Biallelic null variants in PNPLA8 cause microcephaly by reducing the number of basal radial glia.

Patatin-like phospholipase domain-containing lipase 8 (PNPLA8), one of the calcium-independent phospholipase A2 enzymes, is involved in various physiological processes through the maintenance of membrane phospholipids. Biallelic variants in PNPLA8 have been associated with a range of paediatric neurodegenerative disorders. However, the phenotypic spectrum, genotype-phenotype correlations and the underlying mechanisms are poorly understood. Here, we newly identified 14 individuals from 12 unrelated families with biallelic ultra-rare variants in PNPLA8 presenting with a wide phenotypic spectrum of clinical features. Analysis of the clinical features of current and previously reported individuals (25 affected individuals across 20 families) showed that PNPLA8-related neurological diseases manifest as a continuum ranging from variable developmental and/or degenerative epileptic-dyskinetic encephalopathy to childhood-onset neurodegeneration. We found that complete loss of PNPLA8 was associated with the more profound end of the spectrum, with congenital microcephaly. Using cerebral organoids generated from human induced pluripotent stem cells, we found that loss of PNPLA8 led to developmental defects by reducing the number of basal radial glial cells and upper-layer neurons. Spatial transcriptomics revealed that loss of PNPLA8 altered the fate specification of apical radial glial cells, as reflected by the enrichment of gene sets related to the cell cycle, basal radial glial cells and neural differentiation. Neural progenitor cells lacking PNPLA8 showed a reduced amount of lysophosphatidic acid, lysophosphatidylethanolamine and phosphatidic acid. The reduced number of basal radial glial cells in patient-derived cerebral organoids was rescued, in part, by the addition of lysophosphatidic acid. Our data suggest that PNPLA8 is crucial to meet phospholipid synthetic needs and to produce abundant basal radial glial cells in human brain development.

Potential clinical applications of advanced genomic analysis in cerebral palsy.

Cerebral palsy (CP) has historically been attributed to acquired insults, but emerging research suggests that genetic variations are also important causes of CP. While microarray and whole-exome sequencing based studies have been the primary methods for establishing new CP-gene relationships and providing a genetic etiology for individual patients, the cause of their condition remains unknown for many patients with CP. Recent advancements in genomic technologies offer additional opportunities to uncover variations in human genomes, transcriptomes, and epigenomes that have previously escaped detection. In this review, we outline the use of these state-of-the-art technologies to address the molecular diagnostic challenges experienced by individuals with CP. We also explore the importance of identifying a molecular etiology whenever possible, given the potential for genomic medicine to provide opportunities to treat patients with CP in new and more precise ways.

Development and validation of a stakeholder-driven, self-contained electronic informed consent platform for trio-based genomic research studies.

Increasingly long and complex informed consents have yielded studies demonstrating comparatively low participant comprehension and satisfaction with traditional face-to-face approaches. In parallel, interest in electronic consents for clinical and research genomics has steadily increased, yet limited data are available for trio-based genomic discovery studies. We describe the design, development, implementation, and validation of an electronic iConsent application for trio-based genomic research deployed to support genomic studies of cerebral palsy. iConsent development incorporated stakeholder perspectives including researchers, patient advocates, institutional review board members, and genomic data-sharing considerations. The iConsent platform integrated principles derived from prior electronic consenting research and elements of multimedia learning theory. Participant comprehension was assessed in an interactive teachback format. The iConsent application achieved nine of ten proposed desiderata for effective patient-focused electronic consenting for genomic research. Overall, participants demonstrated high comprehension and retention of key human subjects' considerations. Enrollees reported high levels of satisfaction with the iConsent, and we found that participant comprehension, iConsent clarity, privacy protections, and study goal explanations were associated with overall satisfaction. Although opportunities exist to optimize iConsent, we show that such an approach is feasible, can satisfy multiple stakeholder requirements, and can realize high participant satisfaction and comprehension while increasing study reach.

