Prevalence of comorbidities in individuals with neurodevelopmental disorders from the aggregated phenomics data of 51,227 pediatric individuals.

The prevalence of comorbidities in individuals with neurodevelopmental disorders (NDDs) is not well understood, yet these are important for accurate diagnosis and prognosis in routine care and for characterizing the clinical spectrum of NDD syndromes. We thus developed PhenomAD-NDD, an aggregated database containing the comorbid phenotypic data of 51,227 individuals with NDD, all harmonized into Human Phenotype Ontology (HPO), with in total 3,054 unique HPO terms. We demonstrate that almost all congenital anomalies are more prevalent in the NDD population than in the general population, and the NDD baseline prevalence allows for an approximation of the enrichment of symptoms. For example, such analyses of 33 genetic NDDs show that 32% of enriched phenotypes are currently not reported in the clinical synopsis in the Online Mendelian Inheritance in Man (OMIM). PhenomAD-NDD is open to all via a visualization online tool and allows us to determine the enrichment of symptoms in NDD.

Refining the 9q34.3 microduplication syndrome reveals mild neurodevelopmental features associated with a distinct global DNA methylation profile.

Precise regulation of gene expression is important for correct neurodevelopment. 9q34.3 deletions affecting the EHMT1 gene result in a syndromic neurodevelopmental disorder named Kleefstra syndrome. In contrast, duplications of the 9q34.3 locus encompassing EHMT1 have been suggested to cause developmental disorders, but only limited information has been available. We have identified 15 individuals from 10 unrelated families, with 9q34.3 duplications <1.5 Mb in size, encompassing EHMT1 entirely. Clinical features included mild developmental delay, mild intellectual disability or learning problems, autism spectrum disorder, and behavior problems. The individuals did not consistently display dysmorphic features, congenital anomalies, or growth abnormalities. DNA methylation analysis revealed a weak DNAm profile for the cases with 9q34.3 duplication encompassing EHMT1, which could segregate the majority of the affected cases from controls. This study shows that individuals with 9q34.3 duplications including EHMT1 gene present with mild non-syndromic neurodevelopmental disorders and DNA methylation changes different from Kleefstra syndrome.

A recurrent de novo MAX p.Arg60Gln variant causes a syndromic overgrowth disorder through differential expression of c-Myc target genes.

Cyclin D2 (CCND2) stabilization underpins a range of macrocephaly-associated disorders through mutation of CCND2 or activating mutations in upstream genes encoding PI3K-AKT pathway components. Here, we describe three individuals with overlapping macrocephaly-associated phenotypes who carry the same recurrent de novo c.179G>A (p.Arg60Gln) variant in Myc-associated factor X (MAX). The mutation, located in the b-HLH-LZ domain, causes increased intracellular CCND2 through increased transcription but it does not cause stabilization of CCND2. We show that the purified b-HLH-LZ domain of MAXArg60Gln (Max∗Arg60Gln) binds its target E-box sequence with a lower apparent affinity. This leads to a more efficient heterodimerization with c-Myc resulting in an increase in transcriptional activity of c-Myc in individuals carrying this mutation. The recent development of Omomyc-CPP, a cell-penetrating b-HLH-LZ-domain c-Myc inhibitor, provides a possible therapeutic option for MAXArg60Gln individuals, and others carrying similar germline mutations resulting in dysregulated transcriptional c-Myc activity.

Epilepsy is an important feature of KBG syndrome associated with poorer developmental outcome.

