Neurodevelopmental disorders (NDDs) affect 2-4% of the population, are predominantly genetic and remain unsolved in ~50% of individuals. We show that rare biallelic variants in RNU2-2 are enriched and over-transmitted in individuals with unresolved NDDs. We define a recessive RNU2-2 syndrome, delineate its unique genetic architecture and show that it manifests clinically as a severe developmental and epileptic encephalopathy. We find that candidate biallelic variants are significantly correlated with reduced U2-2 abundance, implicating compromised transcript stability as a probable pathomechanism. We identify a decreased ratio of U2-2 to its paralog U2-1 as a potential diagnostic biomarker for this condition. We show that the recessive RNU2-2 syndrome is genetically, clinically and mechanistically distinct from the dominant RNU2-2 disorder. Within our cohort, the recessive RNU2-2 syndrome emerges as by far the most frequent recessive NDD, greatly disproportionate to the small genomic footprint of this non-protein-coding gene.

Cytoskeletal organization, cell adhesion, and cell motility are key to neuronal development and functional synapses. Obg-like ATPase 1 (OLA1) regulates cell-matrix adhesion by modulating focal adhesion kinase (FAK) levels, therefore regulating cytoskeletal dynamics and cell motility. To date, however, no Mendelian phenotypes in humans have been linked to OLA1. We identified fourteen individuals from nine families in whom hypermobility-neurodevelopmental disorder with distinct facies is linked to bi-allelic deleterious variants in OLA1. The hypermobility phenotype evoked a diagnosis of Ehlers-Danlos syndrome (EDS) in some affected individuals. The loss-of-function nature of these variants is confirmed in proband-derived fibroblasts, recapitulating the impaired migration and proliferation phenotype previously described in OLA1-deficient cells. To explore the pathogenesis of abnormal neurodevelopment in our probands, we investigated neurons derived from proband fibroblasts and identified impaired adhesion and cytoskeletal control. Modeling ola-1 deficiency in C. elegans revealed reduced neurite numbers compared to the wild type. Additionally, transcriptomic analysis of the ola-1-deficient worms suggested that dysregulation of key signaling pathways results in suppression of microtubule dynamics and axon regrowth, ultimately crippling the regenerative competence of mutant animals compared to wild-type controls. Our results support an autosomal-recessive OLA1-related hypermobility-neurodevelopmental disorder and suggest that dysregulation of key signaling pathways results in the suppression of microtubule dynamics as a potential underlying mechanism.

This retrospective study on X-linked PDHA1-related pyruvate dehydrogenase complex (PDHc) deficiency combined a systematic literature review with a multicentre survey exploring genotypes, phenotypes and survival. Data from 891 individuals (45% unpublished) were included. Of note, 53% of cases were females. Median age at last assessment was 6 years (range 0-80 years, n = 622). We detected 331 different (118 unpublished) PDHA1 variants, of which 75% (305/405) had occurred de novo. Variants in this study were uploaded to ClinVar (SCV006297015-SCV006297345). The 10 most frequent variants accounted for 36% of the diagnoses. Sixty-nine per cent of the variants were private; missense (50%) and frameshift (20%) variants were most common. Frameshift/nonsense (FS/N) variants in males (44/401, 11%) were confined to regions escaping nonsense-mediated decay (NMD) and were significantly less frequent than in females (151/461, 33%). Neonatal or infantile (405/529, 77%) presentations were most frequent, with pre/perinatal abnormalities reported in 47% (159/342). FS/N variants in the NMD-predicted region 3.9 [95% confidence interval (CI) 1.54-11.04] times increased the odds of fetal findings. Females presented significantly earlier [2 months, interquartile range (IQR) 7.0, n = 224] than males (8 months, IQR 16.6, n = 233), with increased risk of neonatal presentation [odds ratio (OR) 3.01 (95% CI 1.279-7.616)] when harbouring FS/N variants in the NMD-predicted region. The overall (n = 242) mean survival time was 10.9 (95% CI 9.9-11.9) years. On average, females survived 4.5 (95% CI 2.62-6.40) years longer than males despite presenting more severe phenotypes. Poor survival was associated with male sex [hazard ratio (HR) 3.3 (95% CI 1.95-5.62)], neonatal presentation [HR 5.5 (95% CI 2.17-14.09)], FS/N variants in the NMD-predicted region [HR 4.0 (95% CI 1.78, 9.16)] and splice variants [HR 2.3 (95% CI 1.15, 4.59)]. More severe clinical phenotypes were predicted by neonatal or infantile presentations and by female sex. Developmental delay (DD), intellectual disability (ID), muscle hypotonia, abnormal movements, seizures, feeding difficulties and microcephaly were the most frequent phenotypes, all occurring in more than half. Corpus callosum or basal ganglia alterations and cerebral atrophy were common. Four per cent (36/891) were reported to have mild phenotypes with no DD nor ID (25/36 males). This is the largest dataset on a nuclear-encoded defect of mitochondrial energy metabolism. The genotypic and phenotypic details further defines the disease landscape and can be used for variant interpretation. The correlations between genotypes, sex, phenotypes and survival, adds substantial improvement to counselling.

