Grazia Maria Simonetta Mancini

Charcot-Marie-Tooth (CMT) disease is a clinically and genetically heterogeneous group of hereditary neuropathies. Despite progress in genetic sequencing, for around a quarter of patients the disease has lacked a genetic explanation. Here, we identified 16 recessive variants in the RhoGTPase activating protein 19 gene ( ARHGAP19 ) causing motor-predominant neuropathy in 25 individuals from 20 unrelated families. The ARHGAP19 protein acts as a negative regulator of the RhoA GTPase. In vitro biochemical and cellular assays revealed that patient variants impair the GTPase-activating protein (GAP) activity of ARHGAP19 and reduce ARHGAP19 protein levels. Through the use of patient lines, in vitro GAP assays and in silico molecular modeling, we provided evidence that CMT-associated ARHGAP19 variants act through a loss-of-function (LOF) mechanism. LOF in ARHGAP19 orthologues in Drosophila melanogaster and Danio rerio induced motor defects in axonal and synaptic morphology. Similar cellular phenotypes were observed in ARHGAP19 patient-derived motoneurons. Transcriptomic studies further demonstrated that ARHGAP19 regulates cellular pathways associated with motor proteins and the cell cycle. Taken together, our findings establish ARHGAP19 variants as a cause of inherited neuropathy acting through a LOF mechanism.

Individuals with monoallelic gain-of-function variants in the histone lysine methyltransferase DOT1L display global developmental delay and varying congenital anomalies. However, the impact of monoallelic loss of DOT1L remains unclear. Here, we sought to define the effects of partial DOT1L loss by applying bulk and single-nucleus RNA-sequencing, ChIP-sequencing, imaging, multielectrode array recordings and behavioural analysis of zebrafish and multiple mouse models. We present a cohort of 16 individuals (12 females, 4 males) with neurodevelopmental disorders and monoallelic DOT1L variants, including a frameshift deletion, an in-frame deletion, a nonsense, and missense variants clustered in the catalytic domain. We demonstrate that specific variants cause loss of methyltransferase activity. In primary cortical neurons, Dot1l knockdown disrupts transcription of synaptic genes, neuron branching, expression of a synaptic protein and neuronal activity. Further in the cortex of heterozygous Dot1l mice, Dot1l loss causes sex-specific transcriptional responses and H3K79me2 depletion, including within downregulated genes. Lastly, using both zebrafish and mouse models, we found behavioural disruptions that include developmental deficits and sex-specific social behavioural changes. Overall, we define how DOT1L loss leads to neurological dysfunction by demonstrating that partial Dot1l loss impacts neuronal transcription, neuron morphology and behaviour across multiple models and systems.

Brain malformations represent a heterogeneous group of abnormalities of neural morphogenesis, often associated with aberrations of neuronal connectivity and brain volume. Prenatal detection of brain malformations requires a clear understanding of embryology and developmental morphology through the various stages of gestation. This expert panel review is written with the central aim of providing an easy-to-understand road map to improve prenatal detection and characterization of structural malformations based on the current understanding of normal and aberrant brain development. For every developmental stage, the utility of each available neuroimaging modality, including prenatal multiplanar neuro sonography, anatomical MRI and advanced MRI techniques, as well as further insights from post-mortem imaging, has been highlighted.

