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Around 60% of individuals with neurodevelopmental disorders (NDD) remain undiagnosed after comprehensive genetic testing, primarily of protein-coding genes 1 . 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 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 spliceosome 2 . We identify an 18 base pair 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 115 individuals with NDD. Most individuals (77.4%) have the same highly recurrent single base insertion (n.64_65insT). In 54 individuals in whom it could be determined, the de novo variants were all on the maternal allele. We demonstrate that RNU4-2 is highly expressed in the developing human brain, in contrast to RNU4-1 and other U4 homologues. Using RNA sequencing, we show how 5' splice-site use is systematically disrupted in individuals with RNU4-2 variants, consistent with the known role of this region during spliceosome activation. Finally, we estimate that variants in this 18 base pair region explain 0.4% of individuals with NDD. This work underscores the importance of non-coding genes in rare disorders and will provide a diagnosis to thousands of individuals with NDD worldwide., (© 2024. The Author(s).)

Alternating hemiplegia of childhood (AHC) is a rare neurodevelopment disorder that is typically characterized by debilitating episodic attacks of hemiplegia, seizures, and intellectual disability. Over 85% of individuals with AHC have a de novo missense variant in ATP1A3 encoding the catalytic α3 subunit of neuronal Na +/ K + ATPases. The remainder of the patients are genetically unexplained. Here, we used next-generation sequencing to search for the genetic cause of 26 ATP1A3-negative index patients with a clinical presentation of AHC or an AHC-like phenotype. Three patients had affected siblings. Using targeted sequencing of exonic, intronic, and flanking regions of ATP1A3 in 22 of the 26 index patients, we found no ultra-rare variants. Using exome sequencing, we identified the likely genetic diagnosis in 9 probands (35%) in five genes, including RHOBTB2 (n = 3), ATP1A2 (n = 3), ANK3 (n = 1), SCN2A (n = 1), and CHD2 (n = 1). In follow-up investigations, two additional ATP1A3-negative individuals were found to have rare missense SCN2A variants, including one de novo likely pathogenic variant and one likely pathogenic variant for which inheritance could not be determined. Functional evaluation of the variants identified in SCN2A and ATP1A2 supports the pathogenicity of the identified variants. Our data show that genetic variants in various neurodevelopmental genes, including SCN2A, lead to AHC or AHC-like presentation. Still, the majority of ATP1A3-negative AHC or AHC-like patients remain unexplained, suggesting that other mutational mechanisms may account for the phenotype or that cases may be explained by oligo- or polygenic risk factors., (© 2023. The Author(s), under exclusive licence to European Society of Human Genetics.)

BRAT1 biallelic variants are associated with rigidity and multifocal seizure syndrome, lethal neonatal (RMFSL), and neurodevelopmental disorder associating cerebellar atrophy with or without seizures syndrome (NEDCAS). To date, forty individuals have been reported in the literature. We collected clinical and molecular data from 57 additional cases allowing us to study a large cohort of 97 individuals and draw phenotype-genotype correlations. Fifty-nine individuals presented with BRAT1-related RMFSL phenotype. Most of them had no psychomotor acquisition (100%), epilepsy (100%), microcephaly (91%), limb rigidity (93%), and died prematurely (93%). Thirty-eight individuals presented a non-lethal phenotype of BRAT1-related NEDCAS phenotype. Seventy-six percent of the patients in this group were able to walk and 68% were able to say at least a few words. Most of them had cerebellar ataxia (82%), axial hypotonia (79%) and cerebellar atrophy (100%). Genotype-phenotype correlations in our cohort revealed that biallelic nonsense, frameshift or inframe deletion/insertion variants result in the severe BRAT1-related RMFSL phenotype (46/46; 100%). In contrast, genotypes with at least one missense were more likely associated with NEDCAS (28/34; 82%). The phenotype of patients carrying splice variants was variable: 41% presented with RMFSL (7/17) and 59% with NEDCAS (10/17)., (© 2023. The Author(s), under exclusive licence to European Society of Human Genetics.)

Hereditary spastic paraplegias (HSP) are heterogeneous disorders, with more than 70 causative genes. Variants in SPAST are the most frequent genetic etiology and are responsible for spastic paraplegia type 4 (SPG4). Age at onset can vary, even between patients from the same family, and incomplete penetrance is described. Somatic mosaicism is extremely rare with only three patients reported in the literature. We report here SPAST mosaic variants in four unrelated patients. We confirm that mosaicism in SPAST is a very rare event with only four identified cases on more than 300 patients with a SPAST variant previously described by our clinical diagnostic laboratory., (© 2021. The Author(s), under exclusive licence to European Society of Human Genetics.)

