Resolving the Diagnostic Odyssey in Inherited Retinal Dystrophies Through Long-Read Genome Sequencing

Inherited retinal dystrophies (IRDs) inherited are visually disabling monogenic diseases with remarkable genetic and phenotypic heterogeneity. Mutations in more than 300 different genes have been identified as disease-causing. The genetic diagnosis of IRDs has significantly advanced with the integration of Next Generation Sequencing (NGS) technologies. Currently, the molecular diagnostic yield for IRDs using NGS is approximately 60%. Negative results can often be attributed to variants that are not typically detected by standard short-read NGS, including those located in low complexity, uncovered, or high GC content regions and structural variants (SVs). Long-read genome sequencing (LR-GS) is an emerging technology that produces 10-20 kb reads and is expected to overcome short-read sequencing limitations, thus improving the diagnostic yield in heterogeneous diseases such as IRDs. In this work, we describe LR-GS utility in three probands with IRDs and previous inconclusive genetic testing with NGS (exome or gene panel). Whole genome libraries were prepared using the SMRTbell prep kit. Sequencing was performed on the PacBio Revio system. A definite diagnosis was established in all cases by identifying a homozygous deep intronic variant in USH2A (c.4885+740A>T) in a proband with Usher syndrome, a homozygous intragenic deletion involving EYS exon 24 in a proband with retinitis pigmentosa, and USH2A compound heterozygous variants (a deep intronic variant and a duplication involving exons 22-32) in a proband with Usher syndrome. Our case series shows the successful application of LR-GS to detect disease-causing variants in these three patients.