July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Characterization of raifteirí, a novel zebrafish inherited retinal dystrophy model.
Author Affiliations & Notes
  • Breandan N Kennedy
    Sch of Biomolecular and Biomedical Sci, University College Dublin, Dublin, Ireland
  • Milton English
    National Eye Institute, Bethesda, Maryland, United States
  • Sanamjeet Virdi
    Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Karlsruhe, Germany
  • Margaret Starostik
    Johns Hopkins Institute, Baltimore, Maryland, United States
  • Stephen Carter
    Sch of Biomolecular and Biomedical Sci, University College Dublin, Dublin, Ireland
  • Robert Geisler
    Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Karlsruhe, Germany
  • Alison Reynolds
    Sch of Biomolecular and Biomedical Sci, University College Dublin, Dublin, Ireland
  • Anand Swaroop
    National Eye Institute, Bethesda, Maryland, United States
  • Husvinee Sundaramurthi
    Sch of Biomolecular and Biomedical Sci, University College Dublin, Dublin, Ireland
  • Footnotes
    Commercial Relationships   Breandan Kennedy, None; Milton English, None; Sanamjeet Virdi, None; Margaret Starostik, None; Stephen Carter, None; Robert Geisler, None; Alison Reynolds, None; Anand Swaroop, None; Husvinee Sundaramurthi, None
  • Footnotes
    Support  HRB/MRCG Research Project Grant
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 2348. doi:https://doi.org/
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    • Get Citation

      Breandan N Kennedy, Milton English, Sanamjeet Virdi, Margaret Starostik, Stephen Carter, Robert Geisler, Alison Reynolds, Anand Swaroop, Husvinee Sundaramurthi; Characterization of raifteirí, a novel zebrafish inherited retinal dystrophy model.. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2348. doi: https://doi.org/.

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

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Abstract

Purpose : The zebrafish raifteirí, (raf) model was identified in an N-ethyl-N-nitrosourea mutagenesis screen. raf-/- present with severly compromised visual behavioral. We sought to characterize the raf phenotype and identify the causative gene.

Methods : Electroretinography measure visual capacity. Light, electron and confocal microscopy analyzed retinal histology. RNA isolated from eyes of 5 days old raf larvae (n=5). The RNA samples were processed using ‘TruSeq Stranded mRNA Sample Preparation’ protocol. The cDNA libraries were sequenced by Illumina Sequencer. Data obtained were processed and ran through the Mutation Mapping Analysis Pipeline for Pooled RNA-seq (MMAPPR) pipeline. Genomic DNA was isolated from 20 pooled, 5 days old raf larval samples and whole genome sequencing (WGS) and bioinformatics analysis performed.

Results : raf mutants have an average of 0.2 saccades/min OKR response while siblings have an average of 15 saccades/min. Histological analysis of raf-/- retina revealed the photoreceptor layer was thinner and failure to develop the rod and cone outer segments properly. Additionally, gaps were present in the dorsal and ventral side of the lens and vacuoles were observed within the RPE. Immunostaining with rod and cone photoreceptor specific markers indicated that rod photoreceptors were largely absent in the retina and cone photoreceptors were aberrantly formed in the mutants. RNA sequencing (RNAseq) was performed to identify the ‘raf‘ mutant allele and to understand the molecular mechanisms of disease. The MMAPPR pipeline, was ran to call out single nucleotide polymorphisms (SNPs), insertions and deletions (indels). The results were suggestive of the mutant allele being localized to chromosome 23. Approximately 40 potential candidate genes in chromosome 23 have been identified by variant calling and are currently being validated. A total of 118 differentially expressed genes (with a log foldchange of < and ≥ 2 and an adjusted p value of 0.05) were identified in raf samples. WGS sequencing revealed 477,656 SNPs, indels and structural variants inclusive of intronic regions, which are currently being assessed.

Conclusions : raf mutants are visually impaired and fail to form proper photoreceptor outer segments, indicative of potentially a ciliary mutation. The raf mutant allele is localized to chromosome 23, further analysis needs to be undertaken to identify raf mutant allele.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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