Cause of complex dyskinesia remains elusive in some patients. A homozygous missense variant leading to drastic decrease of PDE2A enzymatic activity was reported in one patient with childhood-onset choreodystonia preceded by paroxysmal dyskinesia and associated with cognitive impairment and interictal EEG abnormalities. Here, we report three new cases with biallelic PDE2A variants identified by trio whole-exome sequencing. Mitochondria network was analyzed after Mitotracker™ Red staining in control and mutated primary fibroblasts. Analysis of retrospective video of patients' movement disorder and refinement of phenotype was carried out. We identified a homozygous gain of stop codon variant c.1180C>T; p.(Gln394*) in PDE2A in siblings and compound heterozygous variants in young adult: a missense c.446C>T; p.(Pro149Leu) and splice-site variant c.1922+5G>A predicted and shown to produce an out of frame transcript lacking exon 22. All three patients had cognitive impairment or developmental delay. The phenotype of the two oldest patients, aged 9 and 26, was characterized by childhood-onset refractory paroxysmal dyskinesia initially misdiagnosed as epilepsy due to interictal EEG abnormalities. The youngest patient showed a proven epilepsy at the age of 4 months and no paroxysmal dyskinesia at 15 months. Interestingly, analysis of the fibroblasts with the biallelic variants in PDE2A variants revealed mitochondria network morphology changes. Together with previously reported case, our three patients confirm that biallelic PDE2A variants are a cause of childhood-onset refractory paroxysmal dyskinesia with cognitive impairment, sometimes associated with choreodystonia and interictal baseline EEG abnormalities or epilepsy.

TBR1, a T-box transcription factor expressed in the cerebral cortex, regulates the expression of several candidate genes for autism spectrum disorders (ASD). Although TBR1 has been reported as a high-confidence risk gene for ASD and intellectual disability (ID) in functional and clinical reports since 2011, TBR1 has only recently been recorded as a human disease gene in the OMIM database. Currently, the neurodevelopmental disorders and structural brain anomalies associated with TBR1 variants are not well characterized. Through international data sharing, we collected data from 25 unreported individuals and compared them with data from the literature. We evaluated structural brain anomalies in seven individuals by analysis of MRI images, and compared these with anomalies observed in TBR1 mutant mice. The phenotype included ID in all individuals, associated to autistic traits in 76% of them. No recognizable facial phenotype could be identified. MRI analysis revealed a reduction of the anterior commissure and suggested new features including dysplastic hippocampus and subtle neocortical dysgenesis. This report supports the role of TBR1 in ID associated with autistic traits and suggests new structural brain malformations in humans. We hope this work will help geneticists to interpret TBR1 variants and diagnose ASD probands.

Determining pathogenicity of genomic variation identified by next-generation sequencing techniques can be supported by recurrent disruptive variants in the same gene in phenotypically similar individuals. However, interpretation of novel variants in a specific gene in individuals with mild-moderate intellectual disability (ID) without recognizable syndromic features can be challenging and reverse phenotyping is often required. We describe 24 individuals with a de novo disease-causing variant in, or partial deletion of, the F-box only protein 11 gene (FBXO11, also known as VIT1 and PRMT9). FBXO11 is part of the SCF (SKP1-cullin-F-box) complex, a multi-protein E3 ubiquitin-ligase complex catalyzing the ubiquitination of proteins destined for proteasomal degradation. Twenty-two variants were identified by next-generation sequencing, comprising 2 in-frame deletions, 11 missense variants, 1 canonical splice site variant, and 8 nonsense or frameshift variants leading to a truncated protein or degraded transcript. The remaining two variants were identified by array-comparative genomic hybridization and consisted of a partial deletion of FBXO11. All individuals had borderline to severe ID and behavioral problems (autism spectrum disorder, attention-deficit/hyperactivity disorder, anxiety, aggression) were observed in most of them. The most relevant common facial features included a thin upper lip and a broad prominent space between the paramedian peaks of the upper lip. Other features were hypotonia and hyperlaxity of the joints. We show that de novo variants in FBXO11 cause a syndromic form of ID. The current series show the power of reverse phenotyping in the interpretation of novel genetic variances in individuals who initially did not appear to have a clear recognizable phenotype.

Epilepsy is a neurological condition that affects 1% of the world population. Conventional treatments of epilepsy use drugs targeting neuronal excitability, inhibitory or excitatory transmission. Yet, one third of patients presents an intractable form of epilepsy and fails to respond to pharmacological anti-epileptic strategies. The ketogenic diet is a well-established non-pharmacological treatment that has been proven to be effective in reducing seizure frequency in the pharmaco-resistant patients. This dietary solution is however extremely restrictive and can be associated with complications caused by the high [fat]:[carbohydrate + protein] ratio. Recent advances suggest that the traditional 4:1 ratio of the ketogenic diet is not a requisite for its therapeutic effect. We show here that combining nutritional strategies targeting specific amino-acids, carbohydrates and fatty acids with a low [fat]:[proteins + carbohydrates] ratio also reduces excitatory drive and protects against seizures to the same extent as the ketogenic diet. Similarly, the morphological and molecular correlates of temporal lobe seizures were reduced in animals fed with the combined diet. These results provide evidence that low-fat dietary strategies more palatable than the ketogenic diet could be useful in epilepsy.

