Clustered mutations in the GRIK2 kainate receptor subunit gene underlie diverse neurodevelopmental disorders.

Kainate receptors (KARs) are glutamate-gated cation channels with diverse roles in the central nervous system. Bi-allelic loss of function of the KAR-encoding gene GRIK2 causes a nonsyndromic neurodevelopmental disorder (NDD) with intellectual disability and developmental delay as core features. The extent to which mono-allelic variants in GRIK2 also underlie NDDs is less understood because only a single individual has been reported previously. Here, we describe an additional eleven individuals with heterozygous de novo variants in GRIK2 causative for neurodevelopmental deficits that include intellectual disability. Five children harbored recurrent de novo variants (three encoding p.Thr660Lys and two p.Thr660Arg), and four children and one adult were homozygous for a previously reported variant (c.1969G>A [p.Ala657Thr]). Individuals with shared variants had some overlapping behavioral and neurological dysfunction, suggesting that the GRIK2 variants are likely pathogenic. Analogous mutations introduced into recombinant GluK2 KAR subunits at sites within the M3 transmembrane domain (encoding p.Ala657Thr, p.Thr660Lys, and p.Thr660Arg) and the M3-S2 linker domain (encoding p.Ile668Thr) had complex effects on functional properties and membrane localization of homomeric and heteromeric KARs. Both p.Thr660Lys and p.Thr660Arg mutant KARs exhibited markedly slowed gating kinetics, similar to p.Ala657Thr-containing receptors. Moreover, we observed emerging genotype-phenotype correlations, including the presence of severe epilepsy in individuals with the p.Thr660Lys variant and hypomyelination in individuals with either the p.Thr660Lys or p.Thr660Arg variant. Collectively, these results demonstrate that human GRIK2 variants predicted to alter channel function are causative for early childhood development disorders and further emphasize the importance of clarifying the role of KARs in early nervous system development.

CASK pathogenic variant which expands the clinical spectrum for MICPCH syndrome in an adult patient.

The CASK gene and its product protein kinase have been associated with microcephaly with pontine and cerebellar hypoplasia (MICPCH) syndrome and various other neurodevelopmental disorders. Clinical presentation is highly variable and generally includes intellectual disability, neurological disorders, and dysmorphic features, at a minimum. We present the case of one of the oldest known currently living patients with MICPCH syndrome with additional features not previously described in the literature (midface retrusion, macroglossia, dental crowding, adolescent-onset contractures at large joints, laxity at finger joints, and prominent wrist dystonia). Progressive hypertonicity throughout the patient's life has been managed with serial botulinum toxin injections. A comprehensive multimodal care team including physiatry, physical therapy, exercise therapy, and audiology has been assisting her with hearing deficits, communication skills, and mobility. This potentially expands the phenotype of MICPCH syndrome and provides information about the management of this condition into adulthood.

Clinical delineation, sex differences, and genotype-phenotype correlation in pathogenic KDM6A variants causing X-linked Kabuki syndrome type 2.

PURPOSE: The variant spectrum and the phenotype of X-linked Kabuki syndrome type 2 (KS2) are poorly understood. METHODS: Genetic and clinical details of new and published individuals with pathogenic KDM6A variants were compiled and analyzed. RESULTS: Sixty-one distinct pathogenic KDM6A variants (50 truncating, 11 missense) from 80 patients (34 males, 46 females) were identified. Missense variants clustered in the TRP 2, 3, 7 and Jmj-C domains. Truncating variants were significantly more likely to be de novo. Thirteen individuals had maternally inherited variants and one had a paternally inherited variant. Neonatal feeding difficulties, hypoglycemia, postnatal growth retardation, poor weight gain, motor delay, intellectual disability (ID), microcephaly, congenital heart anomalies, palate defects, renal malformations, strabismus, hearing loss, recurrent infections, hyperinsulinism, seizures, joint hypermobility, and gastroesophageal reflux were frequent clinical findings. Facial features of over a third of patients were not typical for KS. Males were significantly more likely to be born prematurely, have shorter stature, and severe developmental delay/ID. CONCLUSION: We expand the KDM6A variant spectrum and delineate the KS2 phenotype. We demonstrate that the variability of the KS2 phenotypic depends on sex and the variant type. We also highlight the overlaps and differences between the phenotypes of KS2 and KS1.

Expanding the molecular spectrum and the neurological phenotype related to CAMTA1 variants.

