ANK2 loss-of-function variants are associated with epilepsy, and lead to impaired axon initial segment plasticity and hyperactive network activity in hiPSC-derived neuronal networks.

PURPOSE: To characterize a novel neurodevelopmental syndrome due to loss-of-function (LoF) variants in Ankyrin 2 (ANK2), and to explore the effects on neuronal network dynamics and homeostatic plasticity in human-induced pluripotent stem cell-derived neurons. METHODS: We collected clinical and molecular data of 12 individuals with heterozygous de novo LoF variants in ANK2. We generated a heterozygous LoF allele of ANK2 using CRISPR/Cas9 in human-induced pluripotent stem cells (hiPSCs). HiPSCs were differentiated into excitatory neurons, and we measured their spontaneous electrophysiological responses using micro-electrode arrays (MEAs). We also characterized their somatodendritic morphology and axon initial segment (AIS) structure and plasticity. RESULTS: We found a broad neurodevelopmental disorder (NDD), comprising intellectual disability, autism spectrum disorders and early onset epilepsy. Using MEAs, we found that hiPSC-derived neurons with heterozygous LoF of ANK2 show a hyperactive and desynchronized neuronal network. ANK2-deficient neurons also showed increased somatodendritic structures and altered AIS structure of which its plasticity is impaired upon activity-dependent modulation. CONCLUSIONS: Phenotypic characterization of patients with de novo ANK2 LoF variants defines a novel NDD with early onset epilepsy. Our functional in vitro data of ANK2-deficient human neurons show a specific neuronal phenotype in which reduced ANKB expression leads to hyperactive and desynchronized neuronal network activity, increased somatodendritic complexity and AIS structure and impaired activity-dependent plasticity of the AIS.

TNPO2 variants associate with human developmental delays, neurologic deficits, and dysmorphic features and alter TNPO2 activity in Drosophila.

Transportin-2 (TNPO2) mediates multiple pathways including non-classical nucleocytoplasmic shuttling of >60 cargoes, such as developmental and neuronal proteins. We identified 15 individuals carrying de novo coding variants in TNPO2 who presented with global developmental delay (GDD), dysmorphic features, ophthalmologic abnormalities, and neurological features. To assess the nature of these variants, functional studies were performed in Drosophila. We found that fly dTnpo (orthologous to TNPO2) is expressed in a subset of neurons. dTnpo is critical for neuronal maintenance and function as downregulating dTnpo in mature neurons using RNAi disrupts neuronal activity and survival. Altering the activity and expression of dTnpo using mutant alleles or RNAi causes developmental defects, including eye and wing deformities and lethality. These effects are dosage dependent as more severe phenotypes are associated with stronger dTnpo loss. Interestingly, similar phenotypes are observed with dTnpo upregulation and ectopic expression of TNPO2, showing that loss and gain of Transportin activity causes developmental defects. Further, proband-associated variants can cause more or less severe developmental abnormalities compared to wild-type TNPO2 when ectopically expressed. The impact of the variants tested seems to correlate with their position within the protein. Specifically, those that fall within the RAN binding domain cause more severe toxicity and those in the acidic loop are less toxic. Variants within the cargo binding domain show tissue-dependent effects. In summary, dTnpo is an essential gene in flies during development and in neurons. Further, proband-associated de novo variants within TNPO2 disrupt the function of the encoded protein. Hence, TNPO2 variants are causative for neurodevelopmental abnormalities.

CAMTA1-related disorder: Phenotypic and molecular characterization of 26 new individuals and literature review.

