Around 60% of individuals with neurodevelopmental disorders (NDD) remain undiagnosed after comprehensive genetic testing, primarily of protein-coding genes1. Increasingly, large genome-sequenced cohorts are improving our ability to discover new diagnoses in the non-coding genome. Here, we identify the non-coding RNA RNU4-2 as a novel syndromic NDD gene. RNU4-2 encodes the U4 small nuclear RNA (snRNA), which is a critical component of the U4/U6.U5 tri-snRNP complex of the major spliceosome2. We identify an 18 bp region of RNU4-2 mapping to two structural elements in the U4/U6 snRNA duplex (the T-loop and Stem III) that is severely depleted of variation in the general population, but in which we identify heterozygous variants in 119 individuals with NDD. The vast majority of individuals (77.3%) have the same highly recurrent single base-pair insertion (n.64_65insT). We estimate that variants in this region explain 0.41% of individuals with NDD. We demonstrate that RNU4-2 is highly expressed in the developing human brain, in contrast to its contiguous counterpart RNU4-1 and other U4 homologs, supporting RNU4-2s role as the primary U4 transcript in the brain. Overall, this work underscores the importance of non-coding genes in rare disorders. It will provide a diagnosis to thousands of individuals with NDD worldwide and pave the way for the development of effective treatments for these individuals.

NW receives research funding from Novo Nordisk and has consulted for ArgoBio studio. SJS receives research funding from BioMarin Pharmaceutical. AODL is on the scientific advisory board for Congenica, was a paid consultant for Tome Biosciences and Ono Pharma USA Inc., and received reagents from PacBio to support rare disease research. HLR has received support from Illumina and Microsoft to support rare disease gene discovery and diagnosis. MHW has consulted for Illumina and Sanofi and received speaking honoraria from Illumina and GeneDx. SBM is an advisor for BioMarin, Myome and Tenaya Therapeutics. SMS has received honoraria for educational events or advisory boards from Angelini Pharma, Biocodex, Eisai, Zogenix/UCB and institutional contributions for advisory boards, educational events or consultancy work from Eisai, Jazz/GW Pharma, Stoke Therapeutics, Takeda, UCB and Zogenix. The Department of Molecular and Human Genetics at Baylor College of Medicine receives revenue from clinical genetic testing completed at Baylor Genetics Laboratories. JMMH is a full-time employee of Novo Nordisk and holds shares in Novo Nordisk A/S. DGM is a paid consultant for GlaxoSmithKline, Insitro, and Overtone Therapeutics and receives research support from Microsoft.

YC is supported by a studentship from Novo Nordisk. NW is supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (220134/Z/20/Z). GL was supported by the Fonds de recherche en sante du Quebec (FRQS), VSG by NIAMS K23AR083505, and SLS by a fellowship from Manton Center for Orphan Disease Research at Boston Childrens Hospital. The research was supported by grant funding from Novo Nordisk and the Rosetrees Trust (PGL19-2/10025 to NW), the Simons Foundation Autism Research Initiative (SFARI #736613, SJS), the NIMH (R01 MH129751 to SJS), the HDR-UK Molecules to Health Records Driver Programme (SJS), the Australian National Health and Medical Research Council (1164479, 1155244, GNT2001513), the Australian NHMRC Centre for Research Excellence in Neurocognitive Disorders (NHMRC-RG172296), the Australian Medical Research Future Fund (MRF2007677, GHFM76747), NHGRI (U01HG011762, U01HG011745, U24HG011746, UM1HG008900, U01HG011755, R21HG012397, and R01HG009141), NINDS (U01NS134358, U01NS106845, U54NS115052, 1U24NS131172), the Chan Zuckerberg Initiative Donor-Advised Fund at the Silicon Valley Community Foundation (funder DOI 10.13039/100014989) grants 2019-199278, 2020-224274, (https://doi.org/10.37921/236582yuakxy), the US Department of Defense Congressionally Directed Medical Research Programs (PR170396), the National Institute of Neurological Disorders and Stroke of the National Institutes of Health (U01HG007709, U01HG007942, and U01HG010217), and the Clinical Translational Core of the Baylor College of Medicine IDDRC (P50HD103555) from the Eunice Kennedy Shriver National Institute of Child Health and Human Development. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Eunice Kennedy Shriver National Institute of Child Health and Human Development or the National Institutes of Health. The Rare Disease Flagship acknowledges financial support from the Royal Childrens Hospital Foundation, the Murdoch Childrens Research Institute and the Harbig Foundation. Massimos Mission acknowledges funding support from the Australian Government Department of Health and Aged Care (EPCD000034). Sequencing and analysis were supported by the Deutsche Forschungsgemeinschaft (DFG) Research Infrastructure West German Genome Center (project 407493903) as part of the Next Generation Sequencing Competence Network (project 423957469). Short-read genome sequencing was carried out at the production site Cologne (Cologne Center for Genomics; CCG). CD received the DFG 458099954 as part of the DFG Sequencing call #3. SMS is supported by the Epilepsy Society.

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