SLC4A10 is a plasma-membrane bound transporter that utilizes the Na⁺ gradient to drive cellular HCO₃⁻ uptake, thus mediating acid extrusion. In the mammalian brain, SLC4A10 is expressed in principal neurons and interneurons, as well as in epithelial cells of the choroid plexus, the organ regulating the production of CSF. Using next generation sequencing on samples from five unrelated families encompassing nine affected individuals, we show that biallelic SLC4A10 loss-of-function variants cause a clinically recognizable neurodevelopmental disorder in humans. The cardinal clinical features of the condition include hypotonia in infancy, delayed psychomotor development across all domains and intellectual impairment. Affected individuals commonly display traits associated with autistic spectrum disorder including anxiety, hyperactivity and stereotyped movements. In two cases isolated episodes of seizures were reported in the first few years of life, and a further affected child displayed bitemporal epileptogenic discharges on EEG without overt clinical seizures. While occipitofrontal circumference was reported to be normal at birth, progressive postnatal microcephaly evolved in 7 out of 10 affected individuals. Neuroradiological features included a relative preservation of brain volume compared to occipitofrontal circumference, characteristic narrow sometimes ‘slit-like’ lateral ventricles and corpus callosum abnormalities. Slc4a10^−/− mice, deficient for SLC4A10, also display small lateral brain ventricles and mild behavioural abnormalities including delayed habituation and alterations in the two-object novel object recognition task. Collapsed brain ventricles in both Slc4a10^−/− mice and affected individuals suggest an important role of SLC4A10 in the production of the CSF. However, it is notable that despite diverse roles of the CSF in the developing and adult brain, the cortex of Slc4a10^−/− mice appears grossly intact. Co-staining with synaptic markers revealed that in neurons, SLC4A10 localizes to inhibitory, but not excitatory, presynapses. These findings are supported by our functional studies, which show the release of the inhibitory neurotransmitter GABA is compromised in Slc4a10^−/− mice, while the release of the excitatory neurotransmitter glutamate is preserved. Manipulation of intracellular pH partially rescues GABA release. Together our studies define a novel neurodevelopmental disorder associated with biallelic pathogenic variants in SLC4A10 and highlight the importance of further analyses of the consequences of SLC4A10 loss-of-function for brain development, synaptic transmission and network properties.

BACKGROUND AND PURPOSE: Medullary tegmental cap dysplasia is a rare brainstem malformation, first described and defined by James Barkovich in his book Pediatric Neuroimaging from 2005 as an anomalous mass protruding from the posterior medullary surface. We describe the neuroimaging, clinical, postmortem, and genetic findings defining this unique malformation. MATERIALS AND METHODS: This is a multicenter, international, retrospective study. We assessed the patients’ medical records, prenatal ultrasounds, MR images, genetic findings, and postmortem results. We reviewed the medical literature for all studies depicting medullary malformations and evaluated cases in which a dorsal medullary protuberance was described. RESULTS: We collected 13 patients: 3 fetuses and 10 children. The medullary caps had multiple characteristics. Associated brain findings were a rotated position of the medulla, a small and flat pons, cerebellar anomalies, a molar tooth sign, and agenesis of the corpus callosum. Systemic findings included the following: polydactyly, hallux valgus, large ears, and coarse facies. Postmortem analysis in 3 patients revealed that the cap contained either neurons or white matter tracts. We found 8 publications describing a dorsal medullary protuberance in 27 patients. The syndromic diagnosis was Joubert-Boltshauser syndrome in 11 and fibrodysplasia ossificans progressiva in 14 patients. CONCLUSIONS: This is the first study to describe a series of 13 patients with medullary tegmental cap dysplasia. The cap has different shapes: distinct in Joubert-Boltshauser syndrome and fibrodysplasia ossificans progressive. Due to the variations in the clinical, imaging, and postmortem findings, we conclude that there are multiple etiologies and pathophysiology. We suggest that in some patients, the pathophysiology might be abnormal axonal guidance.