Calmodulin-binding transcriptional activator 1 (CAMTA1) is highly expressed in the brain and plays a role in cell cycle regulation, cell differentiation, regulation of long-term memory, and initial development, maturation, and survival of cerebellar neurons. The existence of human neurological phenotypes, including cerebellar dysfunction with variable cognitive and behavioral abnormalities (CECBA), associated with CAMTA1 variants, has further supported its role in brain functions. In this study, we phenotypically and molecularly characterize the largest cohort of individuals (n = 26) with 23 novel CAMTA1 variants (frameshift-7, nonsense-6, splicing-1, initiation codon-1, missense-5, and intragenic deletions-3) and compare the findings with all previously reported cases (total = 53). We show that the most notable phenotypic findings are developmental delay/intellectual disability, unsteady or uncoordinated gait, hypotonia, behavioral problems, and eye abnormalities. In addition, there is a high incidence of dysarthria, dysgraphia, microcephaly, gastrointestinal abnormalities, sleep difficulties, and nonspecific brain MRI findings; a few of which have been under-reported. More than one third of the variants in this cohort were inherited from an asymptomatic or mildly affected parent suggesting reduced penetrance and variable expressivity. Our cohort provides a comprehensive characterization of the spectrum of phenotypes and genotypes among individuals with CECBA and the large data will facilitate counseling and formulating management plans and surveillance recommendations for these individuals.

In-depth characterisation of a cohort of individuals with missense and loss-of-function variants disrupting FOXP2.

BACKGROUND: Heterozygous disruptions of FOXP2 were the first identified molecular cause for severe speech disorder: childhood apraxia of speech (CAS), and yet few cases have been reported, limiting knowledge of the condition. METHODS: Here we phenotyped 28 individuals from 17 families with pathogenic FOXP2-only variants (12 loss-of-function, five missense variants; 14 males; aged 2 to 62 years). Health and development (cognitive, motor, social domains) were examined, including speech and language outcomes with the first cross-linguistic analysis of English and German. RESULTS: Speech disorders were prevalent (23/25, 92%) and CAS was most common (22/25, 88%), with similar speech presentations across English and German. Speech was still impaired in adulthood, and some speech sounds (eg, 'th', 'r', 'ch', 'j') were never acquired. Language impairments (21/25, 84%) ranged from mild to severe. Comorbidities included feeding difficulties in infancy (10/26, 38%), fine (13/26, 50%) and gross (13/26, 50%) motor impairment, anxiety (5/27, 19%), depression (6/27, 22%) and sleep disturbance (10/24, 42%). Physical features were common (22/27, 81%) but with no consistent pattern. Cognition ranged from average to mildly impaired and was incongruent with language ability; for example, seven participants with severe language disorder had average non-verbal cognition. CONCLUSIONS: Although we identify an increased prevalence of conditions like anxiety, depression and sleep disturbance, we confirm that the consequences of FOXP2 dysfunction remain relatively specific to speech disorder, as compared with other recently identified monogenic conditions associated with CAS. Thus, our findings reinforce that FOXP2 provides a valuable entry point for examining the neurobiological bases of speech disorder.

Deep phenotyping of the neuroimaging and skeletal features in KBG syndrome: a study of 53 patients and review of the literature.

BACKGROUND: KBG syndrome is caused by haploinsufficiency of ANKRD11 and is characterised by macrodontia of upper central incisors, distinctive facial features, short stature, skeletal anomalies, developmental delay, brain malformations and seizures. The central nervous system (CNS) and skeletal features remain poorly defined. METHODS: CNS and/or skeletal imaging were collected from molecularly confirmed individuals with KBG syndrome through an international network. We evaluated the original imaging and compared our results with data in the literature. RESULTS: We identified 53 individuals, 44 with CNS and 40 with skeletal imaging. Common CNS findings included incomplete hippocampal inversion and posterior fossa malformations; these were significantly more common than previously reported (63.4% and 65.9% vs 1.1% and 24.7%, respectively). Additional features included patulous internal auditory canal, never described before in KBG syndrome, and the recurrence of ventriculomegaly, encephalic cysts, empty sella and low-lying conus medullaris. We found no correlation between these structural anomalies and epilepsy or intellectual disability. Prevalent skeletal findings comprised abnormalities of the spine including scoliosis, coccygeal anomalies and cervical ribs. Hand X-rays revealed frequent abnormalities of carpal bone morphology and maturation, including a greater delay in ossification compared with metacarpal/phalanx bones. CONCLUSION: This cohort enabled us to describe the prevalence of very heterogeneous neuroradiological and skeletal anomalies in KBG syndrome. Knowledge of the spectrum of such anomalies will aid diagnostic accuracy, improve patient care and provide a reference for future research on the effects of ANKRD11 variants in skeletal and brain development.