OBJECTIVE: The aim of this study was to describe the epilepsy phenotype in a large international cohort of patients with KBG syndrome and to study a possible genotype-phenotype correlation. METHODS: We collected data on patients with ANKRD11 variants by contacting University Medical Centers in the Netherlands, an international network of collaborating clinicians, and study groups who previously published about KBG syndrome. All patients with a likely pathogenic or pathogenic ANKRD11 variant were included in our patient cohort and categorized into an "epilepsy group" or "non-epilepsy group". Additionally, we included previously reported patients with (likely) pathogenic ANKRD11 variants and epilepsy from the literature. RESULTS: We included 75 patients with KBG syndrome of whom 26 had epilepsy. Those with epilepsy more often had moderate to severe intellectual disability (42.3% vs 9.1%, RR 4.6 [95% CI 1.7-13.1]). Seizure onset in patients with KBG syndrome occurred at a median age of 4 years (range 12 months - 20 years), and the majority had generalized onset seizures (57.7%) with tonic-clonic seizures being most common (23.1%). The epilepsy type was mostly classified as generalized (42.9%) or combined generalized and focal (42.9%), not fulfilling the criteria of an electroclinical syndrome diagnosis. Half of the epilepsy patients (50.0%) were seizure free on anti-seizure medication (ASM) for at least 1 year at the time of last assessment, but 26.9% of patients had drug-resistant epilepsy (failure of ≥2 ASM). No genotype-phenotype correlation could be identified for the presence of epilepsy or epilepsy characteristics. SIGNIFICANCE: Epilepsy in KBG syndrome most often presents as a generalized or combined focal and generalized type. No distinctive epilepsy syndrome could be identified. Patients with KBG syndrome and epilepsy had a significantly poorer neurodevelopmental outcome compared with those without epilepsy. Clinicians should consider KBG syndrome as a causal etiology of epilepsy and be aware of the poorer neurodevelopmental outcome in individuals with epilepsy.

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.

CERT1 mutations perturb human development by disrupting sphingolipid homeostasis.

Neural differentiation, synaptic transmission, and action potential propagation depend on membrane sphingolipids, whose metabolism is tightly regulated. Mutations in the ceramide transporter CERT (CERT1), which is involved in sphingolipid biosynthesis, are associated with intellectual disability, but the pathogenic mechanism remains obscure. Here, we characterize 31 individuals with de novo missense variants in CERT1. Several variants fall into a previously uncharacterized dimeric helical domain that enables CERT homeostatic inactivation, without which sphingolipid production goes unchecked. The clinical severity reflects the degree to which CERT autoregulation is disrupted, and inhibiting CERT pharmacologically corrects morphological and motor abnormalities in a Drosophila model of the disease, which we call ceramide transporter (CerTra) syndrome. These findings uncover a central role for CERT autoregulation in the control of sphingolipid biosynthetic flux, provide unexpected insight into the structural organization of CERT, and suggest a possible therapeutic approach for patients with CerTra syndrome.

All-in-one whole exome sequencing strategy with simultaneous copy number variant, single nucleotide variant and absence-of-heterozygosity analysis in fetuses with structural ultrasound anomalies: A 1-year experience.

OBJECTIVE: We performed a 1-year evaluation of a novel strategy of simultaneously analyzing single nucleotide variants (SNVs), copy number variants (CNVs) and copy-number-neutral Absence-of-Heterozygosity from Whole Exome Sequencing (WES) data for prenatal diagnosis of fetuses with ultrasound (US) anomalies and a non-causative QF-PCR result. METHODS: After invasive diagnostics, whole exome parent-offspring trio-sequencing with exome-wide CNV analysis was performed in pregnancies with fetal US anomalies and a non-causative QF-PCR result (WES-CNV). On request, additional SNV-analysis, restricted to (the) requested gene panel(s) only (with the option of whole exome SNV-analysis afterward) was performed simultaneously (WES-CNV/SNV) or as rapid SNV-re-analysis, following a normal CNV analysis. RESULTS: In total, 415 prenatal samples were included. Following a non-causative QF-PCR result, WES-CNV analysis was initially requested for 74.3% of the chorionic villus (CV) samples and 45% of the amniotic fluid (AF) samples. In case WES-CNV analysis did not reveal a causative aberration, SNV-re-analysis was requested in 41.7% of the CV samples and 17.5% of the AF samples. All initial analyses could be finished within 2 weeks after sampling. For SNV-re-analysis during pregnancy, turn-around-times (TATs) varied between one and 8 days. CONCLUSION: We show a highly efficient all-in-one WES-based strategy, with short TATs, and the option of rapid SNV-re-analysis after a normal CNV result.

A Solve-RD ClinVar-based reanalysis of 1522 index cases from ERN-ITHACA reveals common pitfalls and misinterpretations in exome sequencing.