Biallelic NDC1 variants that interfere with ALADIN binding are associated with neuropathy and triple A-like syndrome.

Nuclear pore complexes (NPCs) regulate nucleocytoplasmic transport and are anchored in the nuclear envelope by the transmembrane nucleoporin NDC1. NDC1 is essential for post-mitotic NPC assembly and the recruitment of ALADIN to the nuclear envelope. While no human disorder has been associated to one of the three transmembrane nucleoporins, biallelic variants in AAAS, encoding ALADIN, cause triple A syndrome (Allgrove syndrome). Triple A syndrome, characterized by alacrima, achalasia, and adrenal insufficiency, often includes progressive demyelinating polyneuropathy and other neurological complaints. In this report, diagnostic exome and/or RNA sequencing was performed in seven individuals from four unrelated consanguineous families with AAAS-negative triple A syndrome. Molecular and clinical studies followed to elucidate the pathogenic mechanism. The affected individuals presented with intellectual disability, motor impairment, severe demyelinating with secondary axonal polyneuropathy, alacrima, and achalasia. None of the affected individuals has adrenal insufficiency. All individuals presented with biallelic NDC1 in-frame deletions or missense variants that affect amino acids and protein domains required for ALADIN binding. No other significant variants associated with the phenotypic features were reported. Skin fibroblasts derived from affected individuals show decreased recruitment of ALADIN to the NE and decreased post-mitotic NPC insertion, confirming pathogenicity of the variants. Taken together, our results implicate biallelic NDC1 variants in the pathogenesis of polyneuropathy and a triple A-like disorder without adrenal insufficiency, by interfering with physiological NDC1 functions, including the recruitment of ALADIN to the NPC.

Refining the phenotype of SINO syndrome: A comprehensive cohort report of 14 novel cases.

BACKGROUND: SINO syndrome (Spastic paraplegia, Intellectual disability, Nystagmus and Obesity) is a rare autosomal dominant condition caused by heterozygous variants in KIDINS220. A total of 12 individuals are reported, comprising eight with SINO and four with an autosomal recessive condition attributed to bi-allelic KIDINS220 variants. METHODS: In our international cohort, we have comprised 14 individuals, carrying 13 novel pathogenic KIDINS220 variants in heterozygous form. We assessed clinical and molecular data of our cohort and previously reported individuals and based on functional experiments reached a better understanding of the pathogenesis behind KIDINS220-related disease. RESULTS: Using fetal tissue and in vitro assays, we demonstrate that the variants generate KIDINS220 truncated forms that mislocalize in punctate intracellular structures, with decreased levels of the full-length protein, suggesting a trans-dominant negative effect. 92% had their diagnosis within three years, with symptoms of developmental delay, spasticity, hypotonia, lack of eye contact and nystagmus. We identified a KIDINS220 variant associated with fetal hydrocephalus and show that 58% of examined individuals present brain ventricular dilatation. We extend the phenotypic spectrum of SINO syndrome to behavioral manifestations not previously highlighted. CONCLUSION: Our study provides further insights into the clinical spectrum, etiology and predicted functional impact of KIDINS220 variants.

Antibodies Against ZSCAN1 in Pediatric and Adult Patients With Non-Paraneoplastic ROHHAD Syndrome.

OBJECTIVES: To report the association of zinc finger and SCAN domain containing 1 antibodies (ZSCAN1-abs) with rapid-onset obesity, hypothalamic dysfunction, hypoventilation, and autonomic dysregulation (ROHHAD) syndrome in patients without tumor. METHODS: Patients with symptoms compatible with ROHHAD syndrome but without an associated tumor were selected from our database. Serum and CSF samples were examined for the presence of ZSCAN1-abs by an in-house cell-based assay. In addition, samples from 149 patients with several inflammatory and noninflammatory disorders and 50 healthy participants served as controls. RESULTS: Thirteen patients with ROHHAD syndrome were identified. Of these, we had paired serum/CSF samples from 6 patients and only serum from the other 7. Five of 6 patients (83.3%) with paired serum/CSF (4 children, 1 adult) had ZSCAN-abs only in CSF and 1 had antibodies in serum and CSF. ZSCAN1-abs were not detected in the remaining 7 patients with ROHHAD with only serum available or in any of the 199 control samples. DISCUSSION: Patients with ROHHAD syndrome should be investigated for the presence of ZSCAN1-abs in CSF. The antibodies do not necessarily predict the presence of a tumor. The detection of ZSCAN1-abs in an adult patient suggests that this condition also occurs beyond the pediatric age.

