September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Identifying elusive molecular defects underlying inherited retinal diseases employing stem cell technology and transcriptomics
Author Affiliations & Notes
  • Frans P Cremers
    Raboud university medical center, Nijmegen, Netherlands
  • Footnotes
    Commercial Relationships   Frans Cremers, None
  • Footnotes
    Support  Foundation Fighting Blindness USA grant C-GE-0811-0545-RAD01 (to F.P.M.C.), the FP7-PEOPLE-2012-ITN programme EyeTN, agreement 317472 (to F.P.M.C.), the Macula Vision Research Foundation (to F.P.M.C.), and the Nijmeegse Oogonderzoek Stichting (to F.P.M.C. and C.B.H.).
Investigative Ophthalmology & Visual Science September 2016, Vol.57, No Pagination Specified. doi:
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      Frans P Cremers; Identifying elusive molecular defects underlying inherited retinal diseases employing stem cell technology and transcriptomics. Invest. Ophthalmol. Vis. Sci. 201657(12):.

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

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Abstract

Presentation Description : More than 80% of the genes underlying inherited retinal diseases (IRDs) have been identified. Mutation analysis in IRDs thus far has been performed by sequence analysis of protein coding elements. Whole exome sequencing and gene panel-based analysis in this way yielded causal variants in ~65% of IRD cases. In a significant proportion of IRDs, one variant was found in a gene previously implicated in autosomal recessive RD. We hypothesized that many of these cases carry deep-intronic variants that affect RNA splicing. The majority of IRD-associated genes is expressed very low in lymphoblasts and fibroblasts. In order to analyse retina-specific transcripts, we reprogrammed patient-derived fibroblasts to induced pluripotent stem cell, which were then differentiated into photoreceptor progenitor cells (PPCs).
We have focussed these studies on the ABCA4 gene, mutations in which are associated with Stargardt disease (STGD1) and cone-rod dystrophy (CRD). In ~25% of STGD cases, only one causal variant was previously identified. To identify the missing mutations residing in introns we performed gene locus sequencing in 50 cases. In 10 patients with STGD1 or CRD carrying one causal ABCA4 mutation, we cultured PPCs and performed RNA analysis to detect splice defects. Several rare intronic variants were identified, some of which are predicted to create new splice sites. Employing minigene constructs these candidate splicing defects are tested further. RNA analysis of PPCs yielded abnormal cDNA fragments which are being validated. This analysis possibly is hampered by nonsense-mediated decay of mRNAs carrying truncating mutations. In addition, as many of the missing mutations are considered ‘mild’ alleles, their effect on splicing may be very subtle and escape attention by reverse-transcription-PCR analysis. Using this technology we identified the genetic basis of a few frequent non-canonical splice site variants in ABCA4. Deep-intronic splice sites can be targeted by antisense oligonucleotides, thereby restoring normal splicing.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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