The CAMTA1-associated phenotype was initially defined in patients with intragenic deletions and duplications who showed nonprogressive congenital ataxia, with or without intellectual disability. Here, we describe 10 individuals with CAMTA1 variants: nine previously unreported (likely) pathogenic variants comprising one missense, four frameshift and four nonsense variants, and one missense variant of unknown significance. Six patients were diagnosed following whole exome sequencing and four individuals with exome-based targeted panel analysis. Most of them present with developmental delay, manifesting in speech and motor delay. Other frequent findings are hypotonia, cognitive impairment, cerebellar dysfunction, oculomotor abnormalities, and behavioral problems. Feeding problems occur more frequently than previously observed. In addition, we present a systematic review of 19 previously published individuals with causal variants, including copy number, truncating, and missense variants. We note a tendency of more severe cognitive impairment and recurrent dysmorphic features in individuals with a copy number variant. Pathogenic variants are predominantly observed in and near the N- and C- terminal functional domains. Clinical heterogeneity is observed, but 3'-terminal variants seem to associate with less pronounced cerebellar dysfunction.

High Rate of Recurrent De Novo Mutations in Developmental and Epileptic Encephalopathies.

Developmental and epileptic encephalopathy (DEE) is a group of conditions characterized by the co-occurrence of epilepsy and intellectual disability (ID), typically with developmental plateauing or regression associated with frequent epileptiform activity. The cause of DEE remains unknown in the majority of cases. We performed whole-genome sequencing (WGS) in 197 individuals with unexplained DEE and pharmaco-resistant seizures and in their unaffected parents. We focused our attention on de novo mutations (DNMs) and identified candidate genes containing such variants. We sought to identify additional subjects with DNMs in these genes by performing targeted sequencing in another series of individuals with DEE and by mining various sequencing datasets. We also performed meta-analyses to document enrichment of DNMs in candidate genes by leveraging our WGS dataset with those of several DEE and ID series. By combining these strategies, we were able to provide a causal link between DEE and the following genes: NTRK2, GABRB2, CLTC, DHDDS, NUS1, RAB11A, GABBR2, and SNAP25. Overall, we established a molecular diagnosis in 63/197 (32%) individuals in our WGS series. The main cause of DEE in these individuals was de novo point mutations (53/63 solved cases), followed by inherited mutations (6/63 solved cases) and de novo CNVs (4/63 solved cases). De novo missense variants explained a larger proportion of individuals in our series than in other series that were primarily ascertained because of ID. Moreover, these DNMs were more frequently recurrent than those identified in ID series. These observations indicate that the genetic landscape of DEE might be different from that of ID without epilepsy.

A multiparametric analysis of white matter maturation during late childhood and adolescence.

White matter development has been well described using diffusion tensor imaging (DTI), but the microstructural processes driving development remain unclear due to methodological limitations. Here, using neurite orientation dispersion and density imaging (NODDI), inhomogeneous magnetization transfer (ihMT), and multicomponent driven equilibrium single-pulse observation of T1/T2 (mcDESPOT), we describe white matter development at the microstructural level in a longitudinal cohort of healthy 6-15 year olds. We evaluated age and gender-related trends in fractional anisotropy (FA), mean diffusivity (MD), neurite density index (NDI), orientation dispersion index (ODI), quantitative ihMT (qihMT), myelin volume fraction (VFm ), and g-ratio. We found age-related increases of VFm in most regions, showing ongoing myelination in vivo during late childhood and adolescence for the first time. No relationship was observed between qihMT and age, suggesting myelin volume increases are driven by increased water content. Age-related increases were observed for NDI, suggesting axonal packing is also occurring during this time. g-ratio decreased with age in the uncinate fasciculus, implying changes in communication efficiency are ongoing in this region. FA increased and MD decreased with age in most regions. Gender effects were present in the left cingulum for FA, and an age-by-gender interaction was found for MD in the left uncinate fasciculus. These findings suggest that FA and MD remain useful markers of gender-related processes, and gender differences are likely driven by factors other than myelin. We conclude that white matter development during late childhood and adolescence is driven by a combination of axonal packing and myelin volume increases.

MAGEL2-related disorders: A study and case series.

Pathogenic MAGEL2 variants result in the phenotypes of Chitayat-Hall syndrome (CHS), Schaaf-Yang syndrome (SYS) and Prader-Willi syndrome (PWS). We present five patients with mutations in MAGEL2, including the first patient reported with a missense variant, adding to the limited literature. Further, we performed a systematic review of the CHS and SYS literature, assess the overlap between CHS, SYS and PWS, and analyze genotype-phenotype correlations among them. We conclude that there is neither a clinical nor etiological difference between CHS and SYS, and propose that the two syndromes simply be referred to as MAGEL2-related disorders.

