c.3219 G > C, (p.(Ser1073Arg)) in a male child with a milder phenotype. Using in silico analysis in the context of the recently published structure of human Polymerase δ holoenzyme, we compared these and other variants which lie in close proximity but result in differing degrees of severity and varying features. We hypothesise that the c.3219 G > C, (p.(Ser1073Arg)) substitution likely causes reduced binding of the iron-sulphur cluster without significant disruption of protein structure, while the previously reported c.3209 T > A (p.(Ile1070Asn)) variant likely has a more profound impact on structure and folding in the region. Our analysis supports a central role for the CysB region in regulating POLD1 activity in health and disease.

Defining causal variants in rare epilepsies: an essential team effort between biomedical scientists, geneticists and epileptologists.

In the last few years, with the advent of next generation sequencing (NGS), our knowledge of genes associated with monogenic epilepsies has significantly improved. NGS is also a powerful diagnostic tool for patients with epilepsy, through gene panels, exomes and genomes. This has improved diagnostic yield, reducing the time between the first seizure and a definitive molecular diagnosis. However, these developments have also increased the complexity of data interpretation, due to the large number of variants identified in a given patient and due to the phenotypic variability associated with many of the epilepsy-related genes. In this paper, we present examples of variant classification in "real life" clinic situations. We emphasize the importance of accurate phenotyping of the epilepsies including recognising variable/milder phenotypes and expansion of previously described phenotypes. There are some important issues specific to rare epilepsies - mosaicism and reduced penetrance - which affect genetic counselling. These challenges may be overcome through multidisciplinary meetings including epileptologists, pediatric neurologists, and clinical and molecular geneticists, in which every specialist learns from the others in a process which leads to for rapid and accurate diagnosis. This is an important milestone to achieve as targeted therapiesbased on the functional effects of pathogenic variants become available.

Recessive PRDM13 mutations cause fatal perinatal brainstem dysfunction with cerebellar hypoplasia and disrupt Purkinje cell differentiation.

Pontocerebellar hypoplasias (PCHs) are congenital disorders characterized by hypoplasia or early atrophy of the cerebellum and brainstem, leading to a very limited motor and cognitive development. Although over 20 genes have been shown to be mutated in PCHs, a large proportion of affected individuals remains undiagnosed. We describe four families with children presenting with severe neonatal brainstem dysfunction and pronounced deficits in cognitive and motor development associated with four different bi-allelic mutations in PRDM13, including homozygous truncating variants in the most severely affected individuals. Brain MRI and fetopathological examination revealed a PCH-like phenotype, associated with major hypoplasia of inferior olive nuclei and dysplasia of the dentate nucleus. Notably, histopathological examinations highlighted a sparse and disorganized Purkinje cell layer in the cerebellum. PRDM13 encodes a transcriptional repressor known to be critical for neuronal subtypes specification in the mouse retina and spinal cord but had not been implicated, so far, in hindbrain development. snRNA-seq data mining and in situ hybridization in humans show that PRDM13 is expressed at early stages in the progenitors of the cerebellar ventricular zone, which gives rise to cerebellar GABAergic neurons, including Purkinje cells. We also show that loss of function of prdm13 in zebrafish leads to a reduction in Purkinje cells numbers and a complete absence of the inferior olive nuclei. Altogether our data identified bi-allelic mutations in PRDM13 as causing a olivopontocerebellar hypoplasia syndrome and suggest that early deregulations of the transcriptional control of neuronal fate specification could contribute to a significant number of cases.

The EPIGENE network: A French initiative to harmonize and improve the nationwide diagnosis of monogenic epilepsies.

