June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Towards the identification of deep-intronic ABCA4 mutations in Stargardt patients by using induced pluripotent stem cell-derived photoreceptor progenitor cells
Author Affiliations & Notes
  • Silvia Albert
    Human Genetics, Radboud Universit Medical Centre, Nijmegen, Netherlands
  • Riccardo Sangermano
    Human Genetics, Radboud Universit Medical Centre, Nijmegen, Netherlands
  • Nathalie Bax
    Ophthalmology, Radboud University Medical Center, Nijmegen, Netherlands
  • Susanne Roosing
    Pediatric Brain Diseases, The Rockefeller University, New York, NY
  • L. I. van den Born
    The Rotterdam Eye Hospital, Rotterdam, Netherlands
  • Anke den Engelsman-van Dijk
    Human Genetics, Radboud Universit Medical Centre, Nijmegen, Netherlands
  • Angelique Ramlal
    Human Genetics, Radboud Universit Medical Centre, Nijmegen, Netherlands
  • Edwin M Stone
    Ophthalmology and Visual Sciences, University of Iowa Carver College of Medicine, Iowa, IA
    Howard Hughes Medical Institute, Iowa, IA
  • Carel C B Hoyng
    Ophthalmology, Radboud University Medical Center, Nijmegen, Netherlands
  • Frans Cremers
    Human Genetics, Radboud Universit Medical Centre, Nijmegen, Netherlands
    Ophthalmology, Radboud University Medical Center, Nijmegen, Netherlands
  • Footnotes
    Commercial Relationships Silvia Albert, None; Riccardo Sangermano, None; Nathalie Bax, None; Susanne Roosing, None; L. van den Born, None; Anke den Engelsman-van Dijk, None; Angelique Ramlal, None; Edwin Stone, None; Carel Hoyng, None; Frans Cremers, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 3174. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Silvia Albert, Riccardo Sangermano, Nathalie Bax, Susanne Roosing, L. I. van den Born, Anke den Engelsman-van Dijk, Angelique Ramlal, Edwin M Stone, Carel C B Hoyng, Frans Cremers; Towards the identification of deep-intronic ABCA4 mutations in Stargardt patients by using induced pluripotent stem cell-derived photoreceptor progenitor cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3174.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose: We previously found that ~40% of persons with autosomal recessive Stargardt disease (STGD1) carry only one, and ~10% no ABCA4 mutations. We hypothesized that the lacking mutations are either heterozygous deletions not detected by exon PCR and Sanger sequencing, or located deep in the introns, most likely affecting ABCA4 mRNA. We have shown previously that ABCA4 mRNA is expressed at very low levels in lymphoblasts and fibroblasts but robustly in photoreceptor cells that have differentiated from human stem cells. We propose to perform sequence analysis of ABCA4 mRNA isolated from photoreceptor progenitor cells (PPCs) that were differentiated from reprogrammed patient-specific induced pluripotent stem cells (iPSCs).

Methods: We performed Sanger sequencing of the ABCA4 promoter region in a cohort of 45 patients with STGD1 or STGD1 with cone-rod dystrophy phenotypes carrying one ABCA4 mutation, multiplex ligation-dependent probe amplification (MLPA) of the 50 ABCA4 exons in 27 cases of the cohort, and Sanger sequencing of 20 weak deep-intronic splice sites in 45 cases. Skin biopsy fibroblasts were cultured from eight STGD1 patients and reprogrammed into iPSCs. iPSCs were differentiated into PPCs, and the presence of the ABCA4 transcript was evaluated by quantitative RT-PCR (qRT-PCR) and immunocytochemistry (ICC). The ABCA4 transcript was also analysed by RT-PCR and Sanger sequencing to identify insertions or deletions.

Results: MLPA and Sanger sequencing revealed heterozygous deletions in two maculopathy families, deep-intronic variants in six families, and a combination of a heterozygous deletion and a deep-intronic variant in one family. Patient-derived fibroblasts were successfully reprogrammed into iPSCs, and differentiated into PPCs, in seven and five probands, respectively. qRT-PCR and ICC of the cultured cells confirmed their differentiation into PPCs. The ABCA4 transcripts produced revealed the presence of several abnormal cDNA fragments.

Conclusions: Heterozygous deletions and deep-intronic variants were identified in nine of 45 (20%) maculopathy families illustrating that deep-intronic variants are a significant cause of macular disease. To identify additional deep-intronic variants, we successfully obtained cultures of PPCs that strongly expressed ABCA4 after four weeks of differentiation.

×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×