Transverse relaxometry with transmit field-constrained stimulated echo compensation.

OBJECTIVE: Purely exponential decay is rarely observed in conventional mono-exponential T2 mapping due to transmit field inhomogeneity and calibration errors, which collectively introduce stimulated and indirect echo pathways. Stimulated echo correction (SEC) requires an additional fit parameter for the transmit field, resulting in greater uncertainty in T2 relative to mono-exponential fitting. The aim of this study was to develop an accurate and precise method for T2 mapping using SEC. METHODS: The proposed method, called two-step SEC (tSEC), leverages spatial correlations in the transmit field to reduce the number of fully independent fitting parameters from three to two. The method involves a two-pass fit: the first pass involves a fast but standard SEC fit. The initially estimated transmit field is smoothed and provided as a fixed input to the second pass. RESULTS: Simulations and in vivo experiments demonstrated up to 38% and 27% decreases in relative T2 variance with tSEC relative to SEC. Average T2 values were unchanged between tSEC and SEC fits. The proposed method uses the same input data as SEC and exponential fits, so it is applicable to existing data. DISCUSSION: The proposed method generates reliable and reproducible quantitative T2 maps and should be considered for future relaxometry studies.

Two cases of Legg-Perthes and intellectual disability in Tricho-Rhino-Phalangeal syndrome type 1 associated with novel TRPS1 mutations.

Tricho-Rhino-Phalangeal syndrome is a rare autosomal dominant genetic disorder caused by mutations in the TRPS1 gene. This malformation syndrome is characterized by distinctive craniofacial features including sparse scalp hair, bulbous tip of the nose, long flat philtrum, thin upper vermilion border, and protruding ears. Skeletal abnormalities include cone-shaped epiphyses at the phalanges, hip malformations, and short stature. In this report, we describe two patients with the physical manifestations and genotype of TRPS type I but with learning/intellectual disability not typically described as part of the syndrome. The first patient has a novel heterozygous two-base-pair deletion of nucleotides at 3198-3199 (c.3198-3199delAT) in the TRPS1 gene causing a translational frameshift and subsequent alternate stop codon. The second patient has a 3.08 million base-pair interstitial deletion at 8q23.3 (113,735,487-116,818,578), which includes the TRPS1 gene and CSMD3. Our patients have characteristic craniofacial features, Legg-Perthes syndrome, various skeletal abnormalities including cone shaped epiphyses, anxiety (first patient), and intellectual disability, presenting unusual phenotypes that add to the clinical spectrum of the disease.

De Novo Truncating Mutations in the Kinetochore-Microtubules Attachment Gene CHAMP1 Cause Syndromic Intellectual Disability.

A rare syndromic form of intellectual disability with impaired speech was recently found associated with mutations in CHAMP1 (chromosome alignment-maintaining phosphoprotein 1), the protein product of which is directly involved in microtubule-kinetochore attachment. Through whole-exome sequencing in six unrelated nonconsanguineous families having a sporadic case of intellectual disability, we identified six novel de novo truncating mutations in CHAMP1: c.1880C>G p.(Ser627*), c.1489C>T; p.(Arg497*), c.1876_1877delAG; p.(Ser626Leufs*4), c.1043G>A; p.(Trp348*), c.1002G>A; p.(Trp334*), and c.958_959delCC; p.(Pro320*). Our clinical observations confirm the phenotypic homogeneity of the syndrome, which represents therefore a distinct clinical entity. Besides, our functional studies show that CHAMP1 protein variants are delocalized from chromatin and are unable to bind to two of its direct partners, POGZ and HP1. These data suggest a pathogenic mechanism of the CHAMP1-associated intellectual disability syndrome mediated by direct interacting partners of CHAMP1, several of which are involved in chromo/kinetochore-related disorders.

A human mitochondrial poly(A) polymerase mutation reveals the complexities of post-transcriptional mitochondrial gene expression.