Hyperprolinemia type II (HPII) is a rare inherited metabolic disorder caused by the ALDH4A1 gene variant. Herein, we report a case of a preschool-aged Saudi girl who was born from consanguineous parents and presented with global developmental delay. The patient was clinically diagnosed with autism spectrum disorder with associated disruptive behaviors. Metabolic investigations revealed markedly elevated plasma and urinary proline levels, suggestive of a proline metabolism disorder. Whole-exome sequencing identified a homozygous variant of uncertain significance in the ALDH4A1 gene, which is associated with autosomal recessive HPII. Genetic testing of the patient's family members showed that all individuals had carrier status with varying zygosity. This case underscores the importance of metabolic and genetic evaluation in children with neurodevelopmental disorders and highlights that HPII can present with a clinical phenotype that overlaps substantially with ASD.

TMEM17 encodes a transition zone protein essential for ciliary function. Three cases with homozygous variants in TMEM17 in primary ciliopathies (Joubert and Oral-Facial-Digital syndrome) have been reported. We investigated whether biallelic TMEM17 variants contribute to primary ciliopathies. We queried our Biodatabank and evaluated the gene-disease relationship (GDR) according to the ClinGen recommendations. Four unrelated patients (four families) were identified with a clinical diagnosis of Meckel-Gruber syndrome (MGS) and novel homozygous variants: NM_198276.3:c.4del p.(Glu2Serfs*58); NM_198276.3:c.366dup p.(Pro123Thrfs*9); and NM_198276.3:c.368C>G p.(Pro123Arg). A fifth family lost three foetuses with MGS phenotype, both parents are heterozygote carriers (NM_198276.3:c.4del p.(Glu2Serfs*58)) but biological material from the foetuses was not available. The cases in this study had a severe prenatal phenotype, including encephalocele, polycystic kidney dysplasia, and polydactyly, leading to early lethality. This study strengthens the gene-disease association of TMEM17, upgrading it from "limited" to "moderate." We expand the phenotypic spectrum, ranging from MGS-with prenatal onset and early lethality-to Oral-Facial-Digital and Joubert syndromes. Our findings indicate that loss-of-function variants may underlie the most severe TMEM17 ciliopathy manifestations, suggesting a potential genotype-phenotype correlation.

We report an autosomal recessive neurodevelopmental disorder exclusively affecting females carrying biallelic loss-of-function variants in CIZ1, a gene encoding a nuclear matrix protein essential for the maintenance of X-chromosome inactivation. Eight unrelated affected females were identified, whereas male siblings with biallelic variants were asymptomatic. We showed that loss of CIZ1 compromises the maintenance of X-chromosome inactivation, leading to an abnormal overexpression of a subset of X-linked genes. ### Competing Interest Statement S.A. is a co-founder, and X.B. and E.R., are employees of Medigenome. F.A., B.A. and K.B. are employees of Lifera Omics. G.O. and A.R. are employees of Arcensus GmbH. ### Funding Statement This work was carried out within the framework of the FHU GenOMedS with the support of the Health Cooperation Group of University Hospitals of the Great West (GCS HUGO). This work was also supported by the Groupama Foundation. G.S. and G.MN. received funding from the Medical Research Council (MRC) project grant (UKRI577). M.A, R.A., received funding from the King Salman Center for Disability Research through Excellence Research Center (No. KSCDR-ERC-2024-02). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics committee of CHU de Nantes (Advisory Committee on Information Processing in Research; number CCTIRS: 14.556) gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