PURPOSE: Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the "ClinVar low-hanging fruit" reanalysis, reasons for the failure of previous analyses, and lessons learned. METHODS: Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. RESULTS: We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). CONCLUSION: The "ClinVar low-hanging fruit" analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock.

Modulating effects of FGF12 variants on NaV1.2 and NaV1.6 being associated with developmental and epileptic encephalopathy and Autism spectrum disorder: A case series.

OBJECTIVE: Fibroblast Growth Factor 12 (FGF12) may represent an important modulator of neuronal network activity and has been associated with developmental and epileptic encephalopathy (DEE). We sought to identify the underlying pathomechanism of FGF12-related disorders. METHODS: Patients with pathogenic variants in FGF12 were identified through published case reports, GeneMatcher and whole exome sequencing of own case collections. The functional consequences of two missense and two copy number variants (CNVs) were studied by co-expression of wildtype and mutant FGF12 in neuronal-like cells (ND7/23) with the sodium channels NaV1.2 or NaV1.6, including their beta-1 and beta-2 sodium channel subunits (SCN1B and SCN2B). RESULTS: Four variants in FGF12 were identified for functional analysis: one novel FGF12 variant in a patient with autism spectrum disorder and three variants from previously published patients affected by DEE. We demonstrate the differential regulating effects of wildtype and mutant FGF12 on NaV1.2 and NaV1.6 channels. Here, FGF12 variants lead to a complex kinetic influence on NaV1.2 and NaV1.6, including loss- as well as gain-of function changes in fast and slow inactivation. INTERPRETATION: We could demonstrate the detailed regulating effect of FGF12 on NaV1.2 and NaV1.6 and confirmed the complex effect of FGF12 on neuronal network activity. Our findings expand the phenotypic spectrum related to FGF12 variants and elucidate the underlying pathomechanism. Specific variants in FGF12-associated disorders may be amenable to precision treatment with sodium channel blockers. FUNDING: DFG, BMBF, Hartwell Foundation, National Institute for Neurological Disorders and Stroke, IDDRC, ENGIN, NIH, ITMAT, ILAE, RES and GRIN.

KCNQ2 R144 variants cause neurodevelopmental disability with language impairment and autistic features without neonatal seizures through a gain-of-function mechanism.

BACKGROUND: Prior studies have revealed remarkable phenotypic heterogeneity in KCNQ2-related disorders, correlated with effects on biophysical features of heterologously expressed channels. Here, we assessed phenotypes and functional properties associated with KCNQ2 missense variants R144W, R144Q, and R144G. We also explored in vitro blockade of channels carrying R144Q mutant subunits by amitriptyline. METHODS: Patients were identified using the RIKEE database and through clinical collaborators. Phenotypes were collected by a standardized questionnaire. Functional and pharmacological properties of variant subunits were analyzed by whole-cell patch-clamp recordings. FINDINGS: Detailed clinical information on fifteen patients (14 novel and 1 previously published) was analyzed. All patients had developmental delay with prominent language impairment. R144Q patients were more severely affected than R144W patients. Infantile to childhood onset epilepsy occurred in 40%, while 67% of sleep-EEGs showed sleep-activated epileptiform activity. Ten patients (67%) showed autistic features. Activation gating of homomeric Kv7.2 R144W/Q/G channels was left-shifted, suggesting gain-of-function effects. Amitriptyline blocked channels containing Kv7.2 and Kv7.2 R144Q subunits. INTERPRETATION: Patients carrying KCNQ2 R144 gain-of-function variants have developmental delay with prominent language impairment, autistic features, often accompanied by infantile- to childhood-onset epilepsy and EEG sleep-activated epileptiform activity. The absence of neonatal seizures is a robust and important clinical differentiator between KCNQ2 gain-of-function and loss-of-function variants. The Kv7.2/7.3 channel blocker amitriptyline might represent a targeted treatment. FUNDING: Supported by FWO, GSKE, KCNQ2-Cure, Jack Pribaz Foundation, European Joint Programme on Rare Disease 2020, the Italian Ministry for University and Research, the Italian Ministry of Health, the European Commission, the University of Antwerp, NINDS, and Chalk Family Foundation.