The p.N478D missense mutation in human mitochondrial poly(A) polymerase (mtPAP) has previously been implicated in a form of spastic ataxia with optic atrophy. In this study, we have investigated fibroblast cell lines established from family members. The homozygous mutation resulted in the loss of polyadenylation of all mitochondrial transcripts assessed; however, oligoadenylation was retained. Interestingly, this had differential effects on transcript stability that were dependent on the particular species of transcript. These changes were accompanied by a severe loss of oxidative phosphorylation complexes I and IV, and perturbation of de novo mitochondrial protein synthesis. Decreases in transcript polyadenylation and in respiratory chain complexes were effectively rescued by overexpression of wild-type mtPAP. Both mutated and wild-type mtPAP localized to the mitochondrial RNA-processing granules thereby eliminating mislocalization as a cause of defective polyadenylation. In vitro polyadenylation assays revealed severely compromised activity by the mutated protein, which generated only short oligo(A) extensions on RNA substrates, irrespective of RNA secondary structure. The addition of LRPPRC/SLIRP, a mitochondrial RNA-binding complex, enhanced activity of the wild-type mtPAP resulting in increased overall tail length. The LRPPRC/SLIRP effect although present was less marked with mutated mtPAP, independent of RNA secondary structure. We conclude that (i) the polymerase activity of mtPAP can be modulated by the presence of LRPPRC/SLIRP, (ii) N478D mtPAP mutation decreases polymerase activity and (iii) the alteration in poly(A) length is sufficient to cause dysregulation of post-transcriptional expression and the pathogenic lack of respiratory chain complexes.

The complex behavioral phenotype of 15q13.3 microdeletion syndrome.

BACKGROUND: Chromosome 15q13.3 represents a hotspot for genomic rearrangements due to repetitive sequences mediating nonallelic homologous recombination. Deletions of 15q13.3 have been identified in the context of multiple neurological and psychiatric disorders, but a prospective clinical and behavioral assessment of affected individuals has not yet been reported. METHODS: Eighteen subjects with 15q13.3 microdeletion underwent a series of behavioral assessments, along with clinical history and physical examination, to comprehensively define their behavioral phenotypes. RESULTS: Cognitive deficits are the most prevalent feature in 15q13.3 deletion syndrome, with an average nonverbal IQ of 60 among the patients studied. Autism spectrum disorder was highly penetrant, with 31% of patients meeting clinical criteria and exceeding cutoff scores on both ADOS-2 and ADI-R. Affected individuals exhibited a complex pattern of behavioral abnormalities, most notably hyperactivity, attention problems, withdrawal, and externalizing symptoms, as well as impairments in functional communication, leadership, adaptive skills, and activities of daily living. CONCLUSIONS: The 15q13.3 deletion syndrome encompasses a heterogeneous behavioral phenotype that poses a major challenge to parents, caregivers, and treating providers. Further work to more clearly delineate genotype-phenotype relationships in 15q13.3 deletions will be important for anticipatory guidance and development of targeted therapies.Genet Med 18 11, 1111-1118.

CLTC as a clinically novel gene associated with multiple malformations and developmental delay.

Diagnostic exome sequencing has recently emerged as an invaluable tool in determining the molecular etiology of cases involving dysmorphism and developmental delay that are otherwise unexplained by more traditional methods of genetic testing. Our patient was large for gestational age at 35 weeks, delivered to a 27-year-old primigravid Caucasian whose pregnancy was complicated by preeclampsia. Neonatal period was notable for hypoglycemia, apnea, bradycardia, hyperbilirubinemia, grade I intraventricular hemorrhage, subdural hematoma, laryngomalacia, hypotonia, and feeding difficulties. The patient had numerous minor dysmorphic features. At three and a half years of age, she has global developmental delays and nystagmus, and is being followed for a mediastinal neuroblastoma that is currently in remission. Karyotype and oligo-microarray were normal. Whole-exome, next generation sequencing (NGS) coupled to bioinformatic filtering and expert medical review at Ambry Genetics revealed 14 mutations in 9 genes, and these genes underwent medical review. A heterozygous de novo frameshift mutation, c.2737_2738dupGA p.D913Efs*59, in which two nucleotides are duplicated in exon 17 of the CLTC gene, results in substitution of glutamic acid for aspartic acid at position 913 of the protein, as well as a frame shift that results in a premature termination codon situated 58 amino acids downstream. Clathrin Heavy Chain 1 (CHC1) has been shown to play an important role in the brain for vesicle recycling and neurotransmitter release at pre-synaptic nerve terminals. There is also evidence implicating it in the proper development of the placenta during the early stages of pregnancy. The CLTC alteration identified herein is likely to provide an explanation for the patient's adverse phenotype. Ongoing functional studies will further define the impact of this alteration on CHC1 function and consequently, human disease.