Clinical and radiological assessment of scoliosis in Koolen-de Vries syndrome.

The Koolen-de Vries syndrome (KdVS) is a multisystem disorder characterized by developmental delay, intellectual disability, characteristic facial features, epilepsy, cardiovascular and urogenital malformations, and various musculoskeletal disorders. Scoliosis is a common feature. The aim of this study is to fill the gap in the current knowledge about scoliosis in individuals with KdVS and to provide recommendations for management and follow-up. In total, 54 individuals with KdVS were included in the study, with a mean age of 13.6 years (range 1.9-38.8 years). Spine radiographs, MR scans, and corresponding radiology reports were analyzed retrospectively for scoliosis and additional anomalies. The presence of scoliosis-related clinical conditions was assessed in participants' medical records and by use of a parent survey. Scoliosis was present in 56% of the participants (30/54) with a mean age of onset of 10.6 years and curve progression during the growth spurt. Prevalence at age 6, 10, and 18 years was, respectively, 9%, 41%, and 65%. Most participants were diagnosed with a single curve (13/24, 54%), of which five participants had a long C-curve type scoliosis. No significant risk factors for development of scoliosis could be identified. Severity was mostly classified as mild, although 29% (7/24) of the curves were larger than 30° at last follow-up. Bracing therapy was received in 13% (7/54), and surgical spinal fusion was warranted in 6% (3/54). Remarkably, participants with scoliosis received less often physical therapy compared to participants without scoliosis (P = 0.002). Scoliosis in individuals with KdVS should be closely monitored and radiologic screening for scoliosis and vertebrae abnormalities is recommended at diagnosis of KdVS, and the age of 10 and 18 years.

Familial Bainbridge-Ropers syndrome: Report of familial ASXL3 inheritance and a milder phenotype.

De novo truncating and splicing pathogenic variants in the Additional Sex Combs-Like 3 (ASXL3) gene are known to cause neurodevelopmental delay, intellectual disability, behavioral difficulties, hypotonia, feeding problems and characteristic facial features. We previously reported 45 patients with ASXL3-related disorder including three individuals with a familial variant. Here we report the detailed clinical and molecular characteristics of these three families with inherited ASXL3-related disorder. First, a father and son with c.2791_2792del p.Gln931fs pathogenic variant. The second, a mother, daughter and son with c.4534C > T, p.Gln1512Ter pathogenic variant. The third, a mother and her daughter with c.4441dup, p.Leu1481fs maternally inherited pathogenic variant. This report demonstrates intrafamilial phenotypic heterogeneity and confirms heritability of ASXL3-related disorder.

A mutational hotspot in AMOTL1 defines a new syndrome of orofacial clefting, cardiac anomalies, and tall stature.

AMOTL1 encodes angiomotin-like protein 1, an actin-binding protein that regulates cell polarity, adhesion, and migration. The role of AMOTL1 in human disease is equivocal. We report a large cohort of individuals harboring heterozygous AMOTL1 variants and define a core phenotype of orofacial clefting, congenital heart disease, tall stature, auricular anomalies, and gastrointestinal manifestations in individuals with variants in AMOTL1 affecting amino acids 157-161, a functionally undefined but highly conserved region. Three individuals with AMOTL1 variants outside this region are also described who had variable presentations with orofacial clefting and multi-organ disease. Our case cohort suggests that heterozygous missense variants in AMOTL1, most commonly affecting amino acid residues 157-161, define a new orofacial clefting syndrome, and indicates an important functional role for this undefined region.