Loss-of-function variants in SRRM2 cause a neurodevelopmental disorder.

PURPOSE: SRRM2 encodes the SRm300 protein, a splicing factor of the SR-related protein family characterized by its serine- and arginine-enriched domains. It promotes interactions between messenger RNA and the spliceosome catalytic machinery. This gene, predicted to be highly intolerant to loss of function (LoF) and very conserved through evolution, has not been previously reported in constitutive human disease. METHODS: Among the 1000 probands studied with developmental delay and intellectual disability in our database, we found 2 patients with de novo LoF variants in SRRM2. Additional families were identified through GeneMatcher. RESULTS: Here, we report on 22 patients with LoF variants in SRRM2 and provide a description of the phenotype. Molecular analysis identified 12 frameshift variants, 8 nonsense variants, and 2 microdeletions of 66 kb and 270 kb. The patients presented with a mild developmental delay, predominant speech delay, autistic or attention-deficit/hyperactivity disorder features, overfriendliness, generalized hypotonia, overweight, and dysmorphic facial features. Intellectual disability was variable and mild when present. CONCLUSION: We established SRRM2 as a gene responsible for a rare neurodevelopmental disease.

Human KCNQ5 de novo mutations underlie epilepsy and intellectual disability.

We identified six novel de novo human KCNQ5 variants in children with motor/language delay, intellectual disability (ID), and/or epilepsy by whole exome sequencing. These variants, comprising two nonsense and four missense alterations, were functionally characterized by electrophysiology in HEK293/CHO cells, together with four previously reported KCNQ5 missense variants (Lehman A, Thouta S, Mancini GM, Naidu S, van Slegtenhorst M, McWalter K, Person R, Mwenifumbo J, Salvarinova R; CAUSES Study; EPGEN Study; Guella I, McKenzie MB, Datta A, Connolly MB, Kalkhoran SM, Poburko D, Friedman JM, Farrer MJ, Demos M, Desai S, Claydon T. Am J Hum Genet 101: 65-74, 2017). Surprisingly, all eight missense variants resulted in gain of function (GOF) due to hyperpolarized voltage dependence of activation or slowed deactivation kinetics, whereas the two nonsense variants were confirmed to be loss of function (LOF). One severe GOF allele (P369T) was tested and found to extend a dominant GOF effect to heteromeric KCNQ5/3 channels. Clinical presentations were associated with altered KCNQ5 channel gating: milder presentations with LOF or smaller GOF shifts in voltage dependence [change in voltage at half-maximal conduction (ΔV50) = ∼-15 mV] and severe presentations with larger GOF shifts in voltage dependence (ΔV50 = ∼-30 mV). To examine LOF pathogenicity, two Kcnq5 LOF mouse lines were created with CRISPR/Cas9. Both lines exhibited handling- and thermal-induced seizures and abnormal cortical EEGs consistent with epileptiform activity. Our study thus provides evidence for in vivo KCNQ5 LOF pathogenicity and strengthens the contribution of both LOF and GOF mutations to global pediatric neurological impairment, including ID/epilepsy.NEW & NOTEWORTHY Six novel de novo human KCNQ5 variants were identified from children with neurodevelopmental delay, intellectual disability, and/or epilepsy. Expression of these variants along with four previously reported KCNQ5 variants from a similar cohort revealed GOF potassium channels, negatively shifted in V50 of activation and/or delayed deactivation kinetics. GOF is extended to KCNQ5/3 heteromeric channels, making these the predominant channels affected in heterozygous de novo patients. Kcnq5 LOF mice exhibited seizures, consistent with in vivo pathogenicity.

Inherited variants in CHD3 show variable expressivity in Snijders Blok-Campeau syndrome.

