Resolving the diagnostic odyssey in inherited retinal dystrophies through long-read genome sequencing

Background Inherited Retinal Dystrophies (IRDs) are visually disabling monogenic diseases with remarkable genetic and phenotypic heterogeneity. Mutations in more than 300 different genes have been identified as disease causing. Genetic diagnosis of IRDs has been greatly improved thanks to the incorporation of Next Generation Sequencing (NGS) approaches. However, the current IRD molecular diagnosis yield using NGS is approximately 60% and negative cases can be explained by variants that are not usually identified by the widely used short reads-NGS such as structural variants (SVs) or by variants located in uncovered, low complexity, repetitive, highly homologous, or GC-rich regions. 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 in the clinical context, thus improving the diagnostic yield in heterogeneous diseases as IRDs. Objective To describe LR-GS utility in 3 unrelated, previously unsolved IRD cases. Material & Methods LR-GS was performed on 3 probands with IRDs and previous inconclusive genetic testing with NGS (either exome or gene panel sequencing). Whole genome libraries were prepared using SMRTbell® prep kit. Sequencing was performed on the PacBio Revio system. Results A definite diagnosis was established in the 3 cases. A homozygous deep intronic variant c.4885+740A>T in USH2A was identified in a proband with Usher syndrome; A homozygous intragenic deletion involving EYS exon 24 was found in a proband with Retinitis pigmentosa. Finally, a proband with Usher syndrome was found to be a compound heterozygous for a USH2A deep intronic variant and a multiexonic duplication involving USH2A exons 22-32. Conclusion Our case series show the efficiency in a clinical setting of LR-GS to detect disease-causing variants that were missed by current NGS techniques, improving thus the molecular diagnosis rate in genetically heterogeneous diseases as IRDs