GenIDA: an international participatory database to gain knowledge on health issues related to genetic forms of neurodevelopmental disorders.

Intellectual disability with or without manifestations of autism and/or epilepsy affects 1-2% of the population, and it is estimated that more than 30-50% of these cases have a single genetic cause. More than 1000 genes and recurrent chromosomal abnormalities are involved in these genetic forms of neurodevelopmental disorders, which often remain insufficiently described in terms of clinical spectrum, associated medical problems, etc., due to their rarity and the often-limited number of patients' phenotypes reported. GenIDA is an international online participatory database that aims to better characterise the clinical manifestations and natural histories of these rare diseases. Clinical information is reported by parents of affected individuals using a structured questionnaire exploring physical parameters, cognitive and behavioural aspects, the presence or absence of neurological disorders or problems affecting major physiological functions, as well as autonomy and quality of life. This strengthens the implication in research of the concerned families. GenIDA aims to construct international cohorts of significant size of individuals affected by a given condition. As of July 2022, GenIDA counts some 1545 documented patient records from over 60 nationalities and collaborates with clinicians and researchers around the world who have access to the anonymized data collected to generate new, medically meaningful information to improve patient care. We present the GenIDA database here, together with an overview of the possibilities it offers to affected individuals, their families, and professionals in charge of the management of genetic forms of neurodevelopmental disorders. Finally, case studies of cohorts will illustrate the usefulness of GenIDA.

De novo variants in MPP5 cause global developmental delay and behavioral changes.

Membrane Protein Palmitoylated 5 (MPP5) is a highly conserved apical complex protein essential for cell polarity, fate and survival. Defects in cell polarity are associated with neurologic disorders including autism and microcephaly. MPP5 is essential for neurogenesis in animal models, but human variants leading to neurologic impairment have not been described. We identified three patients with heterozygous MPP5 de novo variants (DNV) and global developmental delay (GDD) and compared their phenotypes and magnetic resonance imaging (MRI) to ascertain how MPP5 DNV leads to GDD. All three patients with MPP5 DNV experienced GDD with language delay/regression and behavioral changes. MRI ranged from normal to decreased gyral folding and microcephaly. The effects of MPP5 depletion on the developing brain were assessed by creating a heterozygous conditional knock out (het CKO) murine model with central nervous system (CNS)-specific Nestin-Cre drivers. In the het CKO model, Mpp5 depletion led to microcephaly, decreased cerebellar volume and cortical thickness. Het CKO mice had decreased ependymal cells and Mpp5 at the apical surface of cortical ventricular zone compared with wild type. Het CKO mice also failed to maintain progenitor pools essential for neurogenesis. The proportion of cortical cells undergoing apoptotic cell death increased, suggesting that cell death reduces progenitor population and neuron number. Het CKO mice also showed behavioral changes, similar to our patients. To our knowledge, this is the first report to show that variants in MPP5 are associated with GDD, behavioral abnormalities and language regression/delay. Murine modeling shows that neurogenesis is likely altered in these individuals, with cell death and skewed cellular composition playing significant roles.

Phenotype based prediction of exome sequencing outcome using machine learning for neurodevelopmental disorders.

PURPOSE: Although the introduction of exome sequencing (ES) has led to the diagnosis of a significant portion of patients with neurodevelopmental disorders (NDDs), the diagnostic yield in actual clinical practice has remained stable at approximately 30%. We hypothesized that improving the selection of patients to test on the basis of their phenotypic presentation will increase diagnostic yield and therefore reduce unnecessary genetic testing. METHODS: We tested 4 machine learning methods and developed PredWES from these: a statistical model predicting the probability of a positive ES result solely on the basis of the phenotype of the patient. RESULTS: We first trained the tool on 1663 patients with NDDs and subsequently showed that diagnostic ES on the top 10% of patients with the highest probability of a positive ES result would provide a diagnostic yield of 56%, leading to a notable 114% increase. Inspection of our model revealed that for patients with NDDs, comorbid abnormal (lower) muscle tone and microcephaly positively correlated with a conclusive ES diagnosis, whereas autism was negatively associated with a molecular diagnosis. CONCLUSION: In conclusion, PredWES allows prioritizing patients with NDDs eligible for diagnostic ES on the basis of their phenotypic presentation to increase the diagnostic yield, making a more efficient use of health care resources.