MDGA2 encodes a membrane-associated protein that is critical for regulating glutamatergic synapse development, modulating neuroligins (Nlgns), and maintaining the balance between excitatory and inhibitory synapses. Although MDGA2 has been extensively studied in murine and cellular models, its association with human developmental disorders has not been established to date. Through exome sequencing we identified six distinct homozygous loss-of-function MDGA2 variants in eight individuals from six consanguineous families, all presenting with developmental and epileptic encephalopathy (DEE). Clinically, these patients exhibited infantile hypotonia, severe neurodevelopmental delay, intractable seizures, progressive brain atrophy, and consistent dysmorphic features, including high anterior hairline, high-arched eyebrows, broad nasal ridge, tented upper lip, and large, low-set ears. Functional studies of three representative nonsense variants in mammalian expression systems and hippocampal cultured neurons revealed impaired MDGA2 membrane trafficking and disrupted Nlgn1 interaction, leading to defective excitatory synapse formation, synaptic transmission, and synaptic strength. Altogether, our findings definitively establish MDGA2 as a novel gene for autosomal recessive DEE subtype, with the pathogenesis explained by loss-of-function mechanism. This discovery underscores the previously unrecognized role of MDGA2 in human synaptic development and regulation, significantly expanding our understanding of the genetic architecture of DEE. ### Competing Interest Statement VAY is founder, shareholder and managing director of OmicsDiscoveries GmbH. ### Funding Statement H.M. was supported by the Wellcome Trust grant 220906/Z/20/Z and UCL Global Engagement Fund scheme 2023. H.K. was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science and Future Planning (RS-2024-00339642). J.W.U. was supported by the NRF funded by the Ministry of Science and Future Planning (RS-2023-NR076948). J.K. was supported by the NRF funded by the Ministry of Science and Future Planning (RS-2022-NR070708). M.Z. was funded by GERF-STDF: 33650, STDF, Egypt. H.H. was funded by the Wellcome Trust, MRC, MSA Trust, National Institute for Health Research University College London Hospitals Biomedical Research Centre (NIHR-BRC), Michael J Fox Foundation (MJFF), Fidelity Trust, Rosetrees Trust, Dolby Family Fund, Alzheimer's Research UK (ARUK), MSA Coalition, Parkinson's Disease Society, Parkinson's Foundation, Guarantors of Brain, Cerebral Palsy Alliance, FARA, EAN, Victoria Brain Bank, NIH NeuroBioBank, Queen Square BrainBank, and MRC Brainbank Network. V.A.Y. and J.G. were funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) via the project NFDI 1/1 "GHGA - German Human Genome-Phenome Archive" (#441914366). ERDERA has received funding from the European Union Horizon Europe research and innovation programme under grant agreement No. 101156595. The TUM IT infrastructure was co-funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) , Project-ID 461264291 ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Research Ethics Committee, Institute of Neurology, University College London (ION UCL) (07/Q0512/26) I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The data that support the findings of this study are available within the paper and in the supplemental information. Whole exome sequencing data are not publicly available due to privacy or ethical restrictions.