PURPOSE: Common diagnostic next-generation sequencing strategies are not optimized to identify inherited variants in genes associated with dominant neurodevelopmental disorders as causal when the transmitting parent is clinically unaffected, leaving a significant number of cases with neurodevelopmental disorders undiagnosed. METHODS: We characterized 21 families with inherited heterozygous missense or protein-truncating variants in CHD3, a gene in which de novo variants cause Snijders Blok-Campeau syndrome. RESULTS: Computational facial and Human Phenotype Ontology-based comparisons showed that the phenotype of probands with inherited CHD3 variants overlaps with the phenotype previously associated with de novo CHD3 variants, whereas heterozygote parents are mildly or not affected, suggesting variable expressivity. In addition, similarly reduced expression levels of CHD3 protein in cells of an affected proband and of healthy family members with a CHD3 protein-truncating variant suggested that compensation of expression from the wild-type allele is unlikely to be an underlying mechanism. Notably, most inherited CHD3 variants were maternally transmitted. CONCLUSION: Our results point to a significant role of inherited variation in Snijders Blok-Campeau syndrome, a finding that is critical for correct variant interpretation and genetic counseling and warrants further investigation toward understanding the broader contributions of such variation to the landscape of human disease.

A Patient with Moderate Intellectual Disability and 49, XXXYY Karyotype.

Klinefelter syndrome is a chromosomal disorder in which one extra X chromosome is present (47,XXY). Several other numeric variants of this syndrome are described that comprise one or more additional sex chromosomes such as 48,XXXY, 48,XXYY and 49,XXXXY. These rare conditions are often associated with increased risk for congenital malformations, additional medical problems, and a more complex psychological phenotype. Since 1963, apart from two infants, only four adult patients with a XXXYY pentasomy have been published as case report. The present paper critically reviews the existing literature and provides detailed assessments of a 25-year-old male with intellectual disability and autism. For the first time, this very rare pentasomy is now recorded using all information about developmental history as well as findings from genetic, somatic, endocrinological and neuropsychological examination. It is concluded that children born with abnormalities of the external genitalia should always be evaluated for genetic abnormalities in order to avoid unwanted delay of appropriately designed multidisciplinary medical and psychological treatment.

Expanding the genotype and phenotype spectrum of SYT1-associated neurodevelopmental disorder.

PURPOSE: Synaptotagmin-1 (SYT1) is a critical mediator of neurotransmitter release in the central nervous system. Previously reported missense SYT1 variants in the C2B domain are associated with severe intellectual disability, movement disorders, behavioral disturbances, and electroencephalogram abnormalities. In this study, we expand the genotypes and phenotypes and identify discriminating features of this disorder. METHODS: We describe 22 individuals with 15 de novo missense SYT1 variants. The evidence for pathogenicity is discussed, including the American College of Medical Genetics and Genomics/Association for Molecular Pathology criteria, known structure-function relationships, and molecular dynamics simulations. Quantitative behavioral data for 14 cases were compared with other monogenic neurodevelopmental disorders. RESULTS: Four variants were located in the C2A domain with the remainder in the C2B domain. We classified 6 variants as pathogenic, 4 as likely pathogenic, and 5 as variants of uncertain significance. Prevalent clinical phenotypes included delayed developmental milestones, abnormal eye physiology, movement disorders, and sleep disturbances. Discriminating behavioral characteristics were severity of motor and communication impairment, presence of motor stereotypies, and mood instability. CONCLUSION: Neurodevelopmental disorder-associated SYT1 variants extend beyond previously reported regions, and the phenotypic spectrum encompasses a broader range of severities than initially reported. This study guides the diagnosis and molecular understanding of this rare neurodevelopmental disorder and highlights a key role for SYT1 function in emotional regulation, motor control, and emergent cognitive function.

Delayed Analysis of Hydrogen-Methane Breath Samples.