Characterization of SETD1A haploinsufficiency in humans and Drosophila defines a novel neurodevelopmental syndrome.

Defects in histone methyltransferases (HMTs) are major contributing factors in neurodevelopmental disorders (NDDs). Heterozygous variants of SETD1A involved in histone H3 lysine 4 (H3K4) methylation were previously identified in individuals with schizophrenia. Here, we define the clinical features of the Mendelian syndrome associated with haploinsufficiency of SETD1A by investigating 15 predominantly pediatric individuals who all have de novo SETD1A variants. These individuals present with a core set of symptoms comprising global developmental delay and/or intellectual disability, subtle facial dysmorphisms, behavioral and psychiatric problems. We examined cellular phenotypes in three patient-derived lymphoblastoid cell lines with three variants: p.Gly535Alafs*12, c.4582-2_4582delAG, and p.Tyr1499Asp. These patient cell lines displayed DNA damage repair defects that were comparable to previously observed RNAi-mediated depletion of SETD1A. This suggested that these variants, including the p.Tyr1499Asp in the catalytic SET domain, behave as loss-of-function (LoF) alleles. Previous studies demonstrated a role for SETD1A in cell cycle control and differentiation. However, individuals with SETD1A variants do not show major structural brain defects or severe microcephaly, suggesting that defective proliferation and differentiation of neural progenitors is unlikely the single underlying cause of the disorder. We show here that the Drosophila melanogaster SETD1A orthologue is required in postmitotic neurons of the fly brain for normal memory, suggesting a role in post development neuronal function. Together, this study defines a neurodevelopmental disorder caused by dominant de novo LoF variants in SETD1A and further supports a role for H3K4 methyltransferases in the regulation of neuronal processes underlying normal cognitive functioning.

KCNT1-related epilepsies and epileptic encephalopathies: phenotypic and mutational spectrum.

Variants in KCNT1, encoding a sodium-gated potassium channel (subfamily T member 1), have been associated with a spectrum of epilepsies and neurodevelopmental disorders. These range from familial autosomal dominant or sporadic sleep-related hypermotor epilepsy to epilepsy of infancy with migrating focal seizures (EIMFS) and include developmental and epileptic encephalopathies. This study aims to provide a comprehensive overview of the phenotypic and genotypic spectrum of KCNT1 mutation-related epileptic disorders in 248 individuals, including 66 previously unpublished and 182 published cases, the largest cohort reported so far. Four phenotypic groups emerged from our analysis: (i) EIMFS (152 individuals, 33 previously unpublished); (ii) developmental and epileptic encephalopathies other than EIMFS (non-EIMFS developmental and epileptic encephalopathies) (37 individuals, 17 unpublished); (iii) autosomal dominant or sporadic sleep-related hypermotor epilepsy (53 patients, 14 unpublished); and (iv) other phenotypes (six individuals, two unpublished). In our cohort of 66 new cases, the most common phenotypic features were: (i) in EIMFS, heterogeneity of seizure types, including epileptic spasms, epilepsy improvement over time, no epilepsy-related deaths; (ii) in non-EIMFS developmental and epileptic encephalopathies, possible onset with West syndrome, occurrence of atypical absences, possible evolution to developmental and epileptic encephalopathies with sleep-related hypermotor epilepsy features; one case of sudden unexplained death in epilepsy; (iii) in autosomal dominant or sporadic sleep-related hypermotor epilepsy, we observed a high prevalence of drug-resistance, although seizure frequency improved with age in some individuals, appearance of cognitive regression after seizure onset in all patients, no reported severe psychiatric disorders, although behavioural/psychiatric comorbidities were reported in ∼50% of the patients, sudden unexplained death in epilepsy in one individual; and (iv) other phenotypes in individuals with mutation of KCNT1 included temporal lobe epilepsy, and epilepsy with tonic-clonic seizures and cognitive regression. Genotypic analysis of the whole cohort of 248 individuals showed only missense mutations and one inframe deletion in KCNT1. Although the KCNT1 mutations in affected individuals were seen to be distributed among the different domains of the KCNT1 protein, genotype-phenotype considerations showed many of the autosomal dominant or sporadic sleep-related hypermotor epilepsy-associated mutations to be clustered around the RCK2 domain in the C terminus, distal to the NADP domain. Mutations associated with EIMFS/non-EIMFS developmental and epileptic encephalopathies did not show a particular pattern of distribution in the KCNT1 protein. Recurrent KCNT1 mutations were seen to be associated with both severe and less severe phenotypes. Our study further defines and broadens the phenotypic and genotypic spectrums of KCNT1-related epileptic conditions and emphasizes the increasingly important role of this gene in the pathogenesis of early onset developmental and epileptic encephalopathies as well as of focal epilepsies, namely autosomal dominant or sporadic sleep-related hypermotor epilepsy.