Motor network dynamic resting state fMRI connectivity of neurotypical children in regions affected by cerebral palsy.

Background: Normative childhood motor network resting-state fMRI effective connectivity is undefined, yet necessary for translatable dynamic resting-state-network-informed evaluation in pediatric cerebral palsy. Methods: Cross-spectral dynamic causal modeling of resting-state-fMRI was investigated in 50 neurotypically developing 5- to 13-year-old children. Fully connected six-node network models per hemisphere included primary motor cortex, striatum, subthalamic nucleus, globus pallidus internus, thalamus, and contralateral cerebellum. Parametric Empirical Bayes with exhaustive Bayesian model reduction and Bayesian modeling averaging informed the model; Purdue Pegboard Test scores of hand motor behavior were the covariate at the group level to determine the effective-connectivity-functional behavior relationship. Results: Although both hemispheres exhibited similar effective connectivity of motor cortico-basal ganglia-cerebellar networks, magnitudes were slightly greater on the right, except for left-sided connections of the striatum which were more numerous and of opposite polarity. Inter-nodal motor network effective connectivity remained consistent and robust across subjects. Age had a greater impact on connections to the contralateral cerebellum, bilaterally. Motor behavior, however, affected different connections in each hemisphere, exerting a more prominent effect on the left modulatory connections to the subthalamic nucleus, contralateral cerebellum, primary motor cortex, and thalamus. Discussion: This study revealed a consistent pattern of directed resting-state effective connectivity in healthy children aged 5-13 years within the motor network, encompassing cortical, subcortical, and cerebellar regions, correlated with motor skill proficiency. Both hemispheres exhibited similar effective connectivity within motor cortico-basal ganglia-cerebellar networks reflecting inter-nodal signal direction predicted by other modalities, mainly differing from task-dependent studies due to network differences at rest. Notably, age-related changes were more pronounced in connections to the contralateral cerebellum. Conversely, motor behavior distinctly impacted connections in each hemisphere, emphasizing its role in modulating left sided connections to the subthalamic nucleus, contralateral cerebellum, primary motor cortex, and thalamus. Motor network effective connectivity was correlated with motor behavior, validating its physiological significance. This study is the first to evaluate a normative effective connectivity model for the pediatric motor network using resting-state functional MRI correlating with behavior and serves as a foundation for identifying abnormal findings and optimizing targeted interventions like deep brain stimulation, potentially influencing future therapeutic approaches for children with movement disorders.

Expanding the spectrum of phenotypes for MPDZ: Report of four unrelated families and review of the literature.

MPDZ, a gene with diverse functions mediating cell-cell junction interactions, receptor signaling, and binding multivalent scaffold proteins, is associated with a spectrum of clinically heterogeneous phenotypes with biallelic perturbation. Despite its clinical relevance, the mechanistic underpinnings of these variants remain elusive, underscoring the need for extensive case series and functional investigations. In this study, we conducted a systematic review of cases in the literature through two electronic databases following the PRISMA guidelines. We selected nine studies, including 18 patients, with homozygous or compound heterozygous variants in MPDZ and added five patients from four unrelated families with novel MPDZ variants. To evaluate the role of Mpdz on hearing, we analyzed available auditory electrophysiology data from a knockout murine model (Mpdzem1(IMPC)J/em1(IMPC)J) generated by the International Mouse Phenotyping Consortium. Using exome and genome sequencing, we identified three families with compound heterozygous variants, and one family with a homozygous frameshift variant. MPDZ-related disease is clinically heterogenous with hydrocephaly, vision impairment, hearing impairment and cardiovascular disease occurring most frequently. Additionally, we describe two unrelated patients with spasticity, expanding the phenotypic spectrum. Our murine analysis of the Mpdzem1(IMPC)J/em1(IMPC)J allele showed severe hearing impairment. Overall, we expand understanding of MPDZ-related phenotypes and highlight hearing impairment and spasticity among the heterogeneous phenotypes.