Phenotypic heterogeneity of genomic disorders and rare copy-number variants.

BACKGROUND: Some copy-number variants are associated with genomic disorders with extreme phenotypic heterogeneity. The cause of this variation is unknown, which presents challenges in genetic diagnosis, counseling, and management. METHODS: We analyzed the genomes of 2312 children known to carry a copy-number variant associated with intellectual disability and congenital abnormalities, using array comparative genomic hybridization. RESULTS: Among the affected children, 10.1% carried a second large copy-number variant in addition to the primary genetic lesion. We identified seven genomic disorders, each defined by a specific copy-number variant, in which the affected children were more likely to carry multiple copy-number variants than were controls. We found that syndromic disorders could be distinguished from those with extreme phenotypic heterogeneity on the basis of the total number of copy-number variants and whether the variants are inherited or de novo. Children who carried two large copy-number variants of unknown clinical significance were eight times as likely to have developmental delay as were controls (odds ratio, 8.16; 95% confidence interval, 5.33 to 13.07; P=2.11×10(-38)). Among affected children, inherited copy-number variants tended to co-occur with a second-site large copy-number variant (Spearman correlation coefficient, 0.66; P<0.001). Boys were more likely than girls to have disorders of phenotypic heterogeneity (P<0.001), and mothers were more likely than fathers to transmit second-site copy-number variants to their offspring (P=0.02). CONCLUSIONS: Multiple, large copy-number variants, including those of unknown pathogenic significance, compound to result in a severe clinical presentation, and secondary copy-number variants are preferentially transmitted from maternal carriers. (Funded by the Simons Foundation Autism Research Initiative and the National Institutes of Health.).

Gastrointestinal manifestations in diploid/triploid mixoploidy.

A girl infant was delivered by cesarean section at 32 weeks of gestation because of growth arrest and poor movement patterns. The infant had feeding problems, which were based on gastroesophageal reflux, laryngomalacia, and decreased gut motility. Hypotonia was notable from the outset, and the patient eventually displayed significant delays in both motor and cognitive milestones. Meanwhile, lymphocytes had yielded a normal karyotype (46,XX), but at 2 years of age the patient underwent a skin biopsy and mosaicism because a 68,XX cell line was discovered in fibroblasts. At the age 6.4 years, the patient is short of stature below the 3rd percentile but has a weight at the 42nd percentile and head circumference above the 97th percentile. Other phenotypic features include low-set ears, piebald irides and scalp hair, eyelid ptosis, strabismus, broad nasal bridge, anteverted nares, upswept eyebrows, hypoplastic teeth, pectus excavatum, hypoplastic labia, scoliosis, 3-4 finger syndactyly, and 2-3 toe syndactyly. We present this case with a review of the literature for mixoploidy (the rare event of mosaicism for diploid and triploid cell lines). We add to the existing data on the clinical features of diploid/triploid mixoploidy. The complexities of the gastrointestinal problems make this case unusual.

Highly accelerated dynamic contrast enhanced imaging.

PURPOSE: Dynamic contrast-enhanced imaging provides unique physiological information, notably the endothelial permeability (K(trans)), and may improve the diagnosis and management of multiple pathologies. Current acquisition methods provide limited spatial-temporal resolution and field-of-view, often preventing characterization of the entire pathology and precluding measurement of the arterial input function. We present a method for highly accelerated dynamic imaging and demonstrate its utility for dynamic contrast-enhanced modeling. METHODS: We propose a novel Poisson ellipsoid sampling scheme and enforce multiple spatial and temporal l1-norm constraints during image reconstruction. Retrospective and prospective analyses were performed to validate the approach. RESULTS: Retrospectively, no mean bias or diverging trend was observed as the acceleration rate was increased from 3× to 18×; less than 10% error was measured in K(trans) at any individual rates in this range. Prospectively accelerated images at a rate of 36× enabled full brain coverage with 0.94 × 0.94 × 1.9 mm(3) spatial and 4.1 s temporal resolutions. Images showed no visible degradation and provided accurate K(trans) values when compared to a clinical population. CONCLUSION: Highly accelerated dynamic MRI using compressed sensing and parallel imaging provides accurate permeability modeling and enables full brain, high resolution acquisitions.

Glutamate alterations associated with transcranial magnetic stimulation in youth depression: a case series.

