Cas9 targeted long-read nanopore sequencing of ABCA4 and the OPN1LW/OPN1MW gene array for investigation of inherited retinal disease

Abstract

Purpose : Short, paired-end sequencing (next generation sequencing, NGS) enables interrogation of up to ~95% of the human genome. Several clinically relevant genes for inherited retinal disease (eg: OPN1LW/OPN1MW) and variants (eg: complex rearrangements) cannot be resolved by NGS technology including whole genome sequencing (WGS). We sought to investigate the use of Cas9 targeting of chromosomal segments (CATCH) with Oxford Nanopore Technologies (ONT) long-read sequencing for a WGS-intractable suspected ABCA4-retinopathy case and to sequence the NGS-intractable OPN1LW/OPN1MW gene array.

Methods : CATCH was performed for ABCA4 and the OPN1LW/OPN1MW array using CRISPR guide RNAs flanking the regions of interest followed by separation of high molecular weight DNA fragments using the SageHLS system (SAGE science). ONT MinION long-read sequencing was performed on resultant enriched DNA fragments.

Results : One patient with Stargardt disease was unresolved following WGS. A single heterozygous variant was identified (c.5715+5G>A) with no candidate non-coding or structural variants that could account for the second allele. CATCH-nanopore sequencing using CRISPR-guides flanking ABCA4 generated 337 on-target reads (chr1:93,992,834-94,121,148) and a maximal read depth of 50x. Interrogation of reads revealed a complex structural rearrangement comprising insertion of 689bp from the long non-coding RNA, LINC01500 at chr14q23.1, and an ~800bp long interspersed nuclear element (LINE) into intron 1 of ABCA4. Splice prediction (nnsplice) showed a potential 14bp pseudoexonwithin the inserted sequence.
Sequencing ofthe OPN1LW/OPN1MW gene array following CATCH enrichment for a control female subject generated a maximal read depth of 36x, and for a control male, 25x read depth, including full-length reads of up to ~233kb spanning the entire fragment enabling accurate haplotyping of the opsin arrays.

Conclusions : This study demonstrates that CATCH-nanopore sequencing is effective for cases intractable to NGS. We were able to generate significant enrichment for target regions and ultra-long reads spanning the entire targets. This study identified a complex structural rearrangement, missed by short-read WGS, in ABCA4 that may lead to cryptic splicing. Effective sequencing of the NGS-intractable OPN1LW/OPN1MW gene array is possible for variant detection in males with blue cone monochromacy.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.