To unravel missing genetic causes underlying monogenic disorders with recurrence in sibling, we explored the hypothesis of parental germline mosaic mutations in familial forms of malformation of cortical development (MCD). Interestingly, four families with parental germline variants, out of 18, were identified by whole-exome sequencing (WES), including a variant in a new candidate gene, syntaxin 7. In view of this high frequency, revision of diagnostic strategies and reoccurrence risk should be considered not only for the recurrent forms, but also for the sporadic cases of MCD.

Variants in the WD repeat 45 (WDR45) gene in human Xp11.23 have recently been identified in patients suffering from neurodegeneration with brain iron accumulation, a genetically and phenotypically heterogeneous condition. WDR45 variants cause a childhood-onset encephalopathy accompanied by neurodegeneration in adulthood and iron accumulation in the basal ganglia. They have been almost exclusively found in females, and male lethality was suggested. Here we describe a male patient suffering from a severe and early neurological phenotype, initially presenting early-onset epileptic spasms in clusters associated with an abnormal interictal electroencephalography showing slow background activity, large amplitude asynchronous spikes and abnormal neurological development. This patient is a carrier of a 19.9-kb microdeletion in Xp11.23 containing three genes, including WDR45. These findings reveal that males with WDR45 deletions are viable, and can present with early-onset epileptic encephalopathy without brain iron accumulation.

Prenatal betamethasone (Celestene) therapy reduces the incidence of brain damage, whereas prenatal or neonatal dexamethasone (Soludecadron) increases the risk of brain lesions or neuromotor deficits. To determine whether this increase is ascribable to the sulfites used as preservatives in Soludecadron, we investigated the effects of 12 h of exposure to pure dexamethasone, Soludecadron, pure betamethasone, Celestene, and sulfites on in vitro and in vivo death of neurons cultured under basal conditions or with excitotoxic agents (N-methyl-D-aspartate or (S)-5-bromowillardiine) or hypoxia. Apoptotic features were quantitated using a fluorescent chromatin stain (Hoechst 33258). Neuronal viability was unaffected by pure dexamethasone, pure betamethasone, or Celestene. Soludecadron or sulfites significantly increased neuronal loss. Pure dexamethasone or pure betamethasone produced a 40-50% decrease in neuronal death induced by N-methyl-D-aspartate, (S)-5-bromowillardiine, or hypoxia, whereas Soludecadron had no effect and sulfites significantly increased the neurotoxicity of excitotoxic agents. In in vivo experiments involving terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling after several i.p. injections of fluorinated glucocorticoids, Soludecadron, but not pure dexamethasone, significantly increased the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-stained cells in neocortical layers and thalamus. These experimental findings suggest that injectable dexamethasone should be used with caution during the perinatal period.

Elevated mean IL-9 serum levels have been observed in human neonates who will later develop cerebral palsy. In earlier studies, using a newborn mouse model of excitotoxic lesions mimicking those described in human cerebral palsy, we found that IL-9 pretreatment exacerbated brain damage produced by intracerebral injections of the glutamatergic analog ibotenate. Among its different cell targets, the Th2 cytokine IL-9 is a mast cell growth and differentiation factor that can cause mast cells to release various substances including histamine. In the present study, we sought to determine whether the deleterious effects of IL-9 in our mouse model were mediated by mast cells through histamine release. All mouse pups were pretreated with intraperitoneal injections of IL-9 or saline between postnatal days (P) P1 and P5. Immunohistochemistry for murine mast cell protease-1 performed on P5 showed an increased density of labeled cells in the neopallium of IL-9-treated Swiss pups as compared with controls. Western blot analysis confirmed the increased murine mast cell protease-1 brain content of IL-9-treated Swiss mice. IL-9 pretreatment had no significant effect on ibotenate-induced excitotoxic brain lesions in mast cell-deficient P5 pups (WBB6F1/J kit(W/W-v)), whereas IL-9 exacerbated these lesions in the control littermates with normal mast cell populations. Finally, cromoglycate or antihistamine drugs significantly reduced ibotenate-induced brain lesions in IL-9-treated Swiss pups. Taken together, these data suggest that recruitment of cerebral mast cells with histamine release may contribute to the exacerbation of neonatal excitotoxic brain lesions produced by IL-9. Neuroprotective strategies targeting mast cells may be useful in some neonates at risk for cerebral palsy.

Neuronal migration appears as a complex ontogenic step occurring early during embryonic and fetal development. Control of neuronal migration involves different cell populations including Cajal-Retzius neurons, subplate neurons, neuronal precursors or radial glia. The integrity of multiple molecular mechanisms, such as cell cycle control, cell-cell adhesion, interaction with extracellular matrix protein, neurotransmitter release, growth factor availability, platelet-activating factor degradation or transduction pathways seems to be critical for normal neuronal migration. The complexity and the multiplicity of these mechanisms probably explain the clinical, radiologic and genetic heterogeneity of human disorders of neuronal migration. The present review will be focused on mechanisms and disturbances of migration of neurons destined to the neocortex. New insights gained from the analysis of animal models as well as from the study of human diseases will be included.