A diagnostic blind spot: an intronic SVA_E insertion as the most common cause of Canavan disease

Biography Danny Miller is an Assistant Professor in the Department of Pediatrics, Division of Genetic Medicine, and the Department of Laboratory Medicine and Pathology at the University of Washington and is an attending physician at Seattle Children's Hospital. His laboratory is developing long-read sequencing- Abstract Canavan disease (CD) is a devastating leukodystrophy caused by biallelic loss-of-function variants in ASPA. Despite comprehensive short-read molecular testing, many patients with clinical and biochemical diagnoses of CD remain unsolved. We used long-read sequencing to identify a deep intronic SINE-VNTR-Alu, subfamily-E (SVA_E) retrotransposon insertion in ASPA that was missed by standard clinical testing. We sequenced eight individuals with confirmed biochemical and clinical diagnoses of CD, but inconclusive or partially inconclusive genetic findings. We used both whole-genome and targeted long-read sequencing (LRS) on Oxford Nanopore Technologies PromethION. Barcoded samples were sequenced in parallel and LRS data were complemented with short-read genome sequencing analyses. We further integrated RNA sequencing (RNA-seq) with and without cycloheximide treatment in fibroblasts to characterize splicing and confirm the molecular consequences of the insertion. In all eight individuals, we identified a 2.6 kb SVA_E insertion in intron 4 of ASPA. This insertion was either homozygous or in trans with a known pathogenic variant. RNA-seq data revealed that the retrotransposon insertion introduces a novel splice acceptor site, resulting in aberrant mRNA splicing, nonsense-mediated decay, and loss of function. Reanalysis of short-read genome data indicated that the insertion is readily detectable using tools specifically designed to identify transposable element events but is missed by standard pipelines. Unexpectedly, population database queries (gnomAD v4) suggest that this variant is the most common pathogenic allele in ASPA. The use of LRS allowed us to completely sequence the SVA_E insertion and develop an antisense oligonucleotide to block the splicing defect created by the insertion. Our study identifies a long-standing diagnostic blind spot in Canavan disease. The SVA_E insertion, which was difficult to detect using short-read methods, is the most prevalent pathogenic ASPA variant. We highlight how targeted nanopore sequencing with adaptive sampling enables robust detection of intronic structural variants such as this and emphasizes the necessity of integrating specialized bioinformatic tools or long-read approaches into routine diagnostics. By illuminating the mechanism and wide prevalence of this elusive retrotransposon insertion, our findings underscore the power of nanopore-based sequencing to resolve complex genetic etiologies, thereby paving the way for accurate diagnoses and expanded therapeutic access for the CD community.