Intronic variants in ABCA4 significantly contribute to the pathogenesis of Stargardt disease and cone-rod dystrophy in Ireland.

Abstract

Purpose : The Target 5000 study aims to genetically characterise ~5000 people in Ireland affected by an inherited retinal degeneration (IRD). Over 1000 patients have been sequenced for variants in ~250 IRD-associated genes. A proportion of unresolved cases have been clinically diagnosed with Stargardt disease (STGD1) or cone-rod dystrophy (CRD). The aim is to improve variant detection by sequencing the entirety of the ABCA4 gene to identify intronic variants not previously captured.

Methods : Target 5000 affiliated ophthalmologists clinically diagnose potential participants. Once informed consent is obtained, patient samples are provided to a laboratory in Trinity College Dublin. Target 5000 employs target capture next-generation sequencing as a preliminary diagnostic measure. Cases that remain unresolved undergo whole gene sequencing of ABCA4 using a NextSeq 500. 3866 single-molecule molecular inversion probes (smMIPs) were employed to capture 110nt increments of both the sense and antisense strands of the ABCA4 gene, as well as 40 kb of flanking sequences.

Results : In this ongoing study, unresolved STGD1 and CRD cases have undergone targeted ABCA4 whole-gene sequencing. With robust coverage of at least 97.4% of the 128-kb ABCA4 gene and 700x average read coverage per nucleotide, all tested coding and non-coding single nucleotide variants as well as known deletions and duplications, were detected. Among Irish STGD1/CRD samples, causal variants were found in 16/36 cases. ABCA4 c.4539+2028C>T, resulting in a 345-nt pseudoexon inclusion, was identified in 5 of these cases. Upon retrospective analysis of other unresolved cases and inclusion of this region in the target capture panel, a further 5 incidences of this variant have been identified, suggesting enrichment of this variant in the Irish IRD cohort.

Conclusions : It is evident that intronic variants significantly contribute to the pathogenesis of IRDs. Possible enrichment of ABCA4 c.4539+2028C>T in this cohort further elucidates the genetic architecture of STGD1/CRD in Ireland. The results support the view that whole gene sequencing represents the optimal approach when establishing a genetic diagnosis in previously unresolved IRD cases. The identification of causal variants is becoming increasingly important as patients require an accurate genetic diagnosis to access clinical trials or approved treatments where appropriate.

This is a 2020 ARVO Annual Meeting abstract.