Congenital anomalies of the kidney and urinary tract (CAKUT) are the predominant cause for chronic kidney disease below age 30 years. Many monogenic forms have been discovered due to comprehensive genetic testing like exome sequencing. However, disease-causing variants in known disease-associated genes only explain a proportion of cases. Here, we aim to unravel underlying molecular mechanisms of syndromic CAKUT in three unrelated multiplex families with presumed autosomal recessive inheritance. Exome sequencing in the index individuals revealed three different rare homozygous variants in FOXD2, encoding a transcription factor not previously implicated in CAKUT in humans: a frameshift in the Arabic and a missense variant each in the Turkish and the Israeli family with segregation patterns consistent with autosomal recessive inheritance. CRISPR/Cas9-derived Foxd2 knockout mice presented with a bilateral dilated kidney pelvis accompanied by atrophy of the kidney papilla and mandibular, ophthalmologic, and behavioral anomalies, recapitulating the human phenotype. In a complementary approach to study pathomechanisms of FOXD2-dysfunction-mediated developmental kidney defects, we generated CRISPR/Cas9-mediated knockout of Foxd2 in ureteric bud-induced mouse metanephric mesenchyme cells. Transcriptomic analyses revealed enrichment of numerous differentially expressed genes important for kidney/urogenital development, including Pax2 and Wnt4 as well as gene expression changes indicating a shift toward a stromal cell identity. Histology of Foxd2 knockout mouse kidneys confirmed increased fibrosis. Further, genome-wide association studies suggest that FOXD2 could play a role for maintenance of podocyte integrity during adulthood. Thus, our studies help in genetic diagnostics of monogenic CAKUT and in understanding of monogenic and multifactorial kidney diseases.

Introduction: Rare diseases (RDs) create a massive burden for governments and families because sufferers of these diseases are required to undergo long-term treatment or rehabilitation to maintain a normal life. In Saudi Arabia (SA), the prevalence of RDs is high as a result of cultural and socio-economic factors. This study, however, aims to shed light on the genetic component of the prevalence of RDs in SA. Methodology: A retrospective study was conducted between September 2020 and December 2021 at King Saud Medical City, a tertiary hospital of the Ministry of Health (MOH), SA. A total of 1080 individuals with 544 potentially relevant variants were included. The index was 738, and the samples were tested in a commercialized laboratory using different molecular techniques, including next-generation sequencing. Result: A total of 867 molecular genetics tests were conducted on 738 probands. These tests included 610 exome sequencing (ES) tests, four genome sequencing (GS) tests, 82 molecular panels, 106 single nucleotide polymorphism (SNP) array, four methylation studies, 58 single-gene studies and three mitochondrial genome sequencing tests. The diagnostic yield among molecular genetics studies was 41.8% in ES, 24% in panels, 12% in SNP array and 24% in single gene studies. The majority of the identified potential variants (68%) were single nucleotide variants (SNV). Other ascertained variants included frameshift (11%), deletion (10%), duplication (5%), splicing (9%), in-frame deletion (3%) and indels (1%). The rate of positive consanguinity was 56%, and the autosomal recessive accounted for 54%. We found a significant correlation between the ES detection rate and positive consanguinity. We illustrated the presence of rare treatable conditions in DNAJC12 , SLC19A3 , and ALDH7A1, and the presence of the founder effect variant in SKIC2. Neurodevelopmental disorders were the main phenotype for which genetics studies were required (35.7%). Conclusion: This is the sixth-largest local study reporting next-generation sequencing. The results indicate the influence of consanguineous marriages on genetic disease and the burden it causes for the Kingdom of SA. This study highlights the need to enrich our society’s knowledge of genetic disorders. We recommend utilising ES as a first-tier test to establish genetic diagnosis in a highly consanguineous population.

Eukaryotic initiation factor-4A2 (EIF4A2) is an ATP-dependent RNA helicase and a member of the DEAD-box protein family that recognizes the 5' cap structure of mRNAs, allows mRNA to bind to the ribosome, and plays an important role in microRNA-regulated gene repression. Here, we report on 15 individuals from 14 families presenting with global developmental delay, intellectual disability, hypotonia, epilepsy, and structural brain anomalies, all of whom have extremely rare de novo mono-allelic or inherited bi-allelic variants in EIF4A2. Neurodegeneration was predominantly reported in individuals with bi-allelic variants. Molecular modeling predicts these variants would perturb structural interactions in key protein domains. To determine the pathogenicity of the EIF4A2 variants in vivo, we examined the mono-allelic variants in Drosophila melanogaster (fruit fly) and identified variant-specific behavioral and developmental defects. The fruit fly homolog of EIF4A2 is eIF4A, a negative regulator of decapentaplegic (dpp) signaling that regulates embryo patterning, eye and wing morphogenesis, and stem cell identity determination. Our loss-of-function (LOF) rescue assay demonstrated a pupal lethality phenotype induced by loss of eIF4A, which was fully rescued with human EIF4A2 wild-type (WT) cDNA expression. In comparison, the EIF4A2 variant cDNAs failed or incompletely rescued the lethality. Overall, our findings reveal that EIF4A2 variants cause a genetic neurodevelopmental syndrome with both LOF and gain of function as underlying mechanisms.