Evolving understanding of CP phenotypes: the importance of dystonia.

Cerebral palsy (CP) is the core neurodevelopmental disorder affecting movement. Several distinct movement disorders can occur in people with cerebral palsy. Dystonia is a movement disorder that causes non-velocity-dependent hypertonia and/or abnormal, often repetitive, twisting movements, and/or postures. Dystonia occurs more frequently in patients with CP than has been recognized previously, and is treated differently than other aspects of CP. Dystonia is an important cause of chronic pain, hospitalization, and musculoskeletal complications. We describe recent advances in dystonia diagnosis in patients with cerebral palsy and highlight focus areas for ongoing research and clinical care. IMPACT: Dystonia is a movement disorder that is more common in people with cerebral palsy (CP) than previously thought. Dystonia contributes to hospitalization, chronic pain, and complications in CP patients. People with dystonic CP require different tools to diagnose and treat their condition. We summarize current state of the art in dystonia in CP and identify areas of focus for future work.

eIF2α phosphorylation evokes dystonia-like movements with D2-receptor and cholinergic origin and abnormal neuronal connectivity.

Dystonia is the 3rd most common movement disorder. Dystonia is acquired through either injury or genetic mutations, with poorly understood molecular and cellular mechanisms. Eukaryotic initiation factor alpha (eIF2α) controls cell state including neuronal plasticity via protein translation control and expression of ATF4. Dysregulated eIF2α phosphorylation (eIF2α-P) occurs in dystonia patients and models including DYT1, but the consequences are unknown. We increased/decreased eIF2α-P and tested motor control and neuronal properties in a Drosophila model. Bidirectionally altering eIF2α-P produced dystonia-like abnormal posturing and dyskinetic movements in flies. These movements were also observed with expression of the DYT1 risk allele. We identified cholinergic and D2-receptor neuroanatomical origins of these dyskinetic movements caused by genetic manipulations to dystonia molecular candidates eIF2α-P, ATF4, or DYT1, with evidence for decreased cholinergic release. In vivo, increased and decreased eIF2α-P increase synaptic connectivity at the NMJ with increased terminal size and bouton synaptic release sites. Long-term treatment of elevated eIF2α-P with ISRIB restored adult longevity, but not performance in a motor assay. Disrupted eIF2α-P signaling may alter neuronal connectivity, change synaptic release, and drive motor circuit changes in dystonia.

Heterozygous loss-of-function variants in DOCK4 cause neurodevelopmental delay and microcephaly.

Neurons form the basic anatomical and functional structure of the nervous system, and defects in neuronal differentiation or formation of neurites are associated with various psychiatric and neurodevelopmental disorders. Dynamic changes in the cytoskeleton are essential for this process, which is, inter alia, controlled by the dedicator of cytokinesis 4 (DOCK4) through the activation of RAC1. Here, we clinically describe 7 individuals (6 males and one female) with variants in DOCK4 and overlapping phenotype of mild to severe global developmental delay. Additional symptoms include coordination or gait abnormalities, microcephaly, nonspecific brain malformations, hypotonia and seizures. Four individuals carry missense variants (three of them detected de novo) and three individuals carry null variants (two of them maternally inherited). Molecular modeling of the heterozygous missense variants suggests that the majority of them affect the globular structure of DOCK4. In vitro functional expression studies in transfected Neuro-2A cells showed that all missense variants impaired neurite outgrowth. Furthermore, Dock4 knockout Neuro-2A cells also exhibited defects in promoting neurite outgrowth. Our results, including clinical, molecular and functional data, suggest that loss-of-function variants in DOCK4 probable cause a variable spectrum of a novel neurodevelopmental disorder with microcephaly.

