July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
ABCA4 midigenes reveal the full splice spectrum of all reported and newly identified non-canonical splice site variants in Stargardt disease
Author Affiliations & Notes
  • Mubeen Khan
    Human genetics, Radboudumc, Nijmegen, Gelderland, Netherlands
    Donders Insitute of Brain, Cognition and Behaviour, Nijmegen, Gelderland, Netherlands
  • Riccardo Sangermano
    Human genetics, Radboudumc, Nijmegen, Gelderland, Netherlands
    Donders Insitute of Brain, Cognition and Behaviour, Nijmegen, Gelderland, Netherlands
  • Stéphanie Cornelis
    Human genetics, Radboudumc, Nijmegen, Gelderland, Netherlands
    Donders Insitute of Brain, Cognition and Behaviour, Nijmegen, Gelderland, Netherlands
  • Valerie Richelle
    Human genetics, Radboudumc, Nijmegen, Gelderland, Netherlands
  • Duaa Elmelik
    Human genetics, Radboudumc, Nijmegen, Gelderland, Netherlands
  • Silvia Albert
    Human genetics, Radboudumc, Nijmegen, Gelderland, Netherlands
    Donders Insitute of Brain, Cognition and Behaviour, Nijmegen, Gelderland, Netherlands
  • Alex Garanto
    Human genetics, Radboudumc, Nijmegen, Gelderland, Netherlands
    Donders Insitute of Brain, Cognition and Behaviour, Nijmegen, Gelderland, Netherlands
  • Raheel Qamar
    Comsats Institute of information technology, Islamabad, Pakistan
  • Dorien Lugtenberg
    Human genetics, Radboudumc, Nijmegen, Gelderland, Netherlands
  • Ingeborgh Born
    Rotterdam eye Hospital, Rotterdam, Netherlands
    Rotterdam, opthalamic institute, Rotterdam, Netherlands
  • Rob W J Collin
    Human genetics, Radboudumc, Nijmegen, Gelderland, Netherlands
    Donders Insitute of Brain, Cognition and Behaviour, Nijmegen, Gelderland, Netherlands
  • Frans P Cremers
    Human genetics, Radboudumc, Nijmegen, Gelderland, Netherlands
    Donders Insitute of Brain, Cognition and Behaviour, Nijmegen, Gelderland, Netherlands
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 4503. doi:
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      Mubeen Khan, Riccardo Sangermano, Stéphanie Cornelis, Valerie Richelle, Duaa Elmelik, Silvia Albert, Alex Garanto, Raheel Qamar, Dorien Lugtenberg, Ingeborgh Born, Rob W J Collin, Frans P Cremers; ABCA4 midigenes reveal the full splice spectrum of all reported and newly identified non-canonical splice site variants in Stargardt disease. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4503.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Stargardt disease (STGD1) is caused by variants in the ABCA4 gene, a considerable part of which are non-canonical splice site (NCSS) variants. In vitro minigene splice assays enable a cost-effective assessment of putative splice defects. Small minigenes that lack the proper genomic context sometimes show inaccurate in vitro splice results. It was our aim to generate a complete library of wild-type midigenes for ABCA4 which would allow us to systematically test all published NCSS variants for splice abnormalities.

Methods : Wild-type multi-exonic ABCA4 splice vectors were generated by employing a BAC clone harboring the ABCA4 gene. We extracted all published NCSS variants from the Leiden Open Variant Database (www.LOVD.nl/ABCA4). In addition, we identified several other NCSS variants through ABCA4 genotyping of patients with maculopathies. The putative effect on splicing of these NCSS variants in ABCA4 was assessed by using 5 different splicing algorithms via Alamut Visual. Through site-directed mutagenesis, NCSS variants were inserted into the wild-type midigenes and tested in HEK293T splice assays.

Results : Twenty-nine wild-type ABCA4 midigenes (insert sizes: 4.7–11.7 kb) were generated that cover 48 of 50 exons. Forty-three exons were flanked by at least one exon. The majority of the NCSS variants showed single or multi-exon skipping, ~17% showed different types of splice defects and 6% showed exon elongation. Based on the fraction of correctly spliced mRNA, all tested NCSS variants were classified to have mild, moderate or severe effects on ABCA4 function. For all NCSS variants for which a phenotype-genotype correlation could be established, we observed concordance between the in vitro splice assay result and the patients’ phenotypes.

Conclusions : The generation of a complete set of wild-type ABCA4 splice vectors allowed us to gain insight into the pathogenicity of the poorly explored class of NCSS ABCA4 variants on a gene-wide scale. By assessing the functional consequences of putative splice defects, STGD1 patients can obtain a more accurate molecular diagnosis and thereby become eligible for novel therapies. This ABCA4 midigene vector library can also be used for medium-throughput analysis of deep-intronic variants.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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