Introduction: Physicians face diagnostic dilemmas upon reports indicating disease variants of unknown significance (VUS). The most puzzling cases are patients with rare diseases, where finding another matched genotype and phenotype to associate their results is challenging. This study aims to prove the value of updating patient files with new classifications, potentially leading to better assessment and prevention. Methodology: We recruited retrospective phenotypic and genotypic data from King Saud Medical City, Riyadh, Kingdom of Saudi Arabia. Between September 2020 and December 2021, 1,080 patients' genetic profiles were tested in a College of American Pathologists accredited laboratory. We excluded all confirmed pathogenic variants, likely pathogenic variants and copy number variations. Finally, we further reclassified 194 VUS using different local and global databases, employing in silico prediction to justify the phenotype-genotype association. Results: Of the 194 VUS, 90 remained VUS, and the other 104 were reclassified as follows: 16 pathogenic, 49 likely pathogenic, nine benign, and 30 likely benign. Moreover, most of these variants had never been observed in other local or international databases. Conclusion: Reclassifying the VUS adds value to understanding the causality of the phenotype if it has been reported in another family or population. The healthcare system should establish guidelines for re-evaluating VUS, and upgrading VUS should reflect on individual/family risks and management strategies.

The von Willebrand Factor A domain containing 1 protein, encoded by VWA1, is an extracellular matrix protein expressed in muscle and peripheral nerve. It interacts with collagen VI and perlecan, two proteins that are affected in hereditary neuromuscular disorders. Lack of VWA1 is known to compromise peripheral nerves in a Vwa1 knock-out mouse model. Exome sequencing led us to identify bi-allelic loss of function variants in VWA1 as the molecular cause underlying a so far genetically undefined neuromuscular disorder. We detected six different truncating variants in 15 affected individuals from six families of German, Arabic, and Roma descent. Disease manifested in childhood or adulthood with proximal and distal muscle weakness predominantly of the lower limbs. Myopathological and neurophysiological findings were indicative of combined neurogenic and myopathic pathology. Early childhood foot deformity was frequent, but no sensory signs were observed. Our findings establish VWA1 as a new disease gene confidently implicated in this autosomal recessive neuromyopathic condition presenting with child-/adult-onset muscle weakness as a key clinical feature.

The authors apologize for errors in the author list, Acknowledgements section, and References. These have been corrected.

Spermatogenesis-associated 5 like 1 (SPATA5L1) represents an orphan gene encoding a protein of unknown function. We report 28 bi-allelic variants in SPATA5L1 associated with sensorineural hearing loss in 47 individuals from 28 (26 unrelated) families. In addition, 25/47 affected individuals (53%) presented with microcephaly, developmental delay/intellectual disability, cerebral palsy, and/or epilepsy. Modeling indicated damaging effect of variants on the protein, largely via destabilizing effects on protein domains. Brain imaging revealed diminished cerebral volume, thin corpus callosum, and periventricular leukomalacia, and quantitative volumetry demonstrated significantly diminished white matter volumes in several individuals. Immunofluorescent imaging in rat hippocampal neurons revealed localization of Spata5l1 in neuronal and glial cell nuclei and more prominent expression in neurons. In the rodent inner ear, Spata5l1 is expressed in the neurosensory hair cells and inner ear supporting cells. Transcriptomic analysis performed with fibroblasts from affected individuals was able to distinguish affected from controls by principal components. Analysis of differentially expressed genes and networks suggested a role for SPATA5L1 in cell surface adhesion receptor function, intracellular focal adhesions, and DNA replication and mitosis. Collectively, our results indicate that bi-allelic SPATA5L1 variants lead to a human disease characterized by sensorineural hearing loss (SNHL) with or without a nonprogressive mixed neurodevelopmental phenotype.