Expansion of the neurodevelopmental phenotype of individuals with EEF1A2 variants and genotype-phenotype study.

Translation elongation factor eEF1A2 constitutes the alpha subunit of the elongation factor-1 complex, responsible for the enzymatic binding of aminoacyl-tRNA to the ribosome. Since 2012, 21 pathogenic missense variants affecting EEF1A2 have been described in 42 individuals with a severe neurodevelopmental phenotype including epileptic encephalopathy and moderate to profound intellectual disability (ID), with neurological regression in some patients. Through international collaborative call, we collected 26 patients with EEF1A2 variants and compared them to the literature. Our cohort shows a significantly milder phenotype. 83% of the patients are walking (vs. 29% in the literature), and 84% of the patients have language skills (vs. 15%). Three of our patients do not have ID. Epilepsy is present in 63% (vs. 93%). Neurological examination shows a less severe phenotype with significantly less hypotonia (58% vs. 96%), and pyramidal signs (24% vs. 68%). Cognitive regression was noted in 4% (vs. 56% in the literature). Among individuals over 10 years, 56% disclosed neurocognitive regression, with a mean age of onset at 2 years. We describe 8 novel missense variants of EEF1A2. Modeling of the different amino-acid sites shows that the variants associated with a severe phenotype, and the majority of those associated with a moderate phenotype, cluster within the switch II region of the protein and thus may affect GTP exchange. In contrast, variants associated with milder phenotypes may impact secondary functions such as actin binding. We report the largest cohort of individuals with EEF1A2 variants thus far, allowing us to expand the phenotype spectrum and reveal genotype-phenotype correlations.

Emerging Subspecialties: Pediatric Movement Disorders Neurology.

Pediatric movement disorders (PMD) neurologists care for infants, children, and adolescents with conditions that disrupt typical movement; serving as important subspecialist child neurologists in both academic and private practice settings. In contrast to adult movement disorders neurologists whose "bread and butter" is hypokinetic Parkinson disease, PMD subspecialty practice is often dominated by hyperkinetic movement disorders including tics, dystonia, chorea, tremor, and myoclonus. PMD neurology practice intersects with a variety of subspecialties, including neonatology, developmental pediatrics, rehabilitation medicine, epilepsy, child & adolescent psychiatry, psychology, orthopedics, genetics & metabolism, and neurosurgery. Over the past several decades, significant advancements in the PMD field have included operationalizing definitions for distinct movement disorders, recognizing the spectrum of clinical phenotypes, expanding research on genetic and neuroimmunologic causes of movement disorders, and advancing available treatments. Subspecialty training in PMD provides trainees with advanced clinical, diagnostic, procedural, and management skills that reflect the complexities of contemporary practice. The child neurologist who is fascinated by the intricacies of child motor development, appreciates the power of observation skills coupled with a thoughtful physical examination, and is excited by the challenge of the unknown may be well-suited to a career as a PMD specialist.

Clinical actionability of genetic findings in cerebral palsy.

