Variants in spliceosomal small nuclear RNA (snRNA) genes RNU4-2 (ReNU syndrome), RNU5B-1, and RNU2-2 have recently been linked to dominant neurodevelopmental disorders (NDDs), revealing a major, previously overlooked role for noncoding snRNAs in human disease. Here, we systematically analysed 200 potentially functional snRNA genes in a French cohort comprising 26,911 individuals with rare disorders and through international collaborations. We identify de novo and biallelic variants in RNU2-2 associated with both dominant and recessive NDDs in 126 individuals from 108 unrelated families. Recessive RNU2-2 NDD is at least twice as frequent as the dominant NDD caused by n.4G>A and n.35A>G, and often arises from a de novo variant in trans with an inherited allele, reflecting the high mutability of snRNA genes. Dominant and recessive RNU2-2-NDDs share overlapping clinical features with frequent epilepsy. Blood transcriptomics and DNA methylation analyses revealed subtle, variant-specific effects on splicing and episignatures. Our findings support a gradient-of-impact model and a continuum between dominant and recessive inheritance, establishing RNU2-2 variants as a frequent cause of NDDs, nearly as prevalent as ReNU syndrome.

N.W. receives research funding from Novo Nordisk and Biomarin Pharmaceutical. L.T.D. receives research funding from Stoke Therapeutics. L.G.S. receives funding from the Health Research Council of New Zealand and Cure Kids New Zealand. She has served as a paid consultant of the Epilepsy Study Consortium for consulting work for Epygenix Therapeutics, Ovid Therapeutics, Stoke Therapeutics, Takeda Pharmaceuticals, UCB, and Zogenix. L.G.S. has received research grants and consultancy fees from Zynerba Pharmaceuticals and has served on Takeda and Eisai Pharmaceuticals scientific advisory panels. I.E.S. has served on scientific advisory boards for CAMP4 Therapeutics, Longboard Pharmaceuticals, Mosaica Therapeutics, Takeda Pharmaceuticals, UCB; has received speaker honoraria from Akumentis, Biocodex, Chiesi, Stoke Therapeutics, UCB, Zuellig Pharma; has received funding for travel from Stoke Therapeutics and UCB; has served as an investigator for Anavex Life Sciences, Biohaven Ltd, Bright Minds Biosciences, Cerebral Therapeutics, Cerecin Inc, Cereval Therapeutics, Encoded Therapeutics, EpiMinder Inc, ES-Therapeutics, Longboard Pharmaceuticals, Marinus, Neuren Pharmaceuticals, Neurocrine BioSciences, Praxis Precision Medicines, Shanghai Zhimeng Biopharma, SK Life Science, Supernus Pharmaceuticals, Takeda Pharmaceuticals, UCB, Ultragenyx, Xenon Pharmaceuticals, Zogenix; and has consulted for Biohaven Pharmaceuticals, Eisai, Epilepsy Consortium, Longboard Pharmaceuticals, Praxis, Stoke Therapeutics, UCB; and is a Non-Executive Director of Bellberry Ltd and a Director of the Australian Academy of Health and Medical Sciences. She may accrue future revenue on pending patent WO61/010176 (filed: 2008): Therapeutic Compound; has a patent for SCN1A testing held by Bionomics Inc and licensed to various diagnostic companies; has a patent molecular diagnostic/theranostic target for benign familial infantile epilepsy (BFIE) [PRRT2] 2011904493 & 2012900190 and PCT/AU2012/001321 (TECH ID:2012-009). All other authors declare no competing interests.

Patients from the PFMFG received funding from Plan France Médecine Génomique 2025 (PFMG2025). Cases identified by the Broad Center for Mendelian Genomics (A.O.D-L. and S.L.S.) were supported by the National Human Genome Research Institute (NHGRI) grants UM1HG008900 and U01HG011755. Genetic analyses (cases from Gleeson lab) were supported by the Gabriella Miller Kids First Pediatric Research Program, funded by the Common Fund of the NIH Office of the Director, with sequencing performed at the Broad Institute Sequencing Center (U24HD090743). Genetic analyses of Australian cases were supported by the Genetic Basis of Epilepsy Research program (NHMRC grants GNT1172897, GNT2033247, GNT2006841) and by Victorian State Government Operational Infrastructure Support and the NHMRC IRIISS. M.F.B., M.S.H., and I.E.S. were supported by an Australian Medical Research Future Fund Genomics Health Futures Mission Grant (2007707). Part of the results has been supported by the RNU-SPLICE project, financed by Health philantropic program of Mutuelles AXA dedicated to supporting innovative research projects in France (to C.N.). The structural interpretation was conducted by C. Charenton. and M.A. as part of the ERC Starting Grant project SPLIFEM. A.S. received a grant from Region Normandie and GIRCI Nord Ouest (FHU-A2M2P). This study uses resources generated by the ENCODE Consortium, 1) the ENCODE Data Analysis Center (ZLab at UMass Medical Center), Henry Pratt, Jill Moore, Michael Purcaro, and Zhiping Weng for providing ENCODE cCREs data, 2) Bernstein Lab at the Broad Institute for the H3K27Ac peaks in H1-hESC, 3) Thomas Gingeras lab at Cold Spring Harbor Laboratory for generating the small RNA-seq data from six human embryonic brain regions. N.W. is supported by a Wellcome Career Development Award (grant no. 305292/Z/23/Z) and a research prize from the Lister Institute. HCM is funded by ALSAC of St. Jude Children s Research Hospital. K.Õ. was supported by Estonian Research Council grants PRG471 and PRG2040. Health Research Council of New Zealand and Curekids New Zealand supported the recruitment, phenotyping and sequencing of the New Zealand participants.

