Sequencing of ABCA4 in a large international Stargardt disease cohort reveals novel pathogenic deep intronic variation and a variant enriched in Ireland.

Abstract

Purpose : Stargardt disease (STGD), the most common form of inherited macular disease, is caused by mutations in the ABCA4 gene. Due to its large size, studies focusing only on exonic regions have resulted in a proportion of genetically unresolved cases. This underscores the necessity for effective sequencing and variant analysis to interrogate the entire 128-kb gene.

Methods : Single-molecule molecular inversion probe (smMIP) based sequencing of the entire ABCA4 gene and 40-kb of flanking sequence was employed for 1054 probands with STGD/STGD-like phenotypes. Most probands (833/1054) were prescreened for exonic variants but remained unresolved. Potentially splice-altering variants were assessed in vitro. 36 Irish probands were included in this cohort, with an additional 126 Irish samples having undergone smMIP sequencing of ABCA4 in a subsequent group. Analysis of the entire recruited Irish STGD cohort was carried out to detect known pathogenic deep intronic variants (DIVs). Detailed clinical examination of all individuals carrying c.4539+2028C>T was performed.

Results : 27 DIVs were identified in 448/1054 probands, 13 of which were novel. Two pathogenic variants were identified in 97/162 Irish cases. 22/97 harboured a pathogenic intronic variant, of which 12/22 carried a DIV. c.4539+2028C>T, resulting in a retina specific pseudoexon inclusion, was detected in 7/12 cases. Analysis of the entire Irish STGD cohort identified 25 individuals who carry this DIV, including two homozygotes who exhibit profound macular hypoautofluorescence and are the only homozygotes reported to date. In contrast, 15 other heterozygous incidences have been reported globally in STGD patients, indicating significant enrichment in Ireland. One heterozygous incidence has been found in >800 control alleles in Ireland.

Conclusions : The study highlights the importance of interrogating non-coding regions of disease genes. Unique insights into the genetic architecture of STGD in individual populations can be achieved by sequencing large numbers of international cases. Identification of individuals homozygous for intronic variants is paramount in assessing phenotypic outcome and variant severity. Studies such as this are particularly relevant given recent advances in splice modulating therapeutics, appropriate access to which will only be possible given an accurate genetic diagnosis.

This is a 2021 ARVO Annual Meeting abstract.