OBJECTIVE: We hypothesized an increase in dorsolateral prefrontal cortex (DLPFC) glutamate levels would occur after 3 weeks of repetitive transcranial magnetic stimulation (rTMS) treatment and a decrease in major depressive disorder (MDD) symptoms. METHODS: We report 6 patients (4 females) 15 to 21 years of age with treatment-resistant MDD. Participants had a mean (SD) age of 18.7 (1.95) years and a mean (SD) IQ of 102.3 (3.39). Short echo proton magnetic resonance spectroscopy (¹H-MRS) was used to quantify glutamate levels in the left DLPFC (4.5 cc) before and after rTMS treatment. Repetitive transcranial magnetic stimulation was localized to the left DLPFC and applied for 15 consecutive weekdays (120% resting motor threshold; 40 pulses over 4 seconds [10 Hz]; intertrain interval, 26 seconds; 75 trains; 3000 pulses). Treatment response was defined as a greater than 50% reduction in Hamilton Depression Rating Scale scores. Short echo proton magnetic resonance spectroscopy data were analyzed with LCModel to determine glutamate concentration. RESULTS: After rTMS, treatment responders (n = 4) showed an increase (relative to baseline) in left DLPFC glutamate levels (11%), which corresponded to an improvement in depressive symptom severity (68% Hamilton Depression Rating Scale score reduction). Treatment nonresponders (n = 2) had elevated baseline glutamate levels compared to responders in that same region, which decreased with rTMS (-10%). Procedures were generally well tolerated with no adverse events. CONCLUSIONS: Repetitive transcranial magnetic stimulation is feasible and possibly efficacious in adolescents with MDD. In responders, rTMS may act by induced elevations in elevating DFPLC glutamate levels in the left DLPFC, thereby leading to symptom improvement.

TREX tetramer disruption alters RNA processing necessary for corticogenesis in THOC6 Intellectual Disability Syndrome.

THOC6 variants are the genetic basis of autosomal recessive THOC6 Intellectual Disability Syndrome (TIDS). THOC6 is critical for mammalian Transcription Export complex (TREX) tetramer formation, which is composed of four six-subunit THO monomers. The TREX tetramer facilitates mammalian RNA processing, in addition to the nuclear mRNA export functions of the TREX dimer conserved through yeast. Human and mouse TIDS model systems revealed novel THOC6-dependent, species-specific TREX tetramer functions. Germline biallelic Thoc6 loss-of-function (LOF) variants result in mouse embryonic lethality. Biallelic THOC6 LOF variants reduce the binding affinity of ALYREF to THOC5 without affecting the protein expression of TREX members, implicating impaired TREX tetramer formation. Defects in RNA nuclear export functions were not detected in biallelic THOC6 LOF human neural cells. Instead, mis-splicing was detected in human and mouse neural tissue, revealing novel THOC6-mediated TREX coordination of mRNA processing. We demonstrate that THOC6 is required for key signaling pathways known to regulate the transition from proliferative to neurogenic divisions during human corticogenesis. Together, these findings implicate altered RNA processing in the developmental biology of TIDS neuropathology.

Spliceosome malfunction causes neurodevelopmental disorders with overlapping features.

Pre-mRNA splicing is a highly coordinated process. While its dysregulation has been linked to neurological deficits, our understanding of the underlying molecular and cellular mechanisms remains limited. We implicated pathogenic variants in U2AF2 and PRPF19, encoding spliceosome subunits in neurodevelopmental disorders (NDDs), by identifying 46 unrelated individuals with 23 de novo U2AF2 missense variants (including 7 recurrent variants in 30 individuals) and 6 individuals with de novo PRPF19 variants. Eight U2AF2 variants dysregulated splicing of a model substrate. Neuritogenesis was reduced in human neurons differentiated from human pluripotent stem cells carrying two U2AF2 hyper-recurrent variants. Neural loss of function (LoF) of the Drosophila orthologs U2af50 and Prp19 led to lethality, abnormal mushroom body (MB) patterning, and social deficits, which were differentially rescued by wild-type and mutant U2AF2 or PRPF19. Transcriptome profiling revealed splicing substrates or effectors (including Rbfox1, a third splicing factor), which rescued MB defects in U2af50-deficient flies. Upon reanalysis of negative clinical exomes followed by data sharing, we further identified 6 patients with NDD who carried RBFOX1 missense variants which, by in vitro testing, showed LoF. Our study implicates 3 splicing factors as NDD-causative genes and establishes a genetic network with hierarchy underlying human brain development and function.