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:

This study was conducted in accordance with the ethical standards and regulations of all participating countries. Written informed consent was obtained for all patients from their parents or legal guardians, with an additional consent form for families agreeing to the publication of photographs. For genetic analyses, patient samples were pseudonymized at each participating center. Information on the patients sex (but not gender) was extracted from clinical records. The promoters of this research study are Assistance Publique-Hôpitaux de Paris (AP-HP) for hospitals associated with the SeqOIA laboratory (project ID APHP241333) and Grenoble-Alpes University Hospital (CHU Grenoble-Alpes, research ID 19814188) for hospitals affiliated with the Auragen laboratory. Ethical approval was obtained from the University Hospital Essen (24-12010-BO) and the Comité Éthique et Scientifique pour les Recherches, les Études et les Évaluations dans le domaine de la Santé (CESREES; reference 21082803 Bis / 2038764). AP-HP has received an authorization from the Commission Nationale de l Informatique et des Libertés (CNIL; reference HGTHGT/MFIMFI/AR2426865; request no. 924924336666) for data processing. Additional approvals were obtained from the ethics committee of CHU de Nantes (CCTIRS number 14.556) and from CPP Ouest V (File 06/15, Ref MESR DC 2017 2987; approval date 04/08/2015). For methylation analyses, DNA from patients and controls had been previously collected in a medical context for genetic testing, with written consent including authorization for research use of leftover material. Control samples consisted of individuals without neurodevelopmental disorders, either unaffected relatives or persons tested presymptomatically for other conditions who were found not to carry pathogenic variants. DNA samples used for methylation profiling were stored within the genetics biobank of the CRBi, Rouen, France (collection DC 2008-711, authorization MCRBi/2024/02). The use of these samples was approved by the CERDE ethics committee of Rouen University Hospital (notification E2023-13). Researchers and clinicians from all contributing centers participated throughout the study, from design and implementation to data collection, analysis and manuscript preparation, and are listed as coauthors of this article.

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

Variant details will be submitted to ClinVar for publication. RNA-seq and methylation data will be deposited in the European Genome-phenome Archive (EGA, http://www.ebi.ac.uk/ega), hosted by the EBI. Both datasets will be subject to a Data Processing Agreement, and access requests will be reviewed by a Data Access Committee to ensure compliance with ethical and legal standards. Due to ethical considerations, individual genome data cannot be made publicly available. Controlled access is required to safeguard participant privacy and to comply with data protection regulations, including the GDPR in Europe. Access to genome data from the PFMG2025 cohort is governed by French data protection laws and is only possible via the Collecteur Analyseur de Données (CAD). More details can be found on the PFMG2025 website: https://pfmg2025.fr/le-plan/collecteur-analyseur-de-donnees-cad/.The coordinates of the ENCODE Registry of candidate cis-Regulatory Elements (cCREs) in the human genome23 and bigwig files concerning the peaks of histone H3 acetylation of lysine 27 (H3K27Ac) obtained for the H1 human embryonic stem cell line (H1-hESC)24 were downloaded through the UCSC Table Browser52. Bigwig files concerning small RNA-seq data from six human embryonic brain regions were downloaded from the ENCODE portal53 (http://www.encodeproject.org/) with the following identifiers: ENCFF013RLG, ENCFF029RIV, ENCFF034QAV, ENCFF197SSE, ENCFF221KEN, ENCFF343ZBS, ENCFF405QIN, ENCFF532SOY, ENCFF654ONK, ENCFF738LDD, ENCFF870FMA, ENCFF887TOS, ENCFF106ESQ, ENCFF222WBQ, ENCFF250WEA, ENCFF254UEQ, ENCFF319GRF, ENCFF425WUZ, ENCFF443ONL, ENCFF820JTT, ENCFF897IWP, ENCFF915WAC, ENCFF946YVE and ENCFF965GHD.