The impact of lockdown on young people with genetic neurodevelopmental disabilities: a study with the international participatory database GenIDA.

BACKGROUND: Previous publications suggested that lockdown is likely to impact daily living issues of individuals with intellectual disabilities. The authors notably suspected an intensification of behavioural, eating and sleep problems. METHODS: To test these hypotheses, we conducted an international online survey about the impact of COVID-19-associated first lockdown on people with genetic neurodevelopmental disorders. This survey was carried out using GenIDA, an international participatory database collecting medical information on genetic neurodevelopmental disorders. Patients' relatives took part in this online survey from 30/04/2020 to 09/06/2020. This survey adapted from GenIDA standard questionnaire requested information on diagnosis, lifestyle and was based on yes/no answers to questions regarding behaviour, diet, and sleep, in the 6-months period before lockdown and during lockdown. We also asked relatives to evaluate the intensity of these problems by severity level. Finally, relatives could freely comment in open fields on the medical and/or quality of life problems they had encountered during lockdown. RESULTS: In total 199 participants-144 children and 45 adults-with neurodevelopmental disorders (intellectual disability (79.4%) and/or autism spectrum disorder (21.6%)) of various genetic origins, with near-equal male/female (96/103) contribution and originating mainly from Europe and Northern America, were included. The average lockdown duration at time of the survey was 57 days. We did not find differences in the frequency of behavioural, eating and sleep problems before and during lockdown. Moreover, there was no apparent difference in the intensity of eating and sleep disorders between both periods. However, for persons with behavioural problems at both periods, relatives reported an increase in aggressivity, self-aggressivity, depressiveness, stereotypies, and restricted interests during lockdown, all of which might be interpreted as consequences of a lack of stimulation or a reaction to unexpected changes in daily habits. CONCLUSIONS: Our results support previous studies that suggest that the negative impact of lockdown does not depend on the intellectual disability per se but on the associated comorbidities such as behavioural disorders. This study addresses the need for prevention of behavioural disturbance in the vulnerable population with genetic neurodevelopmental disabilities.

DNA Methylation Signature for JARID2-Neurodevelopmental Syndrome.