BACKGROUND: The EPIGENE network was created in 2014 by four multidisciplinary teams composed of geneticists, pediatric neurologists and neurologists specialized in epileptology and neurophysiology. The ambition of the network was to harmonize and improve the diagnostic strategy of Mendelian epileptic disorders using next-generation sequencing, in France. Over the years, five additional centers have joined EPIGENE and the network has been working in close collaboration, since 2018, with the French reference center for rare epilepsies (CRéER). RESULTS: Since 2014, biannual meetings have led to the design of four successive versions of a monogenic epilepsy gene panel (PAGEM), increasing from 68 to 144 genes. A total of 4035 index cases with epileptic disorders have been analyzed with a diagnostic yield of 31% (n = 1265/4035). The top 10 genes, SCN1A, KCNQ2, STXBP1, SCN2A, SCN8A, PRRT2, PCDH19, KCNT1, SYNGAP1, and GRIN2A, account for one-sixth of patients and half of the diagnoses provided by the PAGEM. CONCLUSION: These results suggest that a gene-panel approach is an efficient first-tier test for the genetic diagnosis of Mendelian epileptic disorders. In a near future, French patients with "drug-resistant epilepsies with seizure-onset in the first two-years of life" can benefit from whole-genome sequencing (WGS), as a second line genetic screening with the implementation of the 2025 French Genomic Medicine Plan. The EPIGENE network has also promoted scientific collaborations on genetic epilepsies within CRéER.

Adaptive behavior and psychiatric comorbidities in KCNB1 encephalopathy.

AIM: KCNB1 encephalopathy encompasses a broad phenotypic spectrum associating intellectual disability, behavioral disturbances, and epilepsies of various severity. Using standardized parental questionnaires, we aimed to capture the heterogeneity of the adaptive and behavioral features in a series of patients with KCNB1 pathogenic variants. METHODS: We included 25 patients with a KCNB1 encephalopathy, aged from 3.2 to 34.1 years (median = 10 years). Adaptive functioning was assessed in all patients using the French version of the Vineland Adaptive Behavior Scales, Second Edition (VABS-II) questionnaire. We screened global behavior with the Childhood Behavioral Check-List (CBCL, Achenbach) and autism spectrum disorder (ASD) with the Social Communication Questionnaire (SCQ). We used a cluster analysis to identify subgroups of adaptive profiles. RESULTS: VABS-II questionnaire showed pathological adaptive behavior in all participants with a severity of adaptive deficiency ranging from mild in 8/20 to severe in 7/20. Eight out of 16 were at risk of Attention Problems at the CBCL and 13/18 were at risk of autism spectrum disorder (ASD). The adaptive behavior composite score significantly decreased with age (Spearman's Rho=-0.72, p<0.001) but not the equivalent ages, suggesting stagnation and slowing but no regression over time. The clustering analysis identified two subgroups of patients, one showing more severe adaptive behavior. The severity of the epilepsy phenotype predicted the severity of the behavioral profile with a sensitivity of 70% and a specificity of 90.9%. CONCLUSION: This study confirms the deleterious consequences of early-onset epilepsy in addition to the impact of the gene dysfunction in patients with KCNB1 encephalopathy. ASD and attention disorders are frequent. Parental questionnaires should be considered as useful tools for early screening and care adaptation.

MYT1L-associated neurodevelopmental disorder: description of 40 new cases and literature review of clinical and molecular aspects.

Pathogenic variants of the myelin transcription factor-1 like (MYT1L) gene include heterozygous missense, truncating variants and 2p25.3 microdeletions and cause a syndromic neurodevelopmental disorder (OMIM#616,521). Despite enrichment in de novo mutations in several developmental disorders and autism studies, the data on clinical characteristics and genotype-phenotype correlations are scarce, with only 22 patients with single nucleotide pathogenic variants reported. We aimed to further characterize this disorder at both the clinical and molecular levels by gathering a large series of patients with MYT1L-associated neurodevelopmental disorder. We collected genetic information on 40 unreported patients with likely pathogenic/pathogenic MYT1L variants and performed a comprehensive review of published data (total = 62 patients). We confirm that the main phenotypic features of the MYT1L-related disorder are developmental delay with language delay (95%), intellectual disability (ID, 70%), overweight or obesity (58%), behavioral disorders (98%) and epilepsy (23%). We highlight novel clinical characteristics, such as learning disabilities without ID (30%) and feeding difficulties during infancy (18%). We further describe the varied dysmorphic features (67%) and present the changes in weight over time of 27 patients. We show that patients harboring highly clustered missense variants in the 2-3-ZNF domains are not clinically distinguishable from patients with truncating variants. We provide an updated overview of clinical and genetic data of the MYT1L-associated neurodevelopmental disorder, hence improving diagnosis and clinical management of these patients.