RT Journal
A1 Sangermano, Riccardo
A1 Khan, Mubeen
A1 Cornelis, Stéphanie S.
A1 Richelle, Valerie
A1 Albert, Silvia
A1 Garanto, Alejandro
A1 Elmelik, Duaa
A1 Qamar, Raheel
A1 Lugtenberg, Dorien
A1 van den Born, L. Ingeborgh
A1 Collin, Rob W.J.
A1 Cremers, Frans P.M.
T1 ABCA4 midigenes reveal the full splice spectrum of all reported noncanonical splice site variants in Stargardt disease
JF Genome Research 
JO Genome Research 
YR 2018 
FD January 01 
VO 28 
IS 1 
SP 100 
OP 110 
DO 10.1101/gr.226621.117 
UL http://genome.cshlp.org/content/28/1/100.abstract 
AB Stargardt disease is caused by variants in the ABCA4 gene, a significant part of which are noncanonical splice site (NCSS) variants. In case a gene of interest is not expressed in available somatic cells, small genomic fragments carrying potential disease-associated variants are tested for splice abnormalities using in vitro splice assays. We recently discovered that when using small minigenes lacking the proper genomic context, in vitro results do not correlate with splice defects observed in patient cells. We therefore 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 three novel NCSS variants on ABCA4 pre-mRNA splicing. Intriguingly, multi-exon skipping events were observed, as well as exon elongation and intron retention. The analysis of all reported NCSS variants in ABCA4 allowed us to reveal the nature of aberrant splicing events and to classify the severity of these mutations based on the residual fraction of wild-type mRNA. Our strategy to generate large overlapping splice vectors carrying multiple exons, creating a toolbox for robust and high-throughput analysis of splice variants, can be applied to all human genes.