JARID2 (Jumonji, AT Rich Interactive Domain 2) pathogenic variants cause a neurodevelopmental syndrome, that is characterized by developmental delay, cognitive impairment, hypotonia, autistic features, behavior abnormalities and dysmorphic facial features. JARID2 encodes a transcriptional repressor protein that regulates the activity of various histone methyltransferase complexes. However, the molecular etiology is not fully understood, and JARID2-neurodevelopmental syndrome may vary in its typical clinical phenotype. In addition, the detection of variants of uncertain significance (VUSs) often results in a delay of final diagnosis which could hamper the appropriate care. In this study we aim to detect a specific and sensitive DNA methylation signature for JARID2-neurodevelopmental syndrome. Peripheral blood DNA methylation profiles from 56 control subjects, 8 patients with (likely) pathogenic JARID2 variants and 3 patients with JARID2 VUSs were analyzed. DNA methylation analysis indicated a clear and robust separation between patients with (likely) pathogenic variants and controls. A binary model capable of classifying patients with the JARID2-neurodevelopmental syndrome was constructed on the basis of the identified episignature. Patients carrying VUSs clustered with the control group. We identified a distinct DNA methylation signature associated with JARID2-neurodevelopmental syndrome, establishing its utility as a biomarker for this syndrome and expanding the EpiSign diagnostic test.

TAOK1 is associated with neurodevelopmental disorder and essential for neuronal maturation and cortical development.

Thousand and one amino-acid kinase 1 (TAOK1) is a MAP3K protein kinase, regulating different mitogen-activated protein kinase pathways, thereby modulating a multitude of processes in the cell. Given the recent finding of TAOK1 involvement in neurodevelopmental disorders (NDDs), we investigated the role of TAOK1 in neuronal function and collected a cohort of 23 individuals with mostly de novo variants in TAOK1 to further define the associated NDD. Here, we provide evidence for an important role for TAOK1 in neuronal function, showing that altered TAOK1 expression levels in the embryonic mouse brain affect neural migration in vivo, as well as neuronal maturation in vitro. The molecular spectrum of the identified TAOK1 variants comprises largely truncating and nonsense variants, but also missense variants, for which we provide evidence that they can have a loss of function or dominant-negative effect on TAOK1, expanding the potential underlying causative mechanisms resulting in NDD. Taken together, our data indicate that TAOK1 activity needs to be properly controlled for normal neuronal function and that TAOK1 dysregulation leads to a neurodevelopmental disorder mainly comprising similar facial features, developmental delay/intellectual disability and/or variable learning or behavioral problems, muscular hypotonia, infant feeding difficulties, and growth problems.

Variants in PUS7 Cause Intellectual Disability with Speech Delay, Microcephaly, Short Stature, and Aggressive Behavior.

We describe six persons from three families with three homozygous protein truncating variants in PUS7: c.89_90del (p.Thr30Lysfs∗20), c.1348C>T (p.Arg450∗), and a deletion of the penultimate exon 15. All these individuals have intellectual disability with speech delay, short stature, microcephaly, and aggressive behavior. PUS7 encodes the RNA-independent pseudouridylate synthase 7. Pseudouridylation is the most abundant post-transcriptional modification in RNA, which is primarily thought to stabilize secondary structures of RNA. We show that the disease-related variants lead to abolishment of PUS7 activity on both tRNA and mRNA substrates. Moreover, pus7 knockout in Drosophila melanogaster results in a number of behavioral defects, including increased activity, disorientation, and aggressiveness supporting that neurological defects are caused by PUS7 variants. Our findings demonstrate that RNA pseudouridylation by PUS7 is essential for proper neuronal development and function.

De Novo Pathogenic Variants in CACNA1E Cause Developmental and Epileptic Encephalopathy with Contractures, Macrocephaly, and Dyskinesias.

