Wild-type and mutant ABCA4 midigenes for in vitro splice assays

Stargardt disease is caused by variants in the ABCA4 gene, a significant part of which are non-canonical splice site (NCSS) variants. We devised a novel strategy in which a bacterial artificial chromosome was employed to generate midigenes, splice vectors of varying lengths (up to 11.7 kb) covering almost the entire ABCA4 gene. These midigenes were used to analyze the effect of all 44 reported and 3 novel NCSS variants on ABCA4 pre-mRNA splicing.