June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Hidden genetic variation in Stargardt disease: novel copy number variations, cis-regulatory and deep-intronic splice variants within the ABCA4 locus
Author Affiliations & Notes
  • Miriam Bauwens
    Center for Medical Genetics, University of Ghent, Ghent, Belgium
  • Riccardo Sangermano
    Department of Human Genetics, RadboudUMC, Nijmegen, Netherlands
  • Timothy Cherry
    Dept. of Pediatrics, University of Washington School of Medicine, Seattle, Washington, United States
    Center for Developmental Biology and Regenerative Medicine, Seattle Children's Research Institute, Seattle, Washington, United States
  • Van Cauwenbergh Caroline
    Center for Medical Genetics, University of Ghent, Ghent, Belgium
  • José Luis Gómez-Skarmeta
    Centro Andaluz de Biología del Desarrollo, CSIC/ Universidad Pablo de Olavide, Sevilla, Spain
  • Nicole Weisschuh
    Institute for Ophthalmic Research, Centre for Ophthalmology, University of Tuebingen, Tuebingen, Germany
  • Susanne Kohl
    Institute for Ophthalmic Research, Centre for Ophthalmology, University of Tuebingen, Tuebingen, Germany
  • Bart Peter Leroy
    Center for Medical Genetics, University of Ghent, Ghent, Belgium
    Dept of Ophthalmology, Ghent University Hospital & Ghent University, Gent, Belgium
  • Frans P Cremers
    Department of Human Genetics, RadboudUMC, Nijmegen, Netherlands
  • Elfride De Baere
    Center for Medical Genetics, University of Ghent, Ghent, Belgium
  • Footnotes
    Commercial Relationships   Miriam Bauwens, None; Riccardo Sangermano, None; Timothy Cherry, None; Van Cauwenbergh Caroline, None; José Luis Gómez-Skarmeta, None; Nicole Weisschuh, None; Susanne Kohl, None; Bart Leroy, None; Frans Cremers, None; Elfride De Baere, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 580. doi:
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      Miriam Bauwens, Riccardo Sangermano, Timothy Cherry, Van Cauwenbergh Caroline, José Luis Gómez-Skarmeta, Nicole Weisschuh, Susanne Kohl, Bart Peter Leroy, Frans P Cremers, Elfride De Baere; Hidden genetic variation in Stargardt disease: novel copy number variations, cis-regulatory and deep-intronic splice variants within the ABCA4 locus. Invest. Ophthalmol. Vis. Sci. 2017;58(8):580.

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

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Abstract

Purpose : A large proportion of Stargardt disease (STGD1) patients have single coding variants in the disease gene ABCA4, suggestive of hidden genetic variations in non-coding regions. We aimed to assess the contribution of copy number variations (CNVs) and non-coding sequence variations in mono-allelic STGD1 patients. Moreover, we attempted to gain more insight into the cis-regulatory landscape of ABCA4.

Methods : A total of 116 monoallelic STGD1 patients underwent targeted resequencing of a conserved block of synteny encompassing the ABCA4 gene (HaloPlex enrichment). Filtering of variants was based on criteria such as publicly available minor allele frequencies and frequency in the total cohort of sequenced patients. Altered splice predictions, presence in cis-regulatory regions of ABCA4 and RegulomeDB data served as a secondary filter. Candidate splice variants were tested by mini-gene assays while putative cis-regulatory variants were investigated by ex vivo electroporation in mouse retinas. 4C-seq was performed on human retinal cells, using an anchor in the ABCA4 promoter region. Customized arrayCGH (arrEYE) was used for CNV analysis of 5 patients.

Results : Mini-gene assays were performed for a subset of 12 intronic variants, confirming a splice effect for 3 variants. The cis-regulatory effect of 3 promoter and 2 deep intronic variants were tested in mouse retinal explants, revealing a significant effect on regulation for 2 of the promoter variants. A chromatin interaction map of the ABCA4 region was generated by 4C-seq in human adult retinal cells. 11 putative cis-regulatory variants are being tested by luciferase assays in Y79 cells and nine putative splice variants are being assessed by mini-gene assays. CNV analysis revealed a novel ABCA4 deletion (ex 40-50) and a duplication (ex 2-6) in 2/5 patients without candidate non-coding variants.

Conclusions : Resequencing of the whole ABCA4 locus uncovered novel deep intronic splice variants and cis-acting regulatory variants in unsolved cases of STGD1, representing the first report of non-coding regulatory ABCA4 variants. In addition, a chromatin interaction dataset for the ABCA4 locus was generated in retinal cells. Finally, apart from a novel deletion, the first duplication was identified in ABCA4, expanding the CNV spectrum which represents a very small fraction of the known mutational load in ABCA4.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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