Background and objectives: Single gene mutations are increasingly recognized as causes of cerebral palsy (CP) phenotypes, yet there is currently no standardized framework for measuring their clinical impact. We evaluated Pathogenic/Likely Pathogenic (P/LP) variants identified in individuals with CP to determine how frequently genetic testing results would prompt changes in care. Methods: We analyzed published P/LP variants in OMIM genes identified in clinical (n = 1,345 individuals) or research (n = 496) cohorts using exome sequencing of CP patients. We established a working group of clinical and research geneticists, developmental pediatricians, genetic counselors, and neurologists and performed a systematic review of existing literature for evidence of clinical management approaches linked to genetic disorders. Scoring rubrics were adapted, and a modified Delphi approach was used to build consensus and establish the anticipated impact on patient care. Overall clinical utility was calculated from metrics assessing outcome severity if left untreated, safety/practicality of the intervention, and anticipated intervention efficacy . Results: We found 140/1,841 (8%) of individuals in published CP cohorts had a genetic diagnosis classified as actionable , defined as prompting a change in clinical management based on knowledge related to the genetic etiology. 58/243 genes with P/LP variants were classified as actionable; 16 had treatment options targeting the primary disease mechanism , 16 had specific prevention strategies , and 26 had specific symptom management recommendations. The level of evidence was also graded according to ClinGen criteria; 44.6% of interventions had evidence class "D" or below. The potential interventions have clinical utility with 97% of outcomes being moderate-high severity if left untreated and 62% of interventions predicted to be of moderate-high efficacy . Most interventions (71%) were considered moderate-high safety/practicality . Discussion: Our findings indicate that actionable genetic findings occur in 8% of individuals referred for genetic testing with CP. Evaluation of potential efficacy , outcome severity , and intervention safety / practicality indicates moderate-high clinical utility of these genetic findings. Thus, genetic sequencing to identify these individuals for precision medicine interventions could improve outcomes and provide clinical benefit to individuals with CP. The relatively limited evidence base for most interventions underscores the need for additional research.

A biallelic loss-of-function variant in TMEM147 causes profound intellectual disability and spasticity.

Intellectual disability (ID), occurring in syndromic or non-syndromic forms, is the most common neurodevelopmental disorder. Although many cases are caused by single gene defects, ID is highly genetically heterogeneous. Biallelic variants in the transmembrane protein TMEM147 have recently been linked to intellectual disability with dysmorphic facial features. TMEM147 is believed to localize to the endoplasmic reticulum membrane and nuclear envelope and also involved in biogenesis of multi-pass membrane proteins. Here, we report two patients born to a consanguineous family with a novel loss-of-function variant; (NM_001242597.2:c.193-197del) in TMEM147 causing intellectual disability and spasticity. Whole exome sequencing and validating Sanger sequencing were utilized to confirm the identified causal variant. Our findings were in line with the previously described patients with TMEM147 variants manifesting intellectual disability as a major clinical sign but also featured spasticity as a phenotypic expansion. This study provides additional evidence for the pathogenicity of TMEM147 mutations in intellectual disability and expands the phenotypic and variant spectrum linked to this gene.

Intrathecal magnesium delivery for Mg++-insensitive NMDA receptor activity due to GRIN1 mutation.

BACKGROUND: Mutations in the NMDA receptor are known to disrupt glutamatergic signaling crucial for early neurodevelopment, often leading to severe global developmental delay/intellectual disability, epileptic encephalopathy, and cerebral palsy phenotypes. Both seizures and movement disorders can be highly treatment-refractory. RESULTS: We describe a targeted ABA n-of-1 treatment trial with intrathecal MgSO4, rationally designed based on the electrophysiologic properties of this gain of function mutation in the GRIN1 NMDA subunit. CONCLUSION: Although the invasive nature of the trial necessitated a short-term, non-randomized, unblinded intervention, quantitative longitudinal neurophysiologic monitoring indicated benefit, providing class II evidence in support of intrathecal MgSO4 for select forms of GRIN disorders.

AGAP1-associated endolysosomal trafficking abnormalities link gene-environment interactions in neurodevelopmental disorders.