PURPOSE: Hydrogen-methane breath tests are used to diagnose carbohydrate malabsorption and small intestinal bacterial overgrowth. The COVID-19 pandemic has driven the modification of procedures as breath tests are potentially aerosol-generating procedures. We assessed the effect of delayed analysis of breath samples, facilitating the at-home performance of breath testing. METHODS: Children provided two breath samples at every step of the lactose breath test. The samples were brought back to the clinic, and one set of samples was analyzed immediately. The second set was stored at room temperature and analyzed 1-4 days later. RESULTS: Out of the 73 "double" lactose breath tests performed at home, 33 (45.8%) were positive. The second samples were analyzed 20 to 117 hours after the first samples (41.7±24.3 hours). There was no significant difference in the hydrogen concentration between the first and second sets (Z=0.49, p=0.62). This was not the case for methane, which had a significantly higher concentration in the second breath samples (Z=7.6). CONCLUSION: Expired hydrogen levels remain stable in plastic syringes if preserved at room temperature for several days. On the other hand, the delayed analysis of methane appeared to be less reliable. Further research is needed to examine the impact of delayed analysis on methane and hydrogen concentrations.

Expanding the phenotype of ASXL3-related syndrome: A comprehensive description of 45 unpublished individuals with inherited and de novo pathogenic variants in ASXL3.

The study aimed at widening the clinical and genetic spectrum of ASXL3-related syndrome, a neurodevelopmental disorder, caused by truncating variants in the ASXL3 gene. In this international collaborative study, we have undertaken a detailed clinical and molecular analysis of 45 previously unpublished individuals with ASXL3-related syndrome, as well as a review of all previously published individuals. We have reviewed the rather limited functional characterization of pathogenic variants in ASXL3 and discuss current understanding of the consequences of the different ASXL3 variants. In this comprehensive analysis of ASXL3-related syndrome, we define its natural history and clinical evolution occurring with age. We report familial ASXL3 pathogenic variants, characterize the phenotype in mildly affected individuals and discuss nonpenetrance. We also discuss the role of missense variants in ASXL3. We delineate a variable but consistent phenotype. The most characteristic features are neurodevelopmental delay with consistently limited speech, significant neuro-behavioral issues, hypotonia, and feeding difficulties. Distinctive features include downslanting palpebral fissures, hypertelorism, tubular nose with a prominent nasal bridge, and low-hanging columella. The presented data will inform clinical management of individuals with ASXL3-related syndrome and improve interpretation of new ASXL3 sequence variants.

Further evidence for de novo variants in SYNCRIP as the cause of a neurodevelopmental disorder.

SYNCRIP encodes for the Synaptotagmin-binding cytoplasmic RNA-interacting protein, involved in RNA-binding and regulation of multiple cellular pathways. It has been proposed as a candidate gene for neurodevelopmental disorders (NDDs) with autism spectrum disorder (ASD), intellectual disability (ID), and epilepsy. We ascertained genetic, clinical, and neuroradiological data of three additional individuals with novel de novo SYNCRIP variants. All individuals had ID. Autistic features were observed in two. One individual showed myoclonic-atonic epilepsy. Neuroradiological features comprised periventricular nodular heterotopia and widening of subarachnoid spaces. Two frameshift variants in the more severely affected individuals, likely result in haploinsufficiency. The third missense variant lies in the conserved RNA recognition motif (RRM) 2 domain likely affecting RNA-binding. Our findings support the importance of RRM domains for SYNCRIP functionality and suggest genotype-phenotype correlations. Our study provides further evidence for a SYNCRIP-associated NDD characterized by ID and ASD sporadically accompanied by malformations of cortical development and myoclonic-atonic epilepsy.

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.

Germline AGO2 mutations impair RNA interference and human neurological development.

ARGONAUTE-2 and associated miRNAs form the RNA-induced silencing complex (RISC), which targets mRNAs for translational silencing and degradation as part of the RNA interference pathway. Despite the essential nature of this process for cellular function, there is little information on the role of RISC components in human development and organ function. We identify 13 heterozygous mutations in AGO2 in 21 patients affected by disturbances in neurological development. Each of the identified single amino acid mutations result in impaired shRNA-mediated silencing. We observe either impaired RISC formation or increased binding of AGO2 to mRNA targets as mutation specific functional consequences. The latter is supported by decreased phosphorylation of a C-terminal serine cluster involved in mRNA target release, increased formation of dendritic P-bodies in neurons and global transcriptome alterations in patient-derived primary fibroblasts. Our data emphasize the importance of gene expression regulation through the dynamic AGO2-RNA association for human neuronal development.