Carlos Dominguez Gonzalez, CGC

A Diagnostic Blind Spot: Deep Intronic SVA_E Insertion Identified as the Most Common Pathogenic Variant Associated with Canavan Disease

Carlos A. Dominguez Gonzalez1,*, Katrina M. Bell2,3,*, Ramakrishnan Rajagopalan4,5, Michelle G. de Silva2,3, Aída Lemes6, Cristina Zabala6, Florencia Pérez7, Alfredo Cerisola6,7, Arastoo Vossough8,9, Matthew T. Whitehead8,9, Chloe Cunningham2,3,10, Natasha J. Brown2,3,10, Rebecca Quin3,11, Cas Simons12,13, Thomas Conway2, Eloise Uebergang2, Rocio Rius10,12,13, Meutia A. Kumaheri14,15 Emma Kotes1, Ananya Vohra1, Miranda PG Zalusky16, Zachary B. Anderson16, Sophie HR Storz16, Sydney A. Ward16, Joy Goffena16, Jonas A. Gustafson16,17, Susan M. White3,10, Adeline Vanderver1,18,&, Danny E. Miller16,19,20,& 1. Division of Neurology, Children's Hospital of Philadelphia, Philadelphia, PA 2. Murdoch Children's Research Institute, Melbourne, Victoria, Australia 3. Victorian Clinical Genetics Services, Murdoch Children's Research Institute, Parkville, Australia 4. Division of Genomic Diagnostics, Department of Pathology and Lab Medicine, Children's Hospital of Philadelphia, Philadelphia, PA 5. Department of Pathology and Lab Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 6. Centro de Referencia Nacional de Defectos Congénitos y Enfermedades Raras (CRENADECER), Banco de Previsión Social, Montevideo, Uruguay 7. Unidad Académica de Neuropediatría, Facultad de Medicina, Universidad de la República, Centro Hospitalario Pereira Rossell, Montevideo, Uruguay 8. Department of Radiology, Children's Hospital of Philadelphia, Philadelphia, PA 9. Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 10. Department of Paediatrics, University of Melbourne, Parkville, Australia 11. RCH Metabolic Department, Royal Children's Hospital, Melbourne, Victoria, Australia 12. Centre for Population Genomics, Murdoch Children's Research Institute, Melbourne, VIC, Australia 13. Centre for Population Genomics, Garvan Institute of Medical Research, and UNSW Darlinghurst, Sydney, NSW, Australia 14. Genomics and Inherited Disease Program, Garvan Institute of Medical Research, Darlinghurst, New South Wales, Australia 15. St Vincent Clinical School, Faculty of Medicine and Health, University of New South Wales, Sydney, NSW, Australia 16. Division of Genetic Medicine, Department of Pediatrics, University of Washington, Seattle, WA 17. Molecular and Cellular Biology Program, University of Washington, Seattle, WA 18. Department of Neurology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19. Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA 20. Brotman Baty Institute for Precision Medicine, University of Washington, Seattle, WA

Background and Objectives Canavan disease (CD) is a neurodegenerative disorder in which biallelic pathogenic variants in ASPA result in spongiform degeneration of the cerebral white matter, leading to progressive and irreversible motor and cognitive decline. Despite comprehensive genetic testing, many individuals with clinical and biochemical diagnoses of CD remain without a definitive molecular diagnosis. This gap hinders access to emerging gene-targeted therapies and limits participation in clinical trials. Our objective was to understand the genetic etiology of eight unsolved individuals with CD. Methods We utilized long-read sequencing (LRS) to investigate eight individuals clinically and biochemically diagnosed with CD but with negative genetic testing. We performed targeted LRS on the Oxford Nanopore Technologies (ONT) platform for three unrelated individuals and PacBio HiFi for an additional individual from our cohort. We performed targeted LRS on barcoded and pooled samples from the remaining affected individuals. To investigate functional impact on gene function, we performed RNA-sequencing (RNA-seq) with and without cycloheximide (CHX) on fibroblasts. We then evaluated the allele frequency in the population using gnomAD. Results We identified a ~2,600 bp SVA_E retrotransposon intronic insertion in ASPA in all eight individuals. The insertion was found to be either homozygous or compound heterozygous trans with a known pathogenic variant in all individuals. RNA-seq indicated that the SVA_E insertion creates a novel splice acceptor site within intron 4 of ASPA that causes aberrant splicing and transcript degradation. Surprisingly, the frequency of this variant in population databases suggests that it is the most common pathogenic variant in ASPA and that it is present across ancestry groups. Discussion Our study identified the most common pathogenic variant in ASPA, which has been overlooked in 25 years of CD research. Considering this, it is important to ensure all testing laboratories can detect this variant in diagnostic testing and carrier screening. Our study highlights a substantial blind spot in standard short-read diagnostic pipelines, which historically have missed or overlooked these types of insertions. It also shows the power of emerging technologies, such as LRS and RNA-seq, to bring new classes of variants into diagnostic utility for Leukodystrophies.