ABCA4 midigenes reveal the full splice spectrum of all reported noncanonical splice site variants in Stargardt disease

Riccardo Sangermano, Mubeen Khan, Stéphanie S Cornelis, Valerie Richelle, Silvia Albert, Alejandro Garanto, Duaa Elmelik, Raheel Qamar, Dorien Lugtenberg, L Ingeborgh van den Born, Rob W J Collin, Frans P M Cremers

Research output: Contribution to journalArticleResearchpeer-review


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.

Original languageEnglish
Pages (from-to)100-110
Number of pages11
JournalGenome Research
Issue number1
Publication statusPublished - Jan 2018


  • ATP-Binding Cassette Transporters/biosynthesis
  • Adult
  • Female
  • Humans
  • Macular Degeneration/congenital
  • Male
  • RNA Precursors/genetics
  • RNA Splice Sites
  • RNA Splicing
  • Stargardt Disease


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