A Diagnostic Blind Spot: Deep intronic SVA_E Insertion identified as the most Common Pathogenic Variant Associated with Canavan Disease

Abstract Canavan disease (CD) is a neurodegenerative disorder caused by biallelic disease-causing variants in the ASPA gene. Here, we utilized long-read sequencing (LRS) to investigate eight individuals clinically diagnosed with Canavan disease but without definitive genetic diagnoses. Our analyses identified a recurring previously unreported intronic SVA_E retrotransposon insertion within ASPA in all eight individuals. Surprisingly, the frequency of this variant in population databases suggests it is the most common pathogenic variant in ASPA and should be evaluated in diagnostic testing and carrier screening for CD. Additionally, this finding has implications for the broader rare disease community, as it highlights a substantial blind spot in standard short-read diagnostic pipelines, which historically have missed or overlooked these types of insertions. This discovery highlights the power of emerging technologies, such as LRS and RNA-sequencing (RNA-seq), to bring new classes of variants into diagnostic utility for genetic disorders like CD. Competing Interest Statement MPGZ and JG have received travel support from ONT. AV receives grant and in-kind support for translational research without personal compensation from Affinia, Biogen, Boehringer Ingelheim, Eli Lilly, Illumina, Ionis, Homology, Myrtelle, Orchard therapeutics, Passage Bio, Sana, Sanofi, Synaptixbio, and Takeda. DEM holds stock options in MyOme, is on a scientific advisory board at Oxford Nanopore Technologies (ONT), is engaged in a research agreement with ONT, and has received travel support from ONT and PacBio. DEM is on a scientific advisory board at Basis Genetics. Funding Statement The Rare Disease Flagship acknowledges financial support from the Royal Childrens Hospital Foundation and the Murdoch Childrens Research Institute, Melbourne, Australia. The research conducted at the Murdoch Childrens Research Institute was supported by the Victorian Government's Operational Infrastructure Support Program. Massimos Mission acknowledges financial support from the Australian Government Department of Health and Aged Care (EPCD000034). AV is funded by the RDCRN (U54TR002823) and by 5U24NS131172. DEM is supported by NIH grant DP5OD033357. 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/IRB of The Children's Hospital of Philadelphia gave ethical approval for this work. Royal Children's Hospital Human Research Ethics Committee 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 Data Availability Additional data that support the findings of this study are available. De-identified genomic and associated data from this study are available on request from the corresponding author through formal data sharing agreements. All variants reported in this study have been deposited in ClinVar (SCV005407762). The web resources used are: ClinVar: https://www.ncbi.nlm.nih.gov/clinvar gnomAD: https://gnomad.broadinstitute.org IGV: https://software.broadinstitute.org/software/igv NCBI RefSeq: https://www.ncbi.nlm.nih.gov/refseq OMIM: https://omim.org Frequency Filter: https://cardiodb.org/allelefrequencyapp/ UCSC: https://genome.ucsc.edu/