AGAP1 is an Arf1 GTPase-activating protein that regulates endolysosomal trafficking. Damaging variants have been linked to cerebral palsy and autism. We report three new cases in which individuals had microdeletion variants in AGAP1. The affected individuals had intellectual disability (3/3), autism (3/3), dystonia with axial hypotonia (1/3), abnormalities of brain maturation (1/3), growth impairment (2/3) and facial dysmorphism (2/3). We investigated mechanisms potentially underlying AGAP1 variant-mediated neurodevelopmental impairments using the Drosophila ortholog CenG1a. We discovered reduced axon terminal size, increased neuronal endosome abundance and elevated autophagy compared to those in controls. Given potential incomplete penetrance, we assessed gene-environment interactions. We found basal elevation in the phosphorylation of the integrated stress-response protein eIF2α (or eIF2A) and inability to further increase eIF2α phosphorylation with subsequent cytotoxic stressors. CenG1a-mutant flies had increased lethality from exposure to environmental insults. We propose a model wherein disruption of AGAP1 function impairs endolysosomal trafficking, chronically activating the integrated stress response and leaving AGAP1-deficient cells susceptible to a variety of second-hit cytotoxic stressors. This model may have broader applicability beyond AGAP1 in instances where both genetic and environmental insults co-occur in individuals with neurodevelopmental disorders.

Chronic striatal cholinergic interneuron excitation induces clinically-relevant dystonic behavior in mice.

Dystonia is common, debilitating, often medically refractory, and difficult to diagnose. The gold standard for both clinical and mouse model dystonia evaluation is subjective assessment, ideally by expert consensus. However, this subjectivity makes translational quantification of clinically-relevant dystonia metrics across species nearly impossible. Many mouse models of genetic dystonias display abnormal striatal cholinergic interneuron excitation, but few display subjectively dystonic features. Therefore, whether striatal cholinergic interneuron pathology causes dystonia remains unknown. To address these critical limitations, we first demonstrate that objectively quantifiable leg adduction variability correlates with leg dystonia severity in people. We then show that chemogenetic excitation of striatal cholinergic interneurons in mice causes comparable leg adduction variability in mice. This clinically-relevant dystonic behavior in mice does not occur with acute excitation, but rather develops after 14 days of ongoing striatal cholinergic interneuron excitation. This requirement for prolonged excitation recapitulates the clinically observed phenomena of a delay between an inciting brain injury and subsequent dystonia manifestation and demonstrates a causative link between chronic striatal cholinergic interneuron excitation and clinically-relevant dystonic behavior in mice. Therefore, these results support targeting striatal ChIs for dystonia drug development and suggests early treatment in the window following injury but prior to dystonia onset. One Sentence Summary: Chronic excitation of dorsal striatal cholinergic interneuron causes clinically-relevant dystonic phenotypes in mice.

Genotype-phenotype correlations in RHOBTB2-associated neurodevelopmental disorders.

PURPOSE: Missense variants clustering in the BTB domain region of RHOBTB2 cause a developmental and epileptic encephalopathy with early-onset seizures and severe intellectual disability. METHODS: By international collaboration, we assembled individuals with pathogenic RHOBTB2 variants and a variable spectrum of neurodevelopmental disorders. By western blotting, we investigated the consequences of missense variants in vitro. RESULTS: In accordance with previous observations, de novo heterozygous missense variants in the BTB domain region led to a severe developmental and epileptic encephalopathy in 16 individuals. Now, we also identified de novo missense variants in the GTPase domain in 6 individuals with apparently more variable neurodevelopmental phenotypes with or without epilepsy. In contrast to variants in the BTB domain region, variants in the GTPase domain do not impair proteasomal degradation of RHOBTB2 in vitro, indicating different functional consequences. Furthermore, we observed biallelic splice-site and truncating variants in 9 families with variable neurodevelopmental phenotypes, indicating that complete loss of RHOBTB2 is pathogenic as well. CONCLUSION: By identifying genotype-phenotype correlations regarding location and consequences of de novo missense variants in RHOBTB2 and by identifying biallelic truncating variants, we further delineate and expand the molecular and clinical spectrum of RHOBTB2-related phenotypes, including both autosomal dominant and recessive neurodevelopmental disorders.