Authors :
Mark Bennett, PhD – Epilepsy Research Centre, Department of Medicine, University of Melbourne, Victoria, Australia
Harshini Thiyagarajah, BS – Epilepsy Research Centre, Department of Medicine, University of Melbourne, Austin Health, Heidelberg, Victoria, Australia
Sian Macdonald, MSc – Epilepsy Research Centre, Department of Medicine, University of Melbourne, Austin Health, Melbourne, Victoria, Australia
Tom Witkowski, BSc (Hons) – Epilepsy Research Centre, Department of Medicine, University of Melbourne, Austin Health, Melbourne, Victoria, Australia
Annie Ting-Gee Chiu, MBBS, FHKCPaed – Epilepsy Research Centre, Department of Medicine, University of Melbourne, Austin Health, Melbourne, Victoria, Australia
Amy Schneider, MGenCouns – Epilepsy Research Centre, Department of Medicine, University of Melbourne, Austin Health, Melbourne, Victoria, Australia
Talia Allan, MSc – Austin Health & The University of Melbourne
Nico Lieffering, BBmedSc (Hons) – Epilepsy Research Centre, Department of Medicine, University of Melbourne, Austin Health, Melbourne, Victoria, Australia
Edith Fuerte, BSc – Department of Cell and Molecular Biology, Center for Pediatric Neurological Disease Research, St. Jude Children's Research Hospital, Memphis, Tennessee, USA
Blake Robinson, BS – Department of Cell and Molecular Biology, Center for Pediatric Neurological Disease Research, St. Jude Children's Research Hospital, Memphis, Tennessee, USA
Christy LaFlamme, MSc – Department of Cell and Molecular Biology, Center for Pediatric Neurological Disease Research, St. Jude Children's Research Hospital, Memphis, Tennessee, USA
Soham Sengupta, PhD – Department of Cell and Molecular Biology, Center for Pediatric Neurological Disease Research, St. Jude Children's Research Hospital, Memphis, Tennessee, USA
Heather Mefford, MD, PhD – Department of Cell and Molecular Biology, Center for Pediatric Neurological Disease Research, St. Jude Children's Research Hospital, Memphis, Tennessee, USA
Piero Perucca, MD, PhD – Epilepsy Research Centre, Department of Medicine, University of Melbourne, Austin Health, Heidelberg, Victoria, Australia
Samuel Berkovic, MD – Epilepsy Research Centre, Department of Medicine, University of Melbourne, Austin Health, Melbourne, Victoria, Australia
Melanie Bahlo, PhD – Genetics and Gene Regulation Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia; Department of Medical Biology, University of Melbourne, Parkville, Victoria, Australia
Michael Hildebrand, PhD – Epilepsy Research Centre, Department of Medicine, University of Melbourne, Austin Health, Melbourne, Victoria, Australia; Neuroscience Group, Murdoch Children’s Research Institute, Parkville, Victoria, Australia
Presenting Author: Ingrid Scheffer, AO MBBS PhD FRACP FAES FAA FRS – Epilepsy Research Centre and Royal Children’s Hospital, The University of Melbourne, Austin Health, Florey and Murdoch Children’s Research Institutes
Rationale:
Small nuclear RNAs (snRNAs) play a critical role in gene splicing. Recurrent variants in the genes RNU4-2 and RNU2-2, encoding snRNAs that play an important role in the major spliceosome, were recently found to be a frequent cause of neurodevelopmental disorders (0.6 and 0.1% respectively).1-4 We investigated a large cohort of unsolved individuals with developmental and epileptic encephalopathies (DEEs) for causal variants in snRNA genes.
Methods:
We searched genome sequencing data and performed targeted Sanger sequencing to search for germline pathogenic variants in snRNA genes in individuals with DEEs without a genetic diagnosis, focusing on RNU4-2, RNU2-2, RNU5A-1, RNU5B-1, RNU5E-1, and RNU5F-1, where de novo variants have recently been reported.1-4 RNA-seq data was interrogated for evidence of abnormal splicing from individuals with pathogenic snRNA variants to understand the disease mechanism.
Results:
We identified recurrent pathogenic RNU2-2 n.4G >A and n.35A >G variants in four individuals with DEEs (4/672; 0.6%). We did not identify any causal variants in RNU4-2, RNU5A-1, RNU5B-1, RNU5E-1, or RNU5F-1. Individuals with pathogenic RNU2-2 variants had a distinctive phenotype including onset of drug-resistant epilepsy at a median age of 24 months and profound to severe developmental impairment. All recurrent variants in individuals with parental samples available for testing were confirmed to be de novo. In addition, two individuals were found with compound heterozygous RNU2-2 variants, suggesting biallelic variants may also contribute to disease. In contrast to RNU4-2, RNA-seq analysis of four individuals with RNU2-2 variants did not reveal a strong signature of widespread abnormal splicing, consistent with recently reported data.3
Conclusions:
Our findings indicate variants in RNU2-2 account for 0.6% of DEEs. We did not identify any causal variants in our DEE cohort in other snRNA genes recently implicated in neurodevelopmental disorders. Pathogenic de novo variants in RNU2-2 cause a DEE with a distinctive phenotype. Biallelic variants may also cause DEEs. Strikingly the diagnostic yield of pathogenic RNU2-2 variants in our cohort with DEEs is six times that for neurodevelopmental disorders.
References
1.Chen Y, Dawes R, Kim HC, Ljungdahl A, et al. De novo variants in the RNU4-2 snRNA cause a frequent neurodevelopmental syndrome. Nature. 2024 632(8026):832-840.
2. Greene D, Thys C, Berry IR, et al. Mutations in the U4 snRNA gene RNU4-2 cause one of the most prevalent monogenic neurodevelopmental disorders. Nat Med. 2024;30(8):2165-2169.
3. Greene D, De Wispelaere K, Lees J, et al. Mutations in the small nuclear RNA gene RNU2-2 cause a severe neurodevelopmental disorder with prominent epilepsy. Nat Genet. Published online April 10, 2025.
4. Nava C, Cogne B, Santini A, et al. Dominant variants in major spliceosome U4 and U5 small nuclear RNA genes cause neurodevelopmental disorders through splicing disruption. Nat Genet. Published online May 16, 2025.
Funding:
Medical Research Future Fund Australia, National Health and Medical Research Council (NHMRC) Australia