Assessment of the splicing-effect, in vivo, of mutations in USH2A using long-read sequencing of cDNA from nasal epithelium

Abstract

Purpose : To identify and characterize the effect of candidate pathogenic variants in the USH2A gene using whole genome sequencing (WGS) followed by cDNA amplicon sequencing in patients with type 2 Usher syndrome (USH2) or non-syndromic retinitis pigmentosa (arRP).

Methods : Four patients with putative biallelic USH2A pathogenic variants identified by WGS were studied (USH2=2, arRP=2). Candidate variants were selected on the basis of SpliceAI scores. Nasal epithelial cells were collected for RNA extraction, RT-PCR was performed using USH2A-specific primers for amplification of a cDNA fragment encompassing the suspected splice alteration. Amplicons were sequenced using the Oxford Nanopore Technologies ligation sequencing kit with Flongle flowcells alongside control specimens. Resulting reads were basecalled (Guppy), filtered (Porechop and Nanofilt) and aligned (GRCh38, Minimap2). Reads were visualised using the Integrative Genome Viewer and sashimi plots generated.

Results : The identified genotypes are shown in table 1. A splice altering effect was observed for all variants examined by the nanopore assay. c.9959-2971C>T introduces a cryptic donor-site in intron 51 leading to inclusion of a 113bp pseudoexon, frameshift and premature termination. The synonymous variant, c.2139C>T, introduces a donor site 30bp upstream of the canonical intron 12 donor leading to in-frame truncation of exon 12 as the major effect. c.3812-3_3837dup was shown to cause in-frame skipping of exon 18 in contrast to its predicted effect (p.Met1280Ter). This functional effect suggests the variant may not be a true loss of function: it was seen in trans with a stop-gain in patient 3 with no hearing loss at age 70. c.8682-654C>G was shown to create a cryptic donor site in intron 43 leading to a 129bp pseudoexon inclusion and premature termination.

Conclusions : We demonstrate the splice altering effect of 4 variants in the USH2A gene and confirm the molecular diagnosis for 4 patients. These include two previously unreported intronic cryptic donor site variants, one previously reported synonymous variant (classified as a variant of uncertain significance at the time of study) and a previously reported splice region duplication, clarifying its effect to in-frame exon skipping. This study demonstrated the clinical utility of a nanopore based assay for splice investigations from patient derived mRNA samples.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.