Biallelic loss-of-function variants in SMPD4 cause a rare and severe neurodevelopmental disorder with progressive congenital microcephaly and early death. SMPD4 encodes a sphingomyelinase that hydrolyses sphingomyelin into ceramide at neutral pH and can thereby affect membrane lipid homeostasis. SMPD4 localizes to the membranes of the endoplasmic reticulum and nuclear envelope and interacts with nuclear pore complexes (NPC). We refine the clinical phenotype of loss-of-function SMPD4 variants by describing five individuals from three unrelated families with longitudinal data due to prolonged survival. All individuals surviving beyond infancy developed insulin-dependent diabetes, besides presenting with a severe neurodevelopmental disorder and microcephaly, making diabetes one of the most frequent age-dependent non-cerebral abnormalities. We studied the function of SMPD4 at the cellular and organ levels. Knock-down of SMPD4 in human neural stem cells causes reduced proliferation rates and prolonged mitosis. Moreover, SMPD4 depletion results in abnormal nuclear envelope breakdown and reassembly during mitosis and decreased post-mitotic NPC insertion. Fibroblasts from affected individuals show deficient SMPD4-specific neutral sphingomyelinase activity, without changing (sub)cellular lipidome fractions, which suggests a local function of SMPD4 on the nuclear envelope. In embryonic mouse brain, knockdown of Smpd4 impairs cortical progenitor proliferation and induces premature differentiation by altering the balance between neurogenic and proliferative progenitor cell divisions. We hypothesize that, in individuals with SMPD4-related disease, nuclear envelope bending, which is needed to insert NPCs in the nuclear envelope, is impaired in the absence of SMPD4 and interferes with cerebral corticogenesis and survival of pancreatic beta cells.

Background: For neurodevelopmental disorders (NDD), a molecular diagnosis is key for predicting outcome, treatment and genetic counseling. Currently, in about half of NDD cases, routine DNA-based testing fails to establish a genetic diagnosis. Transcriptome analysis (RNA-seq) improves the diagnostic yield for some groups of diseases, but has not been applied to NDD in a routine diagnostic setting. Methods: Here, we explored the diagnostic potential of RNA-seq in a cohort of 96 individuals including 67 undiagnosed NDD subjects. We created a user-friendly web-application to analyze RNA-seq data from single individuals’ cultured skin fibroblasts for genic, exonic and intronic expression outliers, based on modified OUTRIDER Z-scores. Candidate pathogenic events were complemented/matched with genomic data and, if required, confirmed with additional functional assays. Results: We identified pathogenic small genomic deletions, mono-allelic expression, aberrant splicing events, deep intronic variants resulting in pseudo-exon insertion, but also synonymous and nonsynonymous variants with deleterious effects on transcription. This approach increased the diagnostic yield for NDD by 12%. Diagnostic pitfalls during transcriptome analysis include detection of splice abnormalities in putative disease genes caused by benign polymorphisms and/or absence of expression of the responsible gene in the tissue of choice. This was misleading in one case and could have led to the wrong diagnosis in the absence of appropriate phenotyping. Conclusions: Nonetheless, our results demonstrate the utility of RNA-seq in molecular diagnostics and stress the importance of multidisciplinary team consultation. In particular, the approach is useful for the identification and interpretation of unexpected pathogenic changes in mRNA processing and expression in NDD.

Background: Malformations of cortical development (MCDs) have been reported in a subset of patients with pathogenic heterozygous variants in GRIN1 or GRIN2B, genes which encode for subunits of the N-methyl-D-aspartate receptor (NMDAR). The aim of this study was to further define the phenotypic spectrum of NMDAR-related MCDs. Methods: We report the clinical, radiological and molecular features of 7 new patients and review data on 18 previously reported individuals with NMDAR-related MCDs. Neuropathological findings for two individuals with heterozygous variants in GRIN1 are presented. We report the clinical and neuropathological features of one additional individual with homozygous pathogenic variants in GRIN1. Results: Heterozygous variants in GRIN1 and GRIN2B were associated with overlapping severe clinical and imaging features, including global developmental delay, epilepsy, diffuse dysgyria, dysmorphic basal ganglia and hippocampi. Neuropathological examination in two fetuses with heterozygous GRIN1 variants suggests that proliferation as well as radial and tangential neuronal migration are impaired. In addition, we show that neuronal migration is also impaired by homozygous GRIN1 variants in an individual with microcephaly with simplified gyral pattern. Conclusion: These findings expand our understanding of the clinical and imaging features of the ‘NMDARopathy’ spectrum and contribute to our understanding of the likely underlying pathogenic mechanisms leading to MCD in these patients.