Developmental and epileptic encephalopathies (DEEs) are severe neurodevelopmental disorders often beginning in infancy or early childhood that are characterized by intractable seizures, abundant epileptiform activity on EEG, and developmental impairment or regression. CACNA1E is highly expressed in the central nervous system and encodes the α1-subunit of the voltage-gated CaV2.3 channel, which conducts high voltage-activated R-type calcium currents that initiate synaptic transmission. Using next-generation sequencing techniques, we identified de novo CACNA1E variants in 30 individuals with DEE, characterized by refractory infantile-onset seizures, severe hypotonia, and profound developmental impairment, often with congenital contractures, macrocephaly, hyperkinetic movement disorders, and early death. Most of the 14, partially recurring, variants cluster within the cytoplasmic ends of all four S6 segments, which form the presumed CaV2.3 channel activation gate. Functional analysis of several S6 variants revealed consistent gain-of-function effects comprising facilitated voltage-dependent activation and slowed inactivation. Another variant located in the domain II S4-S5 linker results in facilitated activation and increased current density. Five participants achieved seizure freedom on the anti-epileptic drug topiramate, which blocks R-type calcium channels. We establish pathogenic variants in CACNA1E as a cause of DEEs and suggest facilitated R-type calcium currents as a disease mechanism for human epilepsy and developmental disorders.

JARID2 haploinsufficiency is associated with a clinically distinct neurodevelopmental syndrome.

PURPOSE: JARID2, located on chromosome 6p22.3, is a regulator of histone methyltransferase complexes that is expressed in human neurons. So far, 13 individuals sharing clinical features including intellectual disability (ID) were reported with de novo heterozygous deletions in 6p22-p24 encompassing the full length JARID2 gene (OMIM 601594). However, all published individuals to date have a deletion of at least one other adjoining gene, making it difficult to determine if JARID2 is the critical gene responsible for the shared features. We aim to confirm JARID2 as a human disease gene and further elucidate the associated clinical phenotype. METHODS: Chromosome microarray analysis, exome sequencing, and an online matching platform (GeneMatcher) were used to identify individuals with single-nucleotide variants or deletions involving JARID2. RESULTS: We report 16 individuals in 15 families with a deletion or single-nucleotide variant in JARID2. Several of these variants are likely to result in haploinsufficiency due to nonsense-mediated messenger RNA (mRNA) decay. All individuals have developmental delay and/or ID and share some overlapping clinical characteristics such as facial features with those who have larger deletions involving JARID2. CONCLUSION: We report that JARID2 haploinsufficiency leads to a clinically distinct neurodevelopmental syndrome, thus establishing gene-disease validity for the purpose of diagnostic reporting.

NEXMIF encephalopathy: an X-linked disorder with male and female phenotypic patterns.

PURPOSE: Pathogenic variants in the X-linked gene NEXMIF (previously KIAA2022) are associated with intellectual disability (ID), autism spectrum disorder, and epilepsy. We aimed to delineate the female and male phenotypic spectrum of NEXMIF encephalopathy. METHODS: Through an international collaboration, we analyzed the phenotypes and genotypes of 87 patients with NEXMIF encephalopathy. RESULTS: Sixty-three females and 24 males (46 new patients) with NEXMIF encephalopathy were studied, with 30 novel variants. Phenotypic features included developmental delay/ID in 86/87 (99%), seizures in 71/86 (83%) and multiple comorbidities. Generalized seizures predominated including myoclonic seizures and absence seizures (both 46/70, 66%), absence with eyelid myoclonia (17/70, 24%), and atonic seizures (30/70, 43%). Males had more severe developmental impairment; females had epilepsy more frequently, and varied from unaffected to severely affected. All NEXMIF pathogenic variants led to a premature stop codon or were deleterious structural variants. Most arose de novo, although X-linked segregation occurred for both sexes. Somatic mosaicism occurred in two males and a family with suspected parental mosaicism. CONCLUSION: NEXMIF encephalopathy is an X-linked, generalized developmental and epileptic encephalopathy characterized by myoclonic-atonic epilepsy overlapping with eyelid myoclonia with absence. Some patients have developmental encephalopathy without epilepsy. Males have more severe developmental impairment. NEXMIF encephalopathy arises due to loss-of-function variants.