Background The spectrum of mitochondrial disease is genetically and phenotypically diverse, resulting from pathogenic variants in over 400 genes, with aerobic energy metabolism defects as a common denominator. Such heterogeneity poses a significant challenge in making an accurate diagnosis, critical for precision medicine. Methods In an international collaboration initiated by the European Network for Mitochondrial Diseases (GENOMIT) we recruited 2,023 pediatric patients at 11 specialist referral centers between October 2010 and January 2021, accumulating exome sequencing and HPO-encoded phenotype data. An exome-wide search for variants in known and potential novel disease genes, complemented by functional studies, followed ACMG guidelines. Results 1,109 cases (55%) received a molecular diagnosis, of which one fifth have potential disease-modifying treatments (236/1,109, 21%). Functional studies enabled diagnostic uplift from 36% to 55% and discovery of 62 novel disease genes. Pathogenic variants were identified within genes encoding mitochondrial proteins or RNAs in 801 cases (72%), while, given extensive phenotype overlap, the remainder involved proteins targeted to other cellular compartments. To delineate genotype-phenotype associations, our data was complemented with registry and literature data to develop “GENOMITexplorer”, an open access resource detailing patient- (n=3,940), gene- (n=427), and variant-level (n=1,492) associations (prokischlab.github.io/GENOMITexplorer/). Conclusions Reaching a molecular diagnosis was essential for implementation of precision medicine and clinical trial eligibility, underlining the need for genome-wide screening given inability to accurately define mitochondrial diseases clinically. Key to diagnostic success were functional studies, encouraging early acquisition of patient- derived tissues and routine integration of high-throughput functional data to improve patient care by uplifting diagnostic rate.

1 Department of Neuromuscular Diseases, University College London, Queen Square, Institute of Neurology, London, UK 2 Institute of Human Genetics, Technical University Munich, Munich, Germany 3 Institute of Human Genetics, Helmholtz Zentrum Munich, Neuherberg, Germany 4 Radboud Center for Mitochondrial Medicine, Department of Pediatrics, Amalia Children’s Hospital, Radboudumc, Nijmegen, The Netherlands 5 University Childrens Hospital, Paracelsus Medical University (PMU), Salzburg, Austria 6 Department of Pharmacology, Dalhousie University, Halifax, Canada 7 Medical Genetics Research Center, Shahid Sadoughi University of Medical Sciences, Yazd, Iran 8 Abortion Research Centre, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran 9 Department of Neurology, Technical University of Munich, School of Medicine, Munich, Germany 10 Provincial Clinical Genetic Counselling Center, Zahedan University of Medical Sciences Zahedan, Iran 11 Genetics Centre, Molecular and Clinical Sciences Institute, St George’s University of London, London, UK

Up to 40% of neurodevelopmental disorders (NDDs) such as intellectual disability, developmental delay, autism spectrum disorder, and developmental motor abnormalities have a documented underlying monogenic defect, primarily due to de novo variants. Still, the overall burden of de novo variants as well as novel disease genes in NDDs await discovery. We performed parent-offspring trio exome sequencing in 231 individuals with NDDs. Phenotypes were compiled using human phenotype ontology terms. The overall diagnostic yield was 49.8% (n = 115/231) with de novo variants contributing to more than 80% (n = 93/115) of all solved cases. De novo variants affected 72 different-mostly constrained-genes. In addition, we identified putative pathogenic variants in 16 genes not linked to NDDs to date. Reanalysis performed in 80 initially unsolved cases revealed a definitive diagnosis in two additional cases. Our study consolidates the contribution and genetic heterogeneity of de novo variants in NDDs highlighting trio exome sequencing as effective diagnostic tool for NDDs. Besides, we illustrate the potential of a trio-approach for candidate gene discovery and the power of systematic reanalysis of unsolved cases.