Dominguez Gonzalez, Carlos A et al.
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 8 unsolved cases of CD.
Methods: We used long-read sequencing (LRS) to investigate 8 individuals clinically and biochemically diagnosed with CD but who had negative genetic testing results. We performed targeted LRS using the Oxford Nanopore Technologies platform for 3 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 on fibroblasts. We then evaluated the allele frequency in the population using gnomAD.
Results: We identified an ∼2,600-bp SVA_E retrotransposon intronic insertion in ASPA in all 8 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 that all testing laboratories can detect this variant through 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 identify new classes of variants for genetic disorders, including CD.
